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Effects of two weeks of dietary calcium depletion followed by one week of dietary calcium repletion on dynamic bone formation histomorphometric parameters of at the endosteal surface of the secondary spongiosia of the distal femur.
Source publication
The present study sought to evaluate the functional role of osteocyte-derived IGF-I in the bone repletion process by determining whether deficient expression of Igf1 in osteocytes would impair the bone repletion response to one week of dietary calcium repletion after two weeks of dietary calcium deprivation. As expected, the two-week dietary calciu...
Citations
... IGF-I expressed in osteoblasts or osteocytes, but not liver hepatocytes, is indispensable for performing mechanical loading-induced bone formation [28]. While osteocyte-derived IGF-I plays an important role in skeletal development, surprisingly, the disruption of the Igf1 gene in osteocytes did not impede but promoted fracture callus remodeling, as well as bone repletion response, in mice [29,30]. Our previous study found that the disruption of the Igf1 gene in chondrocytes led to a significant reduction in cortical bone size measured via peripheral quantitative computed tomography at the mid-diaphysis of the femur [15]. ...
Simple Summary
To investigate the relative importance of IGF-I expression in various types of bone cells for endochondral ossification, we examined the trabecular bone phenotypes at the distal femoral epiphysis and the secondary spongiosa of male mice with a global deletion of the Igf-I gene, as well as the conditional disruption of Igf-I in osteoblasts, chondrocytes, and osteoblasts/chondrocytes and their corresponding control littermates. We demonstrated that the disruption of Igf-I globally attenuated bone size much more severely than conditional abrogation in osteoblasts. Trabecular bone mass was lower in the secondary spongiosa of all four knockout mouse lines studied. Global Igf-I abrogation, but not conditional loss of Igf-I, locally diminished trabecular bone mass in the epiphysis. Our findings provide evidence that local and endocrine IGF-I actions in bone are pleiotropic and dependent on cell type and the bone compartment in which IGF-I acts.
Abstract
To evaluate the relative importance of IGF-I expression in various cell types for endochondral ossification, we quantified the trabecular bone at the secondary spongiosa and epiphysis of the distal femur in 8–12-week-old male mice with a global knockout of the Igf-I gene, as well as the conditional deletion of Igf-I in osteoblasts, chondrocytes, and osteoblasts/chondrocytes and their corresponding wild-type control littermates. The osteoblast-, chondrocyte-, and osteoblast/chondrocyte-specific Igf-I conditional knockout mice were generated by crossing Igf-I floxed mice with Cre transgenic mice in which Cre expression is under the control of either the Col1α2 or Col2α1 promoter. We found that the global disruption of Igf-I resulted in 80% and 70% reductions in bone size, defined as total volume, at the secondary spongiosa and epiphysis of the distal femur, respectively. The abrogation of Igf-I in Col1α2-producing osteoblasts but not Col2α1-producing chondrocytes decreased bone size by 25% at both the secondary spongiosa and epiphysis. In comparison, the deletion of the Igf-I globally or specifically in osteoblasts or chondrocytes reduced trabecular bone mass by 25%. In contrast, the universal deletion of Igf-I in all cells, but not the conditional disruption of Igf-I in osteoblasts and/or chondrocytes reduced trabecular bone mass in the epiphysis. The reduced trabecular bone mass at the secondary spongiosa in osteoblast- and/or chondrocyte-specific Igf-I conditional knockout mice is caused by the reduced trabecular number and increased trabecular separation. Immunohistochemistry studies found that the expression levels of chondrocyte (COL10, MMP13) and osteoblast (BSP) markers were less in the secondary spongiosa and the epiphyses in the global Igf-I deletion mice. Our data indicate that local and endocrine Igf-I act pleiotropically and in a cell type- and bone compartment-dependent manner in bone.
... As mentioned above, while liver is responsible for most of the IGF-1 secretion, osteocytes produce IGF-1 which is critical for bone homeostasis. Yet, work from Lau et al. suggested that depletion of osteocyte-derived IGF-1 was not essential for bone repletion [148]. Meanwhile, the effects of osteocyte-derived IGF-1 on skeletal muscle homeostasis is largely unknown. ...
Maintenance of skeletal health is tightly regulated by osteocytes, osteoblasts, and osteoclasts via coordinated secretion of bone-derived factors, termed osteokines. Disruption of this coordinated process due to aging and metabolic disease promotes loss of bone mass and increased risk of fracture. Indeed, growing evidence demonstrates that metabolic diseases, including type 2 diabetes, liver disease and cancer are accompanied by bone loss and altered osteokine levels. With the persistent prevalence of cancer and the growing epidemic of metabolic disorders, investigations into the role of inter-tissue communication during disease progression are on the rise. While osteokines are imperative for bone homeostasis, work from us and others have identified that osteokines possess endocrine functions, exerting effects on distant tissues including skeletal muscle and liver. In this review we first discuss the prevalence of bone loss and osteokine alterations in patients with type 2 diabetes, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, and cancer. We then discuss the effects of osteokines in mediating skeletal muscle and liver homeostasis, including RANKL, sclerostin, osteocalcin, FGF23, PGE2, TGF-β, BMPs, IGF-1 and PTHrP. To better understand how inter-tissue communication contributes to disease progression, it is essential that we include the bone secretome and the systemic roles of osteokines.
... Given their ubiquitous nature, osteocytes have been recognized as master regulators of both bone formation and resorption in response to various stimuli including physical activity (14-16), disuse (17,18), diet (19), reproductive status (20,21) and injury (22,23). Osteocytes respond to increased mechanical loading by releasing osteoprotegerin (24), which is an inhibitor of osteoclastogenesis, and proosteogenic factors such as insulin-like growth factor 1 (25), parathyroid hormone related protein (PTHrP) (26), polycystin 1 (27) and Wnts (28)(29)(30). They also down-regulate the expression of sclerostin (31,32), an inhibitor of Wnt-signaling, an important regulatory pathway in osteogenic differentiation. ...
Increased physical loading of the skeleton activates new bone formation ensuring its ability to meet mechanical demands over time; however, the capacity of bone to respond to mechanical stimulation diminishes with age. Osteocytes, the cells embedded and dispersed throughout mineralized bone matrix, are master regulators of mechanoadaptation through recruitment of new bone-forming cells, the osteoblasts, via signaling to osteoprogenitors located on bone surfaces. We previously demonstrated that in vivo and in vitro mechanical stimulation significantly upregulated the chemokine C-X-C Motif Chemokine Ligand 12 (CXCL12) and its receptor, CXCR4, in osteocytes and bone lining cells, and that CXCR4 antagonism with AMD3100 attenuated in vivo load-induced bone formation. Here, we extended this work by showing that ablation of CXCL12+ cells and deletion of cxcl12 in late-stage osteoblasts and osteocytes significantly attenuated in vivo load-induced bone formation in the mouse tibia. This bone loading phenotype was rescued by treatment with recombinant CXCL12. To address mechanism, we showed that in vitro deletion of cxcl12 and cxcr4 , separately, in bone marrow stromal cells resulted in significantly reduced osteogenic differentiation. Furthermore, CXCL12 treatment enhanced GSK-3b phosphorylation and β-catenin translocation to the nucleus, the former of which was partially blocked by AMD3100. Finally, CXCL12 synergized Wnt signaling leading to significantly increased total β-catenin protein and Axin2 expression, a Wnt signaling target gene. These findings together demonstrate that CXCL12 expression in late-stage osteoblasts and osteocytes is essential for load-induced bone formation, in part, by regulating osteogenic differentiation through activation of the Wnt signaling pathway.
Significance
Skeletal adaptation to mechanical loading is contingent on the recruitment of new osteoblasts to bone surfaces. CXCL12, a chemokine expressed by osteolineage cells, targets effector cells expressing its receptor CXCR4, including osteoprogenitors. Exogenous mechanical loading of mouse hind limbs upregulates CXCL12 in osteocytes, bone lining cells and marrow cells, while antagonizing CXCR4 led to significantly attenuated load-induced bone formation. Here, we show that CXCL12 expression in late-stage osteoblasts and osteocytes is required for load-induced bone formation. Treatment with recombinant CXCL12 rescued the bone loading phenotype suggesting that the CXCL12/CXCR4 signaling pathway may be a feasible drug target for promoting load-induced bone formation when exercise alone is insufficient to counteract low bone mass and osteoporosis.
... While this result is surprising given the roles of IGF-I and IGFBP-5 in bone physiology, IGF-I availability may not be crucial for recovery after lactation. A previous study found that osteocyte-derived IGF-I is not required for the recovery of bone following dietary calcium deprivation [56]. Pappa2 deletion has previously been shown to affect bone growth in virgin animals, and in the present study we found that it increased cortical area fraction and trabecular thickness. ...
Background
Lactation results in substantial maternal bone loss that is recovered following weaning. However, the mechanisms underlying this recovery, and in particular the role of insulin-like growth factor 1 (IGF-I), is not clear. Furthermore, there is little data regarding whether recovery is affected by advanced maternal age.
Methods
Using micro-computed tomography, we studied bone recovery following lactation in mice at 2, 5 and 7 months of age. We also investigated the effects of reduced IGF-I availability using mice lacking PAPP-A2, a protease of insulin-like growth factor binding protein 5 (IGFBP-5).
Results
In 2 month old mice, lactation affected femoral trabecular and cortical bone, but only cortical bone showed recovery 3 weeks after weaning. This recovery was not affected by deletion of the Pappa2 gene. The amount of trabecular bone was reduced in 5 and 7 month old mice, and was not further reduced by lactation. However, the recovery of cortical bone was impaired at 5 and 7 months compared with at 2 months.
Conclusions
Recovery of the maternal skeleton after lactation is impaired in moderately-aged mice compared with younger mice. Our results may be relevant to the long-term effects of breastfeeding on the maternal skeleton in humans, particularly given the increasing median maternal age at childbearing.
... The immunoexpression of IGF growth factors has been assessed in animal studies [26], but there are no reports in the literature regarding their expression associated to alendronate in craniofacial bone repair; herein, the likely influence of presurgical administration of a low dose of ALN in calvarial bone repair was verified and correlates with the histological frame found on reparative tissue to the immunohistochemical presence of IGF1 and IGF2. In order to better understand the likely action of IGFs on bone development, we also correlated the presence of IGFs with osteopontin (OP) both on the 15th and 30th days postsurgery which are important periods for understanding the cellular differentiation to one repair. ...
Objectives
The aim of this study was to verify the likely influence of presurgical administration of low doses of alendronate sodium in craniofacial bone repair and correlate the histological frame found on reparative tissue to the immunohistochemical presence of IGF1, IGF2, and osteopontin (OP). Materials and methodsIn total, 120 rats were randomly allocated into four groups: group C (control), group OA (autogenous bone), group B (bisphosphonates), and group OA–B (autogenous bone + bisphosphonates). Groups B and OA–B received alendronate sodium (ALN) 0.01 mg/kg subcutaneously on alternate days for 4 weeks. Groups C and OA received saline solution. Critical 5-mm defects were created in rat calvaria, which were filled with blood clot in groups C and B and with autogenous bone in groups OA and OA–B. The animals were euthanized at 15 or 30 days postoperatively. Histological analysis and immunohistochemistry of IGF1, IGF2, and OP proteins was performed. Immunohistochemistry evaluated the expression in cells and extracellular matrix. ResultsGroups C and B revealed healing predominantly characterized by connective tissue. In groups OA and OA–B, healing of connective tissue and neoformation of compact bone was observed. Expression of IGF1 an OP was present in all specimens. IGF1 expression in cells was more pronounced in groups OA and OA–B 15 days postoperatively. The expression of IGF2 was only observed in groups OA and OA–B, with greater intensity in group OA–B 30 days postoperatively. OP expression was only observed in cells and not in the extracellular matrix and was more pronounced in group OA 15 days postoperatively. Conclusions
The application of systemic ALN at a dose of 0.01 mg/kg did not improve cranial bone matrix deposition. Nevertheless, the expression of IGF1 and OP and a slight marking of IGF2 were observed especially in groups OA and OA–B in the wound healing process. Future studies should assess higher doses of ALN to verify its influence on bone repair. Clinical relevanceThe systemic use of ALN 0.01 mg/kg on alternate days 4 weeks prior to surgery did not interfere with bone repair.
... We chose this mouse strain because it is a common background for transgenic mice, and transgenic mice will be the major thrust to further explore the mechanism of bone repletion. This mouse strain exhibits a bone repletion response to a low calcium diet similar to that seen in the other strains of mice we have studied (10,11). The control diet contained 1.2% calcium and 0.4% phosphate. ...
... Plasma PTH, 1,25(OH) 2 D, C-terminal telopeptide of type 1 collagen (CTX), and procollagen type 1 N-terminal propeptide (P1NP) were determined, as described previously (10). Plasma calcium was determined by the Heartland Assays Laboratory (Ames, IA). ...
... Total RNA extraction from the tibia and quantitative reverse transcription polymerase chain reaction (PCR) of the test genes were performed as previously described (10). The sequence of PCR primers for the test genes is presented in Supplemental Table 2. ...
This study was undertaken to determine the mechanism whereby calcitropic hormones and mesenchymal stem cell progeny changes involved in bone repletion, a regenerative bone process that restores the bone lost to calcium deficiency. To initiate depletion, weanling mice with a mixed C57BL/6 (75%) and CD1 (25%) genetic background were fed a calcium-deficient diet (0.01%) for 14 days. For repletion, the mice were re-fed a control diet containing 1.2% calcium for 14 days. Depletion decreased plasma calcium and increased plasma PTH, 1,25(OH)2D, and CTX. These plasma parameters quickly returned toward normal upon repletion. Trabecular bone volume and connectivity decreased drastically during depletion, but were completely restored by the end of repletion. This bone repletion process was largely due to new bone formation. When BrdU was administered in the middle of depletion for 3 days and examined by FACS at 7 days into repletion, significant increases in BrdU incorporation were seen in several CD105 subsets of cells of osteoblastic lineage. When BrdU was administered on days 1-3 of repletion and examined 11 days later, no increases in BrdU in these subsets were seen. Additionally, osteocytes stained positively for BrdU were increased during depletion. In summary, this study establishes a unique regenerative mechanism to initiate bone repair during the bone insult. Calcium homeostatic mechanisms and the bone repletion mechanism are opposing functions but are simultaneously orchestrated in such a way as to optimize both endpoints. These results have potential clinical relevance to disease entities such as type II osteoporosis.
... Before we initiated fracture studies in osteocyte Igf1 cKO mice, we compared basal cortical bone parameters at the mid-shaft as well as basal trabecular bone parameters at the proximal metaphysis of intact tibiae of cKO mice with those of WT littermates by μ-CT. Consistent with the previous reports [29,30,35], the femur length of male osteocyte Igf1 cKO mice at 12 weeks of age was 4.6% shorter than that of corresponding male WT littermates (Fig. 1A). The total cortical tissue (Ct·TV) and bone volume (Ct·BV) in these cKO mice were each reduced by 25%, resulting in no differences in Ct·BV/TV between the two mouse strains (Fig. 1B). ...
... Deficiency in Sost expression or functional activity, via genetic knockout [8,45] or anti-SOST antibody treatment [12,13], has been shown to promote fracture repair. Because our previous studies have reported that young adult (8-week-old) Igf1 osteocyte cKO mutants had 40% lower basal levels of Sost mRNA in their bone [35], we compared the Sost mRNA level in the fracture callus of cKO mutants with that in WT calluses at 14 days post-fracture. The Sost mRNA level was slightly, but not significantly, increased in WT calluses; but it was reduced by~50% in cKO calluses compared to the contralateral intact bone (Fig. 8C). ...
... The Sost mRNA level was slightly, but not significantly, increased in WT calluses; but it was reduced by~50% in cKO calluses compared to the contralateral intact bone (Fig. 8C). This 50% reduction was on top of the already 40% lower basal level in the cKO mutant mice [35]. To confirm these gene expression results, we stained SOST protein by IHC on thin sections of the fracture calluses and found a drastic reduction in the SOST immunostaining (primarily inside the osteocytes) in cKO calluses compared to that in WT fracture calluses (Fig. 8E). ...
This study evaluated the effects of deficient IGF-I expression in osteocytes on fracture healing. Transgenic mice with conditional knockout (cKO) of Igf1 in osteocytes were generated by crossing Dmp1-Cre mice with Igf1 flox mice. Fractures were created on the mid-shaft of tibia of 12-week-old male cKO mice and wild-type (WT) littermates by three-point bending. At 21 and 28 days post-fracture healing, the increases in cortical bone mineral density, mineral content, bone area, and thickness, as well as sub-cortical bone mineral content at the fracture site were each greater in cKO calluses than in WT calluses. There were 85% decrease in the cartilage area and > 2 fold increase in the number of osteoclasts in cKO calluses at 14 days post-fracture, suggesting a more rapid remodeling of endochondral bone. The upregulation of mRNA levels of osteoblast marker genes (cbfa1, alp, Opn, and Ocn) were greater in cKO calluses than in WT calluses. μ-CT analysis suggested an accelerated bony union of the fracture gap in cKO mice. The Sost mRNA level was reduced by 50% and the Bmp2 mRNA level was increased 3-fold in cKO fractures at 14 days post-fracture, but the levels of these two mRNAs in WT fractures were unchanged, suggesting that the accelerated fracture repair may in part act through the Wnt and/or BMP signaling. In conclusion, conditional deletion of Igf1 in osteocytes not only did not impair, but unexpectedly enhanced, bony union of the fracture gap. The accelerated bony union was due in part to upregulation of the Wnt and BMP2 signaling in response to deficient osteocyte-derived IGF-I expression, which in turn favors intramembranous over endochondral bone repair.
... More recently, the link between IGF-I upregulation and bone formation in response to mechanical loading has been more definitively demonstrated through the use of conditional IGF-I gene disruption in mouse osteoblasts expressing type 1α collagen (Kesavan et al., 2011). Osteocyte-derived IGF-I has also been shown to be a key determinant in bone mechanosensitivity, as well as bone growth, remodeling, and regeneration (Lau et al., 2013;Sheng et al., 2014), although it is not required for bone repletion after a lowcalcium challenge (Lau et al., 2015). Regulation of IGF-I expression is critically important and is accomplished a multitude of diverse systemic and local factors and their interactions. ...
The growth hormone/insulin-like growth factor (GH/IGF) axis is critically important for the regulation of bone formation, and deficiencies in this system have been shown to contribute to the development of osteoporosis and other diseases of low bone mass. The GH/IGF axis is regulated by a complex set of hormonal and local factors which can act to regulate this system at the level of the ligands, receptors, IGF binding proteins (IGFBPs), or IGFBP proteases. A combination of in vitro studies, transgenic animal models, and clinical human investigations has provided ample evidence of the importance of the endocrine and local actions of both GH and IGF-I, the two major components of the GH/IGF axis, in skeletal growth and maintenance. GH- and IGF-based therapies provide a useful avenue of approach for the prevention and treatment of diseases such as osteoporosis.
Introduction:
This study was undertaken to gain mechanistic information about bone repair using the bone repletion model in aged Balb/cBy mice.
Materials and methods:
one month-old (young) mice were fed a calcium-deficient diet for 2 weeks and 8 month-old (adult) and 21-25 month-old (aged) female mice for 4 weeks during depletion, which was followed by feeding a calcium-sufficient diet for 16 days during repletion. To determine if prolonged repletion would improve bone repair, an additional group of aged mice were repleted for 4 additional weeks. Control mice were fed calcium-sufficient diet throughout. In vivo bone repletion response was assessed by bone mineral density gain and histomorphometry. In vitro response was monitored by osteoblastic proliferation, differentiation, and senescence.
Results:
There was no significant bone repletion in aged mice even with an extended repletion period, indicating an impaired bone repletion. This was not due to an increase in bone cell senescence or reduction in osteoblast proliferation, but to dysfunctional osteoblastic differentiation in aged bone cells. Osteoblasts of aged mice had elevated levels of cytosolic and ER calcium, which were associated with increased Cav1.2 and CaSR (extracellular calcium channels) expression but reduced expression of Orai1 and Stim1, key components of Stored Operated Ca2+ Entry (SOCE). Activation of Cav1.2 and CaSR leads to increased osteoblastic proliferation, but activation of SOCE is associated with osteoblastic differentiation.
Conclusion:
The bone repletion mechanism in aged Balb/cBy mice is defective that is caused by an impaired osteoblast differentiation through reducedactivation of SOCE.
As explained by Wolff’s law and the mechanostat hypothesis, mechanical stimulation can be used to promote bone formation. Low intensity pulsed ultrasound (LIPUS) is a source of mechanical stimulation that can activate the integrin/phosphatidylinositol 3-OH kinase/Akt pathway and upregulate osteogenic proteins through the production of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). This paper analyzes the results of in vitro and in vivo studies that have evaluated the effects of LIPUS on cell behavior within three-dimensional (3D) titanium, ceramic, and hydrogel scaffolds. We focus specifically on cell morphology and attachment, cell proliferation and viability, osteogenic differentiation, mineralization, bone volume, and osseointegration. As shown by upregulated levels of alkaline phosphatase and osteocalcin, increased mineral deposition, improved cell ingrowth, greater scaffold pore occupancy by bone tissue, and superior vascularization, LIPUS generally has a positive effect and promotes bone formation within engineered scaffolds. Additionally, LIPUS can have synergistic effects by producing the piezoelectric effect and enhancing the benefits of 3D hydrogel encapsulation, growth factor delivery, and scaffold modification. Additional research should be conducted to optimize the ultrasound parameters and evaluate the effects of LIPUS with other types of scaffold materials and cell types.
