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Maintenance of Osteoblastic and Adipocytic Differentiation Potential with Age and Osteoporosis in Human Marrow Stromal Cell Cultures
Abstract
Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the result of enhanced adipogenesis and decreased osteoblastogenesis from the MSCs. Thus, cultures of MSCs were established from young donors (age 18-42, n = 34), elderly healthy donors (age 66-78, n = 20), and patients with OP (age 58-76, n = 15). Cells were cultured for 2 weeks in an adipogenic medium (containing 15% horse serum and 100 nM dexamethasone), osteogenic medium (containing 10% fetal calf serum [FCS] and 10 nM calcitriol), or control medium (10% FCS). The MSCs were identified by their abilities to form colonies. Total number of colonies, osteoblastic colonies stained positive for alkaline phosphatase (AP+), and adipocytic colonies containing adipocytes (Ad+) were quantitated. In addition, steady state mRNA levels of gene markers of adipocytic and osteoblastic phenotypes were determined using reverse-transcriptase polymerase chain reaction (RT-PCR). The adipogenic and osteogenic media induced cell differentiation and the expression of adipocytic and osteoblastic lineage-specific markers, respectively. We found no age-related changes in the osteoblastic or adipocytic colony formation or the steady state levels of mRNA of the adipogenic or osteogenic gene markers. Cells obtained from patients with OP showed a pattern of differentiation similar to those of age-matched controls. In conclusion, MSCs maintain their differentiation potential during aging and in patients with OP. Other mechanisms responsible for age-related decrease in bone formation need to be determined.
... 2,3 Despite the current strong tendency to associate the process of acute bone loss with sex steroid deficiency and the development of osteoporosis, many scholars recognize that bone progenitor cells are also involved in this process. 4 Skeletal homeostasis is maintained by bone marrow mesenchymal stromal cells (BMSCs), 4,5 a major source of bone progenitor cells for renewal due to their multipotent properties and capacity to respond to stimulation by hormones and growth factors. Recent findings suggest that a decline in the quantity or function of BMSCs in the adult skeleton may contribute to human aging and age-related disease. ...
... 20,21 Histomorphometric analyses of bone formation kinetics suggest that osteoblast recruitment from BMSCs is the rate-limiting step and thus plays a great role in age-related bone loss. 4 Thus, understanding the BMSC potentiality in aging individuals may be crucial to understanding the imbalance between bone resorption and bone formation in age-related bone diseases. ...
... Error bars: SD. (c, d) All groups of BMSCs were negative for hematopoietic markers, CD14, CD34, and CD45, and positive for MSC markers, including CD44, CD95, and CD105. BMSC, bone marrow mesenchymal stromal cell; SD, standard deviation.4 WANG ET AL. ...
Bone is a self-renewing tissue. Bone marrow mesenchymal stromal cells (BMSCs) are located in the adult skeleton and are believed to be involved in the maintenance of skeletal homeostasis throughout life. With increasing age, the ability of the skeleton to repair itself decreases, possibly due to the reduced functional capacity of BMSCs. Recent evidence has suggested the existence of at least two populations of BMSCs with different embryonic origins that cannot be interchanged during stem cell recruitment: craniofacial BMSCs (neural crest origin) and appendicular BMSCs (mesoderm origin). Questions arise as to whether the site-specific characteristics alter the effect of aging on the skeleton. In this study, the effects of biological aging on human BMSCs were compared with BMSCs derived from craniofacial bone vs. BMSCs derived from the appendicular skeleton. The phenotype, proliferation and functional characteristics (osteogenic differentiation, cytokine secretion and bone formation in vivo) of the BMSCs were investigated. The results demonstrated that the proliferative capacity and osteogenic differentiation of the BMSCs decrease significantly with age both in vitro and in vivo. For age-matched groups, the osteogenic differentiation capacity of alveolar BMSCs was higher than that of femoral BMSCs in the middle-aged and old groups while there was no significant difference for the young groups. Compared with old alveolar BMSCs, old femoral BMSCs had a significantly longer population doubling time, a smaller colony-forming population and less bone formation in vivo while there was no significant difference for the young and middle-aged groups. Distinct differences in the expression of cytokine factors were also found. In conclusion, human BMSCs display an age-related decrease in functional capacity, and embryonic origins may play a critical role in mediating the aging rate of BMSCs. These data provide novel insights into the skeletal site-specific characteristics of aged BMSCs.
... 8 Some authors documented an inverse relationship between age and CFU-F capacity 9-14 as well as proliferation rate, 11,[15][16][17] while others found no significant evidence for such a relationship. [18][19][20][21] Furthermore, the expression level of some cell surface antigens, such as CD146, 22,23 CD274, 23 SSEA-4 24 and others, 23 was reported to be associated with donor age. ...
... The decline in CFU-F potential of hBMSCs over passages in culture as observed in the present study by comparing CFU-F forming capacity between P1 and P3 is in agreement with studies by other authors and has been associated with accelerated culture-induced in vitro ageing of MSCs, for example by shortening of telomeres and a higher proportion of senescent cells with continued population doublings, as reviewed by Ganguly et al. 1 The present study shows no correlation between donor age and CFU-F capacity, fitting the results obtained by few others. 18 34 This, in turn, might signify that donor age and sex are of less significance when using hBMSCs as therapeutic agents in regenerative strategies. ...
Ageing is often accompanied by an increase in bone marrow fat together with reduced bone volume and diseases of the bone such as osteoporosis. As mesenchymal stem cells (MSCs) are capable of forming bone, cartilage and fat tissue, studying these cells is of great importance to understand the underlying mechanisms behind age-related bone diseases. However, inter-donor variation has been found when handling MSCs. Therefore, the aim of this study was to investigate the effects of donor age and sex by comparing in vitro characteristics of human bone marrow-derived MSCs (hBMSCs) from a large donor cohort (n = 175). For this, hBMSCs were analysed for CFU-F capacity, proliferation, differentiation capacity and surface antigen expression under standardized culture conditions. The results demonstrated a significantly reduced CFU-F number for hBMSCs of female compared to male donors. Furthermore, there was a significant decrease in the proliferation rate, adipogenic differentiation potential and cell surface expression of SSEA-4, CD146 and CD274 of hBMSCs with an increase in donor age. Interestingly, all these findings were exclusive to hBMSCs from female donors. Further research should focus on postmenopausal-related effects on hBMSCs, as the results imply a functional loss and immunophenotypic change of hBMSCs particularly in aged women.
... The influence of donor age on the biological properties of the hBMSCs has been previously examined in both animal and human studies [41], and the reported results have not been consistent. Some studies have reported that the hBMSCs obtained from elderly donors exhibit a reduced expression of osteoblastic gene markers, ALP activity, and decreased mineralized matrix formation [42][43][44], while others reported no age-related effects on the ability of the cells for OB or AD differentiation [23,45,46]. In vivo transplantation studies of hBMSCs have demonstrated similar bone-forming capacity regardless of donor age [25,47,48]. ...
... Based on a number of previous studies, the effect of osteoporosis, on in vitro mineralized matrix formation has not been consistent with some studies reporting no effect [51] or reduced OB differentiation [52][53][54] when comparing cultured hBMSCs isolated from osteoporotic donors and controls. We have also previously reported that hBMSC cultures established from osteoporotic patients exhibited similar proliferation and differentiation capacities as age-matched controls [39,46]. The discrepancy may be explained by the fact that the donor population of the current study exhibited a more severe osteoporotic phenotype and osteoporotic fracture. ...
Background
Transplantation of human bone marrow stromal cells (hBMSCs) is a promising therapy for bone regeneration due to their ability to differentiate into bone forming osteoblastic cells. However, transplanted hBMSCs exhibit variable capacity for bone formation resulting in inconsistent clinical outcome. The aim of the study was to identify a set of donor- and cell-related characteristics that detect hBMSCs with optimal osteoblastic differentiation capacity.
Methods
We collected hBMSCs from 58 patients undergoing surgery for bone fracture. Clinical profile of the donors and in vitro characteristics of cultured hBMSCs were included in uni- and multivariable analysis to determine their predictive value for osteoblastic versus adipocytic differentiation capacity assessed by quantification of mineralized matrix and mature adipocyte formation, respectively.
Results
We identified a signature that explained > 50% of variation in osteoblastic differentiation outcome which included the following positive predictors: donor sex (male), absence of osteoporosis diagnosis, intake of vitamin D supplements, higher fraction of CD146+, and alkaline phosphate (ALP+) cells. With the exception of vitamin D and ALP+ cells, these variables were also negative predictors of adipocytic differentiation.
Conclusions
Using a combination of clinical and cellular criteria, it is possible to predict differentiation outcome of hBMSCs. This signature may be helpful in selecting donor cells in clinical trials of bone regeneration.
... Persisting estrogen deficiency causes significant dysfunction of bone marrow-derived MSCs and depletion of MSC resources [51]. According to the previous study, the MSCs from osteoporotic humans exhibited considerable differences in their cell behaviours such as a decreased proliferation and migration rates compared to age-matched controls [52]. However, cell differentiation capacity of MSCs from osteoporotic patients was not significantly impaired compared to the controls [52]. ...
... According to the previous study, the MSCs from osteoporotic humans exhibited considerable differences in their cell behaviours such as a decreased proliferation and migration rates compared to age-matched controls [52]. However, cell differentiation capacity of MSCs from osteoporotic patients was not significantly impaired compared to the controls [52]. Several previous studies have found that in the role of the bioactive molecules for bone regeneration, PDGF-BB has a major effect on the early cell behaviours, such as proliferation and migration [24,53], whereas BMP-2 has a major effect on the late cell behaviours or osteogenic differentiation [54]. ...
Objectives
We previously suggested an ovariectomy (OVX)-induced osteoporotic rat model showing an impaired alveolar bone defect healing. This study aimed to evaluate and compare the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human platelet-derived growth factor-BB (rhPDGF-BB) on alveolar bone defect healing in OVX-induced osteoporotic rats.Materials and MethodsA total of forty-one female rats were divided into four groups: a collagen group (n=10), a PDGF-BB group (n=11), a BMP-2 group (n=10), and a control group (n=10). Four months after OVX, alveolar bone drill-hole defects were created and grafted with collagen gel, rhPDGF-BB/collagen gel, or rhBMP-2/collagen gel. The defects in the control group were not grafted with any material. Defect healing was evaluated by histological, histomorphometric, and microcomputed tomographic (micro-CT) analyses at 2 and 4 weeks.ResultsAccording to the micro-CT analysis, the BMP-2 group exhibited the greatest bone volume fraction among all groups, while the PDGF-BB group did not show significant differences compared with the collagen group. The histomorphometric analysis showed a significantly larger amount of new bone area in the BMP-2 group than in the control and collagen groups at 4 weeks; however, the PDGF-BB group did not reach significant superiority compared with the other groups.Conclusions
Alveolar bone regeneration was significantly enhanced by the local use of rhBMP-2/collagen gel compared with the use of rhPDGF-BB/collagen gel in OVX-induced osteoporotic rats.Clinical RelevanceA treatment modality using rhBMP-2 may be a promising approach to promote alveolar bone regeneration in patients suffering from postmenopausal osteoporosis.
... Literature reports on age-related declines in CTP prevalence and CTP concentration have been inconsistent. Some studies have demonstrated age-related declines [29][30][31][32][33] , whereas others have not 18,[34][35][36][37][38][39][40] . These discrepancies may be explained by variation in bone marrow harvest location, aspiration technique, processing, or CTP assay as well as the use of small patient cohorts 41,42 . ...
... CTP prevalence and CTP concentration in BMA can be influenced by local bone mass, particularly by the state of active turnover in local bone. Stenderup et al. reported that osteoporosis was not associated with a change in CTPs 34,35 . Other studies have suggested that changes in the biological function of CTPs might lead to osteoporosis 63,64 . ...
Background:
Connective tissue progenitors (CTPs) resident in native tissues serve as biological building blocks in tissue repair and remodeling processes. Methods for analysis and reporting on CTP quantity and quality are essential for defining optimal cell sources and donor characteristics and the impact of cell processing methods for cell therapy applications. The present study examines the influence of donor characteristics and cell concentration (nucleated cells/mL) on CTP prevalence (CTPs/million nucleated cells) and CTP concentration (CTPs/mL) in bone marrow aspirates (BMAs).
Methods:
Iliac crest bone marrow was aspirated from 436 patients during elective total knee or hip arthroplasty. Bone marrow-derived nucleated cells were plated at a density of 1.19 × 105 cells/cm2. Colony-forming unit analysis was performed on day 6.
Results:
Large variation was seen between donors. Age (p < 0.05) and cell concentration (p < 0.001) significantly influenced CTP prevalence and CTP concentration. For every 1-year increase in age, the odds of having at least an average CTP prevalence and CTP concentration decreased by 1.5% and 1.6%, respectively. For every 1 million cells/mL increase in cell concentration, the odds of having at least an average CTP prevalence and CTP concentration increased by 2.2% and 7.9%, respectively. Sex, race, body mass index (BMI), and the presence of osteoporosis did not influence CTP prevalence or CTP concentration.
Conclusions:
BMA-derived CTPs were obtained from all patient groups. CTP prevalence and CTP concentration decreased with age. Cell concentration decreased with age and positively correlated with total CTP prevalence and CTP concentration. The mean CTP concentration in patients >60 years of age was a third of the CTP concentration in patients <30 years of age.
Clinical relevance:
Proper BMA techniques are necessary to obtain a high-quality yield and composition of cells and CTPs. The reduced CTP concentration and CTP prevalence in the elderly may be mitigated by the use of cell processing methods that increase CTP concentration and CTP prevalence (e.g., by removing red blood cells, serum, and non-CTPs or by increasing aspirate volumes). Cell concentration in the BMA can be measured at the point of care and is an appropriate initial assessment of the quality of BMA.
... The literature showed apparent inconsistency in the effect of age on osteoblast differentiation potential of hMSCs. In early studies using the colony assay approach, age-related declines were reported by some investigators [15,16] not found by others [17][18][19], or found only for cells from women by others [20]. Those reports differed in use of marrow aspirates, biopsies, necropsies, frozen cells, growth-factor supplemented media, and different isolation methods and anatomical sites [21]. ...
... There are limited studies comparing hMSCs from osteoporotic (OP) subjects to age-matched controls. Early studies using the colony assay of non-expanded cells reported that hMSCs from iliac crest bone marrow aspirates showed similar proliferative and differentiation capacities whether from OP or age-matched controls [18,19]. Those initial studies were performed on primary cultures of isolated hMSCs and not on passaged cells. ...
Many bone tissue engineering studies use animal or immortalized human mesenchymal stromal cells (hMSCs), which include osteoblast progenitors, because of the abundance of those cells. It is advantageous to study the less plentiful human MSCs in research on bone tissue engineering because of the clinical relevance and because of the importance of discovering how to optimize hMSCs for autogenous use. There is growing evidence that some of the irreproducibility in biological experiments with hMSCs can be attributed to variable clinical characteristics of the subjects from whom they were obtained. Patients who could benefit from bone tissue engineering are likely to have cellular deficits and compromised biochemical milieu, some of which can be managed by customized in vitro treatments. Many donor characteristics are associated with their hMSC in vitro properties. Studies with cohorts with similar characteristics show that some deficits are modifiable in vitro and can be managed with greater understanding of their pathophysiological mechanisms. Even hMSCs from elders can be rejuvenated in vitro with safe agents. Additional value in studying physiology of hMSCs from characterized subjects is to develop rationales for new in vivo therapies to ensure skeletal health throughout the lifespan.
Lay Summary
During this era of intensive tissue engineering research, it is appealing to use human mesenchymal stromal cells (hMSCs) for bone tissue engineering research because of their clinical relevance. Use of the patient’s own cells for therapeutic applications is called autogenous cell-based therapy. Research shows that properties of hMSCs isolated from a subject’s marrow depend on many clinical characteristics and that, in some cases, their osteoblast differentiation potential in cell culture can be optimized safely. These observations also suggest ways to optimize the functions of the skeleton throughout the lifespan.
... In a previous study, the age-associated osteogenic potential of BMSCs was investigated, and a decrease in osteogenic differentiation potential was observed during aging in humans (4). However, another study demonstrated that there were no age-associated changes in the osteoblastic differentiation potential or the steady state levels of messenger RNA (mRNA) of osteogenic gene markers (5). A previous study demonstrated that the capacity of BMSCs to form bone in vivo was maintained with age, which suggests that the observed senescence-associated decrease in bone formation may be due to a defect in bone microenvironment (6). ...
... Furthermore, a reduced chondrogenic and adipogenic differentiation potential was observed in BMSCs isolated from patients with advanced osteoarthritis (9). However, a previous study demonstrated that there were no age-associated changes in the adipocytic colony formation or the steady-state level mRNA expression of adipogenic gene markers in marrow stromal cells isolated from patients with osteoporosis compared with age-matched healthy controls (5). In the current study, no significant differences in the androgenic differentiation potential of BMSCs isolated from participants in the 20s, 30s and 50s age groups were observed. ...
Stem cells have the characteristics of long-term self-renewal and plasticity and the ability to differentiate into specialized cells. Stem cells are widely recognized as potential tools for use in the development of novel therapeutic strategies. The aim of the current study was to investigate the effect of demographic factors on adipogenic and chondrogenic differentiation in bone marrow-derived stem cell (BMSC) spheroids. Age- and gender-associated alterations in the adipogenic and chondrogenic differentiation potential of BMSCs were examined. Human BMSCs were isolated from male and female participants in their 20s, 30s and 50s. Cell morphology and relative values of adipogenesis and chondrogenesis were examined by measuring the relative intensity of oil red O and Alcian blue staining, respectively. Cell morphology alterations in BMSCs isolated from male and female participants in their 20s, 30s and 50s and grown in adipogenic media were very similar. In addition, there were no significant differences in the relative values of adipogenesis in BMSCs for the 20s, 30s and 50s age groups on day 8 and 16. Similarly, no significant differences were observed in the relative values of adipogenesis in BMSCs for the male and female groups on day 8 and 16. Cell morphology changes in BMSCs isolated from male and female participants in their 20s, 30s and 50s and grown in chondrogenic media were very similar. In addition, there were no significant differences in the relative values of chondrogenesis in BMSCs for the 20s, 30s and 50s age groups on day 8, however there was a significant difference observed in the relative values of chondrogenesis in BMSCs on day 16 for the 30s and 50s age groups, compared with the 20s age group. Furthermore, no significant differences were observed in the relative values of adipogenesis in BMSCs for the male and female groups on day 8 and 16. The current study demonstrated that there were no significant differences in the adipogenic and chondrogenic differentiation potential of BMSCs isolated from healthy male donors vs. healthy female donors. Similarly, no significant differences were observed in the adipogenic differentiation potential of BMSCs isolated from different age groups on day 8. However, there was a significant increase in the chondrogenic differentiation potential of BMSCs isolated from participants in their 30s and 50s, compared with BMSCs isolated from participants in their 20s on day 16.
... The ability of MSCs to secrete membrane derived vesicles (MVs), rich in a wide range of growth factors, antiapoptotic factors, and anti-inflammatory molecules, is currently considered as a novel molecular mechanism with significant therapeutic potential [10,11]. Multipotent cells have been isolated from many sources including adipose tissue, bone marrow, and the umbilical cord [12,13]. Traditionally, human MSCs are isolated from an aspirate of bone marrow harvested from the iliac crest or the acetabulum. ...
... Studies conducted over the past few years have suggested that aging impacts various MSC properties [24,25]. This effect is most clearly illustrated by changes in two specific parameters: osteogenic differentiation and proliferation [12,[26][27][28]. However, there is evidence to suggest that the amount of MSCs, their proliferation rate, and population-doubling potential diminish with a patient's age. ...
Tissue regeneration using human adipose derived mesenchymal stem cells (hASCs) has significant potential as a novel treatment for many degenerative bone and joint diseases. Previous studies have established that age negatively affects the proliferation status and the osteogenic and chondrogenic differentiation potential of mesenchymal stem cells. The aim of this study was to assess the age-related maintenance of physiological function and differentiation potential of hASCs in vitro. hASCs were isolated from patients of four different age groups: (1) >20 years (
n
=
7
), (2) >50 years (
n
=
7
), (3) >60 years (
n
=
7
), and (4) >70 years (
n
=
7
). The hASCs were characterized according to the number of fibroblasts colony forming unit (CFU-F), proliferation rate, population doubling time (PDT), and quantified parameters of adipogenic, chondrogenic, and osteogenic differentiation. Compared to younger cells, aged hASCs had decreased proliferation rates, decreased chondrogenic and osteogenic potential, and increased senescent features. A shift in favor of adipogenic differentiation with increased age was also observed. As many bone and joint diseases increase in prevalence with age, it is important to consider the negative influence of age on hASCs viability, proliferation status, and multilineage differentiation potential when considering the potential therapeutic applications of hASCs.
... Studies in older mice [13] and in the senescence-accelerated mouse model (SAMP-6) [29] demonstrated relatively greater adipogenesis and less osteoblastogenesis in murine MPC cultures. Conversely, Justesen et al. 2002 showed that the adipocyte-forming capacity of human MPCs does not change with donor age [30]. However, sera from elderly donors inhibited osteoblast differentiation [31] and enhanced adipogenesis of MPC [32], suggesting that age-related changes in the bone microenvironment may play a role in directing MSC differentiation into osteoblasts and adipocytes. ...
... Studies in older mice [13] and in the senescence-accelerated mouse model (SAMP-6) [29] demonstrated relatively greater adipogenesis and less osteoblastogenesis in murine MPC cultures. Conversely, Justesen et al. 2002 showed that the adipocyte-forming capacity of human MPCs does not change with donor age [30]. However, sera from elderly donors inhibited osteoblast differentiation [31] and enhanced adipogenesis of MPC [32], suggesting that age-related changes in the bone microenvironment may play a role in directing MSC differentiation into osteoblasts and adipocytes. ...
... Although these results differ from the results of previous studies, certain studies support the results of the present study. For example, Justesen et al (42) reported no evidence for enhanced adipogenesis with aging, as the adipocyte forming capacity of BMSCs was similar between younger and older donors (42). ...
... Although these results differ from the results of previous studies, certain studies support the results of the present study. For example, Justesen et al (42) reported no evidence for enhanced adipogenesis with aging, as the adipocyte forming capacity of BMSCs was similar between younger and older donors (42). ...
Bone injury following radiotherapy has been confirmed by epidemiological and animal studies. However, the underlying mechanism remains to be elucidated and no preventive or curative solution has been identified for this bone loss. The present study aimed to investigate the irradiation‑altered osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs). BMSCs were derived and exposed to γ‑irradiation at doses of 0, 0.25, 0.5, 1, 2, 5 and 10 Gy. Cell viability was assessed using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5 diphenyl tetrazolium bromide assay, and clonal expansion in vitro was detected by colony forming unit assessment. The osteogenic differentiation ability was demonstrated by alkaline phosphatase (ALP) activity, ALP staining and mineralization alizarin red staining, and the adipogenic differentiation ability was determined using Oil O red staining. The osteogenesis‑associated genes, RUNX2, ALP, osteocalcin (OCN) and adipogenesis‑associated genes, PPAR‑γ and C/EBPα, were detected using reverse transcription‑quantitative polymerase chain reaction analyses. The protein expression levels of RUNX2, ALP and PPAR‑γ were detected using western blotting. Compared with the control, significant decreases in the proliferation, ALP activity and mineralization ability of the BMSCs were observed in the γ‑irradiation group, with a high level of correlation with the exposure dose. However, no significant changes were observed in the area of Oil red O positive staining. The mRNA levels of RUNX2, ALP and OCN were decreased (P<0.05), however, no significant changes were observed in the levels of C/EBPα and PPAR‑γ. The protein expression levels of RUNX2 and ALP were decreased in the irradiated BMSCs, however, no significant difference was observed in the protein expression of PPAR‑γ. Irradiation inhibited the osteogenic and adipogenic ability of the BMSCs, and the osteogenic differentiation was decreased. The results of the present study provided evidence to assist in further elucidating radiotherapy‑associated side effects on the skeleton.
... The MSCs derived from populations above 60 years show accelerated senescence compared to younger individuals, probably a consequence of the upregulation of gene expression for apoptosis and cellular senescence of MSCs in older subjects. Zhou [37,38]. The BMAC cellular fractions of leukemia patients show lower yields than those of healthy individuals, but no histomorphological changes were observed between leukemia patients and normal individuals [31,39,40]. ...
This study aims to identify the role of subjective factors (age, sex, and comorbidities) and procedure-specific factors (aspiration volume) in influencing the yield of progenitor cells in bone marrow aspiration concentrate (BMAC) harvested from the iliac crest. A retrospective analysis was conducted on 58 patients (male:female = 31:27; mean age: 52.56 ± 18.14 years) who underwent BMAC therapy between January 2020 and June 2021. The factors analyzed include individual factors such as age, sex, and comorbid conditions, and procedural factors such as aspirate volume. The mononuclear cell (MNC) count and colony-forming unit (CFU) assay were used to assess the yield of progenitors in the aspirate. Pearson's correlation test was performed for the age, aspirate volume, and outcome parameters, such as MNC and CFU. We used the chi-square test to analyze the role of sex and comorbidities on cellular yield. The mean volume of aspirate used for BMAC therapy was 66.65 (±17.82) mL. The mean MNC count of the BMAC was 19.94 (±16.34) × 10 6 cells, which formed 11 (±12) CFUs. Evidence of statistically significant positive associations was noted between the CFUs developed from the BMAC and the MNC count within them (r = 0.95, p < 0.001). The sex of the individual did not play any significant role in MNC count (p = 0.092) or CFUs formed (p = 0.448). The age of the individual showed evidence of a statistically significant negative association with the MNC count (r = −0.681, p < 0.001) and CFUs (r = −0.693, p < 0.001), as did the aspiration volume with the MNC count (r = −0.740, p < 0.001) and CFUs (r = −0.629, p < 0.001). We also noted a significant reduction in the MNC count (p = 0.002) and CFUs formed (p = 0.004) when the patients presented comorbidities. Individual factors such as age, comorbid conditions, and procedure factors such as aspirate volume significantly affected the yield of progenitor cells in the BMAC. The sex of the individual did not influence the yield of progenitor cells in BMAC.
... Results from the present study support the hypothesis that increasing donor age is a major variable impacting equine BM-and AT-MSCs in vitro chondrogenic and osteogenic differentiation performance with decreasing capacities following non-linear kinetics, which is comparable to previous reports in other species [21-23, 25, 28-31]. Other research groups have reported no age related changes in MSC differentiation potential [32][33][34], however, their studies differed in culture conditions and lacked very young individuals in the study populations. ...
Background
Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) have shown potential as cell-based therapies for cartilage and bone injuries and are used increasingly in human and veterinary practice to facilitate the treatment of orthopedic conditions. However, human and rodent studies have documented a sharp decline in chondrogenic and osteogenic differentiation potential with increasing donor age, which may be problematic for the important demographic of older orthopedic patients. The aim of this study was to identify the effect of donor age on the chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs in vitro.
BM- and AT-MSCs and dermal fibroblasts (biological negative control) were harvested from horses in five different age groups ( n = 4, N = 60); newborn (0 days), yearling (15–17 months), adult (5–8 years), middle-aged (12–18 years), and geriatric (≥ 22 years). Chondrogenic differentiation performance was assessed quantitatively by measuring pellet size, matrix proteoglycan levels, and gene expression of articular cartilage biomarkers. Osteogenic differentiation performance was assessed quantitatively by measuring alkaline phosphatase activity, calcium deposition, and gene expression of bone biomarkers.
Results
Chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs declined with increasing donor age. BM-MSCs had a higher chondrogenic differentiation performance. AT-MSCs showed minimal chondrogenic differentiation performance in all age groups. For osteogenesis, alkaline phosphatase activity was also higher in BM-MSCs, but BM-MSCs calcium deposition was affected by donor age earlier than AT-MSCs. Chondrogenic and osteogenic differentiation performance of BM-MSCs exhibited a decline as early as between the newborn and yearling samples. Steady state levels of mRNA encoding growth factors, chondrogenic, and osteogenic biomarkers were lower with increasing donor age in both MSC types.
Conclusions
The data showed that chondrogenic and osteogenic differentiation performance of equine BM-MSCs declined already in yearlings, and that AT-MSCs showed minimal chondrogenic potential, but were affected later by donor age with regards to osteogenesis (calcium deposition). The results highlight the importance of donor age considerations and MSC selection for cell-based treatment of orthopedic injuries and will help inform clinicians on when to implement or potentially cryopreserve cells. Moreover, the study provides molecular targets affected by donor age.
... A study using senescent BM-MSCs obtained after long-term culture showed an increased osteogenic differentiation potential after several passages [77]. However, other studies comparing BM-MSCs harvested from young and old donors have shown both a maintenance [78,79] or a decrease in the osteogenic differentiation of oldest BM-MSCs [30,[80][81][82]. In a recent study, authors analyzed the transcriptional profile of freshly isolated BM-MSCs from young and old donors and showed the upregulation of genes implicated in the peroxisome proliferator-activated receptor (PPAR) signaling in the oldest group, suggesting a reinforcement of pro-adipogenic microenvironment with aging [83]. ...
Mesenchymal stromal cells (MSC) are one of the main components of the bone marrow (BM) microenvironment. These cells contribute to homeostasis and modulate the inflammatory response. This regulation is partially mediated by soluble factors and extracellular vesicles (EVs). We aimed to characterize the biological mechanisms regulating the aging of MSC and their immunomodulatory capacities, and their impact on the regulation of macrophage polarization. MSC from BM samples collected from healthy donors aged from 6 months to 85 years were isolated and cultivated in vitro. MSC from aged donors showed a senescence-associated profile: increased expression of markers p16 and p21, production of beta-galactosidase, size and granularity and lower population doubling time. When comparing the expression of genes involved in immunomodulation after inflammatory priming, we observed that aged MSC had a different expression profile for several genes: IL-6, IL-8, TGF-β, GAL1, TSG-6 and COX1. Some of these influence the macrophage polarization. We conducted experiments in which we polarized M0 macrophages towards M1 (with LPS and IFN-γ) or M2 (with IL-4 and IL-13) state in the presence of MSC, using transwells to avoid direct cellular contact: we reported a decreased expression of M1 markers such as TNF-α and CCL2 by macrophages as well as the expression of STAT1, a transcription factor involved in M1 polarization, and to an increased expression of markers of M2 state (TGM2 and IL-10). The inhibition of polarization towards M1 state was significantly lower when using aged MSC. We isolated exosomal miRNA from EVs secreted by young and aged MSC, identifying several miRNAs differentially expressed in the two groups. Among them, miR-193b-3p seems to be particularly interesting, being known to be involved in the regulation of CCL2 and STAT1 expression, two genes expressed by M1 macrophages.
... Besides being an inhibitor of adipogenesis solely, activation of Hh signaling could redirect cell fate, from adipogenic to osteogenic, in mice [73,74]. Since an increase in adipose tissue volume and a decrease in trabecular bone volume in bone marrow has been observed with aging and other osteogenic disorders [75], Hedgehog signaling activators might steer the balance back to bone formation in these conditions. Activation of hedgehog signaling by induced expression of constitutively active Smoothened (SmoM2) or Gli2 (∆NGli2) in the adipocyte lineage of postnatal mice could prevent obesity induced by a high-fat diet, by suppressing WAT and BAT accumulation [70]. ...
Obesity is an increasingly severe public health problem, which brings huge social and economic burdens. Increased body adiposity in obesity is not only tightly associated with type 2 diabetes, but also significantly increases the risks of other chronic diseases including cardiovascular diseases, fatty liver diseases and cancers. Adipogenesis describes the process of the differentiation and maturation of adipocytes, which accumulate in distributed adipose tissue at various sites in the body. The major functions of white adipocytes are to store energy as fat during periods when energy intake exceeds expenditure and to mobilize this stored fuel when energy expenditure exceeds intake. Brown/beige adipocytes contribute to non-shivering thermogenesis upon cold exposure and adrenergic stimulation, and thereby promote energy consumption. The imbalance of energy intake and expenditure causes obesity. Recent interest in epigenetics and signaling pathways has utilized small molecule tools aimed at modifying obesity-specific gene expression. In this review, we discuss compounds with adipogenesis-related signaling pathways and epigenetic modulating properties that have been identified as potential therapeutic agents which cast some light on the future treatment of obesity.
... On the contrary, one study showed MSCs from young and old donors were reported to form similar levels of mineralized matrix in vitro and give rise to similar numbers of adipocytes, indicating that cellular senescence does not affect osteoblastic activity [18]. No age-related changes in gene expression of osteoblastic or adipogenic markers were also observed in a previous study [19]. However, differentiation was not compared between senescent and non-senescent MSCs stimulated with various concentrations of BMP-2. ...
The use of BMP-2 in orthopedic surgery is limited by uncertainty surrounding its effects on the differentiation of mesenchymal stem cells (MSCs) and how this is affected by cellular aging. This study compared the effects of recombinant human BMP-2 (rhBMP-2) on osteogenic and adipogenic differentiation between senescent and non-senescent MSCs. Senescent and non-senescent MSCs were cultured in osteogenic and adipogenic differentiation medium containing various concentrations of rhBMP-2. The phenotypes of these cells were compared by performing a calcium assay, adipogenesis assay, staining, real-time PCR, western blotting, and microarray analysis. rhBMP-2 induced osteogenic differentiation to a lesser extent (P < 0.001 and P = 0.005 for alkaline phosphatase activity and Ca2+ release) in senescent MSCs regardless of dose-dependent increase in both cells. However, the induction of adipogenic differentiation by rhBMP-2 was comparable between them. There was no difference between these two groups of cells in the adipogenesis assay (P = 0.279) and their expression levels of PPARγ were similar. Several genes such as CHRDL1, NOG, SMAD1, SMAD7, and FST encoding transcription factors were proposed to underlie the different responses of senescent and non-senescent MSCs to rhBMP-2 in microarray analyses. Furthermore, inflammatory, adipogenic, or cell death-related signaling pathways such as NF-kB or p38-MAPK pathways were upregulated by BMP-2 in senescent MSCs, whereas bone forming signaling pathways involving BMP, SMAD, and TGF- ß were upregulated in non-senescent MSCs as expected. This phenomenon explains bone forming dominance by non-senescent MSCs and possible frequent complications such as seroma, osteolysis, or neuritis in senescent MSCs during BMP-2 use in orthopedic surgery.
... A study using senescent BM-MSCs obtained after long-term culture showed an increased osteogenic differentiation potential after several passages [77]. However, other studies comparing BM-MSCs harvested from young and old donors have shown both a maintenance [78,79] or a decrease in the osteogenic differentiation of oldest BM-MSCs [30,[80][81][82]. In a recent study, authors analyzed the transcriptional profile of freshly isolated BM-MSCs from young and old donors and showed the upregulation of genes implicated in the peroxisome proliferator-activated receptor (PPAR) signaling in the oldest group, suggesting a reinforcement of pro-adipogenic microenvironment with aging [83]. ...
Simple Summary
As for many other cancers, the risk of developing hematologic malignancies increases considerably as people age. In recent years, a growing number of studies have highlighted the influence of the aging microenvironment on hematopoiesis and tumor progression. Mesenchymal stromal cells are a major player in intercellular communication inside the bone marrow microenvironment involved in hematopoiesis support. With aging, their functions may be altered, leading to hematopoiesis disturbances which can lead to hematologic cancers. A good understanding of the mechanisms involved in mesenchymal stem cell aging and the consequences on hematopoiesis and tumor progression is therefore necessary for a better comprehension of hematologic malignancies and for the development of therapeutic approaches.
Abstract
Aging of bone marrow is a complex process that is involved in the development of many diseases, including hematologic cancers. The results obtained in this field of research, year after year, underline the important role of cross-talk between hematopoietic stem cells and their close environment. In bone marrow, mesenchymal stromal cells (MSCs) are a major player in cell-to-cell communication, presenting a wide range of functionalities, sometimes opposite, depending on the environmental conditions. Although these cells are actively studied for their therapeutic properties, their role in tumor progression remains unclear. One of the reasons for this is that the aging of MSCs has a direct impact on their behavior and on hematopoiesis. In addition, tumor progression is accompanied by dynamic remodeling of the bone marrow niche that may interfere with MSC functions. The present review presents the main features of MSC senescence in bone marrow and their implications in hematologic cancer progression.
... cell to cell communication and communication between bone cells and theirsurrounding environment. Changes in the balance between trabecular bone and fatty tissue, observed in post-menopausal osteoporosis, are thought to arise from a shift in the balance between bone cells and adipocytes(Justesen et al, 2002) and this could be the result of altered cell-matrix communication. Increased resorption of bone by the osteoclast is also associated with osteoporosis. ...
Communication between osteoblasts and the surrounding milieu is important in the regulation of osteoblast behaviour and function. Evidence suggests that the β1 family of integrins is important in the regulation of this relationship. Although trends in integrin expression and function are beginning to emerge, there is little functional evidence using human osteoblasts. In this thesis I have shown, using FACS analysis, that both MG63 cells and mandibular primary human osteoblasts (HOBs) express α2, α3, α4, α5, α6 and β1 integrins. HOBs were also shown to express αv integrin but had low or negative expression of αvβ3. Integrin expression was confirmed by immunocytochemistry. Adhesion assays showed that MG63s and HOBs were adherent to ECM substrates, including plasma fibronectin, type I collagen and the 120kDa plasma fibronectin fragment. Adhesion to plasma fibronectin and type I collagen was reduced by the β1 integrin blocking antibody. RGD blocking peptide reduced adhesion to plasma fibronectin. Boyden chamber migration assays showed that MG63s were highly migratory after 2 hours on plasma fibronectin, type I collagen and the 120kDa plasma fibronectin fragment. HOBs were also migratory but with lower cell numbers migrating. Addition of the α2β1 and α5β1 blocking antibodies resulted in the partial inhibition of MG63 cell and HOB migration on type 1 collagen and plasma fibronectin respectively. Incubation of cells with the MAPKK inhibitor, U0126, also reduced migration of MG63 cells on plasma fibronectin. Preliminary studies into the function of integrins in the differentiation of the C2C12 stromal cell-line was also carried out. This study provides the first evidence for the involvement of integrins and ECM interaction in osteoblast migration. In addition further evidence for the role of integrins in osteoblast behaviour is reported.
... Previous reports showed that donor age did not affect the ability of the cells to form bone in vivo [41] and in earlypassage cultures of MSC obtained from young and old donors treated with osteogenic differentiation medium [48]. However, in iPS-MSC lines, osteogenic differentiation varied [17]. ...
Aging is a predominant risk factor for many chronic conditions. Stem cell dysfunction plays a pivotal role in the aging process. Prelamin A, an abnormal processed form of the nuclear lamina protein lamin A, has been reported to trigger premature senescence. However, the mechanism driving stem cell dysfunction is still unclear. In this study, we found that while passaging subchondral bone mesenchymal stem cells (SCB-MSCs) in vitro , prelamin A accumulation occurred concomitantly with an increase in senescence-associated β -galactosidase (SA- β -Gal) expression. Unlike their counterparts, SCB-MSCs with prelamin A overexpression (MSC/PLA) demonstrated decreased proliferation, osteogenesis, and adipogenesis but increased production of inflammatory factors. In a hind-limb ischemia model, MSC/PLA also exhibited compromised therapy effect. Further investigation showed that exogenous prelamin A triggered abnormal nuclear morphology, DNA and shelterin complex damage, cell cycle retardation, and eventually cell senescence. Changes in gene expression profile were also verified by microarray assay. Interestingly, we found that ascorbic acid or vitamin C (VC) treatment could inhibit prelamin A expression in MSC/PLA and partially reverse the premature aging in MSC/PLA, with reduced secretion of inflammatory factors and cell cycle arrest and resistance to apoptosis. Importantly, after VC treatment, MSC/PLA showed enhanced therapy effect in the hind-limb ischemia model. In conclusion, prelamin A can accelerate SCB-MSC premature senescence by inducing DNA damage. VC can be a potential therapeutic reagent for prelamin A-induced aging defects in MSCs.
... The idea that BM MSCs might "age" in vivo is also supported by the fact that several diseases of the musculoskeletal system such as osteoporosis and osteoarthritis are significantly more prevalent in older individuals [18,19]. To test whether BM MSC numbers decline with age, many studies performed the classical colony-forming unitfibroblast (CFU-F) assay, which has produced, over the years, somewhat conflicting results: decline [20][21][22] or no decline [23][24][25], which can be explained by different protocols used for BM aspiration and processing, as well as variable assay conditions and the differences in colony scoring methods. Even with the assumption that BM MSCs decline with age, it remains unclear whether this possible reduction is due to the preferential loss of the most proliferative cells or is a result of the nonspecific decline in all types of colonyforming progenitors. ...
Uncultured mesenchymal stromal cells (MSCs) are increasingly used in therapies; however, the effects of donor age on their biological characteristics and gene expression remain unclear. The aim of this study was to investigate age-related changes in bone marrow (BM) MSCs following minimal or no culture manipulation. Iliac crest BM was aspirated from 67 healthy donors (19-89 years old) and directly used for the colony-forming unit-fibroblast (CFU-F) assay or CD45lowCD271+ cell enumeration. The colonies were analysed for colony area and integrated density (ID) when grown in standard MSC media or media supplemented with human serum from young (YS) or old (OS) donors. There was a notable age-related decline in the number of MSCs per millilitre of BM aspirate revealed by the CFU-F assay (r=−0.527, p
... Numerous bone marrow derived therapies are being used for musculoskeletal applications without any assessment of their composition at the time of delivery [1,[3][4][5]25]. The quality and composition of BMA has been found to be influenced by a number of factors including patient variables [26,27], aspiration volume per site [28], needle type [29], physician experience level, etc. [30]. Thus it becomes essential to assess the composition and quality of the BMA ...
... Cela a conduit Kim et ses collaborateurs à émettre le concept que l'inhibition de l'adipogenèse pourrait améliorer la formation osseuse(Kim et al., 2012). Les modifications observées au niveau des propriétés des CSM ne seraient non pas une conséquence directe du vieillissement des cellules mais plutôt un conditionnement dû à la modification de la composition du microenvironnement de la MO (i.e., hormones, facteurs de croissance, acides gras)(Justesen et al., 2002). Leurs résultats indiquaient que l'âge des CSM in vitro (nombre de passages en culture) impacterait plus fortement les propriétés des CSM que leur âge in vivo(âge du donneur). ...
Ce travail de thèse avait pour objectif le développement de systèmes de culture cellulaire, en 2D et en 3D, en mettant à profit les propriétés d’un transporteur d’oxygène marin, HEMOXCell®. Notre approche générale était articulée selon deux grands axes : un premier concernant l’évaluation de l’utilisation d’HEMOXCell® dans la culture de deux modèles cellulaires, et un second, utilisant les résultats obtenus à des fins d’ingénierie tissulaire. Dans le premier axe, l’évaluation de l’effet dose-réponse d’HEMOXCell® dans la culture des cellules CHO-S et des cellules souches mésenchymateuses (CSM), a permis de déterminer des concentrations de travail optimales, favorisant la viabilité et la prolifération cellulaire. Le modèle cellulaire CHO-S a contribué à la mise en place d’un test de performance de la molécule, et encouragé son utilisation dans des systèmes de bioproduction. Les essais menés sur les CSM ont quant à eux permis de valider l’innocuité de la molécule à de faibles doses et le maintien de l’état « souche ». L’idée d’associer les CSM à des supports poreux est prometteuse pour des applications d’ingénierie tissulaire, mais est soumise aux problèmes liés à l’oxygénation en profondeur des supports. Dans le second axe de ce projet, nous avons oeuvré à améliorer la colonisation de substituts osseux et méniscaux, en culture statique et dynamique, en présence d’HEMOXCell®. Parallèlement, une étude a été menée pour tenter de caractériser les cellules méniscales. Les analyses de la colonisation des biomatériaux suggèrent un effet bénéfique d’HEMOXCell® lorsqu’il est utilisé en complément des milieux de différenciation cellulaire. Ce travail a contribué à améliorer la compréhension de ce transporteur d’oxygène et à l’élargissement de ses potentiels champs d’utilisation notamment dans un cadre thérapeutique.
... Zhang et al [43] reported that osteogenic differentiation capacity of bone marrow-derived MSCs from mice increases in an age-dependent manner to 18 mo of age and decreases rapidly thereafter. In contrast to these studies, other groups found the MSCs maintained their differentiation potential even in aged donors [53,54] . There is also disagreement in the literature on whether age has an effect on the chondrogenic potential of MSCs. ...
Successful fracture healing requires the simultaneous regeneration of both the bone and vasculature; mesenchymal stem cells (MSCs) are directed to replace the bone tissue, while endothelial progenitor cells (EPCs) form the new vasculature that supplies blood to the fracture site. In the elderly, the healing process is slowed, partly due to decreased regenerative function of these stem and progenitor cells. MSCs from older individuals are impaired with regard to cell number, proliferative capacity, ability to migrate, and osteochondrogenic differentiation potential. The proliferation, migration and function of EPCs are also compromised with advanced age. Although the reasons for cellular dysfunction with age are complex and multidimensional, reduced expression of growth factors, accumulation of oxidative damage from reactive oxygen species, and altered signaling of the Sirtuin-1 pathway are contributing factors to aging at the cellular level of both MSCs and EPCs. Because of these geriatric-specific issues, effective treatment for fracture repair may require new therapeutic techniques to restore cellular function. Some suggested directions for potential treatments include cellular therapies, pharmacological agents, treatments targeting age-related molecular mechanisms, and physical therapeutics. Advanced age is the primary risk factor for a fracture, due to the low bone mass and inferior bone quality associated with aging; a better understanding of the dysfunctional behavior of the aging cell will provide a foundation for new treatments to decrease healing time and reduce the development of complications during the extended recovery from fracture healing in the elderly.
... It has been reported that senior citizens with osteoporosis have more adipogenesis in their bone marrow cavities [20], researchers have also discovered that aging is accompanied by decreased bone regeneration and increased adipocyte differentiation [36]. This imbalance between bone marrow adipogenesis and osteogenesis is due to BMSC aging [37]. In this study, we demonstrated that secretome could modulate cell differentiation by promoting osteogenesis and inhibiting adipogenesis. ...
Senile osteoporosis is closely related to the loss of function of stem cells. In this study, we tried to investigate the potential of secretome from human umbilical cord-derived mesenchymal stem cells (hUCMSCs) in recovering stem cell ability from senescence and then delaying bone loss. We first harvested bone marrow-derived mesenchymal stem cells (BMSCs) from young and old rats and then compared their cellular characteristics such as cell growth, anti-senescence and differentiation. The results showed that these abilities were negatively affected by animal aging. Subsequently, aged BMSCs were exposed to secretome from hUCMSCs, and we found that this loss of cell potential can be modified by secretome treatment. Thereafter, the secretome was loaded into silk fibroin-based hydrogels and used for an in vivo animal study. The results showed that compared to the old untreated group, the bone formation capacity of aged rats was improved by local treatment of secretome-loaded silk fibroin hydrogels. In conclusion, these findings demonstrated that secretome from hUCMSCs has the capacity to recover stem cell potential and delay local bone loss in age-related osteoporosis, which could potentially be applied in osteoporosis therapy in the future.
... In the literature, conflicting results have been reported. While some studies did not find any evidence for age related differences in proliferation capacity of hMSC [36][37][38][39], others demonstrated a decreased proliferation capacity with aging [15][16][17]20,40,41]. On closer examination, the observed age-related decline takes place during childhood and early adolescence. ...
Human mesenchymal stromal cells (hMSCs) are the cellular source of new bone formation and an essential component of autologous bone grafts. Autologous bone graft harvesting is routinely conducted at the iliac crest, although alternative donor sites with lower complication rates are available. Thus, the aim of this study was to compare hMSCs harvested from the iliac crest and the proximal tibia regarding their proliferative and osteogenic differentiation capacity. Furthermore, we investigated the influence of donor age on these biological properties.
HMSCs were isolated from iliac crest or proximal tibia bone grafts of 46 patients. Proliferative capacity was assessed by cumulative population doublings, population doubling time, colony forming units and cell proliferation assays. Osteogenic capacity was assessed by quantification of extracellular calcium deposition and marker gene expression levels. The number of hMSCs per gram harvested tissue was determined. Furthermore, the adipogenic and chondrogenic differentiation capacity were quantified using BODIPY and Safranin Orange staining, respectively. Additional analyses were carried out after grouping young (18–49 years) and aged (≥50 years) donors.
HMSCs derived from the proximal tibia featured a comparable proliferative and osteogenic differentiation capacity. No significant differences were found for any analysis conducted, when compared to hMSCs obtained from the iliac crest. Furthermore, no significant differences could be revealed when comparing young and aged donors. This was equally true for hMSCs from both donor sites after comparison within the same age group.
Our study demonstrates comparable biological properties of hMSCs derived from both donor sites, the iliac crest and the proximal tibia. Furthermore, aging does not alter proliferative and osteogenic differentiation capacity. Consequently, the proximal tibia should be considered more closely as an alternative donor site in patients of all age groups.
... Other studies showed slight agedependent differences in CFU-f formation for BMSCs isolated from young and old donors, but none of them was statistically significant 19,22) . Likewise, no significant age-dependent difference in the cumulative population doublings was observed, which is in accordance with some published characterizations of BMSCs 23,24) . High donor variability for human BMSCs was demonstrated previously, suggesting that other patient-specific factors might influence the regenerative potential of MSCs to a greater extent than age [25][26][27] . ...
Purpose:
Mesenchymal stem cells (MSCs) isolated from the anterior cruciate ligament (ACL) share multiple characteristics of bone marrow-derived mesenchymal stem cells (BMSCs), allowing their use for regenerative therapies. Injuries to the ACL can affect people of all ages. This study assesses whether the regenerative potential of ACL-derived MSCs (ACL-MSCs) from old donors is as high as the potential of ACL-MSCs from young donors.
Materials and methods:
ACL-MSCs were isolated from ACL tissues obtained from young and old donors at the time of ACL reconstruction or arthroplasty. Proliferative capacity, multilineage differentiation potential (chondrogenic, osteogenic, and adipogenic lineages), and transcriptome-wide gene expression were assessed and compared between young and old donors. BMSCs of middle-aged donors served as an additional comparator.
Results:
No substantial differences between ACL-MSCs from young and old donors were observed in their proliferative capacity and multilineage differentiation potential. The latter did not substantially differ between both ACL-MSC groups and BMSCs. Differential expression of genes related to the cytoskeleton and to protein dephosphorylation amongst other pathways was detected between ACL-MSCs from young and old donors.
Conclusions:
Regenerative potential of ACL-MSCs from old donors was not substantially lower than that from young donors, suggesting that regenerative therapies of ACL tears are feasible in both age groups.In vivostudies of the effect of age on the efficacy of such therapies are needed.
... Initial passages of cultured MSCs retain their characteristic spindle-like morphology, but the cells after a few PDs appear enlarged and more granular. 23,38,39 The relative area of BM-MSCs in the late passages increases over 10-fold compared to early passages (from 5 mm 2 up to 50 mm 2 , respectively). 40 This increase in size appears to parallel with the increase in actin stress filaments. ...
Aging at the cellular level is a complex process resulting from accumulation of various damages leading to functional impairment and a reduced quality of life at the level of the organism. With a rise in the elderly population, the worldwide incidence of osteoporosis (OP) and osteoarthritis (OA) has increased in the past few decades. A decline in the number and “fitness” of mesenchymal stromal cells (MSCs) in the bone marrow (BM) niche has been suggested as one of the factors contributing to bone abnormalities in OP and OA. It is well recognized that MSCs in vitro acquire culture-induced aging features such as gradual telomere shortening, increased numbers of senescent cells, and reduced resistance to oxidative stress as a result of serial population doublings. In contrast, there is only limited evidence that human BM-MSCs “age” similarly in vivo. This review compares the various aspects of in vitro and in vivo MSC aging and suggests how our current knowledge on rejuvenating cultured MSCs could be applied to develop future strategies to target altered bone formation processes in OP and OA.
... Advanced age is known to have detrimental effects on blood and BM-MSCs [87][88][89]. In contrast, ADSC yield seemed to be stable across age groups in 12 of 16 LOE-2 studies included in this review ( Table 1). ...
Background:
The applications for fat grafting have increased recently, within both regenerative and reconstructive surgery. Although fat harvesting, processing and injection techniques have been extensively studied and standardised, this has not had a big impact on the variability of outcome following fat grafting. This suggests a possible larger role of patient characteristics on adipocyte and adipose-derived stem cell (ADSC) viability and function. This systematic review aims to collate current evidence on the effect of patient factors on adipocyte and ADSC behaviour.
Methods:
A systematic literature review was performed using MEDLINE, Cochrane Library and EMBASE. It includes outcomes observed in in vitro analyses, in vivo animal studies and clinical studies. Data from basic science work have been included in the discussion to enhance our understanding of the mechanism behind ADSC behaviour.
Results:
A total of 41 papers were included in this review. Accumulating evidence indicates decreased proliferation and differentiation potential of ADSCs with increasing age, body mass index, diabetes mellitus and exposure to radiotherapy and Tamoxifen, although this was not uniformly seen across all studies. Gender, donor site preference, HIV status and chemotherapy did not show a significant influence on fat retention. Circulating oestrogen levels have been shown to support both adipocyte function and graft viability. Evidence so far suggests no significant impact of total cholesterol, hypertension, renal disease, physical exercise and peripheral vascular disease on ADSC yield.
Conclusions:
A more uniform comparison of all factors highlighted in this review, with the application of a combination of tests for each outcome measure, is essential to fully understand factors that affect adipocyte and ADSC viability, as well as functionality. As these patient factors interact, future studies looking at adipocyte viability need to take them into consideration for conclusions to be meaningful. This would provide crucial information for surgeons when deciding appropriate volumes of lipoaspirate to inject, improve patient selection, and counsel patient expectations with regards to outcomes and likelihood for repeat procedures. An improved understanding will also assist in identification of patient groups that would benefit from graft enrichment and cryopreservation techniques.
... ⁎ p b 0.05. Yet, several authors have shown that MSC from persons of different age exhibit similar differentiation potency ( Justesen et al., 2002), albeit with differences in gene expression ( Wagner et al., 2009). Interestingly, it has been found that MSC from old rats increased proliferation and osteogenic differentiation, reducing the adipogenic one, when grown in presence of serum from young rats ( Geißler et al., 2013). ...
Aging may enhance both oxidative stress and bone-marrow mesenchymal stem-cell (MSC) differentiation into adipocytes. That reduces osteoblastogenesis, thus favoring bone-mass loss and fracture, representing an important worldwide health-issue, mainly in countries with aging populations. Intake of antioxidant products may help to retain bone-mass density. Interestingly, a novel olive-pomace physical treatment to generate olive oil also yields by-products rich in functional antioxidants. Thus, diet of postmenopausal women was supplemented for two months with one of such by-products (distillate 6; D6), being rich in squalene. After treatment, serum from such women showed reduced both lipidic peroxidation and oxidized low-density lipoprotein (LDL). Besides, vitamin E and coenzyme Q10 levels increased. Furthermore, culture medium containing 10% of such serum both increased osteoblastogenesis and reduced adipogenesis in human MSC from bone marrow. Therefore, highly antioxidant by-products like D6 may represent a relevant source for development of functional products, for both prevention and treatment of degenerative pathologies associated with aging, like osteoporosis.
... Ahol különbséget találtak, ott 0-18 év, ill. 7-18 év közötti fiatal korosztályokat vizsgáltak a különböző kutatócsoportok (32,4,60,61). ...
In the first part of the two-part article series, based on the literature and their own research and experience, the authors introduce the stem cell types, detailed characteristics of mesenchymal stem cells, as well as the possibility of their veterinary uses and experimental results, particulary regarding the donor's age and the advantages and disadvantages of using allogene or autologue cell source for medical purposes. In the second part their own experimental results presented, the proliferative and the osteogenic differentiation capacity of adipose-derived mesenchymal stem cells from young and older dogs are demonstrated.
... Para expansão, é necessário isolá-las e purificá-las, sendo que ao se multiplicarem em cultura, caracterizam--se por apresentarem aderência ao plástico, ter aspecto de fibroblastos e se diferenciar em células distintas (Yu et al. 2004). Após o isolamento e expansão podem se diferenciar em osteoblastos (Kassel, Roskilde & Erikson 1993), músculo esquelético (Sampaolesi et al. 2005), endotélio (Abe et al. 2004), cardiomiócitos (Lima, Soares & Santos 2009), hepatócitos (Justesen et al. 2002), adipócitos (Jiang et al. 2002), tecido tendíneo (Kwan, Wan & Longaker 2003) e neurônios (Mendez-Otero et al. 2009). ...
The objective of this study was to describe guidelines for the isolation of bone marrow mononuclear cells from rabbits, followed by cell purification by negative depletion with CD45 monoclonal antibody, and further expansion in MesenCult® medium. Ten adult male New Zealand White rabbits, age average of 1.0±0.2 years and weighting 3.5±0.24kg, were used to obtain a standardized method. The mononuclear cells of the bone marrow were isolated with Ficoll-paque® density gradient centrifugation, and the cell purification and acquisition was completed by negative depletion with CD45 monoclonal antibody in immunomagnetic base. The cell population obtained was expanded in MesenCult® medium. Through isolation with Ficoll-paque® density gradient was possible to obtain an average yield of 7.31x106 cells/mL. After purification and acquisiton of potential mesenchymal stem cells by the immunomagnetic base, there was a yield decrease to 2.28x106 cells/mL; however the expansion process was increased in cell culture. The results indicated that cells obtained from the mononuclear fraction of bone marrow and cultivated in vitro were capable to generate adherent cells 24 hours after culture, with predominance of fibroblastoid cells suggestive of mesenchymal stem cells. It can be concluded that mesenchymal stem cells can be achieved with purified rabbit bone marrow mononuclear cells through the immunomagnetic method, as the MesenCult® medium provides a suitable environment for a quick in vitro expansion, and the number of passages exerts negative influence on the morphological characteristics.
... Likewise, in humans, the number of CFU-F decreases during the fi rst decade of life (39). In the later decades of life, between the ages of 20 to 70, the number of CFU-F remains relatively constant (40,41). In conclusion, human studies show that with aging there is maintenance of CFU-F cell population size in the bone marrow, and that the observed decline in the number of CFU-F in early adulthood may represent changes in the skeletal dynamics from a modeling mode characteristic of skeletal growth and consolidation to a remodeling dynamic characteristic of the adult skeleton. ...
This chapter will provide a general overview of the aging process followed by the potential effect that aging may have in bone biology. Three important aspects will be considered: decreased number of osteoblasts, increasing adipogenesis and significant osteoblast/osteocytes apoptosis during the aging process in bone. Other aspects of bone aging has been addressed in recent reviews [1, 2].
... Ahol különbséget találtak, ott 0-18 év, ill. 7-18 év közötti fiatal korosztályokat vizsgáltak a különböző kutatócsoportok (32,4,60,61). ...
SUMMARY
In the first part of the two-part article series, based on the literature and their
own research and experience, the authors introduce the stem cell types, detailed
characteristics of mesenchymal stem cells, as well as the possibility of their veterinary
uses and experimental results, particulary regarding the donor's age and
the advantages and disadvantages of using allogene or autologue cell source for
medical purposes. In the second part their own experimental results presented,
the proliferative and the osteogenic differentiation capacity of adipose-derived
mesenchymal stem cells from young and older dogs are demonstrated.
... 1), co interpretowano jako wynik zmniejszania siê ogólnej liczby komórek mezenchymalnych w szpiku kostnym [10,54]. Z drugiej strony istniej¹ badania, w których nie zaobserwowano ró¿nic pomiêdzy wiekiem dawcy komórek a zdolnoci¹ do tworzenia kolonii in vitro [32]. Nale¿y podkreliae, ¿e ocena morfologii jest trudna w przypadku MSC, gdy¿ komórki te charakteryzuj¹ siê ró¿norodnoci¹ nie tylko miedzygatunkow¹, ale tak¿e miêdzyosobnicz¹. ...
Mesenchymal stem cells (MSC)are considered to be a promising too] for cellular transplantations This population of cells can find potential applications in multiple clinical disorders as a stimulant for regeneration and/or an immuno-modulator for the treated tissue As adult stem cells. MSC allow performance of autologous transplantations Therefore. it is crucial to understand the relationships between ageing and properties of this cell population. This review presents the current knowledge of morphological and Functional changes in bone marrow-derived MSC during their ageing both in vivo and in vitro. This review discusses the mechanisms of cell ageing, such as telomere shortening or free radical effects oil MSC. along with the susceptibility of cells to negative environmental conditions. depending on donor's age The analyzed results often differ from each other. but the majority in vitro studies indicate that the donor's age has an impact on mesenchymal cell properties The data suggest that cells derived from aged individuals display a lower therapeutic potential then the cells isolated from voting organisms Very few clinical trials conducted in humans do not allow drawing, conclusions about the relationship between the donor's age and the transplantation outcome
... The differentiation capacity of human BM-MSCs (hBM-MSCs) is related to in vivo and in vitro BM-MSC aging [5]. Loss of MSC osteogenic and adipogenic potential with aging has been demonstrated in vitro [6,7], but no significant differences in osteogenic and adipogenic potentials were detected in aged versus young MSC in vivo [7,8]. Regarding in vitro chondrogenic differentiation, the potential of hBM-MSCs was enhanced using cells at passages only between 3 and 6, indicating that this type of mesengenic differentiation is strictly influenced by a limited range of culture passage [9]. ...
Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-term
in vitro
culture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions for
in vitro
expansion in order to minimize these epigenetic changes and to standardize safer procedures.
... The occurrence of lipid droplets is compatible with the fact that MSCs have an adipogenic differentiation potential under cell culture conditions per se. This may correlate with the circumstance that in aging, the risk of bone fractures is associated with a progressive decrease of osteoblasts and an increase of adipocytes (Jilka et al., 1996;Justesen et al., 2002). Especially in osteoporotic patients, the decreased osteoblast formation is correlated with an increased adipocyte formation (Beresford et al., 1992;Nuttall et al., 1998;Zhang et al., 2006). ...
Objective: Within the past few years, bone marrow derived mesenchymal stem cells have been used for tissue engineering applications in the treatment of orthopaedic diseases such as lesions of the tendons. As affected animals are routinely pretreated with nonsteroidal antiphlogistics in order to prevent inflammation and pain, the aim of this study was to investigate the influence of NSAID on equnie mesenchymal stem cells (eMSC) in vitro in regards to cell viability, proliferation and differentiation. Materials and methods: After incubation of eMSC with the NSAIDs Flunixine, Phenylbutazone and Meloxicam, cell vitality, cell proliferation and growth characteristics were analysed. By cultivating eMSC in specific induction media for the adipogenic, chon- drogenic and osteogenic differentiation and using histological staining methods, the influence of NSAID on the differentiation potential could be evaluated. Results: Of the three NSAIDs included in this study in therapeutically relevant concentrations, only Flunixine exerted an in- crease of the generation time and thus an inhibition on cell proliferation. Furthermore, Flunixine and Metacam induced an inhibitory effect on the osteogenic differentiation potential, while the adipogenic and chondrogenic differentiation potential remained unaltered by all three NSAIDs. Conclusion: Apart from Flunixine, the tested NSAIDs have no noteworthy influence on cell vitality and proliferation. The osteogenic differentiation of stem cells is liable to high variations, which has also been confirmed with previous data. Clinical relevance: The selection of NSAID used in the equine practice in conjunction with an eMSC treat- ment has to be critically questioned. A pretreatment with Flunixine should be avoided.
... Accumulated data indicate that MSCs from elderly donors or long-term ex vivo cultivation show great difference compared with that of young donors in cell morphology, proliferation potential, differentiation potential, telomerase length and activity, as well as special molecular markers (5,6). Moreover, several researchers found that passages of in vitro culture share equal importance with donor age when considering the proliferative and differential properties of MSCs (7,8). The decline of regenerative capacity of MSCs is predicted to be caused by cellular ageing (9). ...
Mesenchymal stem cells (MSCs) represent an ideal source of autologous cell-based therapy for chronic wounds. Functional characteristics of MSCs may benefit wound healing by exerting their multi-regenerative potential. However, cell ageing resulting from chronic degenerative diseases or donor age could cause inevitable effects on the regenerative abilities of MSCs. A variety of studies have shown the relationship between MSC ageing and age-related dysfunction, but few associate these age-related impacts on MSCs with their ability of repairing chronic wounds, which are common in the elderly population. Here, we discuss the age-associated changes of MSCs and describe the potential impacts on MSC-based therapy for chronic wounds. Furthermore, critical evaluation of the current literatures is necessary for understanding the underlying mechanisms of MSC ageing and raising the corresponding concerns on considering their possible use for chronic wound repair.
... However, invasive donation procedure (although with reduced donor site pain in comparison to traditional autologous bone grafting), as well as reports concerning age-related deterioration in proliferation and differentiation capabilities, may hinder its clinical use (Gimbel et al., 2007;Wu et al., 2014). First studies did not show any differences concerning BM-MSC potential and aging (Stenderup, Justesen, Eriksen, Rattan, & Kassem, 2001;Justesen, Stenderup, Eriksen, & Kassem, 2002;Leskelä et al., 2003), but more recent papers on both human and animal BM-MSC showed that the stem cells do undergo the process of senescence and thus their differentiation and proliferation capabilities decrease with the donor age (Kretlow, Jin, Liu, Zhang, Hong, & Zhou, 2008). Bellows et al., (Bellows, Pei, Jia, & Heersche, 2003) proved reduced selfrenewal capability of rat osteoprogenitors, while Tokalov et al. (2007) showed age-related decrease of MSC population on a rat model. ...
Achieving a successful and well-functioning reconstruction of craniofacial deformities still remains a challenge. As for now, autologous bone grafting remains the gold standard for alveolar cleft reconstruction. However, its aesthetic and functional results often remain unsatisfactory, which carries a long-term psychosocial and medical sequelae. Therefore, searching for novel therapeutic approaches is strongly indicated. With the recent advances in stem cell research, cell-based tissue engineering strategies move from the bench to the patients' bedside. Successful stem cell engineering employs a carefully selected stem cell source, a biodegradable scaffold with osteoconductive and osteoinductive properties, as well as an addition of growth factors or cytokines to enhance osteogenesis. This review highlights recent advances in mesenchymal stem cell tissue engineering, discusses animal models and case reports of stem cell enhanced bone regeneration, as well as ongoing clinical trials.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Changes in bone architecture and metabolism with aging increase the likelihood of osteoporosis and fracture. Age-onset osteoporosis is multifactorial, with contributory extrinsic and intrinsic factors including certain medical problems, specific prescription drugs, estrogen loss, secondary hyperparathyroidism, microenvironmental and cellular alterations in bone tissue, and mechanical unloading or immobilization. At the histological level, there are changes in trabecular and cortical bone as well as marrow cellularity, lineage switching of mesenchymal stem cells to an adipogenic fate, inadequate transduction of signals during skeletal loading, and predisposition toward senescent cell accumulation with production of a senescence-associated secretory phenotype. Cumulatively, these changes result in bone remodeling abnormalities that over time cause net bone loss typically seen in older adults. Age-related osteoporosis is a geriatric syndrome due to the multiple etiologies that converge upon the skeleton to produce the ultimate phenotypic changes that manifest as bone fragility. Bone tissue is dynamic but with tendencies toward poor osteoblastic bone formation and relative osteoclastic bone resorption with aging. Interactions with other aging physiologic systems, such as muscle, may also confer detrimental effects on the aging skeleton. Conversely, individuals who maintain their BMD experience a lower risk of fractures, disability, and mortality, suggesting that this phenotype may be a marker of successful aging. © 2023 American Physiological Society. Compr Physiol 13:4355-4386, 2023.
Our previous study revealed that 3T3-L1 preadipocytes can differentiate to either osteoblasts or adipocytes in response to bone morphogenic protein 9 (BMP9). In the present study, we try to further investigate whether the Wnt/β-catenin signaling plays a crucial role in this process. It was found that BMP9 effectively activated the Wnt/β-catenin signaling, and induced the expression levels of certain canonical Wnt ligands and their receptors in preadipocytes. Exogenous expression of β-catenin, Wnt1, Wnt3a, and Wnt10b potentiated BMP9-induced alkaline phosphatase (ALP) activity, while β-catenin knockdown or Dickkopf 1 (Dkk1) diminished BMP9-induced ALP activity. Moreover, it was demonstrated that β-catenin overexpression promoted BMP9-induced mineralization, and increased the expression levels of late osteogenic markers osteopontin and osteocalcin. Furthermore, β-catenin inhibited BMP9-induced lipid accumulation and the adipogenic marker adipocyte fatty acid binding protein (aP2). The cell-implantation assay results identified that β-catenin not only augmented BMP9-induced ectopic bone formation, but also blocked adipogenesis in vivo. Mechanistically, it was found that β-catenin and BMP9 synergistically stimulated the osteogenic transcription factors runt-related transcription factor 2 (Runx2) and Osterix (OSX). However, BMP9-induced adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα), were inhibited by β-catenin. Therefore, these findings suggested that the Wnt/β-catenin signaling, potentially via the modulation of osteogenic and adipogenic transcriptional factors, exerts an opposite effect on BMP9-induced osteogenic and adipogenic differentiation in preadipocytes.
L’ostéoporose est une maladie fréquente et généralisée du squelette qui se caractérise par une perte de masse osseuse et des altérations de la structure des os. Ces variations des propriétés osseuses, observées à diverses échelles et associées à une augmentation du contenu adipeux de la moelle, témoignent d’une plus grande fragilité du squelette. Ces modifications sont cependant différentes en fonction du site. Cette variation du contenu adipeux médullaire, rapportée notamment dans les os longs du squelette appendiculaire, semble jouer un rôle crucial dans la survenue de l’ostéoporose. Néanmoins, ces altérations ostéo-médullaires, aussi bien quantitatives que qualitatives, sont controversées et peu documentées au niveau des maxillaires, la mandibule en particulier. Le but de notre étude est d’évaluer, au cours du vieillissement et dans un modèle d’ostéoporose (ratte adulte ovariectomisée), les relations entre les variations du contenu adipeux médullaire et la microarchitecture osseuse en site mandibulaire en comparaison avec un site de référence, le tibia. La microarchitecture osseuse, la quantité et la répartition du contenu adipeux médullaire après marquage au tétroxyde d’osmium ont été analysées par microtomographie à rayons X dans la mandibule dentée (os alvéolaire) et non dentée (condyle). Au cours du vieillissement, aucune altération des paramètres osseux et médullaires n’a été mise en évidence. En revanche, le déficit hormonal en oestrogènes induit par ovariectomie conduit à une perte osseuse plus marquée (+35%) dans le tibia que dans la mandibule, associée à des modifications microarchitecturales et adipeuses médullaires site-spécifiques. En effet, en comparaison avec le tibia, un faible contenu adipeux (<10% de moelle) est retrouvé dans le secteur denté (alvéolaire) ainsi que non denté (condyle). A la différence du tibia, au sein duquel un effet de « clustering » des adipocytes est observé à la surface osseuse trabéculaire, le contenu adipeux mandibulaire est réparti de manière homogène dans les espaces médullaires avec très peu de contact avec la surface osseuse trabéculaire. En conclusion, nos résultats indiquent une perte osseuse modérée dans la mandibule par rapport au tibia associée à une augmentation du contenu adipeux médullaire tardive dont la répartition au sein des espaces médullaires n’est pas modifiée à la différence du tibia. Nous émettons l’hypothèse qu’une activité de remodelage et des sollicitations biomécaniques différentes puissent expliquer les spécificités physiopathologiques observées en site mandibulaire. D’autres études seront nécessaires pour préciser l’impact de telles modifications sur la qualité osseuse à l’échelle moléculaire et élémentaire, ainsi que pour préciser les mécanismes de dialogues entre les ostéoblastes et les adipocytes.
Osteoporosis is a disease that leads to a loss of bone mass and to alterations in the bone microarchitecture that occur in a site-specific manner; however it remains controversial in the jaw. The involvement of bone marrow adipose tissue (BMAT) in the bone metabolism has been suggested in several physiopathological contexts, such as in aging and osteoporosis. To test whether the BMAT content is related to mandibular bone loss, this study aimed to investigate the potential correlations between the trabecular bone microarchitecture on one hand and BMAT content and its spatial distribution in relation to bone surface on the other hand during aging and ovariectomy (OVX) during a long-term follow-up in a mature rat model.
No age-related microarchitectural or BMAT changes were observed in the mandible. The OVX-induced bone loss was three-fold lower in the mandible than in the tibia and was observed only in the alveolar bone (not in the condyle). We also report a delayed increase in the mandibular BMAT content that remained 4–6-fold lower compared to tibia. This low BMAT content in the mandible was located at a distance from the trabecular bone surface (only 5% in contact with the bone surface versus 87% in the tibia). These findings highlight a specific mandibular response to OVX, in particular fewer microarchitectural alterations compared to that in the tibia. For the latter, the trabecular bone thickness and surface were correlated with the BMAT content. Oral functions may have a protective effect on the mandibular BMAT conversion in an OVX context.
Background:
Paradoxically, elderly persons with type 2 diabetes mellitus (T2DM) fracture despite having higher bone density than nondiabetics. Systemic factors associated with aging and T2DM may have detrimental, local effects on the skeleton. One such factor could be by altering the microenvironment of the mesenchymal stem cells (MSCs), multipotent progenitors capable of differentiating into adipocytes or osteoblasts.
Methods:
Sera were obtained from four participant groups (n = 40 total, 10 per group): (1) young women with normal glucose tolerance (NGTY), (2) postmenopausal women with NGT), (3) postmenopausal women with impaired glucose tolerance (IGT), and (4) postmenopausal women with T2DM. Sera were incubated with human MSCs for 14 days. Cell proliferation and apoptosis were measured using EdU and TUNEL labeling assays, respectively. MSC differentiation for each group was determined using osteogenic and adipogenic gene expression markers quantified by qRT-PCR, as well as Alizarin Red and Oil Red O staining.
Results:
Expression of adipogenic genes was greater than twofold higher (P < 0.05) in MSCs cultured with T2DM sera compared to those incubated with NGTY, NGT, or IGT sera. The increase in adipogenic gene expression corresponded with increased Oil Red O staining. Despite the increased adipogenic differentiation of MSCs exposed to T2DM sera, cell proliferation and apoptosis rates as well as osteoblastic activity were not significantly different among the four conditions.
Conclusions:
Systemic, circulating factors in the serum of older women with T2DM may promote MSC differentiation into adipocytes versus osteoblasts. Increased differentiation of MSCs into adipocytes is one possible mechanism by which T2DM increases fracture risk.
Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into a variety of cell types under proper stimuli. Bone morphogenetic protein 9 (BMP9) is able to simultaneously induce both adipogenic and osteogenic differentiation of MSCs although the regulatory molecules involved remain to be fully identified and characterized. Heme oxygenase 1 (Hmox1) plays an essential role not only in fat metabolism, but also in bone development. In the present study, we investigated the functional role of Hmox1 in BMP9-induced osteogenic/adipogenic differentiation in MSCs line C3H10T1/2 and probed the possible mechanism involved. We found that BMP9 promoted the endogenous expression of Hmox1 in C3H10T1/2 cells. Overexpression of Hmox1 or cobalt protoporphyrin (CoPP), an inducer of Hmox1, increased BMP9-induced osteogenic differentiation in vitro. Subcutaneous stem cell implantation in nude mice further confirmed that Hmox1 potentiated BMP9-induced ectopic bone formation in vivo. In contrast, Hmox1 reduced BMP9-induced adipogenic differentiation in C3H10T1/2 cells. Although had no obvious effect on BMP9-induced Smad1/5/8 phosphorylation, Hmox1 enhanced phosphorylation of p38 and AKT, while decreased phosphorylation of ERK1/2. Furthermore, Hmox1 increased total β-catenin protein level, and promoted the nuclear translocation of β-catenin in C3H10T1/2 cells. Taken together, our study strongly suggests that Hmox1 is likely to potentiate osteogenic differentiation and yet decrease adipogenic differentiation induced by BMP9 possibly through regulation of multiple signaling pathways. This article is protected by copyright. All rights reserved
Radiation injury occurs after nuclear explosives, radiological or nuclear terrorism, nuclear accidents, and radiation
therapy in combination with surgery, and it is more complicated and difficult to heal than single radiation or wound injuries. The stem cell-based therapy holds promise for radiation related injuries
treatment. In this review, we summarized some representative biological properties of MSCs pre- and post-irradiation, discussed the feasibility of MSCs to apply to the treatment of radiation injuries on animal models and clinical patients, and elucidated the mechanisms of MSCs-based therapy to radiation injuries.
Tissue regeneration using adult stem cells (ASCs) has significant potential as a novel treatment for many degenerative diseases. Previous studies have established that age negatively affects the proliferation status and differentiation potential of ASCs, suggesting a possible limitation in their potential therapeutic use. Therefore, we hypothesized that apple extract might exert beneficial effects on ASCs. The specific objectives were to investigate the proliferative effect of apple ethanol extract on human adipose tissue-derived mesenchymal stem cells (ADSCs) and human cord blood-derived mesenchymal stem cells (CB-MSCs), and identify the possible molecular mechanisms. Apple extract promoted proliferation of ADSCs and CB-MSCs as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Click-iT™ EdU flow cytometry assays. In addition, phosphorylation of p44/42 MAPK (ERK), mammalian target of rapamycin (mTOR), p70 S6 kinase (p70S6K), S6 ribosomal protein (S6RP), eukaryotic initiation factor (eIF) 4B and eIF4E was induced stepwise in ADSCs. Furthermore, apple extract significantly induced the production of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in both ADSCs and CB-MSCs. Similarly, apple extract-induced phosphorylation of the mTOR/p70S6K/S6RP/eIF4B/eIF4E pathway was blocked by pretreatment with PD98059, a specific ERK inhibitor. These results indicate that apple extract-induced proliferation of ADSCs under serum-free conditions is mediated by ERK-dependent cytokine production. Moreover, the beneficial effect of apple extract on proliferation of ASCs may overcome the limitation in therapeutic use of stem cells in tissue regeneration and maintenance of stem cell homeostasis.
Die Alterung von Zellen ist ein generelles Lebensphänomen. Sie betrifft die beiden grundlegenden Fähigkeiten der Zellen, die Fähigkeit zur Vermehrung sowie die Entwicklung und Ausführung zellspezifischer Leistungen nach ihrer Differenzierung. Die Erforschung der Ursachen zellulärer Alterungsvorgänge führt in zwei unterschiedliche Richtungen. Eine Forschungsrichtung begreift Altern als ein genetisches Programm, welches unter anderem beinhaltet, dass die Proliferationskapazität der Zellen geringer wird. Dies hat zur Folge, dass die Regeneration von Geweben durch den regelmäßigen Ersatz differenzierter Zellen im Alter abnimmt, aber auch das Risiko der Entstehung von Tumoren reduziert wird. Die andere Forschungsrichtung führt altersassoziierte Phänomene veränderter zellulärer Funktion vorwiegend darauf zurück, dass oxidative Vorgänge zelluläre Proteine verändern und in ihrer Funktion beeinträchtigen. Solche Phänomene werden auch bei Proteinen beobachtet, die mit dem Knochenstoffwechsel, der Kalziumhomöostase und dem intrazellulären Signalling durch Kalzium verknüpft sind. Die beiden verschiedenen Mechanismen der Zellalterung, auch als replikative und postreplikative Mechanismen bezeichnet, interagieren miteinander und sind im Grunde nicht vollständig voneinander zu trennen (Übersichten bei Campisi 2001, Kuro-o 2001).
Osteoarthritis is the most common cause of chronic disability in older adults. It is a slowly progressive degenerative disorder of articular joints that has multiple etiologies with age being a key factor. Osteoarthritis most commonly affects the hands, knees, hips and spine with knee osteoarthritis representing a major cause of pain and activity limitation. Current therapies are focused on symptom relief while interventions proven to slow or stop the structural progression of the disease are not yet available. No longer considered a “wear and tear” condition that is an inevitable consequence of aging, it is becoming increasingly evident that osteoarthritis has elements of chronic inflammation associated with an imbalance in anabolic and catabolic activity within affected joint tissues. Age-related changes in the cells and tissues of the joint, including cell senescence, oxidative stress, a decline in autophagy, epigenetic alterations, and matrix damage all appear to contribute to the development and progression of osteoarthritis. Which of these may serve as effective targets will be a key question to address in the search for disease modifying interventions.
Human aging is associated with bone loss leading to bone fragility and increased risk for fractures, a disease known as osteoporosis. Osteoporosis is one of the most prevalent and serious diseases affecting the elderly population and constitutes a major public health problem. The cellular and molecular causes of age-related bone loss are current intensive topic of investigation with the aim of identifying new approaches to abolish its negative effects on the skeleton. The aim of this chapter is to give a review on the current understanding of the contribution of aging of the osteoblasts (the bone forming cells) to the phenomenon of age-related boneloss.
The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis.
Mesenchymal stem cells (MSCs) are multipotent precursors with osteogenic, chondrogenic, and adipogenic yet also anti-inflammatory and tissue-protective properties. Due to organism aging, the controlled perpetuation and continuance of developmental principlescomprising cellular, interstitial, and systemic cues, stem cell proliferation, as wellas progenitor differentiation appears to become constricted. Over the recent years, during which abundant information regarding MSC biology has been gathered, also a plethora of information regarding age-associated changes and molecular mechanisms has been collected, which are comprehended in this contribution, in particular regarding insights derived from both in vitro and in vivo analyses which cover alterations in DNA and chromatin modifications, as well as changes in regulatory elements such as miRNAs.
Adipocytes constitute a major part of the bone marrow stroma in vivo and may play an active role in lymphohematopoiesis. Earlier studies had shown that the bone marrow stromal cell clone BMS2 was capable of adipocyte differentiation in vitro, in addition to its well-defined ability to support B lymphopoiesis. We now demonstrate that the process of adipogenesis in this functional bone marrow stromal cell clone can be inhibited by the cytokines interleukin-1 alpha, tumor necrosis factor, and transforming growth factor beta. Exposure of preadipocyte BMS2 cells to these agents blocked the induction of adipocyte differentiation as assessed by morphologic criteria and analysis of the neutral lipid content. Both interleukin-1 alpha and tumor necrosis factor elicited a rapid transient elevation in the steady-state mRNA levels of c-fos, c-jun, and JE. When added to differentiated adipocytes, the three cytokines continued to act as adipogenic antagonists. This was indicated by concentration- and time-dependent decreases in the activity of an adipocyte-specific enzyme, lipoprotein lipase. These changes in enzyme activity correlated directly with a decrease in steady-state levels of lipoprotein lipase mRNA. Another RNA marker of adipocyte differentiation (adipsin) was less influenced by the adipogenic antagonists. This may reflect the longer half-life of this mRNA transcript compared with those of lipoprotein lipase. Our results dramatically demonstrate that the differentiation state of bone marrow stromal cells can be modulated by exogenous factors in vitro. It is also the first report that transformation growth factor beta regulates the activity of lipoprotein lipase. These data suggest potential physiologic actions for these cytokines in vivo within the overall context of lymphohematopoiesis.
Fibroblastic colonies, each of which is derived from a single precursor cell (CFU-F), are formed when suspensions of marrow cells are cultured in vitro. The ability of marrow CFU-F to differentiate in vitro was investigated using the expression of alkaline phosphatase activity as a marker for osteogenic differentiation. In cultures of rabbit marrow cells the colonies formed varied in size, morphology and expression of enzyme activity, indicating that marrow stromal CFU-F are a heterogeneous population. Growth and differentiation of marrow CFU-F can be modified in vitro. Epidermal growth factor increased average colony size and reduced clonal expression of alkaline phosphatase activity to very low levels. Hydrocortisone activated the osteogenic differentiation programme within the cellular progeny of a wide spectrum of CFU-F. The results support the possible development of in vitro clonal methods for the study of differentiation and regulation of the osteogenic and other fibroblastic cell lines of the marrow stromal system.
The monoclonal antibody STRO-1 identifies clonogenic bone marrow stromal cell progenitors (fibroblast colony-forming units [CFU-F]) in adult human bone marrow. These STRO-1+ CFU-F have previously been shown to give rise to cells with the phenotype of fibroblasts, adipocytes, and smooth muscle cells. In this study, the osteogenic potential of CFU-F derived from the STRO-1+ fraction of adult human bone marrow was determined. CFU-F were isolated from normal bone marrow aspirates by fluorescence activated cell sorting, based on their expression of the STRO-1 antigen. Osteogenic differentiation was assessed by the induction of alkaline phosphatase expression, by the formation of a mineralized matrix (hydroxyapatite), and by the production of the bone-specific protein osteocalcin. STRO-1+ cells were cultured in the presence of dexamethasone (DEX; 10(-8) mol/L), ascorbic acid 2-phosphate (ASC-2P; 100 mumol/L), and inorganic phosphate (PO4i; 2.9 mmol/L). After 2 weeks of culture, greater than 90% of the cells in each CFU-F colony stained positive for alkaline phosphatase using a monoclonal antibody specific for bone and liver alkaline phosphatase. Alkaline phosphatase activity was confirmed by histochemistry. A mineralized matrix developed in the CFU-F cultures, after 4 weeks of culture in the presence of DEX, ASC-2P, and PO4i. Mineralization was confirmed by both light and electron microscopy. The mineral was identified as hydroxyapatite by electron dispersive x-ray microanalysis and by x-ray diffraction analysis. In replicate cultures, osteocalcin release was shown after exposure of the cells to 1,25-dihydroxyvitamin D3 (10(-7) mol/L) both by radioimmunoassay and Northern blot analysis. This work provides direct evidence that adult human bone marrow-derived CFU-F are capable of differentiating into functional osteoblasts and that osteoprogenitors are present in the STRO-1+ population.
Glucocorticoid agonists, i.e. dexamethasone or triamcinolone acetonide, rapidly induce expression of CCAAT/enhancer-binding protein (C/EBP) delta and repress expression of C/EBP alpha in fully differentiated 3T3-L1 adipocytes. Within 30 min of glucocorticoid treatment, the cellular level of C/EBP delta rises dramatically, increasing > 100-fold within 6 h. Concurrently, the level of C/EBP alpha decreases, reaching a minimum within 4 h. The dexamethasone concentration dependence and steroid specificity of these responses suggest that both processes are mediated by the glucocorticoid receptor. The reciprocal effects of dexamethasone on the steady-state levels of C/EBP alpha and C/EBP delta can be accounted for kinetically and quantitatively by changes in their mRNA levels and by the transcription rates of their respective genes. The glucocorticoid-induced changes in expression of the C/EBP isoforms are correlated with the transcriptional activation of the SCD1 gene, an adipocyte gene known to be transactivated by C/EBP isoforms. Glucocorticoids also regulate expression of the C/EBP isoforms in vivo. Within 4 h of administration of dexamethasone or triamcinolone acetonide to adult rats, expression of C/EBP delta is induced in white adipose tissue while expression of C/EBP alpha is repressed. Like the response in 3T3-L1 adipocytes, the effects of dexamethasone on C/EBP alpha in white adipose tissue are rapid and transient.
Peroxisome proliferator-activated receptors (PPARs) alpha and gamma are key regulators of lipid homeostasis and are activated by a structurally diverse group of compounds including fatty acids, eicosanoids, and hypolipidemic drugs such as fibrates and thiazolidinediones. While thiazolidinediones and 15-deoxy-Delta12, 14-prostaglandin J2 have been shown to bind to PPARgamma, it has remained unclear whether other activators mediate their effects through direct interactions with the PPARs or via indirect mechanisms. Here, we describe a novel fibrate, designated GW2331, that is a high-affinity ligand for both PPARalpha and PPARgamma. Using GW2331 as a radioligand in competition binding assays, we show that certain mono- and polyunsaturated fatty acids bind directly to PPARalpha and PPARgamma at physiological concentrations, and that the eicosanoids 8(S)-hydroxyeicosatetraenoic acid and 15-deoxy-Delta12,14-prostaglandin J2 can function as subtype-selective ligands for PPARalpha and PPARgamma, respectively. These data provide evidence that PPARs serve as physiological sensors of lipid levels and suggest a molecular mechanism whereby dietary fatty acids can modulate lipid homeostasis.
The osteoblast is the bone-forming cell. The molecular basis of osteoblast-specific gene expression and differentiation is unknown. We previously identified an osteoblast-specific cis-acting element, termed OSE2, in the Osteocalcin promoter. We have now cloned the cDNA encoding Osf2/Cbfa1, the protein that binds to OSE2. Osf2/Cbfa1 expression is initiated in the mesenchymal condensations of the developing skeleton, is strictly restricted to cells of the osteoblast lineage thereafter, and is regulated by BMP7 and vitamin D3. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts. Finally, forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. This study identifies Osf2/Cbfa1 as an osteoblast-specific transcription factor and as a regulator of osteoblast differentiation.
Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as
undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage,
fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated
from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro.
These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages.
Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.
Bone marrow stromal stem cells differentiate into many different types of cells including osteoblasts and adipocytes. Long-term glucocorticoid treatment decreases osteoblastic activity but increases adipocytes. We investigated the mechanism of glucocorticoid-induced PPARgamma2 transcription. Treatment of human bone marrow stromal cells with dexamethasone induced the differentiation of these cells into adipocytes as measured by oil-red O staining, and Northern blot analysis showed that dexamethasone strongly induced PPARgamma2 mRNA expression in cells cultured in adipocyte induction medium. Moreover, the mRNA of C/EBPdelta, an adipocyte-promoting transcription factor, was also induced by dexamethasone in the presence of induction medium. Gel mobility shift assays using purified GST-C/EBPdelta fusion protein showed that C/EBPdelta specifically binds to a 40-base pair DNA element from PPARgamma2 promoter, which was found to contain a tandem repeat of C/EBP binding sites. Transfection studies in mouse mesenchymal C3H10T1/2 cells showed that it is the tandem repeat of the C/EBP binding site in PPARgamma2 promoter region that regulates dexamethasone-mediated PPARgamma2 gene activation. We conclude that glucocorticoid-induced adipogenesis from bone marrow stromal cells is mediated through a reaction cascade in which dexamethasone transcriptionally activates C/EBPdelta; C/EBPdelta then binds to PPARgamma2 promoter and transactivates PPARgamma2 gene expression. This activated master regulator, in turn, initiates the adipocyte differentiation.
In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined
the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular
osteoblasts, by the use of semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR). There is a progressive
and significant reduction of the CBFA1 steady-state mRNA level down to 50% during cellular aging of human osteoblasts. In
comparison to the normal cells, human osteosarcoma cell lines SaOS-2 and KHOS/NP, and the SV40-transformed human lung fibroblast
cell line MRC5V2 have 20 to 40% higher levels of CBFA1 mRNA. Similar levels of CBFA1 mRNA are detectable in normal human skin
fibroblasts, and these cells also exhibit an age-related decline to the same extent. In addition, the expression of topoisomerase
I is reduced by 40% in senescent osteoblasts, and the mRNA levels are significantly higher (40–70%) in transformed osteoblasts
and fibroblasts. These changes in gene expression may be among the causes of impaired osteoblast functions, resulting in reduced
bone formation during aging.
Aging of the human skeleton is characterized by decreased bone formation and bone mass and these changes are more pronounced in patients with osteoporosis. As osteoblasts and adipocytes share a common precursor cell in the bone marrow, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis is the result of enhanced adipognesis versus osteoblastogenesis from precursor cells in the bone marrow. Thus, we examined iliac crest bone biopsies obtained from 53 healthy normal individuals (age 30-100) and 26 patients with osteoporosis (age 52-92). Adipose tissue volume fraction (AV), hematopoietic tissue volume fraction (HV) and trabecular bone volume fraction (BV) were quantitated as a percentage of total tissue volume fraction (TV) (calculated as BV + AV + HV) using the point-counting method. We found an age-related increase in AV/TV (r = 0.53, P < 0.001, n = 53) and an age-related decline in BV/TV (r = -0.46, P < 0.001, n = 53) as well as in the HV/TV (r -0.318, P < 0.05, n = 53). There was an age-related inverse correlation between BV/TV and AV/TV (r = -0.58, P < 0.001). No significant correlation between the AV/TV and the body mass index (r = 0.06, n.s., n = 52) was detectable. Compared with age-matched controls, patients with osteoporosis exhibited an increased AV/TV (P < 0.05) and decreased BV/TV (P < 0.05) but no statistically significant difference in HV/TV. Our data support the hypothesis that with aging and in osteoporosis an enhanced adipogenesis is observed in the bone marrow and that these changes are inversely correlated to decreased trabecular bone volume. The cellular and molecular mechanisms mediating these changes remain to be determined.
Adipose tissue is a major component of normal rabbit marrow. Its physiologic function has not been elucidated, but it is generally believed to be, essentially, a passive space filler. The response of marrow fat cells to lipolytic stimuli was studied by subjecting rabbits to acute starvation. Following 2 weeks of starvation and a weight loss of 28%, the mean marrow fat cell volume was 50.9 ± 9.6 pl (control, 42.8 ± 8.1 pl), and the mean volume of the perinephric fat cell 134.3 ± 87.8 picoliter (pl) (control, 318 ± 89.7 pl). Esterification capacity for labeled palmitate of the marrow fat cells did not change from control; the esterification capacity of the perinephric fat cells fell by about 60%. After 3 weeks of starvation and further weight loss to 34% of initial weight, the perinephric fat cells decreased their mean cell volume to 50.2 ± 33.6 pl. The marrow fat cell volume remained essentially unchanged from control. Esterification capacity of the marrow fat cells again did not differ from that of the control animals, but the esterification capacity of the perinephric fat cells fell further to 19% of that of the marrow fat cells. Despite the urgent energy requirements of the starved animals, the marrow fat cells did not yield their stored fat and continued to esterify free fatty acids at an unaltered rate. Drastic weight loss produced no change in volume, in esterification capacity, or in number of fat cells in the femoral marrow of the rabbit. Marrow adipocytes are metabolically active cells that are involved in the hematopoietic process rather than involved with the total energy needs of the animal.
Serum was obtained from approximately 10 individuals of each sex from each decade of life from 0 to 90 years of age. Most of the serum samples were obtained from the excess remaining after diagnostic procedures performed for hospitalized patients. The individuals were screened according to tentative diagnosis, and overt cases of diseases known to have effects upon essential fatty acid (EFA) metabolism were eliminated. Serum lipids were separated into phospholipids, cholesteryl esters, triglycerides, and free fatty acids. These lipid classes and a sample of the total lipids were subjected to fatty acid analysis by gas chromatography. The data produced were arranged by computer to compare the effects of age upon each fatty acid present and upon each parameter calculated from fatty acid composition for each fatty acid present and upon each parameter calculated from fatty acid composition for each lipid group and each sex. No significant difference between sexes was found for any individual fatty acid in any lipid, but the average total unsaturation of all lipid classes was significantly higher for males than for females at both 0 and 90 years of age. In cholesteryl esters, and to a lesser extent in phospholipids, the total polyunsaturated fatty acids, the total ω6 acids and the sum 18:2ω6 + 20:4ω6 - 20:3ω9 declined more rapidly with age in females than in males, suggesting a trend toward EFA deficiency, although the ratio 20:3ω9/20:4ω6 did not change significantly. The age and sex differences observed and the SD encountered were much smaller than the change observed in EFA deficiency, indicating that this population has utility in comparisons with suggested cases of EFA deficiency. The triene/tetraene ratio, 20:3ω9/20:4ω6 was found to be 0.1 ± 0.08 for male and female populations, indicating that a ratio above 0.2 should be considered the upper limit of normalcy. Between 0 and 4 months of age, a profound change took place in the patterns of fatty acids in serum lipids. At birth, phospholipids were low in 18:2ω6 but 20:4ω6 and 20:3ω9 were elevated. The ratio of 20:3ω9/20:4ω6 was 0.16 at birth and decreased progressively during infancy. Some of these observations suggest that at birth the infant has marginal reserves of EFA. The drastic change in composition of serum lipid shortly after birth probably reflects a change in supply of EFA from one rich in 20:4ω6 and poor in 18:2ω6 to one high in 18:2ω6 and low in 20:4ω6. This may have significance in the nutrition of premature or very young infants.
The differentiation of adipocytic and osteogenic cells has been investigated in cultures of adult rat marrow stromal cells. Adipocytic differentiation was assessed using morphological criteria, changes in expression of procollagen mRNAs, consistent with a switch from the synthesis of predominantly fibrillar (types I and III) to basement membrane (type IV) collagen, and the induction of expression of aP2, a specific marker for differentiation of adipocytes. Osteogenic differentiation was assessed on the basis of changes in the abundance of the mRNAs for the bone/liver/kidney isozyme of alkaline phosphatase and the induction of mRNAs for bone sialoprotein and osteocalcin. In the presence of foetal calf serum and dexamethasone (10(-8) M) there was always differentiation of both adipocytic and osteogenic cells. When the steroid was present throughout primary and secondary culture the differentiation of osteogenic cells predominated. Conversely, when dexamethasone was present in secondary culture only, the differentiation of adipocytes predominated. When marrow stromal cells were cultured in the presence of dexamethasone in primary culture and dexamethasone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10(-8) M) in secondary culture, the differentiation of adipocytes was inhibited whereas the differentiation of osteogenic cells was enhanced, as assessed by an increase in expression of osteocalcin mRNA. The results, therefore, demonstrate an inverse relationship between the differentiation of adipocytic and osteogenic cells in this culture system and are consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.
Stromal colonies with fibroblastic morphology grown from rabbit marrow cells in culture supplemented with foetal calf serum. In this study the same marrow cells cultured with autologous rabbit plasma and hydrocortisone form colonies of a single lineage that express the adipocytic phenotype. A comparison of the potential for differentiation of cloned cell populations grown from fibroblastic and adipocytic colonies has been made using an in vivo diffusion chamber assay. The adipocytic colonies differentiated and grew to a limited size in medium with rabbit plasma and hydrocortisone, but attempts to isolate them and expand them in this medium failed. When the serum supplement was changed to foetal calf serum at day 10 the cells in the adipocytic colonies acquired a less differentiated morphology, there was a large increase in colony growth and cells were produced in sufficient numbers for the diffusion chamber assay. Thirty one fibroblastic colonies and twenty one adipocytic colonies were isolated either by limiting dilution or ring cloning and then expanded. Of these, eleven fibroblastic and eight adipocytic colonies provided enough cells (2 x 10(5) to 2 x 10(6] for implantation and culture in the chambers. Four of the eleven fibroblastic and three of the eight adipocytic colonies formed an osteogenic tissue in the chambers. It was concluded that cells that have differentiated in an adipocytic direction are able to revert to a more proliferative stage and subsequently to differentiate along the osteogenic pathway. Adipocytic and fibroblastic cells cultured in vitro from marrow have, with osteogenic cells, a common precursor in adult marrow.
Osteoblast-like cells are commonly found in the vicinity of osteoclasts formed in long-term human bone marrow cultures, and they are believed to be derived from osteogenic cell precursors belonging to the stromal cell system. This paper describes a new culture method for human osteoblasts from the adherent cell population of long-term human mononuclear bone marrow cultures. The cells obtained exhibited all the classic characteristics of osteoblasts. They contained high intracellular concentrations of alkaline phosphatase and they secreted the osteoblast-specific marker bone Gla protein. Collagen production was mainly (95-98%) procollagen type I propeptide and only minute quantities of procollagen type III propeptide were detectable by radioimmunoassay in the conditioned medium. After eight weeks the cells formed a mineralized matrix on exposure to beta-glycerophosphate and ascorbic acid. This system provides a model for the study of osteoblast differentiation in vitro and may form the basis for the use of defined media in bone cell cultures due to the presence of high concentrations of osteoblast precursors.
It is known that marrow fat content increases in a variety of osteoporoses. This study sought to clarify this phenomenon by combining bone and marrow histomorphometry, and to determine (1) whether the marrow fat increase follows or precedes diminishment of bone volume, and (2) whether the increase in fat volume is due to adipocyte growth or proliferation. The relationship between marrow fat content and bone turnover was studied in the metaphysis and epiphysis of the proximal tibia in 20 ovariectomized and 20 intact 200 gm Sprague-Dawley rats. The results were examined after one and three months. The epiphysis had greater trabecular bone volume than the metaphysis. The metaphysis exhibited a decrease in bone volume with time; the epiphysis did not. Following ovariectomy, the bone volume diminished twice as much in the metaphysis as in the epiphysis. In the epiphysis, as bone volume fraction fell following ovariectomy, the vacated space was filled by both hemopoietic and adipose tissue. In the metaphysis, space-filling was time-dependent: hemopoietic tissue at one month and adipose tissue at three months. Marrow fat content increased with both time and ovariectomy in the metaphysis, but only with time in the epiphysis. Thus, marrow fat increased after bone volume began to decline in the metaphysis, and not at all in the epiphysis. Ovariectomy increased erosion surface in both the epiphysis and the metaphysis, but bone formation rate was increased only in the epiphysis. There was a reciprocal relationship between marrow fat content and bone formation rate.
It was previously shown that 11 months after ovariectomy the volume fraction of trabecular bone in the spine and 11th rib medullary canal of Beagle dogs (6 control, 9 ovariectomized) was significantly reduced. In this paper it is shown that these changes are accompanied by increased marrow fat volume in the 11th rib (59.0 +/- 9.5% vs. 44.3 +/- 10.0%). Conversely, the volume fraction of functional (hematopoietic) cells in the marrow was reduced by ovariectomy. Additionally, variations in marrow fat volume were tested for correlation with 22 other variables pertinent to bone physiology. Marrow fat volume was significantly positively correlated with serum osteocalcin, rib trabecular bone porosity, rib cross-sectional area, and gains in body weight. It was negatively correlated with serum estrogen concentrations and the extent of rib trabecular surfaces labeled with tetracycline.
This article has no abstract; the first 100 words appear below.
OSTEOPOROSIS is an enormous public health problem, responsible for at least 1.2 million fractures in the United States each year. The sites of these fractures are the vertebrae in 538,000 cases, the hip in 227,000, the distal forearm (Colles' fracture) in 172,000, and other limb sites in 283,0001 (and Melton LJ, Riggs BL: unpublished data). One third of women over 65 will have vertebral fractures (Melton LJ, Riggs BL: unpublished data). By extreme old age, one of every three women and one of every six men will have had a hip fracture.² This catastrophic type of fracture is fatal in . . .
Supported by grants (AM-27065, AM-30582, and AG-04875) from the National Institutes of Health, U.S. Public Health Service.
Source Information
From the Endocrine Research Unit, Division of Endocrinology, Metabolism, and Internal Medicine, and the Department of Medical Statistics and Epidemiology, Mayo Clinic and Mayo Foundation, Rochester, Minn. Address reprint requests to Dr. Riggs, Division of Endocrinology and Internal Medicine, Mayo Clinic, Rochester, MN 55905.
Retrospective histologic analyses of bone biopsies and of post mortem samples from normal persons of different age groups, and of bone biopsies of age- and sex-matched groups of patients with primary osteoporosis and aplastic anemia show characteristic age dependent as well as pathologic changes including atrophy of osseous trabeculae and of hematopoiesis, and changes in the sinusoidal and arterial capillary compartments. These results indicate the possible role of a microvascular defect in the pathogenesis of osteoporosis and aplastic anemia.
In monolayer cultures of guinea-pig bone marrow and spleen the development of discrete fibroblast colonies takes place on days 9–12. The linear increase in the number of colonies with increasing numbers of explanted cells and the distribution of male and female cells in mixed cultures support the view that fibroblast colonies are clones. The concentration of colony-forming cells in bone marrow and spleen is approximately 10-5. Bone marrow culture (but not spleen culture) fibroblasts are capable of spontaneous bone formation in diffusion chambers. Fibroblasts from both bone marrow and spleen cultures are inducible to osteogenesis in diffusion chambers in the presence of transitional epithelium.
At several points during the growth of a clone of human embryonic lung fibroblasts in vitro, 100 to 200 cells were removed at random and the proliferative potential of each cell was determined. At each sample point, a wide variation in remaining population doubling ability was observed among the individual cells and the distributions of doubling potentials were distinctly bimodal. Furthermore, the two cells arising from a single mitosis differed in their ability to proliferate by as many as eight population doublings (256-fold in the number of cells produced). The results suggest that a stochastic process is responsible for determining the limited proliferative potential of human embryonic lung fibroblasts.
When freshly isolated rabbit marrow cells were cultured either in vitro or in diffusion chambers in vivo, the hemopoietic cells disappeared and there was a proliferation of the stromal cell population. The colonies formed in vitro were mainly fibroblastic, and this cell type predominated in confluent cultures. Staining for alkaline phosphatase activity and for the Von Kossa reaction was negative in in vitro cultures. However, marrow cell suspensions or fibroblasts harvested from in vitro culture of marrow cells, gave rise to a mixture of bone, cartilage and fibrous tissue in diffusion chambers implanted into the peritoneal cavity. In contrast, only a soft fibrous tissue developed from spleen fibroblasts in diffusion chambers. Differentiation of osteogenic tissue within diffusion chambers fell into two categories: (1) Formation of bone in a fibrous layer surrounding cartilage; (2) intramembranous bone formed directly within fibrous tissue unassociated with cartilage. In both cases alkaline phosphatase activity appeared before the onset of mineralization, and decreased as the first signs of mineral became apparent. The present results suggest that postnatal marrow contains osteogenic precursors with the potential to differentiate via either of the two major paths followed during skeletal development in the embryo. Clonal analysis of the marrow stromal cell population will be required to clarify whether osteo-, chondro-, and fibrogenic cells are the products of one stromal cell line modulated by the microenvironment, or whether there are distinct cell lines for each type.
The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-beta gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.
Peroxisome proliferator-activated receptor gamma 2 (PPAR gamma 2) is an adipocyte-specific nuclear hormone receptor that has recently been identified as a key regulator of two fat cell enhancers. Transcriptional activation by PPAR gamma 2 is potentiated by a variety of lipids and lipid-like compounds, including naturally occurring polyunsaturated fatty acids. We demonstrate here that retroviral expression of PPAR gamma 2 stimulates adipose differentiation of cultured fibroblasts. PPAR activators promote the differentiation of PPAR gamma 2-expressing cells in a dose-dependent manner. C/EBP alpha, a second transcription factor induced during adipocyte differentiation, can cooperate with PPAR gamma 2 to stimulate the adipocyte program dramatically. Our results suggest that the physiologic role of PPAR gamma 2 is to regulate development of the adipose lineage in response to endogenous lipid activators and that this factor may serve to link the process of adipocyte differentiation to systemic lipid metabolism.
Decreased osteoblastic activity seems to play an important role in the pathogenesis of postmenopausal osteoporosis. The aim of the present study was to examine the direct effects of human growth hormone (GH) on proliferation and differentiation of osteoblastic cells obtained from patients with postmenopausal osteoporosis and age-matched normals and to compare the cellular responses induced by GH between the two groups. Osteoblast cultures (human marrow stromal osteoblast-like cells) were established from bone marrow aspirates obtained from 9 osteoporotic patients and 12 age-matched normals. Effects on cell proliferation and cell differentiation markers [alkaline phosphatase (AP)], procollagen type I propeptide (PICP), and osteocalcin] were assessed. GH stimulated 3H-thymidine incorporation into DNA in cell cultures of osteoporotic patients to a maximum of 158 +/- 14% of no-treatment controls (n = 9, P < 0.001) and to 203 +/- 52% (n = 9, P < 0.001) in normals. GH increased cell number as measured by methylene blue (MB) assay in cells of osteoporotic patients to 138 +/- 10% (P < 0.05, n = 7) and in normals to 138 +/- 12 (P < 0.05, n = 7). GH alone reduced cellular AP production: 61 +/- 3.8% (P < 0.05, n = 7) versus 65 +/- 16% (P < 0.05, n = 7) and cellular PICP production: 79 +/- 6% (P < 0.05, n = 7) versus 69 +/- 16% (n.s., n = 7), in cell cultures of osteoporotics and normals, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Adipose tissue fatty acids, it has been proposed, reflect dietary intake. Using data from a validation study preceding a prospective study on diet, cancer, and health in Denmark, we were able to compare fatty acid profiles in adipose tissue biopsies from 86 individuals (23 men and 63 women) aged 40-64 y and dietary intake of fatty acids (as percentage of total fat) assessed by two 7-d weighed-diet records or by a semi-quantitative food frequency questionnaire. Correlation coefficients (Pearson r) between fatty acid concentrations in adipose tissue biopsies (as percentage of total peak area) and dietary intake of fatty acid (percentage of total fat), determined from the diet records for men and women, respectively, were as follows: polyunsaturated fatty acids r = 0.74 and r = 0.46; n - 3 fatty acids of marine origin: eicosapentaenoic acid r = 0.15 and r = 0.61, and docosahexaenoic acid r = 0.47 and r = 0.57. Correlation coefficients obtained by using the food frequency questionnaire were slightly lower for most fatty acids.
The adipocyte is the most abundant stromal cell phenotype in adult human bone marrow. Four hypotheses may explain their function. First, adipocytes may serve a passive role, simply occupying excess space in the bone marrow cavity. Second, they may play an active role in systemic lipid metabolism. Third, adipocytes may provide a localized energy reservoir in the bone marrow. Or fourth, marrow adipocytes may contribute directly to the promotion of hematopoiesis and influence osteogenesis. This article reviews recent findings concerning bone marrow adipocyte morphology and physiology, the transcriptional and cytokine mechanisms regulating their differentiation, and the interrelationships existing between bone marrow adipocytes, hematopoiesis, and osteogenesis. Overall, these data lend support to a "plastic" model of bone marrow stromal cell differentiation; adipocytes may share common functions with stromal stem cells, osteoblasts, and hematopoietic supportive cells.
Routine postmortem examinations and the pathobiological features revealed by systematically designed studies have shown several pathologic phenotypes that are often characteristic enough to differentiate among the various SAM strains: senile amyloidosis in SAMP1, -P2, -P7, -P9, -P10, and -P11; secondary amyloidosis in SAMP2 and -P6; contracted kidney in SAMP1, -P2, -P10, and -P11; immunoblastic lymphoma in SAMR1 and -R4; histiocytic sarcoma in SAMR1 and -R4; ovarian cysts in SAMR1; impaired immune response in SAMP1, -P2, and -P8; hyperinflation of the lungs in SAMP1; hearing impairment in SAMP1; degenerative temporomandibular joint disease in SAMP3; senile osteoporosis in SAMP6; deficits in learning and memory in SAMP8 and -P10; emotional disorders in SAMP8 and -P10; cataracts in SAMP9; and brain atrophy in SAMP10. These are all age-associated pathologies, the incidence and severity of which increase with advancing age. The SAM model in which these pathobiological features have been carefully monitored will be a valuable tool in the clarification of the pathogenic mechanisms of age-associated pathologies and in the research for effective methods to modulate or ameliorate these pathologies.
Populations of marrow stromal fibroblasts (MSFs) can differentiate into functional osteoblasts and form bone in vivo. It is not known, however, what proportion of MSF precursor cells, colony forming units-fibroblast (CFU-Fs), have osteogenic potential. In the present study, analysis of bone formation in vivo by single-colony derived strains of human marrow stromal fibroblasts (HMSFs) has been performed for the first time. Each strain originated from an individual CFU-F and underwent four passages in vitro prior to subcutaneous implantation into immunodeficient mice within vehicles containing hydroxyapatite-tricalcium phosphate ceramic. Multicolony derived HMSF strains were also transplanted to serve as positive controls. After 8 weeks, abundant bone formation was found in the transplants of all multicolony derived HMSF strains, whereas 20 out of 34 (58.8%) single-colony derived strains from four donors formed bone. Immunostaining with antibody directed against human osteonectin and in situ hybridization for human-specific alu sequences demonstrated that cells forming new bone were of human origin and were vital for at least 45 weeks post-transplantation. Both the incidence of bone-forming colonies and the extent of bone formation by single-colony derived HMSF strains were increased by cultivation with dexamethasone and ascorbic acid phosphate. Other factors, including type of transplantation vehicle, morphology, size, and structure of the original HMSF colonies showed no obvious correlation with the incidence or extent of bone formation. Hematopoietic tissue within the newly formed bone was developed in the transplants exhibiting exuberant bone formation. These results provide evidence that individual human CFU-Fs have osteogenic potential and yet differ from each other with respect to their osteogenic capacity.
Human bone marrow was harvested by means of iliac crest aspiration and cultured under conditions that promote an osteoblastic phenotype. Human bone marrow aspirates from 30 normal subjects, ages 8-80 years, with no systemic illness, yielded a mean of 92 +/- 65 x 10(6) nucleated cells per 2 ml of aspirate. The prevalence of potential osteoblastic progenitors was estimated by counting the number of alkaline phosphatase-positive colonies. This assay demonstrated a mean of 43 +/- 28 alkaline phosphatase-positive colonies per 10(6) nucleated cells, which was about one per 23,000 nucleated cells. The prevalence of these colonies was positively correlated with the concentration of nucleated cells in the original aspirate (p = 0.014) and was negatively correlated with donor age (p = 0.020). The population of alkaline phosphatase-positive colonies in this model sequentially exhibited markers of the osteoblastic phenotype; essentially all colonies (more than 99%) stained positively for alkaline phosphatase on day 9. Matrix mineralization, which was associated with the synthesis of bone sialoprotein, was demonstrated on day 17 with alizarin red S staining. On day 45, cells that were stimulated with 1,25-dihydroxyvitamin D3 synthesized and secreted osteocalcin at concentrations consistent with known osteoblastic cell lines. This model provides a useful method for the assay of progenitors of connective tissue from human subjects, examination of the effects of aging and selected disease states on this progenitor population, and investigation into the regulation of human osteoblastic differentiation.
Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. However, little is known about the interrelationship between these two processes. We have previously shown that the senescence accelerated mouse-P6 (SAMP6) exhibits decreased osteoblastogenesis in the bone marrow that is temporally linked with a low rate of bone formation and decreased bone mineral density. Here we report that in contrast to decreased osteoblastogenesis, ex vivo bone marrow cultures from SAMP6 mice exhibited an increase in the number of colony-forming unit adipocytes, as well as an increase in the number of fully differentiated marrow adipocytes, compared with SAMR1 (nonosteopenic) controls. Further, long-term bone marrow cultures from SAMP6 produced an adherent stromal layer more rapidly, generated significantly more myeloid progenitors and produced more IL-6 and colony-stimulating activity. Consistent with this, the number of myeloid cells in freshly isolated marrow from SAMP6 mice was increased, as was the number of granulocytes in peripheral blood. The evidence that SAMP6 mice exhibit decreased osteoblastogenesis, and increased adipogenesis and myelopoiesis, strongly suggests that a switch in the differentiation program of multipotential mesenchymal progenitors may underlie the abnormal phenotype manifested in the skeleton and other tissues of these animals. Moreover, these observations support the contention for the existence of a reciprocal relationship between osteoblastogenesis and adipogenesis that may explain the association of decreased bone formation and the resulting osteopenia with the increased adiposity of the marrow seen with advancing age in animals and humans.
Insulin-like growth factors (IGF)-I and IGF-II are produced by osteoblasts and are important paracrine/autocrine regulators of osteoblast proliferation and differentiation. Estrogen has been reported to increase gene expression of IGF-I in rodent osteoblasts. However, because species differences have been demonstrated in expression of various aspects of the IGF system in bone cells, it is not known whether this action also occurs in human osteoblasts. Thus, we assessed the effects of estrogen treatment on IGF-I and IGF-II gene expression in vitro in a recently developed human fetal osteoblast cell line that has high levels of estrogen receptors. As assessed by a quantitative reverse transcriptase-polymerase chain reaction method, treatment of hFOB/ER9 cells with 17beta-estradiol (E2) increased steady state levels of IGF-I mRNA in a time- and dose- dependent fashion with a maximal increase of 319% +/- 33% (P < 0.01) of control occurring after treatment with 10(-7) M E2 for 48 hours. In contrast, E2 did not alter steady state levels of IGF-II mRNA. The pure (type 2) antiestrogens ICI 182,780 (10(-7) M) and ICI 164,384 (10(-6) M) blocked the E2- induced increase in IGF-I mRNA levels. Interestingly, 4-hydroxytamoxifen (10(-7) M), a documented pure antiestrogen in reproductive tissues, also increased IGF-I mRNA to levels similar to those observed in E2-treated cells. Since E2 was shown to mediate its effects on some target genes through a cAMP-dependent pathway, we studied the interaction between E2 and agents that are known to increase intracellular cAMP. Forskolin (10(-8) M) and dibutyryl cAMP (10(-3) M) increased IGF-I mRNA levels sixfold, and cotreatment with E2 did not affect these changes, consistent with a possible mediation of the estrogen effect on IGF-I gene expression by cAMP. We conclude that in human osteoblastic cells, the IGF-I gene is a target for estrogen action, suggesting that IGF-I may mediate part of the effects of estrogen in human bone.
Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte-like differentiation in human osteosarcoma SaOS-2/B10 and MG-63 cell lines, in rat ROS17/2.8 cells, and in mouse calvaria-derived osteoblastic MB1.8 cells, as evidenced by the accumulation of Oil Red O positive lipid vesicles and the decrease in alkaline phosphatase expression. Both SaOS-2/B10 and MG-63 cells, but not ROS17/2.8 nor MB1.8 cells, express significant levels of PPARgamma mRNA, a member of the peroxisome proliferator activated receptor (PPAR) family that has been implicated in the control of adipocyte differentiation. However, both ROS17/2.8 and MG-63 cells express significant levels of the adipocyte selective marker, aP2 fatty acid binding mRNA, which can be further increased by RS. These cell types express PPARdelta/NUC-1 but not PPARalpha, indicating that cells that do not express either PPARgamma or PPARalpha are capable of differentiating into adipocyte-like cells. Transfection experiments in COS cells showed that compared with fetal bovine serum (FBS), RS is rich in agents that stimulate PPAR-dependent transcription. The stimulatory activity was ethyl acetate extractable and was 35-fold more abundant in RS than in FBS. Purification and analysis revealed that the major components of this extract are free fatty acids. Furthermore, the same fatty acids, a mixture of palmitic, oleic, and linoleic acids, activate the PPARs and induce adipocyte-like differentiation of both ROS17/2.8 and SaOS-2/B10 cells. These findings suggest that fatty acids or their metabolites can initiate the switch from osteoblasts to adipocyte-like cells.
The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by increased marrow adipose tissue formation. Reversal of this process may provide a novel therapeutic approach for osteopenic disorders. We have shown that cells cultured from human trabecular bone are not only osteogenic, but are able also to undergo adipocyte differentiation under defined culture conditions. Osteoblast differentiation was induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and adipocyte differentiation by dexamethasone (dex) plus 3-isobutyl-1-methylxanthine (IBMX) treatment. Adipogenesis was characterized by lineage-specific enzyme and gene activities, alpha-glycerophosphate-3-dehydrogenase activity, fatty acid binding protein, aP2 and lipoprotein lipase expression. Osteoblastogenesis was assessed by osteoblast characteristic 1,25(OH)2D3 induction of alkaline phosphatase activity and osteoblast-specific 1,25(OH)2D3-induced osteocalcin synthesis and release. We provide evidence for a common pluripotent mesenchymal stem cell that is able either to undergo adipogenesis or osteoblastogenesis, using clonal cell lines derived from human trabecular bone cell cultures. Adipogenesis can be induced also by long chain fatty acids and the thiazolidinedione troglitazone. Dex plus IBMX-induced adipogenesis can be inhibited by interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta. Interestingly, and in contrast to extramedullary adipocyte differentiation as shown by mouse 3T3L-1 and a human liposarcoma SW872 cell line, trabecular bone adipogenesis was unaffected by insulin. Also, the formation of fully differentiated adipocytes from trabecular bone cells after troglitazone treatment and long chain fatty acids was dependent on increased expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma2 caused by dex plus IBMX. Specific inhibition of marrow adipogenesis and promotion of osteoblastogenesis of a common precursor cell may provide a novel therapeutic approach to the treatment of osteopenic disorders.
The proliferative capacity and cellular and biochemical characteristics of human trabecular bone osteoblasts were analysed throughout their replicative lifespan in vitro. Like several other cell types, human osteoblasts demonstrated a typical Hayflick phenomenon of cellular aging comprising a period of rapid proliferation until cumulative population doubling level (CPDL) 22 to 24, followed by a phase of slow growth and the final cessation of cell division at CPDL 32 to 34. Comparing young cells (less than 20% lifespan completed) and old cells (more than 90% lifespan completed) revealed a progressive increase in population doubling (PD) time, a decrease in attachment frequency, a decrease in the number of S-phase positive cells, a decrease in the rates of DNA, RNA and protein synthesis, an increase in the protein content per cell and an increased proportion of senescence-specific beta-galactosidase positive cells. While osteoblastic production of collagen type I decreased progressively during aging, alkaline phosphatase activity dropped rapidly after the first few passages and then remained constant during the rest of the proliferative lifespan, Significant morphological changes from thin and spindle-shaped early passage young cells to large, flattened and irregularly shaped late passage old cells full of intracellular debris were observed. In comparison, osteoblasts established from an osteoporotic bone sample showed a maximum CPDL of less than 5, had a longer PD time and exhibited abnormal senescent morphology. Thus, we have demonstrated for the first time that human osteoblasts, like several other diploid cell types, have a limited proliferative capacity in vitro and undergo aging and senescence as measured by various cellular and biochemical markers. In addition, preliminary studies show that cells from osteoporotic bone have a severely reduced proliferative capacity. This model of bone cell aging facilitates study of the molecular mechanisms of osteoblast senescence as well as factors related to osteoblast dysfunction in patients with osteoporosis.
1. Stem and progenitor cells present within bone marrow give rise to colony forming units-fibroblastic (CFU-F) which can differentiate into fibroblastic, osteogenic, myogenic, adipogenic and reticular cells. The decrease in skeletal bone formation and rate of fracture repair observed with ageing and in osteoporosis has been suggested to be due to a decrease in numbers of these progenitors, but human studies are limited.
2. We have tested the potential to form CFU-F in a total of 99 patients undergoing corrective surgery (16 controls, 14–48 years of age) or hip arthroplasty for osteoarthritis (57 patients, 28–87 years of age) or osteoporosis (26 patients, 69–97 years of age). Total colony number, alkaline phosphatase-positive colony number and colony size were determined.
3. No decrease in colony forming efficiency under the culture conditions used was observed in all populations examined irrespective of age, disease or gender, as determined by the lack of correlation between colony formation and age.
3. Examination of colony sizes showed a significant reduction in colony size with age in osteoarthritis and in control populations indicating a change in cellular proliferative potential with age.
4. Examination of number and percentage of alkaline phosphatase-positive CFU-F showed a significant decrease in osteoporotic patients compared with controls and osteoarthritis patients, indicating altered differentiation potential.
5. These results suggest that the reduction in bone mass with ageing may be due to reduction of the proliferative capacity of progenitor cells or their responsiveness to biological factors leading to alteration in subsequent differentiation. The maintenance of CFU-F number and alkaline phosphatase activity in these osteoarthritis patients may, in part, explain the inverse relationship observed for the preservation of bone mass between generalized osteoarthritis and primary osteoporosis.
The variation in marrow colony forming unit-fibroblastic (CFU-F) number in 59 patients (14-87 years of age) undergoing corrective surgery (14 controls; 14-48 years of age) or hip arthroplasty for primary osteoarthritis (45 OA; 46-87 years of age) was examined to determine whether marrow CFU-F, derived from marrow stromal fibroblastic stem cells, are maintained with the development of primary osteoarthritis (OA). Total colony number, colony size as well as alkaline phosphatase-positive colonies were determined. The mean fibroblast colony forming efficiency from the whole patient group was 2.4 x 10(-5) +/- 1.4 x 10(-5). Ageing had no effect on the colony forming efficiency or on the alkaline-phosphatase-positive colony forming efficiency, irrespective of gender. Thus precursor cells with the potential for osteogenic differentiation are maintained in OA with ageing. However, colony size showed a significant reduction with age, implying altered proliferation potential of osteogenic progenitors with ageing. This ageing effect may not be as significant in OA as in the rest of the population as bone mineral density is often preserved in osteoarthritis. As there is no apparent deficit in primitive progenitor cells, this preservation may be the result of altered regulation of osteoprogenitor activity in OA.
Information on the interconversion potential of adipocytes and other end cells characteristic of the stromal fibroblastic cell lineages, key in the understanding of bone turnover in metabolic diseases such as osteoporosis, is limited. The object of the present study was: i) to isolate relatively pure populations of adipocytes from human bone marrow; ii) to clone single adipocytes from these populations; and iii) to examine in vitro the interconversion potential of the progeny of these single-cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cells cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. Cloned adipocytes were found to dedifferentiate into fibroblast-like cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining, and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaining using specific osteocalcin antiserum, and formation of mineralized cell aggregates. These findings demonstrate the extent of plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. We have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and the interconversion potential of human marrow stromal cells in vitro. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor.
Mesenchymal stem cells (MSCs) residing in bone marrow (BM) are the progenitors for osteoblasts and for several other cell types. In humans, the age-related decrease in bone mass could reflect decreased osteoblasts secondary to an age-related loss of osteoprogenitors. To test this hypothesis, BM cells were isolated from vertebral bodies of thoracic and lumbar spine (T1-L5) from 41 donors (16 women and 25 men) of various ages (3-70 years old) after death from traumatic injury. Primary cultures were grown in alpha modified essential medium with fetal bovine serum for 13 days until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP+) were considered to have osteogenic potential. BM nucleated cells were plated (0.5, 1, 2.5, 5, or 10 x 106 cells/10-cm dish) and grown in dexamethasone (Dex), which promotes osteoblastic differentiation. The optimal plating efficiency using BM-derived cells from donors of various ages was 5 x 106 cells/10-cm dish. BM-derived cells were also grown in the absence of Dex at this plating density. At the optimal plating density, in the presence of Dex, the number of CFU-F/ALP+ present in the BM of the younger donors (3-36 years old) was 66.2 +/- 9.6 per 106 cells (mean +/- SEM), but only 14.7 +/- 2.6 per 106 cells in the older donors (41-70 years old). With longer-term culture (4-5 weeks) of these BM cells in medium containing 10 mM beta-glycerophosphate and 100 microg/ml ascorbic acid, the extracellular matrix mineralized, a result consistent with mature osteoblastic function. These results demonstrate that the number of MSCs with osteogenic potential (CFU-F/ALP+) decreases early during aging in humans and may be responsible for the age-related reduction in osteoblast number. Our results are particularly important in that the vertebrae are a site of high turnover osteoporosis and, possibly, the earliest site of bone loss in age-related osteoporosis.
The glucocorticoid dexamethasone (dex) stimulates differentiation of cultured preadipocytes of the 3T3-L1 cell line to adipocytes. The preadipocytes express a gene termed preadipocyte factor 1 (pref-1). The pref-1 gene product is abundant in preadipocytes but completely absent in adipocytes. Constitutive expression of pref-1 blocks differentiation of adipocytes, as does a soluble 50 kDa fragment of the pref-1 protein. It appears that dex mediates adipogenesis by down-regulating expression of pref-1.
Pluripotent mesenchymal stem cells in bone marrow differentiate to osteoblast progenitor cells. When the bone marrow cells are cultured in vitro, they form colony-forming units-fibroblastic (CFU-Fs) with exhibiting osteoblastic features such as expression of alkaline phosphatase (ALP) and formation of calcified nodules ex vivo. This article describes the effect of growth, maturation, and aging of the skeleton on human CFU-Fs harvested from human iliac bone marrow. Human bone marrow cells were harvested from the ilia of 49 women, and were cultured ex vivo for examination. The 49 subjects ranged in age from 4 to 88 years and were without metabolic bone disease. These aspirated bone marrow cells from human ilium exhibited osteoblastic phenotype such as alkaline phosphatase (ALP) activity, expression of osteocalcin (OSC) and parathyroid hormone-receptor (PTH-R) mRNA, and the formation of calcified nodules in vitro. The number of ALP-positive CFU-Fs and the ALP activity were quantified. The highest levels of ALP-positive CFU-Fs were observed in the young group, particularly in those under 10 years of age. The levels of ALP-positive CFU-Fs declined sharply after 10 years of age; those above 20 years of age exhibited a lower number of ALP-positive CFU-Fs, with a gradual decline with increasing age. These results indicate that change in the number of ALP-positive CFU-Fs may be associated with skeletal growth and maturation. The results also show that osteoblastic features such as ALP activity and capability of formation of calcification nodules were maintained even in the older subjects. These findings suggest that decreased activity of bone formation in the aged subjects could be, in part, caused by the decreased number of osteoprogenitor cells differentiating into osteoblasts because the number of ALP-positive CFU-Fs was one of the indices exhibiting bone-forming activity in the human marrow stromal cells.
Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [CFU-Fs]) when cultured in vitro. To examine the effect of aging and osteoporosis on the MSC population, we quantified the number of MSCs and their proliferative capacity in vitro. Fifty-one individuals were studied: 38 normal volunteers (23 young individuals [age, 22-44 years] and 15 old individuals [age, 66-74 years]) and 13 patients with osteoporosis (age, 58-83 years). Bone marrow was aspirated from iliac crest; mononuclear cells were enriched in MSCs by magnetic activated cell sorting (MACS) using STRO-1 antibody. Total CFU-F number, size distribution, cell density per CFU-F, number of alkaline phosphatase positive (ALP+) CFU-Fs, and the total ALP+ cells were determined. In addition, matrix mineralization as estimated by alizarin red S (AR-S) staining was quantified. No significant difference in colony-forming efficiency between young individuals (mean +/- SEM; 87 +/- 12 CFU-Fs/culture), old individuals (99 +/- 19 CFU-Fs/culture), and patients with osteoporosis (129 +/- 13 CFU-Fs/culture; p = 0.20) was found. Average CFU-F size and cell density per colony were similar in the three groups. Neither the percentage of ALP+ CFU-Fs (66 +/- 6%, 65 +/- 7%, and 72 +/- 4% for young individuals, old individuals, and patients with osteoporosis, respectively) nor the percentage of ALP+ cells per culture (34 +/- 5%, 40 +/- 6%, and 41 +/- 4%) differed between groups. Finally, mineralized matrix formation was similar in young individuals, old individuals, and patients with osteoporosis. Our study shows that the number and proliferative capacity of osteoprogenitor cells are maintained during aging and in patients with osteoporosis and that other mechanisms must be responsible for the defective osteoblast (OB) functions observed in these conditions.
Aging is associated with decreased trabecular bone mass and increased adipocyte formation in bone marrow. As osteoblasts and adipocytes share common precursor cells present in the bone marrow stroma, it has been proposed that an inverse relationship exists between adipocyte and osteoblast differentiation. In order to test this hypothesis, we studied mice treated with troglitazone (n = 9) given as a 0.2% of food admixture (2.0 g troglitazone per kg food) for 10 months and control mice (n = 9). Troglitazone is a potent stimulator of adipogenesis acting at the nuclear receptor: peroxisome proliferator activated receptor-gamma (PPARgamma). Histomorphometric analysis of proximal tibia was performed in order to quantitate the amount of trabecular bone volume per total volume (BV/TV %), adipose tissue volume per total volume (AV/TV %), and hematopoietic marrow volume per total volume (HV/TV %) using the point-counting technique. Bone size did not differ between the two groups. In troglitazone-treated mice, AV/TV was significantly higher than in control mice (4.7+/-2.1% vs. 0.2+/-0.3%, respectively, mean +/- SD, P < 0.001). BV/TV was similar in the two groups (16.9+/-5.6% for troglitazone-treated group vs. 14.9+/-4.7% for control group) as well as ash weight of the vertebrae. HV/TV was reduced in troglitazone-treated mice compared with control mice (78.4+/-6.8% vs. 84.9+/-4.7%, respectively, P < 0.05) and the presence of vascular sinusoids was reduced (7.3+/-1.7% vs. 16.1+/-5.6%, respectively, P < 0.05). Our data demonstrate that adipogenesis and osteogenesis can be regulated independently. Troglitazone-induced adipogenesis in the bone marrow may be caused by changes in the bone marrow vascularity.
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