Defective bone repletion in aged Balb/cBy mice was caused by impaired osteoblastic differentiation.

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.

A Mouse Noninvasive Intraarticular Tibial Plateau Compression Loading-Induced Injury Model of Posttraumatic Osteoarthritis.

This study sought to develop a noninvasive, reliable, clinically relevant, and easy-to-implement mouse model that can be used for investigation of the pathophysiology of PTOA and for preclinical testing of new therapies of PTOA. Accordingly, we have established a closed intraarticular tibial plateau compression loading-induced injury model of PTOA in C57BL/6J mice. In this model, a single application of a defined loading force was applied with an indenter to the tibial plateau of the right knee to create injuries to the synovium, menisci, ligaments, and articular cartilage. The limiting loading force was set at 55 N with the loading speed of 60 N/s. This loading regimen limits the distance that the indenter would travel into the joint, but still yields substantial compression loading energy to cause significant injuries to the synovium, meniscus, and articular cartilage. The joint injury induced by this loading protocol consistently yielded evidence for key histological hallmarks of PTOA at 5-11 weeks post-injury, including loss of articular cartilage, disorganization of chondrocytes, meniscal hyperplasia and mineralization, osteophyte formation, and degenerative remodeling of subchondral bone. These arthritic changes were highly reproducible and of a progressive nature. Because 50% of patients with meniscal and/or ligament injuries without intraarticular fractures developed PTOA over time, this intraarticular tibial plateau compression loading-induced injury model is clinically relevant. In summary, we have developed a noninvasive intraarticular tibial plateau compression loading-induced injury model in the mouse that can be used to investigate the pathophysiology of PTOA and for preclinical testing for new therapies.

The EphA4 Signaling is Anti-catabolic in Synoviocytes but Pro-anabolic in Articular Chondrocytes.

The expression and activation of EphA4 in the various cell types in a knee joint was upregulated upon an intraarticular injury. To determine if EphA4 signaling plays a role in osteoarthritis, we determined whether deficient EphA4 expression (in EphA4 knockout mice) or upregulation of the EphA4 signaling (with the EfnA4-fc treatment) would alter cellular functions of synoviocytes and articular chondrocytes. In synoviocytes, deficient EphA4 expression enhanced, whereas activation of the EphA4 signaling reduced, expression and secretion of key inflammatory cytokines and matrix metalloproteases. Conversely, in articular chondrocytes, activation of the EphA4 signaling upregulated, while deficient EphA4 expression reduced, expression levels of chondrogenic genes (e.g., aggrecan, lubricin, type-2 collagen, and Sox9). EfnA4-fc treatment in wildtype, but not EphA4-deficient, articular chondrocytes promoted the formation and activity of acidic proteoglycan-producing colonies. Activation of the EphA4 signaling in articular chondrocytes upregulated Rac1/2 and downregulated RhoA via enhancing Vav1 and reducing Ephexin1 activation, respectively. However, activation of the EphA4 signaling in synoviocytes suppressed the Vav/Rac signaling while upregulated the Ephexin/Rho signaling. In summary, the EphA4 signaling in synoviocytes is largely of anti-catabolic nature through suppression of the expression of inflammatory cytokines and matrix proteases, but in articular chondrocytes the signaling is pro-anabolic in that it promotes the biosynthesis of articular cartilage. The contrasting action of the EphA4 signaling in synoviocytes as opposing to articular chondrocytes may in part be mediated through the opposite differential effects of the EphA4 signaling on the Vav/Rac signaling and Ephexin/Rho signaling in the two skeletal cell types.

Changes in ephrin gene expression during bone healing identify a restricted repertoire of ephrins mediating fracture repair.

To identify the repertoire of ephrin genes that might regulate endochondral bone fracture repair, we examined changes in ephrin ligand and receptor (Eph) gene expression in fracture callus tissues during bone fracture healing. Ephrin and Eph proteins were then localized in the fracture callus tissues present when changes in gene expression were observed. Ephrin gene expression was widespread in fracture tissues, but the repertoire of ephrin genes with significant changes in expression that might suggest a regulatory role in fracture callus development was restricted to the ephrin A family members Epha4, Epha5 and the ephrin B family member Efnb1. After 3 weeks of healing, Epha4 fracture expression was downregulated from 1.3- to 0.8-fold and Epha5 fracture expression was upregulated from 1.2- to 1.5-fold of intact contralateral femur expression, respectively. Efnb1 expression was downregulated from 1.5- to 1.2-fold after 2 weeks post-fracture. These ephrin proteins were localized to fracture callus prehypertrophic chondrocytes and osteoblasts, as well as to the periosteum and fibrous tissues. The observed positive correlation between mRNA levels of EfnB1 with Col10 and Epha5 with Bglap, together with colocalized expression with their respective proteins, suggest that EfnB1 is a positive mediator of prehypertrophic chondrocyte development and that Epha5 contributes to osteoblast-mediated mineralization of fracture callus. In contrast, mRNA levels of Epha4 and Efnb1 correlated negatively with Bglap, thus suggesting a negative role for these two ephrin family members in mature osteoblast functions. Given the number of family members and widespread expression of the ephrins, a characterization of changes in ephrin gene expression provides a basis for identifying ephrin family members that might regulate the molecular pathways of bone fracture repair. This approach suggests that a highly restricted repertoire of ephrins, EfnB1 and EphA5, are the major mediators of fracture callus cartilage hypertrophy and ossification, respectively, and proposes candidates for additional functional study and eventual therapeutic application.

PDGFB-based stem cell gene therapy increases bone strength in the mouse.

Substantial advances have been made in the past two decades in the management of osteoporosis. However, none of the current medications can eliminate the risk of fracture and rejuvenate the skeleton. To this end, we recently reported that transplantation of hematopoietic stem/progenitor cells (HSCs) or Sca1(+) cells engineered to overexpress FGF2 results in a significant increase in lamellar bone matrix formation at the endosteum; but this increase was attended by the development of secondary hyperparathyroidism and severe osteomalacia. Here we switch the therapeutic gene to PDGFB, another potent mitogen for mesenchymal stem cells (MSCs) but potentially safer than FGF2. We found that modest overexpression of PDGFB using a relatively weak phosphoglycerate kinase (PGK) promoter completely avoided osteomalacia and secondary hyperparathyroidism, and simultaneously increased trabecular bone formation and trabecular connectivity, and decreased cortical porosity. These effects led to a 45% increase in the bone strength. Transplantation of PGK-PDGFB-transduced Sca1(+) cells increased MSC proliferation, raising the possibility that PDGF-BB enhances expansion of MSC in the vicinity of the hematopoietic niche where the osteogenic milieu propels the differentiation of MSCs toward an osteogenic destination. Our therapy should have potential clinical applications for patients undergoing HSC transplantation, who are at high risk for osteoporosis and bone fractures after total body irradiation preconditioning. It could eventually have wider application once the therapy can be applied without the preconditioning.

Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling.

Urokinase plasminogen activator (uPA) regulates a proteolytic cascade of extracellular matrix degradation that functions in tissue development and tissue repair. The development and remodeling of the skeletal extracellular matrix during wound healing suggests that uPA might regulate bone development and repair. To determine whether uPA functions regulate bone development and repair, we examined the basal skeletal phenotype and endochondral bone fracture repair in uPA-deficient mice. The skeletal phenotype of uPA knockout mice was compared with that of control mice under basal conditions by dual-energy X-ray absorptiometry and micro-CT analysis, and during femur fracture repair by micro-CT and histological examination of the fracture callus. No effects of uPA gene deficiency were observed in the basal skeletal phenotype of the whole body or the femur. However, uPA gene deficiency resulted in increased fracture callus cartilage abundance during femur fracture repair at 14 days healing. The increase in cartilage corresponded to reduced tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts in the uPA knockout fracture callus at this time, consistent with impaired osteoclast-mediated remodeling of the fracture cartilage. CD31 staining was reduced in the knockout fracture tissues at this time, suggesting that angiogenesis was also reduced. Osteoclasts also colocalized with CD31 expression in the endothelial cells of the fracture tissues during callus remodeling. These results indicate that uPA promotes remodeling of the fracture cartilage by osteoclasts that are associated with angiogenesis and suggest that uPA promotes angiogenesis and remodeling of the fracture cartilage at this time of bone fracture repair.

Sequential application of small molecule therapy enhances chondrogenesis and angiogenesis in murine segmental defect bone repair.

The increasing incidence of physiologic/pathologic conditions that impair the otherwise routine healing of endochondral bone fractures and the occurrence of severe bone injuries necessitate novel approaches to enhance clinically challenging bone fracture repair. To promote the healing of nonunion fractures, we tested an approach that used two small molecules to sequentially enhance cartilage development and conversion to the bone in the callus of a murine femoral segmental defect nonunion model of bone injury. Systemic injections of smoothened agonist 21k (SAG21k) were used to stimulate chondrogenesis through the activation of the sonic hedgehog (SHH) pathway early in bone repair, while injections of the prolyl hydroxylase domain (PHD)2 inhibitor, IOX2, were used to stimulate hypoxia signaling-mediated endochondral bone formation. The expression of SHH pathway genes and Phd2 target genes was increased in chondrocyte cell lines in response to SAG21k and IOX2 treatment, respectively. The segmental defect responded to sequential systemic administration of these small molecules with increased chondrocyte expression of PTCH1, GLI1, and SOX9 in response to SAG and increased expression of hypoxia-induced factor-1α and vascular endothelial growth factor-A in the defect tissues in response to IOX2. At 6 weeks postsurgery, the combined SAG-IOX2 therapy produced increased bone formation in the defect with the bony union over the injury. Clinical significance: This therapeutic approach was successful in promoting cartilage and bone formation within a critical-size segmental defect and established the utility of a sequential small molecule therapy for the enhancement of fracture callus development in clinically challenging bone injuries.

A Novel EphA4 Signaling-Based Therapeutic Strategy for Osteoarthritis in Mice.

This study took advantage of the recent discovery that the EphA4 signaling has anti-catabolic effects on osteoclasts/macrophages/synoviocytes but pro-anabolic effects on articular chondrocytes and sought to develop an EphA4 signaling-based therapeutic strategy for osteoarthritis (OA) using a mouse model of OA/posttraumatic OA (PTOA). The injured joint of C57BL/6J mice received biweekly intraarticular injections of a soluble EphA4-binding ligand (EfnA4-fc) at 1 day after the tibial plateau injury or at 5 weeks post-injury. The animals were euthanized 5 weeks later. The injured right and contralateral uninjured left joints were analyzed for hallmarks of OA by histology. Relative severity was determined by a modified Mankin OA scoring system and serum COMP and CTX-II levels. Tibial plateau injury caused more severe OA in Epha4 null mice than in wild-type (WT) littermates, suggesting a protective role of EphA4 signaling in OA. A prototype strategy of an EphA4 signaling-based strategy involving biweekly injections of EfnA4-fc into injured joints was developed and was shown to be highly effective in preventing OA/PTOA when it was administered at 1 day post-injury and in treating OA/PTOA when it was applied after OA has been established. The efficacy of this prototype was dose- and time-dependent. The effects were not caused by the Fc moiety of EfnA4-fc. Other soluble EfnA ligands of EphA4, ie, EfnA1-fc and EfnA2-fc, were also effective. A prototype of a novel EphA4 signaling-based therapy was developed for OA/PTOA that not only reduces the progressive destruction of articular cartilage but may also promote regeneration of the damaged cartilage. © 2022 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Contrasting effects of Ksr2, an obesity gene, on trabecular bone volume and bone marrow adiposity.

Pathological obesity and its complications are associated with an increased propensity for bone fractures. Humans with certain genetic polymorphisms at the kinase suppressor of ras2 (KSR2) locus develop severe early-onset obesity and type 2 diabetes. Both conditions are phenocopied in mice with Ksr2 deleted, but whether this affects bone health remains unknown. Here we studied the bones of global Ksr2 null mice and found that Ksr2 negatively regulates femoral, but not vertebral, bone mass in two genetic backgrounds, while the paralogous gene, Ksr1, was dispensable for bone homeostasis. Mechanistically, KSR2 regulates bone formation by influencing adipocyte differentiation at the expense of osteoblasts in the bone marrow. Compared with Ksr2's known role as a regulator of feeding by its function in the hypothalamus, pair-feeding and osteoblast-specific conditional deletion of Ksr2 reveals that Ksr2 can regulate bone formation autonomously. Despite the gains in appendicular bone mass observed in the absence of Ksr2, bone strength, as well as fracture healing response, remains compromised in these mice. This study highlights the interrelationship between adiposity and bone health and provides mechanistic insights into how Ksr2, an adiposity and diabetic gene, regulates bone metabolism.

Our bones are living tissues which constantly reshape and renew themselves. This ability relies on stem cells present in the marrow cavity, which can mature into the various types of cells needed to produce new bone material, marrow fat, or other components. Obesity and associated conditions such as type 2 diabetes are often linked to harmful changes in the skeleton. In particular, these metabolic conditions are associated with weight-bearing bones becoming more prone to facture and healing poorly. Mice genetically modified to model obesity and diabetes could help researchers to study exactly how these conditions ­ and the genetic changes that underlie them ­ impact bone health. Gomez et al. aimed to address this question by focusing on KSR2, a gene involved in energy consumption and feeding behavior. Children who carry certain KSR2 mutations are prone to obesity and type 2 diabetes; mice lacking the gene also develop these conditions due to uncontrolled eating. Closely examining mutant mice in which Ksr2 had been deactivated in every cell revealed that the weight-bearing bones of these animals were also more likely to break, and the fractures then healed more slowly. This was the case even though these bones had higher mass and less marrow fat compared to healthy mice. Non-weight bearing bones (such as the spine) did not exhibit these changes. Further experiments revealed that, when expressed normally in the skeleton, Ksr2 skews the stem cell maturation process towards marrow fat cells instead of bone-creating cells. This suggests a new role for Ksr2, which therefore seems to independently regulate both feeding behavior and bone health. In addition, the work by Gomez et al. demonstrate that Ksr2 mutant mice could be a useful model to better understand how obesity and diabetes affect human bones, and to potentially develop new therapies.

Lentiviral-based BMP4 in vivo gene transfer strategy increases pull-out tensile strength without an improvement in the osteointegration of the tendon graft in a rat model of biceps tenodesis.

BACKGROUND: The present study aimed to develop a rat model of biceps tenodesis and to assess the feasibility of a lentiviral (LV)-based bone morphogenetic protein (BMP) 4 in vivo gene transfer strategy for healing of biceps tenodesis. METHODS: A rat model of biceps tenodesis was developed with an interference-fit open surgical technique. A LV vector expressing a BMP4 gene or ß-galactosidase (ß-gal) control gene was applied to the bone tunnel and the tendon graft before its insertion into the bone tunnel. Osteointegration was assessed by histology and pull-out tensile strength was measured by a biomechanical test suitable for small rat biceps tendon grafts. RESULTS: Neo-chondrogenesis was seen at the tendon-bone interface of LV-BMP4-treated but not control rats. The LV-BMP4-treated rats showed 32% (p < 0.05) more newly-formed trabecular bone at the tendon-bone junction than the LV-ß-gal-treated controls after 3 weeks. However, the sites of neo-chondrogenesis and new bone formation in the LV-BMP4-treated tenodesis were highly spotty. Although the LV-BMP4 strategy did not promote bony integration of the tendon graft, it yielded a 29.5 ± 11.8% (p = 0.066) increase in improvement the pull-out strength of rat biceps tendons compared to the LV-ß-gal treatment after 5 weeks. CONCLUSIONS: Although the LV-BMP4 in vivo gene transfer strategy did not enhance osteointegration of the tendon graft, it yielded a marked improvement in the return of the pull-out strength of the tendon graft. This presumably was largely a result of the bone formation effect of BMP4 that traps or anchors the tendon graft onto the bony tunnel.

A segmental defect adaptation of the mouse closed femur fracture model for the analysis of severely impaired bone healing.

OBJECTIVE: To better characterize nonunion endochondral bone healing and evaluate novel therapeutic approaches for critical size defect healing in clinically challenging bone repair, a segmental defect model of bone injury was adapted from the three-point bending closed fracture technique in the murine femur. METHODS: The mouse femur was surgically stabilized with an intramedullary threaded rod with plastic spacers and the defect adjusted to different sizes. Healing of the different defects was analyzed by radiology and histology to 8 weeks postsurgery. To determine whether this model was effective for evaluating the benefits of molecular therapy, BMP-2 was applied to the defect and healing then examined. RESULTS: Intramedullary spacers were effective in maintaining the defect. Callus bone formation was initiated but was arrested at defect sizes of 2.5 mm and above, with no more progress in callus bone development evident to 8 weeks healing. Cartilage development in a critical size defect attenuated very early in healing without bone development, in contrast to the closed femur fracture healing, where callus cartilage was replaced by bone. BMP-2 therapy promoted osteogenesis of the resident cells of the defect, but there was no further callus development to indicate that healing to pre-surgery bone structure was successful. CONCLUSIONS: This segmental defect adaptation of the closed femur fracture model of murine bone repair severely impairs callus development and bone healing, reflecting a challenging bone injury. It is adjustable and can be compared to the closed fracture model to ascertain healing deficiencies and the efficacy of therapeutic approaches.

Unique anabolic action of stem cell gene therapy overexpressing PDGFB-DSS6 fusion protein in OVX osteoporosis mouse model.

In the present study we sought to improve the efficacy and safety of our Sca1+ PDGFB stem cell gene therapy for osteoporosis in ovariectomized (OVX) mouse model. This therapy is administered by marrow transplantation. We established the promise of this approach by previously showing that this therapy in normal mice increase bone density, increased endosteal cortical and trabecular bone formation, caused de novo trabecular bone formation, increased cortical thickness and improve bone strength. In the current study we produced a fusion gene, PDGFB-DSS6. We reasoned that the DSS6, calcium binding protein would trap the PDGFB at the bone surface and thereby limit the amount of PDGFB required to produce an optimal bone formation response, i.e. efficacy with a lower engraftment. The result shows that indeed with a very low level of engraftment we achieved a large increase in bone formation in the OVX model of bone loss. Serum analysis for biochemical marker of new bone formation showed an approximate 75% increase in alkaline phosphatase levels in Sca1+PDGFB-DSS6 group as compared to other groups. Quantitative analysis of bone by microCT showed a massive increase in trabecular bone density and trabecular connectivity of the femur in the metaphysis in Sca1+ PDGFB-DSS6 group. The increased cortical porosity produced by OVX was replaced by the Sca1+ PDGFB-DSS6 therapy but not by the positive control Sca1+ PDGFB. Additionally, an increase in the femur bone strength was also observed specifically in Sca1+ PDGFB-DSS6 as compared to other treatment groups, emphasizing the functional significance of the observed anabolic action is on bone formation. In future work we will focus on nontoxic preconditioning of our marrow transplantation procedure and also on transcriptional control of therapeutic gene expression to avoid excess bone formation.

Cyclooxygenase 2 augments osteoblastic but suppresses chondrocytic differentiation of CD90+ skeletal stem cells in fracture sites.

Cyclooxygenase 2 (COX-2) is essential for normal tissue repair. Although COX-2 is known to enhance the differentiation of mesenchymal stem cells (MSCs), how COX-2 regulates MSC differentiation into different tissue-specific progenitors to promote tissue repair remains unknown. Because it has been shown that COX-2 is critical for normal bone repair and local COX-2 overexpression in fracture sites accelerates fracture repair, this study aimed to determine the MSC subsets that are targeted by COX-2. We showed that CD90+ mouse skeletal stem cells (mSSCs; i.e., CD45-Tie2-AlphaV+ MSCs) were selectively recruited by macrophage/monocyte chemoattractant protein 1 into fracture sites following local COX-2 overexpression. In addition, local COX-2 overexpression augmented osteoblast differentiation and suppressed chondrocyte differentiation in CD90+ mSSCs, which depended on canonical WNT signaling. CD90 depletion data demonstrated that local COX-2 overexpression targeted CD90+ mSSCs to accelerate fracture repair. In conclusion, CD90+ mSSCs are promising targets for the acceleration of bone repair.

Bax deficiency in mice increases cartilage production during fracture repair through a mechanism involving increased chondrocyte proliferation without changes in apoptosis.

This study sought to determine the role of the pro-apoptotic gene, Bax, in fracture healing by comparing femoral fracture healing in Bax knockout (KO) and wild-type C57BL/6J (background strain) mice. Bax KO fractures were larger, had more bone mineral content, had approximately 2-fold larger cartilage area per callus area in the first and second weeks of fracture healing, and showed an increased osteoclast surface area in the third and fourth weeks of fracture healing compared to C57BL/6J fractures. The increased cartilage area in the Bax KO fracture callus was due to increases in number of both pre-hypertropic and hypertropic chondrocytes. TUNEL analysis showed no significant differences in the number of either chondrocyte or non-chondrocyte apoptotic cells between Bax KO and C57BL/6J fractures at 7 or 14 days post-fracture, indicating that the increased number of chondrocytes in Bax KO fractures was not due to reduced apoptosis. Analysis of expression of apoptotic genes revealed that although the expression levels of Bcl-2 and Bcl-xL were not different between the Bax KO and C57BL/6J mice at 7 or 14 days post-fracture, the expression of BH3-domain only Bak and "Bik-like" pro-apoptotic gene increased approximately 1.5-fold and approximately 2-fold, respectively, in Bax KO fractures at 7 and 14 days post-fracture, compared to C57BL/6J fractures, suggesting that up-regulation of the Bak and Bik-like pro-apoptotic genes in Bax KO mice might compensate for the lack of Bax functions in the context of apoptosis. Analysis by in vivo incorporation of bromodeoxyuridine into chondrocytes within the fracture tissues indicated a highly significant increase in chondrocyte proliferation in Bax KO fractures compared to C57BL/6J fractures at day 7. The increased expression of collagen 2alpha1 and 9alpha1 gene in Bax KO fractures during early healing was consistent with an increased chondrocyte proliferation. In conclusion, this study demonstrates for the first time that Bax has an important role in the early stage of fracture healing, and that the increased callus size and cartilage area in Bax KO fractures was due to increased chondrocyte proliferation and not to reduced apoptosis or increased chondrocyte hypertrophy. The unexpected effect of Bax deficiency on chondrocyte proliferation implicates a novel regulatory function for Bax on chondrocyte proliferation during fracture repair.

Fracture healing in mice deficient in plasminogen activator inhibitor-1.

To evaluate the role of plasminogen activator inhibitor (PAI)-1, a key negative regulator of the plasmin system of extracellular matrix proteases in developmental bone growth and fracture repair, the bone phenotype of male adult PAI-1-deficient mice was determined and femoral fracture healing was compared with that of age- and sex-matched wild-type C57BL/6J control mice. Regarding bone phenotype, the length and size (but not cortical thickness) of the femur of male PAI-1-deficient mice were smaller than those of wild-type controls. Although the total bone mineral content of PAI-1-deficient mice was not significantly different from that of wild-type mice, the total bone area in PAI-1-deficient mice was smaller, leading to an increase in total bone mineral density. With respect to fracture healing, PAI-1-deficient mice developed fracture calluses that were larger and more mineralized than those of wild-type mice but only at 14 days postfracture. These changes were even greater given the smaller size of the normal femur in PAI-1-deficient mice. Surprisingly, the larger fracture callus remodeled rapidly to normal size and mineral content by 21 days postfracture. Examination of fracture histology revealed that these changes were associated with a dramatic increase followed by a rapid remodeling of the fracture callus cartilage. The remodeling of fracture callus cartilage in PAI-1-deficient mice also displayed an abnormal pattern. These findings demonstrate for the first time that PAI-1 (and potentially the plasminogen extracellular matrix protease system) is an important regulator of bone size during developmental growth and plays a regulatory role in the determination of fracture callus size, cartilage formation, and resorption during bone fracture repair.

Role of IGF1 and EFN-EPH signaling in skeletal metabolism.

Insulin-like growth factor 1(IGF1) and ephrin ligand (EFN)-receptor (EPH) signaling are both crucial for bone cell function and skeletal development and maintenance. IGF1 signaling is the major mediator of growth hormone-induced bone growth, but a host of different signals and factors regulate IGF1 signaling at the systemic and local levels. Disruption of the Igf1 gene results in reduced peak bone mass in both experimental animal models and humans. Additionally, EFN-EPH signaling is a complex system which, particularly through cell-cell interactions, contributes to the development and differentiation of many bone cell types. Recent evidence has demonstrated several ways in which the IGF1 and EFN-EPH signaling pathways interact with and depend upon each other to regulate bone cell function. While much remains to be elucidated, the interaction between these two signaling pathways opens a vast array of new opportunities for investigation into the mechanisms of and potential therapies for skeletal conditions such as osteoporosis and fracture repair.

1,25-Dihydroxyvitamin D suppresses M1 macrophages and promotes M2 differentiation at bone injury sites.

An indispensable role of macrophages in bone repair has been well recognized. Previous data have demonstrated the copresence of M1 macrophages and mesenchymal stem cells (MSCs) during the proinflammatory stage of bone repair. However, the exact role of M1 macrophages in MSC function and bone repair is unknown. This study aimed to define the role of M1 macrophages at bone injury sites via the function of 1,25-Dihydroxyvitamin D (1,25[OH]2D) in suppressing M1 but promoting M2 differentiation. We showed that 1,25(OH)2D suppressed M1 macrophage-mediated enhancement of MSC migration. Additionally, 1,25(OH)2D inhibited M1 macrophage secretion of osteogenic proteins (i.e., Oncostatin M, TNF-α, and IL-6). Importantly, the 1,25(OH)2D-mediated suppression of osteogenic function in M1 macrophages at the proinflammatory stage was associated with 1,25(OH)2D-mediated reduction of MSC abundance, compromised osteogenic potential of MSCs, and impairment of fracture repair. Furthermore, outside the proinflammatory stage, 1,25(OH)2D treatment did not suppress fracture repair. Accordingly, our data support 2 conclusions: (a) M1 macrophages are important for the recruitment and osteogenic priming of MSCs and, hence, are necessary for fracture repair, and (b) under vitamin D-sufficient conditions, 1,25(OH)2D treatment is unnecessary and can be detrimental if provided during the proinflammatory stage of fracture healing.

Unique Regenerative Mechanism to Replace Bone Lost During Dietary Bone Depletion in Weanling Mice.

The present study was undertaken to determine the mechanism whereby calcitropic hormones and mesenchymal stem cell progeny changes are 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 fed a control diet containing 1.2% calcium for 14 days. Depletion decreased plasma calcium and increased plasma parathyroid hormone, 1,25(OH)2D (calcitriol), and C-terminal telopeptide of type I collagen. These plasma parameters quickly returned toward normal on repletion. The trabecular bone volume and connectivity decreased drastically during depletion but were completely restored by the end of repletion. This bone repletion process largely resulted from the development of new bone formation. When bromodeoxyuridine (BrdU) was administered in the middle of depletion for 3 days and examined by fluorescence-activated cell sorting at 7 days into repletion, substantial increases in BrdU incorporation were seen in several CD105 subsets of cells of osteoblastic lineage. When BrdU was administered on days 1 to 3 of repletion and examined 11 days later, no increases in BrdU were seen in these subsets. Additionally, osteocytes that stained positively for BrdU were increased during depletion. In conclusion, the results of the present study have established 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 such that both endpoints are optimized. These results have potential clinical relevance for disease entities such as type 2 osteoporosis.

Conditional Disruption of miR17~92 in Osteoclasts Led to Activation of Osteoclasts and Loss of Trabecular Bone In Part Through Suppression of the miR17-Mediated Downregulation of Protein-Tyrosine Phosphatase-oc in Mice.

This study sought to understand the regulation of an osteoclastic protein-tyrosine phosphatase (PTP-oc), a positive regulator of osteoclast activaty. Our past studies suggested that PTP-oc is regulated post-transcriptionally. The 3'-UTR of PTP-oc mRNA contains a target site for miR17. During osteoclastic differentiation, there was an inverse relationship between the cellular levels of miR17 (expressed as one of the six cluster genes of miR17~92) and PTP-oc mRNA. Overexpression of pre-miR17~92 in mouse osteoclast precursors reduced PTP-oc mRNA level and the size of the derived osteoclasts; whereas deletion of miR17~92 or inhibition of miR17 resulted in the formation of larger osteoclasts containing more nuclei that expressed higher PTP-oc mRNA levels and created larger resorption pits. Thus, PTP-oc-mediated osteoclast activation is modulated in part by miR17~92, particularly miR17. The miR17~92 osteoclast conditional knockout (cKO) mutants, generated by breeding miR17~92loxp/loxp mice with Ctsk-Cre mice, had lower Tb.BV/TV, Tb.BMD, Tb.Conn-Dens, Tb.N, and Tb.Th, but larger Tb.Sp, and greater bone resorption without a change in bone formation compared to littermate controls. The cKO marrow-derived osteoclasts were twice as large, contained twice as many nuclei, and produced twice as large resorption pits as osteoclasts of littermate controls. The expression of genes associated with osteoclast activation was increased in cKO osteoclasts, suggesting that deletion of miR17~92 in osteoclasts promotes osteoclast activation. The cKO osteoblasts did not show differences in cellular miR17 level, alkaline phosphatase activity, and bone nodule formation ability. In conclusion, miR17-92 negatively regulates the osteoclast activity, in part via the miR17-mediated suppression of PTP-oc in osteoclasts.

Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair.

Microarray analysis of gene expression was performed in the healing femur fractures of 13-week-old male rats during the inflammatory stage of repair, at 3 days post-fracture, and the endochondral bone formation stage of repair, at 11 days post-fracture. Multiple replicate pairs of fracture tissues paired with unfractured tissues, and unfractured control bones that had the stabilizing K-wire were introduced. This approach normalized the marrow contributions to the RNA repertoire. We identified 6555 genes with significant changes in expression in fracture tissues at 3 days and 11 days healing. The repertoire of growth factor genes expressed was also surprisingly restricted at both post-fracture intervals. The large number of Expressed Sequence Tags (ESTs) expressed at both post-fracture times indicates that several molecular pathways yet to be identified regulate fracture repair. The number of genes expressed during immune responses and inflammatory processes was restricted with higher expression largely during the early post-fracture analysis. Several of the genes identified in this study have been associated with regulation of cell and extracellular matrix interactions during scarless healing of fetal skin wounds. These observations suggest that these genes might also regulate the scarless healing characteristic of bone regeneration by similar mechanisms.