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Abstract
Human bone marrow stromal cells were examined for their osteogenic potential in an in vitro cell culture system. Dexamethasone (Dex) treatment induced morphological transformation of these cells from an elongated to a more cuboidal shape, increased their alkaline phosphatase activity and cAMP responses to PTH and prostaglandin E2, and was essential for mineralization of the extracellular matrix. Dex-induced differentiation of human bone marrow stromal cells was apparent after 2-3 days of treatment and reached a maximum at 7-14 days, as judged by alkaline phosphatase activity, although induction of osteocalcin by 1,25-dihydroxyvitamin D3 was attenuated by Dex. Withdrawal of Dex resulted in an enhancement of the 1,25-dihydroxyvitamin D3-induced secretion of osteocalcin, whereas alkaline phosphatase activity and the cAMP response to PTH remained at prewithdrawal levels. The steady state mRNA level of osteonectin was not affected by Dex. Our results, which demonstrate that Dex conditions the differentiation of human bone marrow osteogenic stromal cells into osteoblast-like cells, support the hypothesis of a permissive effect of glucocorticoids in ensuring an adequate supply of mature osteoblast populations. Furthermore, the established human bone marrow stromal cell culture provides a good model of an in vitro system to study the regulation of differentiation of human bone osteoprogenitor cells.
... Dexamethasone (DM) induces osteoblastic differentiation in vitro by increasing alkaline phosphatase activity, osteocalcin expression, and bone sialoprotein levels (5). DM is used for the prevention of inflammation in orthopedic surgery and has a well-known effect on bone density. ...
... Some researchers have reported that DM-loaded HA has an effect on bone regeneration in vitro (5), while the present study was conducted to confirm that it is also effective in vivo. Some researchers have reported that the effective DM concentration to induce osteogenic differentiation of bone marrow stromal cells is in the range of 40-400 ng/ml and that DM has toxic effects at 4,000 ng/ml (20). ...
Background/aim:
Many cases of bone damage are due to trauma and metabolic diseases. This study aimed to evaluate bone regeneration into a porous hydroxyapatite (HA) scaffold using dexamethasone (DM)-loaded polymeric microspheres.
Materials and methods:
Four adult dogs were used to evaluate the in vivo performance of DM-loaded microspheres immobilized on the surfaces of porous HA scaffolds. Two 5-mm drill holes were created in both the left and right femurs of each dog. The experimental groups included a control group (drill holes filled with HA scaffold alone), a DM 20 group (holes filled with DM-loaded HA scaffold with 20 mg DM per scaffold), and a DM 100 group (hole filled with DM-loaded HA scaffold with 100 mg DM per scaffold). Resulting bone volume percentages and bone mineral densities were calculated by examing micro-computed tomographic (CT) images.
Results:
The DM-loaded HA scaffold groups showed a gradual periosteal reaction two weeks after insertion of the HA scaffold into the femoral drill holes. Four weeks after HA scaffold insertion, the periosteal reaction in the femoral drill holes became denser. Eight weeks after insertion of DM-loaded HA scaffolds, clear images of the scaffold were observed in micro-CT images of the femoral drill hole. The DM 100 group had better bone healing tendencies (bone mineral density, bone mass, trabecular volume, bone surface, and trabecular thickness) than the DM 20 group.
Conclusion:
DM-loaded HA scaffolds are suitable platforms for distributing bioactive molecules during osteogenesis in femoral drill holes.
... During fracture healing, bone mesenchymal stromal cells (BMSCs) can differentiate toward adipocytes or osteoblasts under different stimulations 3,4 , and promote bone callus tissue formation 5,6 . A reduction of human BMSC pool and an inhibited BMSC proliferation have been regarded as potential contributors to nonunion during bone healing 7,8 . During BMSC osteogenic differentiation, the expression of timely expressed genes, including Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and Collagen I, and the mineralization of extracellular matrix could be observed 9,10 . ...
... During osteogenic differentiation of BMSCs, the expression of Runx2, ALP, and Col1agen I, as well as the mineralization of the extracellular matrix have been considered as marker events 8,32,33 . Wnt signaling activation promotes this differentiation [13][14][15][16] . ...
The osteogenic differentiation of human bone mesenchymal stromal cells (BMSCs) has been considered as a central issue in fracture healing. Wnt signaling could promote BMSC osteogenic differentiation through inhibiting PPARγ. During atrophic nonunion, Wnt signaling-related factors, WNT5A and FZD3 proteins, were significantly reduced, along with downregulation of Runx2, ALP, and Collagen I and upregulation of PPARγ. Here, we performed a microarray analysis to identify differentially expressed miRNAs in atrophic nonunion tissues that were associated with Wnt signaling through targeting related factors. Of upregulated miRNAs, miR-381 overexpression could significantly inhibit the osteogenic differentiation in primary human BMSCs while increase in PPARγ protein level. Through binding to the 3′UTR of WNT5A and FZD3, miR-381 modulated the osteogenic differentiation via regulating β-catenin nucleus translocation. Moreover, PPARγ, an essential transcription factor inhibiting osteogenic differentiation, could bind to the promoter region of miR-381 to activate its expression. Taken together, PPARγ-induced miR-381 upregulation inhibits the osteogenic differentiation in human BMSCs through miR-381 downstream targets, WNT5A and FZD3, and β-catenin nucleus translocation in Wnt signaling. The in vivo study also proved that inhibition of miR-381 promoted the fracture healing. Our finding may provide a novel direction for atrophic nonunion treatment.
... A wide range of Alizarin red stain, from strong to almost no calcification staining, has been reported for human VICs when cultured in OM without the addition of other calcificationpromoting compounds such as pro-inflammatory stimuli (7-10). Dexamethasone, a component of OM, was initially demonstrated to promote bone osteoblast differentiation (11). OM was subsequently used to induce calcification in vascular smooth muscle cell cultures by promoting osteogenic differentiation (12). ...
... Beyond cells residing in the aortic valve, our proteomics analysis may be relevant to other calcifying cell types, as the media conditions we tested are not exclusive to VICs. This includes smooth muscle cells and bone cells, which can model calcification potential using similar media conditions to those used in our present study (11,12,16,30,33). In the case of human coronary artery and aortic smooth muscle cells cultured in OM, we have observed calcification in passages beyond P1 with higher frequency than we do using the same experimental conditions with VICs (16,33). ...
Aortic valvular interstitial cells (VICs) isolated from patients undergoing valve replacement are commonly used as in vitro models of calcific aortic valve disease (CAVD). Standardization of VIC calcification, however, has not been implemented, which impairs comparison of results from different studies. We hypothesized that different culture methods impact the calcification phenotype of human VICs. We sought to identify the key parameters impacting calcification in primary human VICs to standardize CAVD in vitro research. Here we report that in calcification media containing organic phosphate, termed osteogenic media (OM), primary human VICs exhibited a passage-dependent decrease in calcification potential, which was not observed in calcification media containing inorganic phosphate, termed pro-calcifying media (PM). We used Alizarin red staining to compare the calcification potential of VICs cultured in OM and PM between the first and fourth passages after cell isolation from human CAVD tissues. Human VICs showed consistent Alizarin red stain when cultured with PM in a passage-independent manner. VICs cultured in OM did not exhibit consistent calcification potential between donors in early passages and consistently lacked positive Alizarin red stain in late passages. We performed whole cell, cytoplasmic and nuclear fractionation proteomics to identify factors regulating VIC passage-dependent calcification in OM. Proteomics cluster analysis identified tissue non-specific alkaline phosphatase (TNAP) as a regulator of passage-dependent calcification in OM. We verified an association of TNAP activity with calcification potential in VICs cultured in OM, but not in PM in which VICs calcified independent of TNAP activity. This study demonstrates that media culture conditions and cell passage impact the calcification potential of primary human VICs and should be taken into consideration in cell culture models of CAVD. Our results help standardize CAVD modeling as part of a greater effort to identify disease driving mechanisms and therapeutics for this unmet medical need.
... Note that oestrogens reduce BALP in post-menopausal women which might theoretically disguise subtle elevations in women on hormone replacement therapy (128). Steroids have an unpredictable effect on BALP as although they may be osteogenic in exogenous or endogenous hypercortisolism there may be no noticeable effects or mild suppression (66,129,130). Hyperthyroidism and thyrotoxicosis cause an elevation in ALP, likely by effects on bone metabolism (131,132). ...
... Glucocorticoids are often used in protocols to induce commitment and differentiation of uncommitted MSCs to cells of the osteoblast lineage (Jaiswal, Haynesworth, Caplan, & Bruder, 1997;Pittenger et al., 1999). Moreover, glucocorticoids are also used to stimulate the expression of a range of cellular osteoblast markers such as alkaline phosphatase (ALP) and osteocalcin (Bellows, Aubin, & Heersche, 1987;Buttery et al., 2001;Cheng, Yang, Rifas, Zhang, & Avioli, 1994;Leboy, Beresford, Devlin, & Owen, 1991). Although inconsistent effects of glucocorticoids on cellular proliferation have emerged , high doses have been reported to decrease the proliferation of mature osteoblast-like cells in cultures (Shi et al., 2015). ...
Animal models are fundamental to advance our knowledge of the underlying pathophysiology of bone loss and to study pharmaceutical countermeasures against it. The animal model of post-menopausal osteoporosis from ovariectomy is the most widely used preclinical approach to study skeletal deterioration. However, several other animal models exist, each with unique characteristics such as bone loss from disuse, lactation, glucocorticoid excess, or exposure to hypobaric hypoxia. The present review aimed to provide a comprehensive overview of these animal models to emphasize the importance and significance of investigating bone loss and pharmaceutical countermeasures from perspectives other than post-menopausal osteoporosis only. Hence, the pathophysiology and underlying cellular mechanisms involved in the various types of bone loss are different, and this might influence which prevention and treatment strategies are the most effective. In addition, the review sought to map the current landscape of pharmaceutical countermeasures against osteoporosis with an emphasis on how drug development has changed from being driven by clinical observations and enhancement or repurposing of existing drugs to today's use of targeted anti-bodies that are the result of advanced insights into the underlying molecular mechanisms of bone formation and resorption. Moreover, new treatment combinations or repurposing opportunities of already approved drugs with a focus on dabigatran, parathyroid hormone and abaloparatide, growth hormone, inhibitors of the activin signaling pathway, acetazolamide, zoledronate, and romosozumab are discussed. Despite the considerable progress in drug development, there is still a clear need to improve treatment strategies and develop new pharmaceuticals against various types of osteoporosis. The review also highlights that new treatment indications should be explored using multiple animal models of bone loss in order to ensure a broad representation of different types of skeletal deterioration instead of mainly focusing on primary osteoporosis from post-menopausal estrogen deficiency.
... Among different molecules used to induce cell differentation during cell culture, Dexamethasone (Dex), a synthetic glucocorticoid, is known to be an important regulator of mesenchymal progenitor cell commitment to osteoblast, adipocyte and chondrocyte lineages. Indeed, it has been demonstrated, in many studies, that Dex regulates the osteogenesis of human MSCs and mineralization in vitro (Cheng et al., 1994;Jaiswal et al., 1997;Aubin, 1998;Walsh et al., 2001;Hardy and Cooper, 2011). It is also demonstrated that Dex has a pro-osteogenic effect on mouse MSCs (Edgar et al., 2007). ...
Mechanical stimuli control cell behaviors that are crucial for bone tissue repair. Osteocytes sense extracellular mechanical stimuli then convert them into biochemical signals to harmonize bone remodeling. However, the mechanisms underlying this process remain unclear. Autophagy, which is an evolutionarily preserved process, that occurs at a basal level when stimulated by multiple environmental stresses. We postulated that mechanical stimulation upregulates osteocyte autophagy via AMPK-associated signaling, driving osteocyte-mediated osteogenesis. Using a murine model of orthodontic tooth movement, we show that osteocyte autophagy is triggered by mechanical tension, increasing the quantity of LC3B-positive osteocytes by 4-fold in the tension side. Both in vitro mechanical tension as well as the chemical autophagy agonist enhanced osteocyte Fibroblast growth factor 23 (FGF23) secretion, which is an osteogenenic related cytokine, by 2-and 3-fold, respectively. Conditioned media collected from tensioned osteocytes enhanced osteoblast viability. These results indicate that mechanical tension drives autophagy-mediated FGF23 secretion from osteocytes and promotes osteogenesis. Our findings highlight a potential strategy for accelerating osteogenesis in orthodontic clinical settings.
... GelUPy/nZnHApUPy hydrogel with 15% DEX was chosen for cytotoxicity evaluation because of its relatively long release period, which met both the drug dosage and release period requirements (Cheng et al., 1994;Su et al., 2012). ...
Synthesis of a supramolecular composite hydrogel, a novel class of physical and dynamic hydrogels, is reported. The hydrogel comprised gelatin (Gel), Zn-doped nano-hydroxyapatite (nZnHAp), and dexamethasone disodium phosphate (DEX). nZnHAp was functionalized with ureidopyrimidinone (UPy; quadruple hydrogen-bond-forming groups). The nHAp, nZnHAp, and nZnHApUPy were characterized by FTIR, EDX, SEM, and TGA analysis. Gelatin was also functionalized with UPy groups (GelUPy), and the nanocomposites were prepared in solution (GelUPy/nZnHApUPy). Dexamethasone was added to the composite hydrogels (15, 20, and 25 wt%), as an osteoinductive and anti-inflammation medicine (GelUPy/nZnHApUPy/DEX). The mechanical (in compression mode), rheological, and thermal properties of the GelUPy/nZnHApUPy/DEX were compared with those of GelUPy/nZnHAp/DEX. The GelUPy/nZnHApUPy/DEX series showed the maximum compressive modulus (about 841 KPa) for GelUPy/nZnHApUPy/DEX25% in the swollen state. DSC analysis indicated that Tg of the pure GelUPy decreased significantly upon composite preparation and drug loading from 160°C for GelUPy, down to 107°C for GelUPy/nZnHApUPy/DEX15%. In-vitro drug release studies confirmed sustained release of DEX over a period of 12 days for GelUPy/nZnHApUPy composites with no remarkable initial burst compared to that of the pure GelUPy. Overall, our data suggests that the DEX carrying supramolecular nanocomposite can be used as an osteogenic hydrogel scaffold for bone tissue engineering applications.
... DEX, a synthetic glucocorticoid, is known and used clinically as an anti-inflammatory drug. Previous studies have reported that DEX can induce osteoporosis or even pathological fractures [23], while DEX in vitro promotes osteoblast differentiation and bone mineralization [24][25][26]. In fact, DEX has been commonly used to induce mesenchymal stem cell differentiation and is a key component in osteogenic differentiation. ...
Introduction. In modern dentistry the oral rehabilitation of patients with the help of dental implants has a very high success rate. However, the problem is the situation of patients with serious chronic diseases in which the insertion of dental implants is problematic or dental procedures can complicate or aggravate the patients’ disease.
Case report. We presented the case of a patient with associated chronic diseases who benefited from the insertion of implants with a nanostructured surface. The nanotube surface of the dental implant has been loaded with anti-inflammatory drugs to promote healing and stimulate the process of osteogenesis.
Conclusions. Within the limitations of this study, we consider the therapy with avant-garde nanostructured dental implants to represent a viable treatment option for patients with a medical history with complicated chronic diseases.
... In other experiments, we replaced standard medium with medium inductive for osteoblastic differentiation. This contained α-MEM 10% FCS, 10 -2 M β-glycerol, 5×10 -3 M ascorbic acid and 10 -8 M dexamethasone [14,15] and was done at D0, at the end of the exponential phase of proliferation (week 3) and for 3 additional weeks. MCs were then collected after 1 hr incubation with collagenase and assessed for their alkaline phosphatase (ALP) activity on cytospin. ...
Background:
During major bone substance loss, secured allogeneic bone matrix (ABM) is normally utilized for bone repair. Here, we propose a method to colonize ABM using autologous mesenchymal cells (MCs) to improve their integration. Moreover, in this study, the consequences of in vitro colonization on MCs have been evaluated.
Methods:
After in vitro propagation of MCs, their proliferation kinetics on ABM pre-coated with gelatin, fibronectin, collagen IV and human serum (HS) was monitored, and they were compared with cells cultured without ABM for 8 weeks. The effect of ABM on cell phenotype was also assessed. Lastly, the ability of ABM-colonizing MCs to perform hematopoiesis, a function normally preserved in selected culture conditions, and their differentiation towards osteoblastic lineage were evaluated.
Results:
MC and colony-forming unit-fibroblast proliferated 930- and 590-fold, respectively. The proliferation rate of the expanded MCs was higher, forming a 3-dimensional structure in all ABMs. Pre-coating with HS was the most efficient treatment of ABMs to increase the initial adherence of MCs, and it partly explains the reason for the higher propagation of MCs. Flow cytometry analyses revealed subtle alterations in ABM-colonizing cells; however, the ability of MCs to maintain long-term culture initiating cells proliferation and differentiate into osteoblastic lineage was preserved.
Conclusions:
In this study, the in vitro biocompatibility of bone marrow (BM) MCs with ABMs, the role of HS in scaffold coating, and the possibility of initially using a small BM sample for this approach were demonstrated.
... The synthetic glucocorticoid dexamethasone (dex) is commonly used in vitro in trilineage differentiation protocols for mesenchymal stromal cells (MSCs). In bone research, dex has been used for osteogenic differentiation of bone-marrow-derived MSCs (BMSCs) for almost 30 years [14], and differentiation cocktails containing at least 10 nM dex, together with ascorbic acid and a source of phosphate groups, are the standard in the field [15][16][17]. Dex has been reported to increase the expression of RUNX2 [15,18], which is considered the master regulator of osteogenic differentiation [19]. ...
Despite the huge body of research on osteogenic differentiation and bone tissue engineering , the translation potential of in vitro results still does not match the effort employed. One reason might be that the protocols used for in vitro research have inherent pitfalls. The synthetic glucocor-ticoid dexamethasone is commonly used in protocols for trilineage differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). However, in the case of osteogenic commitment, dexamethasone has the main pitfall of inhibiting terminal osteoblast differentiation, and its pro-adipogenic effect is well known. In this work, we aimed to clarify the role of dexamethasone in the osteogenesis of hBMSCs, with a particular focus on off-target differentiation. The results showed that dexamethasone does induce osteogenic differentiation by inhibiting SOX9 expression, but not directly through RUNX2 upregulation as it is commonly thought. Rather, PPARG is concomitantly and strongly upregulated, leading to the formation of adipocyte-like cells within osteogenic cultures. Limiting the exposure to dexamethasone to the first week of differentiation did not affect the mineralization potential. Gene expression levels of RUNX2, SOX9, and PPARG were simulated using approximate Bayesian computation based on a simplified theoretical model, which was able to reproduce the observed experimental trends but with a different range of responses, indicating that other factors should be integrated to fully understand how dexamethasone influences cell fate. In summary, this work provides evidence that current in vitro differentiation protocols based on dex-amethasone do not represent a good model, and further research is warranted in this field.
... In addition to the overexpression of ALP, BMSCs also synthesized and secreted a large number of bone matrix substituents during osteoblast differentiation, such as OC, Col-I, and Runx2 [32]. The expressions of OC (Fig. 7B), Col-I (Fig. 7C), and Runx2 (Fig. 7D) in the ICA, TP scaffold and ITP scaffolds extracts group increased over time, indicating both ICA and PCL and b-TCP released from scaffold achieved osteoblast differentiation. ...
Despite the accessibility to porous architectures through various biofabrication approaches for tissue engineering, incorporating various active growth regulators within their matrices that act as biochemical cues is also an essential attribute for effective tissue growth. To address these facts, icariin (ICA)-encapsulated polymeric scaffolds are fabricated using a low-temperature extrusion-based three-dimensional (3D) printing technology for efficiently promoting osteogenesis. This approach not only resulted in the generation of porous architectures but also substantially maintained the bio-efficacy of the encapsulated ICA. Moreover, these composite scaffolds based on poly(ε-caprolactone) (PCL) and tricalcium phosphate (β-TCP) encapsulated with ICA (ITP scaffolds) are systematically characterized using various techniques before and after printing. Furthermore, various investigations relevant to biodegradability, biocompatibility, ICA release, and osteogenic ability of the ITP scaffolds are explored. The intact physiochemical properties of the materials, sustained release of ICA from the scaffolds, and high biosafety at various levels ranging from cellular to animal efficiently promoted the proliferation of mouse bone marrow mesenchymal stem cells (BMSCs) and their differentiation to osteoblasts. Together, the utilization of low-temperature extrusion approach provides a convenient and eco-friendly means of fabricating highly porous 3D architectures that supply the required growth regulators in their active form for tissue regeneration.
... Dexamethasone is a classic in vitro osteogenic induction agent for MSCs across species [11,12,14,17,[29][30][31][32][33][34][35][36][37][38], yet its mechanism of action is incompletely understood [35,[38][39][40]. Dexamethasone has varying effects on osteogenic differentiation of MSCs based on concentration and duration of supplementation [39,[41][42][43]. ...
A growing body of work suggests that canine mesenchymal stromal cells (cMSCs) require additional agonists such as BMP-2 for consistent in vitro osteogenic differentiation. BMP-2 is costly and may challenge the translational relevance of the canine model. Dexamethasone enhances osteogenic differentiation of human MSCs and is widely utilized in osteogenic protocols. The aim of this study was to determine the effect of BMP-2 and dexamethasone on early- and late-stage osteogenesis of autologous and iPS-derived cMSCs. Two preparations of marrow-derived cMSCs were selected to represent exceptionally or marginally osteogenic autologous cMSCs. iPS-derived cMSCs were generated from canine fibroblasts. All preparations were evaluated using alkaline phosphatase activity, Alizarin Red staining of osteogenic monolayers, and qPCR. Data were reported as mean +/- standard deviation and compared using one- or two-way ANOVA and Tukey or Sidak post-hoc tests. Significance was established at p<0.05. In early-stage assays, dexamethasone decreased ALP activity for all cMSCs in the presence of BMP-2. In late-stage assays, inclusion of dexamethasone and BMP-2 at Day 1 of culture produced robust monolayer mineralization for autologous cMSCs. Delivering 100 nM dexamethasone at Day 1 improved mineralization and reduced the BMP-2 concentrations required to achieve mineralization of the marginal cMSCs. For iPS-cMSCs, dexamethasone was inhibitory to both ALP activity and monolayer mineralization. There was increased expression of osteocalcin and osterix with BMP-2 in autologous cMSCs but a more modest expression occurred in iPS cMSCs. While autologous and iPS-derived cMSCs respond similarly in early-stage osteogenic assays, they exhibit unique responses to dexamethasone and BMP-2 in late-stage mineralization assays. This study demonstrates that dexamethasone and BMP-2 can be titrated in a time- and concentration-dependent manner to enhance osteogenesis of autologous cMSC preparations. These results will prove useful for investigators performing translational studies with cMSCs while providing insight into iPS-derived cMSC osteogenesis.
... Whilst the precise concentration of dexamethasone to be used for optimal differentiation has been a source of debate 301 , its importance is nevertheless undisputed. Dexamethasone is known to induce the expression of Runx2, Osx and bone matrix proteins [302][303][304] and when combined with ascorbic acid and a source of phosphate, it promotes the osteoblastic differentiation of hMSCs, stimulates the expression of ALP, COL 1 and OCN and promotes mineralisation [305][306][307][308][309] , whilst also inhibiting cell proliferation 305,307,310 . ...
The bone tissue engineering community has been striving to develop novel approaches that mimic natural bone formation. The rapid generation of mineralised bone tissue with a capacity for vascularisation and the selection of highly osteogenic cell sources are still the focus of research today. This study addresses three novel approaches in these key areas. Mineralisation in bone tissue involves stepwise cell-cell and cell-extracellular matrix (ECM) interactions. Regulation of the osteoblast culture microenvironment can manipulate osteoblast proliferation and mineralisation rates and consequently the quality and/or quantity of the final calcified tissue. Therefore, an in vitro model to investigate possible influential factors would be highly sought after. We developed a facile in vitro model through the modification of culture surfaces in which an osteoblast cell line and aggregate culture was used to mimic intramembranous ossification. Conventional monolayer culturing was used as a comparative control. The effects of multiple culture parameters, including culture duration and aggregate size, on mineralisation rates and subsequent mineral quantities and distributions have been examined by numerous well established methods alongside certain innovative techniques. Ultimately, spatial and temporal production of minerals differed depending upon aggregate size with larger aggregates mineralising faster with a distinct gene expression pattern compared to the smaller aggregates. We also demonstrated that mineralisation in the larger aggregates initiated from the periphery, whilst mineralisation in the smaller aggregates initiated from the centre. This implies that aggregate size influences mineral distribution and development over time. An in vivo study using a cell line and primary cell population was conducted to investigate how the observations noted during the short term in vitro studies would affect long term in vivo aggregate survival and bone formation. Both cell types saw similar results. The large aggregates appeared to disintegrate over the course of the experiment, whilst the small aggregates remained intact and ii produced an abundant volume of extracellular material. A monolayer cell sample was again used as a comparative control and generated a lower material volume over the same period. The data obtained from this element of the project produced some invaluable insights into how the specific variables of cellular aggregation might affect possible bone formation in vivo. In addition, a novel substrate, substrate X, was used to identify and investigate the possibility of mesenchymal stem cell (MSC) subpopulations within mixed MSC populations and their donor-dependent variations alongside their subsequent influences upon an individual's osteogenic capacity. Substrate X successfully identified what are thought to be three subpopulations within individual MSC populations from multiple donors through distinct cellular attachments. Each of the subpopulations was shown to hold differing osteogenic capacities and their proportions were also shown to be donor-dependent. Subpopulation proportions were shown to correlate with specific cadherin levels and cellular aggregation potential was also shown to be donor-dependent. Furthermore, the novel aggregation technique developed by this study was pitted against a conventional aggregation technique to assess aggregate vascularisation and mineralisation simultaneously using cellular co-culturing. This study also investigated how mechanical stimulation would affect aggregate vascularisation and mineralisation. The method of aggregation developed earlier in this project was shown to create an inner-aggregate architecture that aided in specific cellular organisation and possible vascularisation more than the conventional aggregation technique. The mechanical stimulation reduced cellular migration from the aggregate body compared to a static culture equivalent but nodule mineralisation within the co-cultured aggregates was inconclusive due to the short culture period. To conclude, simple yet effective substrate chemistry modifications enabled us to evaluate a variety of parameters for refined bone tissue engineering. These included the development of an aggregate model for the study of developing mineralisation, possible MSC subpopulation identification, measurement and assessment and the evaluation of aggregate vascularisation. iii
... 24 Early research on dexamethasone demonstrated that it could rapidly induce osteoblast differentiation of human bone marrow osteogenic stromal cells and rat bone marrow stromal cells. 25,26 However, a high dose of dexamethasone suppresses the proliferation of human osteoblast precursors. 27 Numerous recent studies have suggested that dexamethasone can induce osteoblast cell death, bone loss, and osteoporosis. ...
Purpose
Glucocorticoids are used for the treatment of inflammatory diseases, but glucocorticoid treatment is associated with bone damage. Resveratrol is a phytoalexin found in many plants, and we investigated its protective role on dexamethasone-induced dysfunction in MC3T3-E1 cells and primary osteoblasts.
Materials and Methods
MC3T3-E1 cells and primary osteoblasts were treated with dexamethasone in the presence/absence of different doses of resveratrol for 24 or 48 h. Then, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium (MTT) and lactate dehydrogenase (LDH) assays were used to evaluate cell viability. Apoptosis was analyzed by a flow cytometry. An alkaline phosphatase (ALP) activity assay and Alizarin Red S staining were used to study osteoblast differentiation. Expression of osteoblast-related genes was measured by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The AMP-activated protein kinase (AMPK) signaling pathway and mitochondrial expression of superoxide dismutase were evaluated by Western blotting. Intracellular reactive oxygen species (ROS), adenosine triphosphate (ATP) content, mitochondrial-complex activity, and mitochondrial DNA content were measured to evaluate mitochondrial function.
Results
Resveratrol induced the proliferation and inhibited apoptosis of osteoblasts in the presence of dexamethasone. Resveratrol increased the ALP activity and mineralization of osteoblasts. Resveratrol also attenuated dexamethasone-induced inhibition of mRNA expression of osteogenesis maker genes, including bone morphogenetic protein-2, osteoprotegerin, runt-related transcription factor-2, and bone Gla protein. Resveratrol alleviated dexamethasone-induced mitochondrial dysfunction. Resveratrol strongly stimulated expression of peroxisome proliferator–activated receptor-γ coactivator 1α and sirtuin-3 genes, as well as their downstream target gene superoxide dismutase-2. Resveratrol induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Blockade of AMPK signaling using compound C reversed the protective effects of resveratrol against dexamethasone.
Conclusion
Resveratrol showed protective effects against dexamethasone-induced dysfunction of osteoblasts by activating AMPK signaling.
... In this study, calcium accumulation in DPSCs transfected with SIRT1 was significantly higher rather than in control group after osteogenic differentiation in vitro. Furthermore, ALP is widely used to test the mineralized matrices produced by osteoblast-like cells in early differentiation [29]. Our results indicated that SIRT1 increased the ALP activity significantly. ...
Distraction osteogenesis (DO) is one of the most promising reconstructive methods for repairing large craniofacial defects or growth deficiencies through bone regeneration, but it is also a challenge because of an undesirably long process and its complications, which limit its application in clinical practice. The transplantation of mesenchymal stem cells (MSCs) is regarded as an innovative approach to accelerate bone regeneration. Dental pulp stem cells (DPSCs) have shown some advantages over other human adult MSCs, and DPSCs have been regarded as one of the most promising cell sources used in the endogenous tissue engineering. Furthermore, using stem cells modified by gene engineering in DO has been reported in previous studies. It has been shown that Sirtuin-1 (SIRT1) can directly regulate the differentiation of MSCs into osteoblasts. In this study, DPSCs expressing SIRT1 were prepared and their effects on the new bone formation were further investigated in rabbits with tibia. Rabbits were injected with the adenovirus (Adv)-SIRT1-green fluorescent protein (GFP)-transfected DPSCs (overexpression group, Group OE), Adv-GFP transfected DPSCs (negative control group, Group NC) or physiologic saline (control group, Groups CON) into the distraction gap. The new bone tissues in the distraction gap were harvested 8 weeks later, and subjected to by radiographic examination, micro-CT evaluation, and histological and mechanical testing. The better bone formation, the highest bone mineral density (BMD) and the highest bone mineral content (BMC) were observed in the OE group. In conclusion, SIRT1-modified DPSCs in DO was more effective to promote new bone formation during DO, which provides evidence for further investigation about the role of of SIRT1 in the DO.
... Western blotting also showed that BMP4 and SMAD4 protein expression levels were downregulated in non-union fractures (p < 0.01; Fig. 1c, d). Furthermore, protein levels of ALP, Col I, and RUNX2-important markers of osteogenesis [27][28][29]-were reduced in non-union fractures (p < 0.01; Fig. 1c, d). ALP activity was downregulated in non-union fractures (p < 0.01; Fig. 1e). ...
Background:
Atrophic non-union fractures show no radiological evidence of callus formation within 3 months of fracture. microRNA dysregulation may underlie the dysfunctional osteogenesis in atrophic non-union fractures. Here, we aimed to analyze miR-1323 expression in human atrophic non-union fractures and examine miR-1323's underlying mechanism of action in human mesenchymal stromal cells.
Methods:
Human atrophic non-union and standard healing fracture specimens were examined using H&E and Alcian Blue staining, immunohistochemistry, qRT-PCR, immunoblotting, and ALP activity assays. The effects of miR-1323 mimics or inhibition on BMP4, SMAD4, osteogenesis-related proteins, ALP activity, and bone mineralization were analyzed in human mesenchymal stromal cells. Luciferase reporter assays were utilized to assay miR-1323's binding to the 3'UTRs of BMP4 and SMAD4. The effects of miR-1323, BMP4, and SMAD4 were analyzed by siRNA and overexpression vectors. A rat femur fracture model was established to analyze the in vivo effects of antagomiR-1323 treatment.
Results:
miR-1323 was upregulated in human atrophic non-union fractures. Atrophic non-union was associated with downregulation of BMP4 and SMAD4 as well as the osteogenic markers ALP, collagen I, and RUNX2. In vitro, miR-1323 suppressed BMP4 and SMAD4 expression by binding to the 3'UTRs of BMP4 and SMAD4. Moreover, miR-1323's inhibition of BMP4 and SMAD4 inhibited mesenchymal stromal cell osteogenic differentiation via modulating the nuclear translocation of the transcriptional co-activator TAZ. In vivo, antagomiR-1323 therapy facilitated the healing of fractures in a rat model of femoral fracture.
Conclusions:
This evidence supports the miR-1323/BMP4 and miR-1323/SMAD4 axes as novel therapeutic targets for atrophic non-union fractures.
... Dexamethasone (Dex) is a glucocorticoid that promotes osteogenic differentiation and is used for enhancing bone regeneration [99][100][101]. However, the simultaneous delivery of these therapeutic agents together with bioactive ions using the same vehicle remains a challenge [102]. ...
Nanocomposite hydrogels consist of polymeric network embedded with functional nanoparticles or nanostructures, which not only contribute to the enhanced mechanical properties but also exhibit the bioactivities for regulating cell behavior. Bisphosphonates (BPs) are capable of coordinating with various metal ions and modulating bone homeostasis. Thanks to the inherent dynamic properties of metal–ligand coordination bonds, BP-based nanocomposite hydrogels possess tunable mechanical properties, highly dynamic structures, and the capability to mediate controlled release of encapsulated therapeutic agents, thereby making them highly versatile for various biomedical applications. This review presents the comprehensive overview of recent developments in BP-based nanocomposite hydrogels with an emphasis on the properties of embedded nanoparticles (NPs) and interactions between hydrogel network and NPs. Furthermore, various challenges in the biomedical applications of these hydrogels are discussed to provide an outlook of potential clinical translation.
... The medium was changed every 3-4 days, and cell morphology was assessed visually every day for up to 3 weeks. At the end of differentiation, cells were stained for alkaline phosphatase and with Von Kossa stain [42][43][44]. ...
Preeclampsia is a syndrome characterized by deterioration of either the maternal condition or the fetal condition. The adverse intrauterine environment made by preeclampsia results into intrauterine growth restriction and increased risk of a variety of diseases in future life. Given the adverse environment of fetal circulation made in the preeclamptic condition, and the role of mesenchymal stem cell (MSC) as a multipotent progenitor cell, we hypothesized that MSCs derived from human umbilical cord blood (hUCB-MSCs) obtained from preeclampsia are adversely altered or affected compared with normal pregnancy. The aim of this study was to analyze the biological characteristics and compare the functional abilities and gene expression patterns of hUCB-MSCs originating from pregnant women with and without severe preeclampsia. hUCB-MSCs were isolated and cultured from 28 pregnant women with severe preeclampsia and 30 normal pregnant women. hUCB-MSCs obtained from women with preeclampsia were less proliferative and more senescent and had lower telomerase activity and higher ROS activity than cells from women with normal pregnancy. In addition, many senescence-related differentially expressed genes (DEGs) were identified by analysis of microarray gene expression profiles and significantly associated with the Gene Ontology term cell aging. In conclusion, hUCB-MSCs obtained from women with preeclampsia showed the poorly proliferative, more senescent, and decreased telomerase activity, and these characters may be related with functional impairment of MSC from preeclampsia compared with cells from normal pregnancy.
... Osteogenesis is a complex multistep process, and it is likely that CLCF1 plays a role in cellular transitions. Differentiation of MSCs into osteoblasts can be induced in vitro (12). To further investigate the effect of CLCF1 on osteogenesis, we examined the capacity of CLCF1 to bind, activate JAK/STAT signaling, and regulate osteoblastic differentiation in mouse MSCs. ...
Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into adipocytes, chondrocytes or osteocytes. MSC secrete an array of cytokines and express the LIFRβ (leukemia inhibitory factor receptor) chain on their surface. Mutations in the gene coding for LIFRβ lead to a syndrome with altered bone metabolism. LIFRβ is one of the signalling receptor chains for Cardiotrophin-like cytokine (CLCF1), a neurotrophic factor known to modulate B and myeloid cell functions. We investigated its effect on MSC induced to differentiate into osteocytes in vitro. Our results indicate that CLCF1 binds mouse MSC, triggers STAT1 and 3 phosphorylation, inhibits the upregulation of master genes involved in the control of osteogenesis and markedly prevents osteoblast generation and mineralization. This suggests that CLCF1 could be a target for therapeutic intervention with agents such as cytokine traps or blocking mAbs in bone diseases such as osteoporosis.
... Electric fields can control differentiation by modifying the membrane potential [8,9], which impact voltage-gated channels [10] and the influx of ions to determine the differentiation of embryonic stem cells [11]. Electric fields can also induce cytoskeletal stresses to manipulate osteoblasts and mesenchymal stem cells [12], which was previously possible only by using chemicals [13] or proteins [14]. ...
Low-intensity electric fields can induce changes in cell differentiation and cytoskeletal stresses that facilitate manipulation of osteoblasts and mesenchymal stem cells; however, the application times (tens of minutes) are of the order of physiological mechanisms, which can complicate treatment consistency. Intense nanosecond pulsed electric fields (nsPEFs) can overcome these challenges by inducing similar stresses on shorter timescales while additionally inducing plasma membrane nanoporation, ion transport and intracellular structure manipulation. This paper shows that treating myoblasts and osteoblasts with five 300 ns PEFs with intensities from 1.5 to 25 kV cm-1 increased proliferation and differentiation. While nsPEFs above 5 kV cm-1 decreased myoblast population growth, 10 and 20 kV cm-1 trains increased myoblast population by approximately fivefold 48 h after exposure when all cell densities were set to the same level after exposure. Three trials of the PEF-treated osteoblasts showed that PEF trains between 2.5 and 10 kV cm-1 induced the greatest population growth compared to the control 48 h after treatment. Trains of nsPEFs between 1.5 and 5 kV cm-1 induced the most nodule formation in osteoblasts, indicating bone formation. These results demonstrate the potential utility for nsPEFs to rapidly modulate stem cells for proliferation and differentiation and motivate future experiments to optimize PEF parameters for in vivo applications.
... The oscillation of this parameter can occur primarily in the early stages of culture (Hoemann et al., 2009). Similarly, dexamethasone (10nM or 100nM) stimulates alkaline phosphatase activity during the first week of MSC cultures (Cheng et al., 1994), then promoting the activity of the enzyme after three weeks of suppression (Yin et al., 2006). It is known that caffeine acts in synergy with dexamethasone and activates receptors for glucocorticoids in bone cell cultures (Focking et al., 2005). ...
The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.
... We demonstrated that OIM induced the enhancement of osteogenic differentiation of spheroid-derived HPLSCs, as compared with monolayer-derived HPLSCs. MSCs cultured with OIM are characterized by an increase in ALP at early stages, followed by matrix deposition, maturation, and mineralization at later stages 28,29) . In this study, enhanced staining with ALP was observed in spheroid-derived HPLSCs cultured with OIM. ...
Human periodontal ligament stem cells (HPLSCs) contribute to the regeneration of periodontal tissue because of their multilineage potential. Although monolayer cultures are commonly used in cell cultures, they inadequately overcome their low differentiation capacity. The use of spheroid cultures is expected to overcome the problem of mimicking the in vivo microenvironment. In this study, we assessed whether HPLSC spheroids are susceptible to osteogenic differentiation through the canonical Wnt/β-catenin signaling pathway. HPLSC spheroids were generated using low-binding plates. Osteogenic differentiation of monolayer- and spheroid-derived HPLSCs was induced by osteogenic induction medium. Increased expression levels of osterix and Runx2 and intense staining of alkaline phosphatase (ALP) activity were observed in spheroid-derived HPLSCs, as compared with monolayer-derived cells. During spheroid formation, the integrin pathway of cells composed of spheroids was activated through focal adhesion kinase (FAK), suggesting that this activation may induce susceptibility of the Wnt pathway in HPLSC spheroids. Wnt 3a stimulation increased the expression levels of β-catenin and T-cell factor (TCF), but decreased that of glycogen synthase kinase-3β. Wnt 3a-induced the expression of β-catenin and TCF was effectively decreased by Dickkopf-1 (Dkk-1), a Wnt antagonist. Wnt 3a stimulation also increased the expression levels of osterix and Runx2 which was accompanied by intense ALP staining, in HPLSC spheroids, whereas the addition of Dkk-1 decreased both expression levels and ALP staining. These findings indicate that HPLSC spheroids enhance osteogenic differentiation because cells, composed of spheroids, induce susceptibility of the canonical Wnt pathway, which is mediated by activation of the integrin/FAK pathway during spheroid formation.
... There were no differences in cell morphology and viability between monolayer-derived MSCs and spheroid-derived MSCs. OIM-induced osteogenesis in MSCs is characterized by an increase in ALP at early stages, followed by matrix deposition, matrix maturation, and mineralization at later stages [25][26][27] . BMPs are known to be the most important inducers and stimulators of osteogenic differentiation 8,9) . ...
Spheroid culture systems more accurately recreate the in vivo microenvironment and are susceptible to factors that induce differentiation. In this study, we assessed whether bone morphogenetic protein (BMP)-2 induces enhanced osteogenic differentiation in spheroid-derived mesenchymal stem cells (MSCs). MSC spheroids were generated from human adipose tissue-derived MSCs using low-binding plates. Osteogenic differentiation of monolayer and spheroid-derived MSCs was induced by osteogenesis induction medium (OIM) with or without BMP-2. Increased alkaline phosphatase and Alizarin Red staining were observed in spheroid-derived MSCs treated with a mixture of OIM and BMP-2, compared with monolayer MSCs. Spheroid-derived MSCs had increased mRNA and protein expressions of osteogenic runt-related transcription factor 2 (Runx2) and osterix (OSX). The intranuclear expression of OSX was also observed in spheroid-derived MSCs treated with the mixture of OIM and BMP-2. In addition, spheroid-derived MSCs with BMP-2 treatment showed the upregulation of Smad5 mRNA and phosphorylated Smad1/5, suggesting that the Smad-BMP signaling pathway is enhanced in these cells. Our data indicate that the Smad-dependent BMP signaling pathway accelerates osteogenic differentiation in spheroid-derived MSCs, compared with monolayer MSCs.
... Assessing in vitro differentiation potential of calvarial osteoblast culture is one of the standard systems for studying the regulation of bone cell function 12 . A wide variety of approaches have been developed to study osteoblasts in vitro, including murine primary cell cultures, immortalized osteoblast-like cell lines, and human osteoblasts [12][13][14][15][16] . In combination, these methods have facilitated critical information on the regulation of osteoblast proliferation, differentiation, survival, and function. ...
Osteoblasts are responsible for the maintenance of bone homeostasis. Deregulation of their differentiation is etiologically linked to several bone disorders, making this process an important target for therapeutic intervention. Systemic identification of osteoblast regulators has been hampered by the unavailability of physiologically relevant in vitro systems suitable for efficient RNAi and for differentiation read-outs compatible with fluorescent microscopy-based high-content analysis (HCA). Here, we report a new method for identification of osteoblast differentiation regulators by combining siRNA transfection in physiologically relevant cells with high-throughput screening (HTS). Primary mouse calvarial osteoblasts were seeded in 384-well format and reverse transfected with siRNAs and their cell number and differentiation was assayed by HCA. Automated image acquisition allowed high-throughput analyses and classification of single cell features. The physiological relevance, reproducibility, and sensitivity of the method were validated using known regulators of osteoblast differentiation. The application of HCA to siRNAs against expression of 320 genes led to the identification of five potential suppressors and 60 activators of early osteoblast differentiation. The described method and the associated analysis pipeline are not restricted to RNAi-based screening, but can be adapted to large-scale drug HTS or to small-scale targeted experiments, to identify new critical factors important for early osteoblastogenesis.
... Clinically, DXM has been utilized as an anti-inflammatory drug [15]. Previous studies reported that DXM can induce osteoporosis and even pathological fracture [16], while DXM in vitro promotes osteoblast differentiation and bone mineralization [17][18][19]. Actually, DXM has been routinely used to induce the differentiation of MSCs and is a key component in osteogenic differentiation medium. Nevertheless, differential effects of DXM on undifferentiated MSCs and osteoblasts have been reported [20]. ...
The extracellular microvesicles (MVs) are attracting much attention because they are found to be the key paracrine mediator participating in tissue regeneration. Dexamethasone (DXM) is widely accepted as an important regulator in tailoring the differentiation potential of mesenchymal stem cells (MSCs). However, the effect of DXM on the paracrine signaling of MSCs remains unknown. To this point, we aimed to explore the role of DXM in regulating the paracrine activity of MSCs through evaluating the release and function of MSC-MVs, based on their physicochemical characteristics and support on osteogenic response. Results showed that DXM had no evident impact on the release of MSC-MVs but played a pivotal role in regulating the function of MSC-MVs. MVs obtained from the DXM-stimulated MSCs (DXM-MVs) increased MC3T3 cell proliferation and migration and upregulated Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteopontin (OPN) expression. The repair efficiency of DXM-MVs for femur defects was further investigated in an established rat model. It was found that DXM-MVs accelerated the healing process of bone formation in the defect area. Thus, we conclude that using DXM as stimuli to obtain functional MSCs-MVs could become a valuable tool for promoting bone regeneration.
... Identification of a substance with a positive effect on implant stability could have a significant clinical relevance on implant survival. Recently, many investigations have been carried out on factors which are able to increase the speed and quantity of bone formation around dental implants: [19], [20], [21], [22]; add to many natural products previously evaluated for their involvement in bone remodeling, effects on bone metabolism, structure, and strength [15]. Here, in this study, we tried to use these natural products to test their possible involvement in bone integration around orthodontic implant as represented by their stability value. ...
... Recently, many investigations have been carried out on factors which may be able to increase the speed and quantity of bone formation around dental implants. [22][23][24][25] This study tried to use four types of natural products to test their possible effect on bone integration around orthodontic implant represented by serum biochemistry evaluation. The study condition is differing from previous studies, as there was traumatic injury, inflammation and bone response pathologic remodeling. ...
Background and objective: Bone integration around orthodontic implant is a matter of concern for their stability. This study was conducted to estimate calcium and phosphorous level in after insertion of orthodontic implant.
Methods: Twenty five white mature male rabbits, classified into 5 groups: one control and four experimental, with five rabbits for each group were used. Fifty orthodontic implants were used, 2 micro-implant for each tibia. Four different natural products extract were used in this study that included Curcumin 15mg/kg, Nigella Sativa oil 0.25 ml/kg, Cissus Quadrangularis 500mg/kg and Virgin Coconut oil 1 ml/kg. Each product was given to certain experimental group started from the day of implant insertion for four weeks healing period. The biochemistry evaluation was conducted involving calcium and phosphorus level in serum.
Results: Significant difference in serum calcium levels were detected between Curcumin, Nigella Sativa oil from side and control group on the other side.
Conclusion: Systemic Curcumin and Nigella sativa oil may be used for possibly enhancing bone response around orthodontic implant as reflected by lower serum calcium level as compared to control group.
... This action is dependent upon the duration of the exposure and the dose of GC, and the stage of differentiation of the osteoblasts. Low GC doses or short durations of in vitro administration of embryonic or adult mesenchymal stem cells (MSC), as well as immature osteoblast progenitor cells, promote early osteoblastic differentiation with increases in the mRNA expression of osteoblastic genes [1,93]. In contrast, higher GC doses and longer exposure of cells committed to the osteoblastic lineage suppress differentiation and inhibit the expression of osteoblast markers [94,95]. ...
Glucocorticoids (GC), produced and released by the adrenal glands, regulate numerous physiological processes in a wide range of tissues. Because of their profound immunosuppressive and anti-inflammatory actions, GC are extensively used for the treatment of immune and inflammatory conditions, the management of organ transplantation, and as a component of chemotherapy regimens for cancers. However, both pathologic endogenous elevation and long-term use of exogenous GC are associated with severe adverse effects. In particular, excess GC has devastating effects on the musculoskeletal system. GC increase bone resorption and decrease formation leading to bone loss, microarchitectural deterioration, and fracture. GC also induce loss of muscle mass and strength leading to an increased incidence of falls. The combined effects on bone and muscle account for the increased fracture risk with GC. This review summarizes the advance in knowledge in the last two decades about the mechanisms of action of GC in bone and muscle and the attempts to interfere with the damaging actions of GC in these tissues with the goal of developing more effective therapeutic strategies.
... On the other hand, ALP activity can be influenced by calcium levels, which in small amounts cause the increase of ALP activity and cell differentiation. Moreover, osteogenic supplements, like ascorbic acid and β-glycerophosphate, up-regulate ALP gene expression [36]. Osteogenic supplements in OM, low concentrations of Ca and increasing levels of Si from 21 days to 28 days may create a synergistic effect on the stimulation of ALP activity. ...
This work evaluates in vitro the influence of a new biocompatible porous Si-CaP monophasic (7CaO·P 2 O 5 ·2SiO 2) ceramic on the cellular metabolic activity, morphology and osteogenic differentiation of adult human mesenchymal stem cells (ahMSCs) cultured in basal growth medium and under osteogenic inductive medium. Alamar Blue Assay and FESEM were carried out in order to monitor the cell proliferation and the shape of the cells growing on the Si-CaP monophasic ceramic during the study period. The osteogenic differentiation of ahMSCs was investigated by means of immunofluorescent staining (osteocalcin, osteopontin, heparan sulphate and collagen type I expression), quantitative reverse transcription polymerase chain reaction (qRT-PCR) (integrin-binding sialoprotein, osteocalcin, alkaline phosphatase, osteopontin, osteonectin, runt-related transcription factor 2 and collagen type I) and expression of surface markers (CD73, CD90 and CD105). We could check osteogenic differentiation in ahMSCs growing under the influence of Si-CaP monophasic ceramics itself, but especially when growth medium was replaced by osteogenic medium in the culture conditions. These results allowed us to conclude that the new Si-CaP monophasic scaffold greatly enhanced ahMSCs proliferation and osteogenic differentiation; therefore, it may be considered to be employed as a new bone graft substitute or scaffold for bone tissue engineering.
... 17 As one of the earliest known and readily available osteogenic inducers for mesenchymal stem cells (MSCs), DEX plays a key role in controlling the expression of osteogenic marker genes. 18 MSCs cultured with continuous DEX treatment exhibit higher expression levels of osteogenic markers and a higher positive rate of osteogenic colony formation than those without DEX treatment. [19][20][21] To date, several DEX delivery methods have been developed, ranging from systemic injection, 22 eluting coating, [23][24][25] and loading within biodegradable polymer carriers. ...
As an earliest known and readily available osteogenic inducer for stem cells, dexamethasone (DEX) plays a key role in affecting cell functions and cellular processes, especially for cell proliferation and differentiation. However, the clinical application of DEX has been limited because of its uncontrolled release. An ideal carrier is desired to control DEX release for osteogenic differentiation of stem cells and bone tissue engineering. Biphasic calcium phosphate nanoparticles (BCP-NPs) should be a potential carrier for DEX due to their osteoconductive property and good biocompatibility as a bone graft biomaterial. In this study, DEX-loaded BCP-NPs were prepared by two methods: (1) immersion of BCP-NPs in a DEX solution (defined as DEX/BCP-NPs), (2) DEX incorporation during BCP-NPs formation in a calcifying solution (defined as DEX@BCP-NPs). The DEX@BCP-NPs showed higher DEX loading amount and more sustainable DEX release than did the DEX/BCP-NPs. The DEX@BCP-NPs were used for culture of human bone marrow-derived mesenchymal stem cells (hMSCs) and showed a promotive effect on proliferation of hMSCs. Furthermore, the DEX@BCP-NPs significantly increased the alkaline phosphatase (ALP) activity, calcium deposition and gene expressions of osteogenic markers of hMSCs when compared to BCP-NPs without DEX loading. The results demonstrated BCP-NPs were a good carrier for DEX loading and the DEX@BCP-NPs should be useful guidance for bone tissue engineering.
... The roles of GR activation on osteogenesis have also been reported. Low glucocorticoids (GC) concentrations promote mesenchymal stem cell commitment and osteoblastogenic differentiation (Cheng et al., 1994;Hong et al., 2009); while high concentrations and long term treatments inhibit osteoblasts maturation and terminal differentiation (Ishida & Heersche, 1998;Ito et al., 2007). Even though we could not detect GR activation using MMTV-Luc reporter assays, knockdown of GR clearly attenuated the suppression of ALP mRNA, potentiated the suppression of OPN mRNA, and had no effects on suppression on OCA mRNA, by butylparaben. ...
... Finally, dexamethasone is often used as an osteogenic factor, because it is essential for the full differentiation of hBMSCs into mineral-producing osteoblasts as shown by increased ALP activity [49,50]. However, in the present study, hBMSCs cultured in OM with dexamethasone on TCP ceramics showed reduced levels of Col I, OCN, and OPN gene expression compared to cells in BM. ...
Statement of significance:
The surface topography of synthetic biomaterials plays important roles in material-driven osteogenesis. The data presented herein have shown that the surface topography of calcium phosphate ceramics regulates mesenchymal stromal cells (e.g., human bone marrow mesenchymal stromal cells, hBMSCs) with respect to morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation in vitro. Together with bone formation in vivo, our results suggested a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery by the bioengineering control of osteogenesis via primary cilia modulation.
... A indução química de diferenciação em osteoblastos foi deflagrada pela adição de dexametasona (Sigma) 10 -7 M, 5g/mL de ácido ascórbico (Gibco) e 2,16 g/mL de glicerolfosfato (Sigma) ao meio de cultura DMEM/F12 suplementado com 15% de SBF. Estas condições de cultura induzem a diferenciação osteoblástica (ROSA, BELOTI, 2003;COELHO et al., 2000;PITTENGER et al., 1999;CHENG et al., 1994). ...
MicroRNAs (miRNAs) are able to direct the repression of translation of target mRNAs, generating significant changes in gene expression. They are important regulators of many biological processes including stem cell differentiation, among which mesenchymal cells (MSCs). The commitment of MSCs to osteoblasts is a process defined by four stages: cell commitment, proliferation, maturation and matrix mineralization. The differentiation of osteoblasts is a key step involving the activation of several signaling pathways, including TGFb, BMP, Wnt and transcription factors, which are tightly regulated by miRNAs. In this work we did use of the methodological potential of microarrays along with the robustness of bioinformatics analysis (hierarchical data clustering and post-transcriptional interactions) to study the regulatory mechanisms involved in the control of osteoblastic differentiation of stem cells of human dental pulp. This enabled the study of both transcriptional (mRNA expression) and post-transcriptional (miRNA-mRNA interactions), searching for those miRNAs and mRNAs differentially expressed under the influence of compounds that induce in vitro the osteogenesis of MSCs (dexamethasone, ascorbic acid and beta -glycerol phosphate), antagonists of miRNAs (antisense oligos that specifically bind to miRNAs) and miRNAs (sense oligos that are identical to miRNAs or miRNA mimics). The expression profile of miRNAs and mRNAs during the differentiation of MSCs was analyzed by hybridization with Agilent microarrays and the interactions between miRNAs and their targets were analyzed using GenMir ++ algorithm and reconstruction of networks of miRNA-mRNA interaction. A set of six miRNAs were identified (miR-29b, miR-29c-5p, miR-17-3p, miR-28-5p, miR-450a-5p, miR-145-3p), which were analyzed in detail. We found that the interactions between miR-450a-5p and the mRNAs that encode respectively STAT1 and PTGS2 represent important nodes during the post-transcriptional control of osteoblast differentiation of human MSCs.
Biomimetic silica deposition is an in-situ immobilization method for bioactive molecules under biocompatible conditions. The osteoinductive P4 peptide derived from the knuckle epitope of bone morphogenetic protein (BMP), which binds to BMP receptor-II (BMPRII), has been newly found to contain silica formation ability. We found that the two lysine residues at the N-terminus of P4 played a vital role in silica deposition. The P4 peptide co-precipitated with silica during P4-mediated silicification, yielding P4/silica hybrid particles (P4@Si) with a high loading efficiency of 87%. P4 was released from P4@Si at a constant rate for over 250 h, representing a zero-order kinetic model. In flow cytometric analysis, P4@Si showed a 1.5-fold increase in the delivery capacity to MC3T3 E1 cells than the free form of P4. Furthermore, P4 was found anchored to hydroxyapatite (HA) through a hexa-glutamate tag, followed by P4-mediated silicification, yielding P4@Si coated HA. This suggested a superior osteoinductive potential compared to silica or P4 alone coated HA in the in vitro study. In conclusion, the co-delivery of the osteoinductive P4 peptide and silica by P4-mediated silica deposition is an efficient method for capturing and delivering its molecules and inducing synergistic osteogenesis.
Glucocorticoid (GC) is one of the most prescribed medicines to treat various inflammatory and autoimmune diseases. However, high doses and long-term use of GCs lead to multiple adverse effects, particularly glucocorticoid-induced osteoporosis (GIO). Excessive GCs exert detrimental effects on bone cells, including osteoblasts, osteoclasts, and osteocytes, leading to impaired bone formation and resorption. The actions of exogenous GCs are considered to be strongly cell-type and dose dependent. GC excess inhibits the proliferation and differentiation of osteoblasts and enhances the apoptosis of osteoblasts and osteocytes, eventually contributing to reduced bone formation. Effects of GC excess on osteoclasts mainly include enhanced osteoclastogenesis, increased lifespan and number of mature osteoclasts, and diminished osteoclast apoptosis, which result in increased bone resorption. Furthermore, GCs have an impact on the secretion of bone cells, subsequently disturbing the process of osteoblastogenesis and osteoclastogenesis. This review provides timely update and summary of recent discoveries in the field of GIO, with a particular focus on the effects of exogenous GCs on bone cells and the crosstalk among them under GC excess.
Understanding the optimal conditions required for bone healing can have a substantial impact to target the problem of non–unions and large bone defects. The combination of bioactive factors, regenerative progenitor cells and biomaterials to form a tissue engineered (TE) complex is a promising solution but translation to the clinic has been slow. We hypothesized the typical material testing algorithm used is insufficient and leads to materials being mischaracterized as promising. In the first part of this study, human bone marrow – derived mesenchymal stromal cells (hBM-MSCs) were embedded in three commonly used biomaterials (hyaluronic acid methacrylate, gelatin methacrylate and fibrin) and combined with relevant bioactive osteogenesis factors (dexamethasone microparticles and polyphosphate nanoparticles) to form a TE construct that underwent in vitro osteogenic differentiation for 28 days. Gene expression of relevant transcription factors and osteogenic markers, and von Kossa staining were performed. In the second and third part of this study, the same combination of TE constructs were implanted subcutaneously (cell containing) in T cell-deficient athymic Crl:NIH-Foxn1rnu rats for 8 weeks or cell free in an immunocompetent New Zealand white rabbit calvarial model for 6 weeks, respectively. Osteogenic performance was investigated via MicroCT imaging and histology staining. The in vitro study showed enhanced upregulation of relevant genes and significant mineral deposition within the three biomaterials, generally considered as a positive result. Subcutaneous implantation indicates none to minor ectopic bone formation. No enhanced calvarial bone healing was detected in implanted biomaterials compared to the empty defect. The reasons for the poor correlation of in vitro and in vivo outcomes are unclear and needs further investigation. This study highlights the discrepancy between in vitro and in vivo outcomes, demonstrating that in vitro data should be interpreted with extreme caution. In vitro models with higher complexity are necessary to increase value for translational studies.
Statement of Significance
: Preclinical testing of newly developed biomaterials is a crucial element of the development cycle. Despite this, there is still significant discrepancy between in vitro and in vivo test results. Within this study we investigate multiple combinations of materials and osteogenic stimulants and demonstrate a poor correlation between the in vitro and in vivo data. We propose rationale for why this may be the case and suggest a modified testing algorithm.
The standard procedure to treat oronasal fistula in dogs requires tooth extraction to close the fistula; hence, the subject would lose its tooth. In this study, trafermin was applied to four dog models with oronasal fistula to investigate the periodontal tissue regenerative effects of trafermin in the treatment without tooth extraction. A fistula was created along the palatal side of each upper canine tooth. One of the fistulae was filled with trafermin, whereas that on the contralateral side was left unfilled as a control. The results showed a significant decrease in the non-calcified periodontal tissue volume on the trafermin side after the fourth week. In addition, oronasal fistula closure was visually and histologically confirmed at the eighth week on the trafermin side of all four models.
The bone marrow is composed of the non-adherent hematopoietic and adherent stromal cell compartments. This adherent bone marrow stromal cell fraction contains pluripotent mesenchymal stem cells and differentiated mesenchymal bone marrow stromal cells. In vivo the mesenchymal stem cells self-renew by proliferation while maintaining their stem-cell phenotype and give rise to the differentiated stromal cells which belong to different lineages. This experimental study demonstrate that mesenchymal stem cells from dog bone marrow had the in vitro capability to differentiate in osteogenic and chondrogenic lineages after culture in proper media and these properties can be used in osteoarticular reconstruction if the differentiated cells had a suitable support.
The bone marrow is composed of the non-adherent hematopoietic and adherent stromal cell compartments. This adherent bone marrow stromal cell fraction contains pluripotent mesenchymal stem cells and differentiated mesenchymal bone marrow stromal cells. In vivo the mesenchymal stem cells self-renew by proliferation while maintaining their stem-cell phenotype and give rise to the differentiated stromal cells which belong to different lineages. This experimental study demonstrate that mesenchymal stem cells from dog bone marrow had the in vitro capability to differentiate in osteogenic and chondrogenic lineages after culture in proper media and these properties can be used in osteoarticular reconstruction if the differentiated cells had a suitable support.
The purpose of this study was to evaluate the bioactivity and cell response of a well-characterized Nurse´s A-phase (7CaO•P2O5•2SiO2) ceramic and his effect compared to a control (tissue culture polystyrene-TCPS) on the adhesion, viability, proliferation and osteogenic differentiation of ahMSCs in vitro. Cell proliferation (Alamar Blue Assay), Alizarin Red-S (AR-s) staining, alkaline phosphatase (ALP) activity, osteocalcin (OCN) and collagen I (Col I) were evaluated. Also, field emission scanning electron microscopy (FESEM) images were acquired in order to visualise the cells and the topography of the material. The proliferation of cells growing in a direct contact with the material was slower at early stages of the study because of the new environmental conditions. However, the entire surface was colonized after 28 days of culture in growth medium (GM). Osteoblastic differentiation markers were significantly enhanced in cells growing on Nurse´s A phase ceramic and cultured with osteogenic medium (OM), probably due to the role of silica to stimulate the differentiation of ahMSCs. Moreover, calcium nodules were formed under the influence of ceramic material. Therefore, it is predicted that Nurse´s A-phase ceramic would present high biocompatibility and osteoinductive properties being a good candidate to be used as a biomaterial for bone tissue engineering.
This chapter describes methodologies based on the use of fluorescent indicators employed to explore intracellular events in live bone cells. The development of fluorescent probes sensitive to the concentration of ions, and more recently to cyclic adenosine monophosphate (cAMP) has enabled investigators to study real-time rapid events occurring after hormonal stimulation, or after changes in the extracellular environment. Fluorescence techniques are also used to analyze the mechanisms of cell–cell communication through gap junctions. Application of these techniques to bone biology has led to fundamental discoveries in several areas, including signal transduction of calciotropic hormones, regulation of intracellular pH homeostasis, cell coupling, and signaling by cell–matrix contact. There are two general experimental approaches to the application of fluorescence technique: One involves the use of bulk cell suspensions, and the other applies light microscopy to adherent cell layers. In the one involving the use of bulk cell suspensions, the signal reported by the fluorescent probe represents the average of a phenomenon occurring in a large number of cells, whereas in the latter, events can be monitored and recorded in individual cells. The two methods offer features that can be exploited for particular experimental needs. This chapter reviews the application of fluorescence techniques to bone biology, with emphasis on the peculiar methodological issues imposed by the biologic characteristic of such specialized cell systems. The investigator who approaches this field for the first time may find the introductory overview of the currently available models of bone cells useful to devise the most appropriate experimental system.
Dexamethasone (DEX) is a glucocorticoid commonly used as an in vitro osteogenic inducer of mesenchymal stem/progenitor cells (abbreviated MSCs). However, several studies investigating the effects of glucocorticoids on bone regeneration through systemic injections have demonstrated negative impacts of the drugs at high concentration on the healing of hard tissues. These contrasting evidences suggest that application of glucocorticoids should be limited to low dosages but at the same time a long enough treatment period is preferred, which prompted us to evaluate the effects of different local release systems of DEX on MSC differentiation and bone repair. Two types of DEX-loaded β-cyclodextrin (CD) complexes, including CD/DEX and CD/AD-DEX, were fabricated via host-guest interactions and characterized by FT-IR, 1H-NMR, MS-ESI, and UV-vis. The results demonstrated that these CD-based assemblies released DEX in differentiated profiles, with CD/DEX releasing significantly faster than CD/AD-DEX. Although CD/DEX were slightly more powerful than CD/AD-DEX in inducing rat bone marrow MSCs (rBMSCs) into osteogenic lineage in vitro, CD/AD-DEX was advantageous over CD/DEX in accelerating bone regeneration over a time period of 4 weeks in a rat tibia defect model. The results suggest that DEX-loaded assemblies via host-guest interactions are flexible in modulating DEX release patterns and have great potential in bone tissue engineering.
Background
The periodontal ligament cells (PDLCs) contain heterogeneous cell populations and possess stem-cell-like properties. PDLCs have attracted considerable attention as an option for periodontal regeneration. However, the osteogenic differentiation of PDLCs remains obscure owing to variable osteo-inductive methods and whether PDLCs could be directly used for periodontal regeneration without stem cell enrichment is uncertain. The aim of the present study was to clarify the osteogenic differentiation capacity of PDLCs and test PDLCs as an alternative to stem cells for periodontal regeneration.
Methods
We tested the performance of human PDLCs in osteo-inductive culture and transplantation in vivo while taking human bone marrow derived mesenchymal stem cells (hMSCs) as positive control. Proliferation of PDLCs and hMSCs in osteo-inductive condition were examined by MTT assay and colony formation assay. The osteogenic differentiations of PDLCs and hMSCs were assessed by Alkaline phosphatase (ALP) activity measurement, von Kossa staining, Alizarin red S staining and quantitative RT-PCR of osteogenic marker gene including RUNX2, ALP, OCN, Col I, BSP, OPN. We transplanted osteo-inductive PDLCs and hMSCs with hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds to immunodeficient mice to explore their biological behaviors in vivo by histological staining and immunohistochemical evaluation.
Results
After 14 days of osteo-induction, PDLCs exhibited significantly higher proliferation rate but lower colony-forming ability comparing with hMSCs. PDLCs demonstrated lower ALP activity and generated fewer mineralized nodules than hMSCs. PDLCs showed overall up-regulated expression of RUNX2, ALP, OCN, Col I, BSP, OPN after osteo-induction. Col I level of PDLCs in osteo-inductive group was significantly higher while RUNX2, ALP, OCN were lower than that of hMSCs. Massive fiber bundles were produced linking or circling the scaffold while the bone-like structures were limited in the PDLCs-loaded HA/TCP samples. The fiber bundles displayed strong positive Col I, but weak OCN and OPN staining. The in vivo results were consistent with the in vitro data, which confirmed strong collagen forming ability and considerable osteogenic potential of PDLCs.
Conclusion
It is encouraging to find that PDLCs exhibit higher proliferation, stronger collagen fiber formation capacity, but lower osteogenic differentiation ability in comparison with hMSCs. This characteristic is essential for the successful periodontal reconstruction which is based on the synchronization of fiber formation and bone deposition. Moreover, PDLCs have advantages such as good accessibility, abundant source, vigorous proliferation and evident osteogenic differentiation capacity when triggered properly. They can independently form PDL-like structure in vivo without specific stem cell enrichment procedure. The application of PDLCs may offer a novel cytotherapeutic option for future clinical periodontal reconstruction.
Damit die Knochenregeneration lege artis abläuft ist ein sensibles und komplexes Zusammenspiel einer Reihe von Faktoren notwendig. Neben bestimmten Zelltypen, die für die Knochenregeneration essentiell sind, sind auch eine Reihe von Wachstumsfaktoren notwendig um die Kommunikation zwischen den Zellverbänden zu gewährleisten und die einzelnen Entwicklungsstadien zu steuern und zu regulieren. Zur Untersuchung der Möglichkeit, sowohl die Osteokonduktion als auch Vaskularisation eines Scaffolds zu initiieren, wurden in der vorliegenden Arbeit verschiedene Untersuchungsmethoden eingesetzt. Damit wurden adulte humane mesenchymale Stammzellen untersucht, die zur Differenzierung mit den Wachstumsfaktoren bone morphogenetic protein 2 (BMP 2) und/oder vascular endothelial grwoth factor (VEGF) inkubiert und auf Glas- oder Bruschitoberflächen kultiviert wurden. Die Experimente wurden mittels immunologischer Methoden wie Immunfluoreszenz (IF) und Westernblot (WB), sowie über Rasterelektronenmikroskopie (REM) analysiert. Es konnte mit diesen Methoden gezeigt werden, dass die humanen mesenchymalen Stammzellen (hMSC) auf den verschiedenen Substraten adhärierten und proliferierten. Darüber hinaus konnte in der Arbeit nachgewiesen werden, dass unter diesen bestimmten Bedingungen sowohl knöcherne als auch vaskuläre Zellbildung angeregt werden kann. So konnte sowohl auf Glas als auch auf Bruschit mittels IF und REM zum Teil aus hMSC differenzierte Osteoblasten detektiert werden. Diese zeigten die für Osteoblasten typischen Zellfortsätze, mit denen die Osteoblasten mit den Nachbarzellen in Kontakt stehen. Die beginnende Differenzierung zu Osteoblasten bzw. Endothelzellen konnte auch durch Detektion spezfischer Marker, wie z.B. alkalische Phosphatase und PECAM mittels WB gezeigt werden. Jedoch war die in dieser Arbeit zur Untersuchung der ortsgerichteten Differenzierung auf Bruschitsubstrat eingesetzte Methodik nicht geeignet, eindeutige Aussagen zu treffen. Daher müssen zur Untersuchung dieses Vorganges alternative Methoden entwickelt und optimiert werden. Bei der vorliegenden Arbeit handelt es sich um eine reine in vitro Studie. Dennoch könnten diese Ergebnisse Hinweise auf das Verhalten von hMSC unter Stimulation mit osteogenen und endothelialen Wachstumsfaktoren in vivo im Tierversuch oder im Menschen liefern. Allerdings wird es bei der Übertragung auf eine kombiniertes in vitro- in vivo Vorgehen hinauslaufen, da das ungerichtete Wachstum von Gewebsformationen eine Hürde für in vivo Studien darstellt.
Osteoporosis associated with long-term glucocorticoid therapy remains a common and serious bone disease. In addition, in recent years it has become clear that more subtle states of endogenous glucocorticoid excess may have a major impact on bone health. Adverse effects can be seen with mild systemic glucocorticoid excess but there is also evidence of tissue-specific regulation of glucocorticoid action within bone as a mechanism of disease. This review article will examine a) the role of endogenous glucocorticoids in normal bone physiology, b) the skeletal effects of endogenous glucocorticoid excess in the context of endocrine conditions such as Cushing’s disease/syndrome and autonomous cortisol secretion (subclinical Cushing’s syndrome), and c) the actions of therapeutic (exogenous) glucocorticoids on bone. We will review the extent to which the effect of glucocorticoids on bone is influenced by variations in tissue metabolising enzymes and glucocorticoid receptor expression and sensitivity. We will consider how the effects of therapeutic glucocorticoids on bone are complicated by the effects of the underlying inflammatory disease being treated. We will also examine the impact that glucocorticoid replacement regimens have on bone in the context of primary and secondary adrenal insufficiency. We conclude that even subtle excess of endogenous or moderate doses of therapeutic glucocorticoids are detrimental to bone. However in patients with inflammatory disorders there is a complex interplay between glucocorticoid treatment and underlying inflammation, with the underlying condition frequently representing the major component underpinning bone damage.
Cell replacement therapy holds a promising future in the treatment of degenerative diseases related to neuronal, cardiac and bone tissues. In such kind of diseases, there is a progressive loss of specific types of cells. Currently the most upcoming and trusted cell candidate is Mesenchymal Stem Cells (MSCs) as these cells are easy to isolate from the tissue, easy to maintain and expand and no ethical concerns are linked. MSCs can be obtained from a number of sources like bone marrow, umbilical cord blood, umbilical cord, dental pulp, adipose tissues, etc. MSCs help in tissue repair and regeneration by various mechanisms of action like cell differentiation, immunomodulation, paracrine effect, etc. The future of regenerative medicine lies in tissue engineering and exploiting various properties to yield maximum output. In the current review article, we have targeted the repair and regeneration mechanisms of MSCs in neurodegenerative diseases, cardiac diseases and those related to bones. Yet there is a lot to understand, discover and then understand again about the molecular mechanisms of MSCs and then applying this knowledge in developing the therapy to get maximum repair and regeneration of concerned tissue and in turn the recovery of the patient.
In this study, an injectable and thermo-sensitive alginate/β-tricalcium phosphate hydrogel (TSAH/β-TCP) was prepared for aspirin release to a bone defect. Aspirin was dissolved into a mixture of poly(N-isopropylacrylamide) (PNIPAAm), an aminated alginate-g-PNIPAAm co-polymer, and β-TCP powders. Scanning electron microscopy showed that TSAH/β-TCP had an interconnected porous microstructure with a porosity of 86.78%. The cross-linked hydrogel released approximately 40% of the aspirin in the first 3 days and then slowly released the remainder. At a low concentration (≤100 μg/mL), aspirin did not promote cell proliferation, but enhanced the alkaline phosphatase activity, and osteocalcin (OCN) and collagen I expression of human bone marrow-derived mesenchymal stem cells. The TSAH/β-TCP/aspirin hydrogel was injected into the periosteum of the rat cranial bone, and its in vivo bone-forming ability was evaluated at 12 weeks. A bone morphogenetic protein 2 (BMP-2)-loaded TSAH/β-TCP hydrogel was injected as a control. Micro-computed tomography showed that the percentage of mineralized tissue in the TSAH/β-TCP/BMP-2 and TSAH/β-TCP/aspirin groups were similar and significantly higher than that in the TSAH/β-TCP group. Immunohistochemical staining showed considerable expression of OCN, especially in the TSAH/β-TCP/BMP-2 and TSAH/β-TCP/aspirin groups. These results suggest that the injectable TSAH/β-TCP/aspirin hydrogel has great potential for bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017.
In the present study, the in vitro osteogenic differentiating potential of rat dental pulp stem cells was examined. The induction was carried out under the same inducing system as used for bone marrow cells. Alkaline phosphatase (ALP) activity and mineralization were measured at 1, 2, 3, and 6 weeks after the induction. The level of ALP activity was very high at 1 week after induction and the level was maintained for 3 weeks, however the level of ALP activity was dramatically decreased (to 50%) after 6 weeks. Although mineralization was detected at low levels after 3 weeks, it became expanded to up to 29 nodules mineralized at 6 weeks after induction, as determined by von Kossa staining. It was shown that in vitro osteogenic differentiation of rat dental pulp stem cells was induced following the increase of the ALP activity at an early stage of the induction.
Over the past decade, stem cell therapy has been extensively studied for clinical application for heart diseases. Among various stem cells, adipose tissue-derived stem cell (ADSC) is still an attractive stem cell resource due to its abundance and easy accessibility. In vitro studies showed the multipotent differentiation potentials of ADSC, even differentiation into cardiomyocytes. Many pre-clinical animal studies have also demonstrated promising therapeutic results of ADSC. Furthermore, there were several clinical trials showing the positive results in acute myocardial infarction using ADSC. The present article covers the brief introduction, the suggested therapeutic mechanisms, application methods including cell dose and delivery, and human clinical trials of ADSC for myocardial regeneration.
