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GNAS encodes the stimulatory G protein alpha-subunit (Gsα) and its large variant XLαs. Studies have suggested that XLαs is expressed exclusively paternally. Thus, XLαs deficiency is considered to be responsible for certain findings in patients with paternal GNAS mutations, such as pseudo-pseudohypoparathyroidism, and the phenotypes associated with maternal uniparental disomy of chromosome 20, which comprises GNAS. However, a study of bone marrow stromal cells (BMSC) suggested that XLαs could be biallelically expressed. Aberrant BMSC differentiation due to constitutively activating GNAS mutations affecting both Gsα and XLαs is the underlying pathology in fibrous dysplasia of bone. To investigate allelic XLαs expression, we employed next-generation sequencing and a polymorphism common to XLαs and Gsα, as well as A/B, another paternally expressed GNAS transcript. In mouse BMSCs, Gsα transcripts were 48.4 ± 0.3% paternal, while A/B was 99.8 ± 0.2% paternal. In contrast, XLαs expression varied among different samples, paternal contribution ranging from 43.0 to 99.9%. Sample-to-sample variation in paternal XLαs expression was also detected in bone (83.7–99.6%) and cerebellum (83.8 to 100%) but not in cultured calvarial osteoblasts (99.1 ± 0.1%). Osteoblastic differentiation of BMSCs shifted the paternal XLαs expression from 83.9 ± 1.5% at baseline to 97.2 ± 1.1%. In two human BMSC samples grown under osteoinductive conditions, XLαs expression was also predominantly monoallelic (91.3 or 99.6%). Thus, the maternal GNAS contributes significantly to XLαs expression in BMSCs but not osteoblasts. Altered XLαs activity may thus occur in certain cell types irrespective of the parental origin of a GNAS defect.

In the United States, 5–12% of adults have at least one symptom of temporomandibular joint (TMJ) disorders, including TMJ osteoarthritis (TMJ-OA). However, there is no chondroprotective agent that is approved for clinical application. We showed that LOXL2 is elevated in the regenerative response during fracture healing in mice and has a critical role in chondrogenic differentiation. Indeed, LOXL2 is an anabolic effector that attenuates pro-inflammatory signaling in OA cartilage of the TMJ and knee joint, induces chondroprotective and regenerative responses, and attenuates NF-kB signaling. The specific goal of the study was to evaluate if adenoviral delivery of LOXL2 is anabolic to human and mouse TMJ condylar cartilage in vivo and evaluate the protective and anabolic effect on cartilage-specific factors. We employed two different models to assess TMJ-OA. In one model, clinical TMJ-OA cartilage from 5 different samples in TMJ-OA cartilage plugs were implanted subcutaneously in nude mice. Adenovirus LOXL2 -treated implants showed higher mRNA levels of LOXL2, ACAN, and other anabolic genes compared to the adenovirus-Empty-treated implants. Further characterization by RNA-seq analysis showed LOXL2 promotes proteoglycan networks and extracellular matrix in human TMJ-OA cartilage implants in vivo. In order to evaluate if LOXL2-induced functional and sex-linked differences, both male and female four-month-old chondrodysplasia (Cho/+) mice, which develop progressive TMJ-OA due to a point mutation in the Col11a1 gene, were subjected to intraperitoneal injection with Adv-RFP-LOXL2 every 2 weeks for 12 weeks. The data showed that adenovirus delivery of LOXL2 upregulated LOXL2 and aggrecan (Acan), whereas MMP13 expression was slightly downregulated. The fold change expression of Acan and Runx2 induced by Adv-RFP-LOXL2 was higher in females compared to males. Interestingly, Adv-RFP-LOXL2 injection significantly increased Rankl expression in male but there was no change in females, whereas VegfB gene expression was increased in females, but not in males, as compared to those injected with Adv-RFP-Empty in respective groups. Our findings indicate that LOXL2 can induce specifically the expression of Acan and other anabolic genes in two preclinical models in vivo. Further, LOXL2 has beneficial functions in human TMJ-OA cartilage implants and promotes gender-specific anabolic responses in Cho/+ mice with progressive TMJ-OA, suggesting its merit for further study as an anabolic therapy for TMJ-OA.

The failure of bony union following a fracture, termed a fracture nonunion, has severe patient morbidity and economic consequences. This review describes current consensuses and future directions of investigation for determining why, detecting when, and effective treatment if this complication occurs. Current nonunion investigation is emphasizing an expanded understanding of the biology of healing. This has led to assessments of the immune environment, multiple cytokines and morphogenetic factors, and the role of skeletogenic stem cells in the development of nonunion. Detecting biological markers and other objective diagnostic criteria is also a current objective of nonunion research. Treatment approaches in the near future will likely be dominated by the development of specific adjunct therapies to the nonunion surgical management, which will be informed by an expanded mechanistic understanding of nonunion biology. Current consensus among orthopedists is that improved diagnosis and treatment of nonunion hinges first on discoveries at the bench side with later translation to the clinic.

The retinoid X receptors (RXR), peroxisome proliferator activated receptor gamma (PPARγ), and liver X receptors (LXR) all have been shown to regulate bone homeostasis. Tributyltin (TBT) is an environmental contaminant that is a dual RXRα/β and PPARγ agonist. TBT induces RXR, PPARγ, and LXR-mediated gene transcription and suppresses osteoblast differentiation in vitro. Bone marrow multipotent mesenchymal stromal cells derived from female C57BL/6J mice were more sensitive to suppression of osteogenesis by TBT than those derived from male mice. In vivo, oral gavage of 12 week old female, C57Bl/6J mice with 10 mg/kg TBT for 10 weeks resulted in femurs with a smaller cross-sectional area and thinner cortex. Surprisingly, TBT induced significant increases in trabecular thickness, number, and bone volume fraction. TBT treatment did not change the Rankl:Opg RNA ratio in whole bone, and histological analyses showed that osteoclasts in the trabecular space were minimally reduced. In contrast, expression of cardiotrophin-1, an osteoblastogenic cytokine secreted by osteoclasts, increased. In primary bone marrow macrophage cultures, TBT marginally inhibited the number of osteoclasts that differentiated, in spite of significantly suppressing expression of osteoclast markers Nfatc1, Acp5, and Ctsk and resorptive activity. TBT induced expression of RXR- and LXR-dependent genes in whole bone and in vitro osteoclast cultures. However, only an RXR antagonist, but not an LXR antagonist, significantly inhibited TBTs ability to suppress osteoclast differentiation. These results suggest that TBT has distinct effects on cortical versus trabecular bone, likely resulting from independent effects on osteoblast and osteoclast differentiation that are mediated through RXR.

Diabetes increases the likelihood of fracture, interferes with fracture healing and impairs angiogenesis. The latter may be significant due to the critical nature of angiogenesis in fracture healing. Although it is known that diabetes interferes with angiogenesis the mechanisms remain poorly defined. We examined fracture healing in normoglycemic and streptozotocin-induced diabetic mice and quantified the degree of angiogenesis with antibodies to three different vascular markers, CD34, CD31 and Factor VIII. The role of diabetes-enhanced inflammation was investigated by treatment of the TNFα-specific inhibitor, pegsunercept starting 10days after induction of fractures. Diabetes decreased both angiogenesis and VEGFA expression by chondrocytes. The reduced angiogenesis and VEGFA expression in diabetic fractures was rescued by specific inhibition of TNF in vivo. In addition, the TNF inhibitor rescued the negative effect of diabetes on endothelial cell proliferation and endothelial cell apoptosis. The effect of TNFα in vitro was enhanced by high glucose and an advanced glycation endproduct to impair microvascular endothelial cell proliferation and tube formation and to stimulate apoptosis. The effect of TNF, high glucose and an AGE was mediated by the transcription factor FOXO1, which increased expression of p21 and caspase-3. These studies indicate that inflammation plays a major role in diabetes-impaired angiogenesis in endochondral bone formation through its effect on microvascular endothelial cells and FOXO1.

Phosphate plays a critical role in chondrocyte maturation and skeletal mineralization. Studies examining the consequences of dietary phosphate restriction in growing mice demonstrated not only the development of rickets, but also a dramatic decrease in bone accompanied by increased marrow adipose tissue (MAT). Thus studies were undertaken to determine the effects of dietary phosphate restriction on bone formation and bone marrow stromal cell (BMSC) differentiation. Acute phosphate restriction of 28-day-old mice profoundly inhibited bone formation within 48 hours. It also resulted in increased mRNA expression of the early osteolineage markers Sox9 and Runt-related transcription factor 2 (Runx2), accompanied by decreased expression of the late osteolineage markers Osterix and Osteocalcin in BMSCs and osteoblasts, suggesting that phosphate restriction arrests osteoblast differentiation between Runx2 and Osterix. Increased expression of PPARγ and CEBPα, key regulators of adipogenic differentiation, was observed within 1 week of dietary phosphate restriction and was followed by a 13-fold increase in MAT at 3 weeks of phosphate restriction. In vitro phosphate restriction did not alter BMSC osteogenic or adipogenic colony formation, implicating aberrant paracrine or endocrine signaling in the in vivo phenotype. Because BMP signaling regulates the transition between Runx2 and Osterix, this pathway was interrogated. A dramatic decrease in pSmad1/5/9 immunoreactivity was observed in the osteoblasts of phosphate-restricted mice on day 31 (d31) and d35. This was accompanied by attenuated expression of the BMP target genes Id1, KLF10, and Foxc2, the latter of which promotes osteogenic and angiogenic differentiation while impairing adipogenesis. A decrease in expression of the Notch target gene Hey1, a BMP-regulated gene that governs angiogenesis, was also observed in phosphate-restricted mice, in association with decreased metaphyseal marrow vasculature. Whereas circulating phosphate levels are known to control growth plate maturation and skeletal mineralization, these studies reveal novel consequences of phosphate restriction in the regulation of bone formation and osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.

A soluble form of BMP receptor type 1A (mBMPR1A-mFC) acts as an antagonist to endogenous BMPR1A and has been shown to increase bone mass in mice. The goal of this study was to examine the effects of mBMPR1A-mFC on secondary fracture healing. Treatment consisted of 10 mg/kg intraperitoneal injections of mBMPR1A-mFC twice weekly in male C57BL/6 mice. Treatment beginning at 1, 14, and 21 days post-fracture assessed receptor function during endochondral bone formation, at the onset of secondary bone formation, and during coupled remodeling, respectively. Control animals received saline injections. mBMPR1A-mFC treatment initiated on day 1 delayed cartilage maturation in the callus and resulted in large regions of fibrous tissue. Treatment initiated on day 1 also increased the amount of mineralized tissue and up-regulated many bone-associated genes (p = 0.002) but retarded periosteal bony bridging and impaired strength and toughness at day 35 (p < 0.035). Delaying the onset of treatment to day 14 or 21 partially mitigated these effects and produced evidence of accelerated coupled remodeling. These results indicate that inhibition of the BMPR1A-mediated signaling has negative effects on secondary fracture healing that are differentially manifested at different stages of healing and within different cell populations. These effects are most pronounced during the endochondral period and appear to be mediated by selective inhibition of BMPRIA signaling within the periosteum. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2096-2105, 2016.

Background: The goal of this study was to determine if adenovirus-delivered LOXL2 protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action, and determine if the overexpression of LOXL2 in transgenic mice can protect against the development of OA-related cartilage damage and joint disability. Methods: Four-month-old Cho/+ male and female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes were examined by immunostaining and qRT-PCR. The effect of LOXL2 expression on signaling was tested via the pro-inflammatory cytokine IL1&beta, in the cartilage cell line ATDC5. Finally, the OA by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and assessment of allodynia. Results: The adenovirus treatment upregulated LOXL2, Sox9, Acan and Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection, but not the empty vector injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression. LOXL2 attenuated IL-1&beta, induced phospho-NF-&kappa, B/p65 and rescued chondrogenic lineage-related genes in ATDC5 cells, demonstrating one potential protective mechanism. LOXL2 attenuated phospho-NF-&kappa, B independent of its enzymatic activity. Finally, LOXL2-overexpressing transgenic mice were protected from MIA-induced OA-related functional changes, including the time and distance traveled on the treadmill and allodynia. Conclusion: Our study demonstrates that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.

Organotins are members of the environmental obesogen class of contaminants because they activate peroxisome proliferator-activated receptor γ (PPARγ), the essential regulator of adipogenesis. Exposure to thiazolidinediones (PPARγ ligands used to treat type 2 diabetes) is associated with increased fractures. Diminished bone quality likely results from PPARγ's role in promoting adipogenesis while suppressing osteogenesis of bone marrow multipotent mesenchymal stromal cells (BM-MSC). We hypothesized that tributyltin (TBT) would be a potent modifier of BM-MSC differentiation and a negative regulator of bone formation. Organotins interact with both PPARγ and retinoid X receptors (RXR), suggesting that they activate multiple nuclear receptor pathways. To investigate the role of RXR in the actions of TBT, the effects of PPARγ (rosiglitazone) and RXR (bexarotene, LG100268) agonists were compared to the effects of TBT in BMS2 cells and primary mouse BM-MSC cultures. In BMS2 cells, TBT induced the expression of Fabp4, Abca1, and Tgm2 in an RXR-dependent manner. All agonists suppressed osteogenesis in primary mouse BM-MSC cultures, based on decreased alkaline phosphatase activity, mineralization, and expression of osteoblast-related genes. While rosiglitazone and TBT strongly activated adipogenesis, based on lipid accumulation and expression of adipocyte-related genes, the RXR agonists did not. Extending these analyses to other RXR heterodimers showed that TBT and the RXR agonists activated the liver X receptor pathway, whereas rosiglitazone did not. Application of either a PPARγ antagonist (T0070907) or an RXR antagonist (HX531) significantly reduced rosiglitazone-induced suppression of bone nodule formation. Only the RXR antagonist significantly reduced LG100268- and TBT-induced bone suppression. The RXR antagonist also inhibited LG100268- and TBT-induced expression of Abca1, an LXR target gene, in primary BM-MSC cultures. These results provide novel evidence that TBT activates multiple nuclear receptor pathways in BM-MSCs, activation of RXR is sufficient to suppress osteogenesis, and TBT suppresses osteogenesis largely through its direct interaction with RXR.

Tendon healing is characterized by the formation of fibrovascular scar tissue, as tendon has very little intrinsic regenerative capacity. This creates a substantial clinical challenge in the setting of large, chronic tears seen clinically. Interest in regenerative healing seen in amphibians and certain strains of mice has arisen in response to the biological behavior of tendon tissue. Bone is also a model of tissue regeneration as healing bone will achieve the mechanical and histologic characteristics of the original tissue. The ultimate goal of the study of genes and mechanisms that contribute to true tissue regeneration is to ultimately attempt to manipulate the expression of those genes and activate these mechanisms in the setting of tendon injury and repair. Clearly, further research is needed to bring this to the forefront, however, study of scarless healing has potential to have meaningful application to tendon healing.

Fractures are one of the most common large-organ, traumatic injuries in humans, and osteoporosis-related fractures are the fastest growing health care problem of aging. Elective orthopedic surgeries of the bones and joints also represent some of most common forms of elective surgeries performed. Optimal repair of skeletal tissues is necessary for successful outcomes of these many different orthopedic surgical treatments. Research focused on post-natal skeletal repair is therefore of immense clinical importance and of particular relevance in situations in which bone tissue healing is compromised due to the extent of tissue trauma or specific medical co-morbidities. Three commonly used murine surgical models of bone healing, closed fracture with intramedullary fixation, distraction osteogenesis (DO), and marrow ablation by reaming are presented. The biological aspects of these models are contrasted and the types of research questions that may be addressed with these models are presented.

Three commonly used murine surgical models of bone healing [closed fracture with intramedullary fixation, distraction osteogenesis (DO), and marrow ablation by reaming] are presented. Detailed surgical protocols for each model are outlined. The nature of the regenerative processes and the types of research questions that may be addressed with these models are briefly outlined. The relative strengths and weaknesses of these models are compared to a number of other surgical models that are used to address similar research questions.

A targeted proteomic analysis of murine serum over a 35-day course of fracture healing was carried out to determine if serum proteomic changes could be used to monitor the biological progression of fracture healing. Transverse, closed femoral fractures where generated and stabilized with intramedullary fixation. A single stranded DNA aptamer-based multiplexed proteomic approach was used to assay 1,310 proteins. The transcriptomic profiles for genes matching the 1,310 proteins were obtained by microarray analysis of callus mRNA. Of the 1,310 proteins analyzed, 850 proteins showed significant differences among the time points (p-value

Radiographic Union Score for Tibia (RUST) and modified RUST (mRUST) are radiographic tools for quantitatively evaluating fracture healing using a cortical scoring system. This tool has high intra-class correlation coefficients (ICCs); however, little evidence has evaluated the scores against the physical properties of bone healing. Closed, stabilized fractures were made in the femora of C3H/HeJ male mice (8-12 week-old) of two dietary groups: a control and a phosphate restricted diet group. Micro-computed tomography (μCT) and torsion testing were carried out at post-operative days (POD) 14, 21, 35, and 42 (n=10-16) per group time-point. Anteroposterior and lateral radiographic views were constructed from the μCT scans and scored by five raters. The raters also indicated if the fracture were healed. ICCs were 0.71 (mRUST) and 0.63 (RUST). Both RUST scores were positively correlated with callus bone mineral density (BMD) (r = 0.85 and 0.80, p 0.318). Clinical relevance of this study is both RUST scores showed high correlation to physical properties of healing and generally distinguished healed vs. non-healed fractures. This article is protected by copyright. All rights reserved.

Background Lysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. Our previous studies showed that LOXL2 is elevated during mouse fracture healing. The goal of this study was to evaluate the potential of LOXL2 to act as an anabolic agent in cartilage affected by osteoarthritis (OA). Methods LOXL2 was visualized in tissues from human knee and hip joints and temporomandibular joints (TMJ) by immunofluorescence. The activity of LOXL2 in human articular and TMJ chondrocytes was assessed by cell-based assays, microarray analysis, and RT-qPCR, and LOXL2-mediated activation of NF-κB and extracellular signal-related kinase (ERK) signaling pathways was measured by western blotting. To examine LOXL2-induced effect in vivo, we implanted Matrigel-imbedded human chondrocytes into nude mice and exposed them to exogenous LOXL2 for 6 weeks. Finally, LOXL2-induced effects on collagen type 2 α1 (COL2A1) and phospho-SMAD2/3 were evaluated by immunofluorescence analysis. Results LOXL2 staining was detected in damaged regions of human TMJ, hip and knee joints affected by OA. Stimulation with transforming growth factor (TGF)-β1 upregulated LOXL2 expression, while pro-inflammatory cytokines IL-1β and TNF-α downregulated LOXL2, in human chondrocytes. Viral transduction of LOXL2 in OA chondrocytes increased the mRNA levels of chondroitin sulfate proteoglycan (CSPG4), aggrecan (ACAN), sex determining region Y-box containing gene 9 (SOX9), and COL2A1 but reduced the levels of extracellular matrix (ECM)-degrading enzymes matrix metalloproteinase (MMP)1, MMP3, and MMP13. Further, forced expression of LOXL2 promoted chondrogenic lineage-specific gene expression, increased the expression of COL2A1 in the presence of TNF-α, and inhibited chondrocyte apoptosis. LOXL2 expression also inhibited IL-1β-induced phospho-NF-κB/p65 and TGF-β1-induced ERK1/2 phosphorylation. Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed anabolic responses after LOXL2 transduction, including increased expression of SOX9, ACAN, and COL2A1. Finally, immunofluorescence staining revealed co-localization of LOXL2 with SOX9 in the nuclei of cells in the implants, decreased phospho-SMAD2/3, and increased COL2A1 staining. Conclusion Our results suggest that although LOXL2 is upregulated in cartilage affected by OA, this may be a protective response that promotes anabolism while inhibiting specific catabolic responses in the pathophysiology of OA. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1388-8) contains supplementary material, which is available to authorized users.

Injury to the growth plate is associated with growth disturbances, most notably premature cessation of growth. The goal of this study was to identify spatial changes in the structure and composition of the growth plate in response to injury to provide a foundation for developing therapies that minimize the consequences for skeletal development. We used contrast-enhanced microcomputed tomography (CECT) and histological analyses of a murine model of growth plate injury to quantify changes in the cartilaginous and osseous tissue of the growth plate. To distinguish between local and global changes, the growth plate was divided into regions of interest near to and far from the injury site. We noted increased thickness and CECT attenuation (a measure correlated with glycosaminoglycan (GAG) content) near the injury, and increased tissue mineral density (TMD) of bone bridges within the injury site, compared to outside the injury site and contralateral growth plates. Furthermore, we noted disruption of the normal zonal organization of the physis. The height of the hypertrophic zone was increased at the injury site, and the relative height of the proliferative zone was decreased across the entire injured growth plate. These results indicate that growth plate injury leads to localized disruption of cellular activity and of endochondral ossification. These local changes in tissue structure and composition may contribute to the observed retardation in femur growth. In particular, the changes in proliferative and hypertrophic zone heights seen following injury may impact growth and could be targeted when developing therapies for growth plate injury.

Bone formation and aging are sexually dimorphic. Yet, definition of the intrinsic molecular differences between male and female multipotent mesenchymal stromal cells (MSC) in bone is lacking. This study assessed sex-linked differences in MSC differentiation in 3-, 6-, and 9-month-old C57BL/6J mice. Analysis of tibiae showed that female mice had lower bone volume fraction and higher adipocyte content in the bone marrow compared to age-matched males. While both males and females lost bone mass in early aging, the rate of loss was higher in males. Similar expression of bone- and adipocyte-related genes was seen in males and females at 3 and 9 months, while at 6 months, females exhibited a two-fold greater expression of these genes. Under osteogenic culture conditions, bone marrow MSCs from female 3- and 6-month-old mice expressed similar levels of bone-related genes, but significantly greater levels of adipocyte-related genes, than male MSCs. Female MSCs also responded to rosiglitazone-induced suppression of osteogenesis at a 5-fold lower (10 nM) concentration than male MSCs. Female MSCs grown in estrogen-stripped medium showed similar responses to rosiglitazone as MSCs grown in serum containing estrogen. MSCs from female mice that had undergone ovariectomy before sexual maturity also were sensitive to rosiglitazone-induced effects on osteogenesis. These results suggest that female MSCs are more sensitive to modulation of differentiation by PPARγ and that these differences are intrinsic to the sex of the animal from which the MSCs came. These results also may explain the sensitivity of women to the deleterious effects of rosiglitazone on bone.

Previous studies showed that loss of tumor necrosis factor α (TNFα) signaling delayed fracture healing by delaying chondrocyte apoptosis and cartilage resorption. Mechanistic studies showed that TNFα induced Fas expression within chondrocytes; however, the degree to which chondrocyte apoptosis is mediated by TNFα alone or dependent on the induction of Fas is unclear. This question was addressed by assessing fracture healing in Fas-deficient B6.MRL/Faslpr/J mice. Loss of Fas delayed cartilage resorption but also lowered bone fraction in the calluses. The reduced bone fraction was related to elevated rates of coupled bone turnover in the B6.MRL/Faslpr/J calluses, as evidenced by higher osteoclast numbers and increased osteogenesis. Analysis of the apoptotic marker caspase 3 showed fewer positive chondrocytes and osteoclasts in calluses of B6.MRL/Faslpr/J mice. To determine if an active autoimmune state contributed to increased bone turnover, the levels of activated T cells and Treg cells were assessed. B6.MRL/Faslpr/J mice had elevated Treg cells in both spleens and bones of B6.MRL/Faslpr/J but decreased percentage of activated T cells in bone tissues. Fracture led to ∼30% to 60% systemic increase in Treg cells in both wild-type and B6.MRL/Faslpr/J bone tissues during the period of cartilage formation and resorption but either decreased (wild type) or left unchanged (B6.MRL/Faslpr/J) the numbers of activated T cells in bone. These results show that an active autoimmune state is inhibited during the period of cartilage resorption and suggest that iTreg cells play a functional role in this process. These data show that loss of Fas activity specifically in chondrocytes prolonged the life span of chondrocytes and that Fas synergized with TNFα signaling to mediate chondrocyte apoptosis. Conversely, loss of Fas systemically led to increased osteoclast numbers during later periods of fracture healing and increased osteogenesis. These findings suggest that retention of viable chondrocytes locally inhibits osteoclast activity or matrix proteolysis during cartilage resorption. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

The relationship between BMP2 expression and the recruitment of skeletogenic stem cells was assessed following bone marrow reaming. BMP2 expression was examined using transgenic mice in which β-galactosidase had been inserted into the coding region of BMP2. Stem cell mobilization was analyzed by FACS analysis using CD73, a marker associated with bone marrow stromal stem cells. BMP2 expression was induced in endosteal lining cells, cortical osteocytes and periosteal cells in both the reamed and in contralateral bones. BMP2 mRNA expression in the reamed bone showed an early peak within the first 24 h of reaming followed by a later peak at 7 days, while contralateral bones only showed the 7 days peak of expression. FACS analysis sorting on CD73 positive cells showed a 50% increase of these cells at 3 and 14 days in the marrow of the injured bone and a single peak at 14 days of the marrow cell population of the contralateral bone. A ∼20% increase of CD73 positive cells was seen in the peripheral blood 2 days after reaming. These data showed that traumatic bone injury caused a systemic induction of BMP2 expression and that this increase is correlated with the mobilization of CD73 positive cells.

Runx2 and Runx3 are known to be expressed in the growth plate during endochondral bone formation. Here we addressed the functional role of Runx3 as distinct from Runx2 by using two models of postnatal bone repair: fracture healing that proceeds by an endochondral process and marrow ablation that proceeds by only an intramembranous process. Both Runx2 and Runx3 mRNAs were differentially up regulated during fracture healing. In contrast, only Runx2 showed increased expression after marrow ablation. During fracture healing, Runx3 was expressed earlier than Runx2, was concurrent with the period of chondrogenesis, and coincident with maximal aggrecan expression a protein associated with proliferating and permanent cartilage. Immunohistological analysis showed Runx3 protein was also expressed by chondrocytes in vivo. In contrast, Runx2 was expressed later during chondrocyte hypertrophy, and primary bone formation. The functional activities of Runx3 during chondrocyte differentiation were assessed by examining its regulatory actions on aggrecan gene expression. Aggrecan mRNA levels and aggrecan promoter activity were enhanced in response to the over-expression of either Runx2 and Runx3 in ATDC5 chondrogenic cell line, while sh-RNA knocked down of each Runx protein showed that only Runx3 knock down specifically suppressed aggrecan mRNA expression and promoter activity. ChIP assay demonstrated that Runx3 interactions were selective to sites within the aggrecan promoter and were only observed during early periods of chondrogenesis before hypertrophy. Our studies suggest that Runx3 positively regulates aggrecan expression and suggest that its function is more limited to cartilage development than to bone. In aggregate these data further suggest that the various members of the Runx transcription factors are involved in the coordination of chondrocyte development, maturation, and hypertrophy during endochondral bone formation.

Activin A receptor type I or activin receptor-like kinase 2 (ACVRI/ALK2) belongs to type I TGF-β family and plays an important role in bone development. Activating mutations of ALK2 containing the R206 to H mutation, are present in 95% in the rare autosomal genetic disease fibrodysplasiaossificansprogressiva (FOP), which leads to the development of ectopic bone formation in muscle. The effect of AMP-activated protein kinase (AMPK) activation on ALK2R206H-mediated signaling in fibroblast obtained from a FOP patient was assessed in the present study. The activity of the mutated ALK2 was suppressed by pharmacological AMPK activators such as metformin and aspirin, while their actions were blocked by the dominant negative mutant of AMPK and mimicked by the constitutively active mutant of AMPK. Furthermore, activation of AMPK upregulated Smad6 and Smurf1 and thereby enhanced their interactions, resulting in its proteosome-dependent degradation of ALK2. In contrast, knockdown of Smad6 or Smurf1 prevented metformin-induced reduction of ALK2. To evaluate the biological relevance of AMPK action on ALK2 activity, we induced FOP fibroblasts within iPS cells and found that their osteogenic differentiation in vitro was inhibited by metformin. Our studies provide novel insight into potential approaches to treatment of FOP, since several AMPK activators (e.g. metformin, berberine, and aspirin, etc.) are already in clinical use for the treatment of diabetes and metabolic syndromes.

Background: Donor site morbidity and limited volume remain primary drawbacks of using bone graft from the iliac crest and an impetus for finding other sources of autologous bone-graft material. The Synthes Reamer/Irrigator/Aspirator (RIA) has beenfoundtohavevalueasanautologousbone-graftharvestingdevice.Thepurposeofthisstudywastocomparethecellular and biochemical characteristics of bone grafts obtained with use of the RIA and from the iliac crest of the same patient. Methods: A prospective study was performed on a consecutive series of ten skeletally mature patients presenting for repair of nonunited tibial or femoral fractures. Graft material was harvested from both the iliac crest (in the standard fashion) and the medullary canal of the femur or tibia (with use of the RIA) of each patient. Portions of each autologous graft sample were assessed histologically and by genomewide transcriptional profiling for biochemical markers known to be expressed during fracture-healing. Results: Principal-component analysis comparing the messenger RNA expression profiles in the RIA and iliac crest samples showed that the expression profile at each harvest site was unique and independent of patient, age, sex, or any identified comorbidity. Transcriptional analysis showed that the RIA samples had greater levels of expression of genes associated with vascular, skeletal, and hematopoietic tissues. Additionally, stem cell markers and growth factors that act early in the osteogenic cascade were more abundant in the RIA samples compared with the iliac crest samples. Conclusions: This is the first study to directly compare the histological and molecular profiles of bone grafts from reamingdebrisand theiliaccrestof thesamepatient. Thedebrisgeneratedduringintramedullaryreaming,harvestedwith use of the RIA technique, and the bone graft harvested from the iliac crest possessed a similar transcriptional profile for genes known to act in the early stages of bone repair and formation. This suggests that reaming debris may be a viable alternative to iliac crest bone graft when autologous cancellous graft is needed.

Vascular endothelial growth factor (VEGF) is an endothelial cell survival factor and is required for effective coupling of angiogenesis and osteogenesis. Although central to bone homeostasis, repair and the pathobiology that affect these processes, the precise mechanisms coupling endothelial cell function within bone formation and remodelling remain unclarified. This review will (i) focus on the potential directionality of VEGF signalling in adult bone by identifying the predominant source of VEGF within the bone microenvironment, (ii) will summarize current VEGF receptor expression studies by bone cells and (iii) will provide evidence for a role for VEGF signalling during postnatal repair and osteoporosis. A means of understanding the directionality of VEGF signalling in adult bone would allow us to most effectively target angiogenic pathways in diseases characterized by changes in bone remodelling rates and enhance bone repair when compromised.

The differentiation of osteoblasts from their precursors, mesenchymal stem cells, is an important component of bone homeostasis as well as fracture healing. The A2B adenosine receptor (A2BAR) is a Gαs/αq-protein-coupled receptor that signals via cAMP. cAMP-mediated signaling has been demonstrated to regulate the differentiation of mesenchymal stem cells (MSCs) into various skeletal tissue lineages. Here, we studied the role of this receptor in the differentiation of MSCs to osteoblasts. In vitro differentiation of bone marrow-derived MSCs from A2BAR KO mice resulted in lower expression of osteoblast differentiation transcription factors and the development of fewer mineralized nodules, as compared with WT mice. The mechanism of effect involves, at least partially, cAMP as indicated by experiments involving activation of the A2BAR or addition of a cAMP analog during differentiation. Intriguingly, in vivo, microcomputed tomography analysis of adult femurs showed lower bone density in A2BAR KO mice as compared with WT. Furthermore, A2BAR KO mice display a delay in normal fracture physiology with lower expression of osteoblast differentiation genes. Thus, our study identified the A2BAR as a new regulator of osteoblast differentiation, bone formation, and fracture repair.

Whilst the majority of fractures heal normally, it is estimated that ∼10% of fractures exhibit some level of delayed or impaired healing. Although radiography is the primary diagnostic tool to assess the progression of fracture healing, radiographic features only qualitatively correlate with tissue level increases in mineral content and do not quantitatively measure underlying biological processes that are associated with the progression of healing. Specific metaloproteinases have been shown to be essential to processes of both angiogenesis and mineralised cartilage resorption and bone remodelling at different phases of fracture healing. The aim of this study was to determine the potential of using a simple urine based assay of the activity of two MMPs as a means of assessing the biological progression of fracture healing through the endochondral phase of healing. Using a standard mid-diaphyseal murine model of femoral fracture, MMP9 and MMP13 proteins and enzymatic activity levels were quantified in the urine of mice across the time-course of fracture healing and compared to the mRNA and protein expression profiles in the calluses. Both urinary MMP9 and MMP13 protein and enzymatic activity levels, assessed by Western blot, zymogram and specific MMP fluorometric substrate assays, corresponded to mRNA expression and immunohistologic assays of the proteins within callus tissues. These studies suggest that urinary levels of MMP9 and MMP13 may have potential as metabolic markers to monitor the progression of fracture healing.

Fractures initiate one round of endochondral bone formation in which callus cells differentiate in a synchronous manner that temporally phenocopies the spatial variation of endochondral development of a growth plate. During fracture healing C57BL/6J (B6) mice initiate chondrogenesis earlier and develop more cartilage than bone, whereas C3H/HeJ (C3H) mice initiate osteogenesis earlier and develop more bone than cartilage. Comparison of the transcriptomes of fracture healing in these strains of mice identified the genes that showed differences in timing and quantitative expression and encode for the variations in endochondral bone development of the two mouse strains. The complement of strain-dependent differences in gene expression was specifically associated with ontologies related to both skeletal and vascular formation. Moreover, the differences in gene expression associated with vascular tissue formation during fracture healing were correlated with the underlying differences in development and function of the cardiovascular systems of these two strains of mice. Significant differences in gene expression associated with bone morphogenetic protein/transforming growth factor β (BMP/TGF-β) signal-transduction pathways were identified between the two strains, and a network of differentially expressed genes specific to the MAP kinase cascade was further defined as a subset of the genes of the BMP/TGF-β pathways. Other signal-transduction pathways that showed significant strain-specific differences in gene expression included the RXR/PPAR and G protein-related pathways. These data identify how bone and vascular regeneration are coordinated through expression of common sets of transcription and morphogenetic factors and suggest that there is heritable linkage between vascular and skeletal tissue development during postnatal regeneration.

BACKGROUND:: The acute-phase response (APR) is critical to the body's ability to successfully respond to injury. A murine model of closed unilateral femur fractures and bilateral femur fracture were used to study the effect of injury magnitude on this response. METHODS:: Standardized unilateral femur fracture and bilateral femur fracture in mice were performed. The femur fracture sites, livers, and serum were harvested over time after injury. Changes in mRNA expression of cytokines, hepatic acute-phase proteins, and serum cytokines overtime were measured. RESULTS:: There was a rapid and short-lived hepatic APR to fracture injuries. The overall pattern in both models was similar. Both acute-phase proteins' mRNA (fibrinogen-gamma and serum amyloid A-3) showed increased mRNA expression over baseline within the first 48 hours and their levels positively correlated with the extent of injury. However, increased severity of injury resulted in a delayed induction of the APR. A similar effect on the gene expression of cytokines (interleukin [IL]-1beta, IL-6, and tumor necrosis factor-alpha) at the fracture site was seen. Serum IL-6 levels increased with increased injury and showed no delay between injury models. CONCLUSIONS:: Greater severity of injury resulted in a delayed induction of the liver's APR and a diminished expression of cytokines at the fracture site. Serum IL-6 levels were calibrated to the extent of the injury, and changes may represent mechanisms by which the local organ responses to injury are regulated by the injury magnitude. Language: en

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human bone morphogenetic protein-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess bone healing, the genetically modified ("gene activated") tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as 10 days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed bone were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal bone and cartilage more expeditiously, and at lower cost, than is presently possible.

Non-invasive characterization of fracture callus structure and composition may facilitate development of surrogate measures of the regain of mechanical function. As such, quantitative computed tomography- (CT-) based analyses of fracture calluses could enable more reliable clinical assessments of bone healing. Although previous studies have used CT to quantify and predict fracture healing, it is unclear which of the many CT-derived metrics of callus structure and composition are the most predictive of callus mechanical properties. The goal of this study was to identify the changes in fracture callus structure and composition that occur over time and that are most closely related to the regain of mechanical function. Micro-computed tomography (microCT) imaging and torsion testing were performed on murine fracture calluses (n=188) at multiple post-fracture timepoints and under different experimental conditions that alter fracture healing. Total callus volume (TV), mineralized callus volume (BV), callus mineralized volume fraction (BV/TV), bone mineral content (BMC), tissue mineral density (TMD), standard deviation of mineral density (sigma(TMD)), effective polar moment of inertia (J(eff)), torsional strength, and torsional rigidity were quantified. Multivariate statistical analyses, including multivariate analysis of variance, principal components analysis, and stepwise regression were used to identify differences in callus structure and composition among experimental groups and to determine which of the microCT outcome measures were the strongest predictors of mechanical properties. Although calluses varied greatly in the absolute and relative amounts of mineralized tissue (BV, BMC, and BV/TV), differences among timepoints were most strongly associated with changes in tissue mineral density. Torsional strength and rigidity were dependent on mineral density as well as the amount of mineralized tissue: TMD, BV, and sigma(TMD) explained 62% of the variation in torsional strength (p

Fracture healing involves multiple stages of repair and coordinated actions of multiple cell types. Consequently, it may be possible to enhance healing through treatment strategies that target more than one repair process or cell type. The goal of this study was to determine the combined effects of recombinant human bone morphogenetic protein 7 (rhBMP-7) and parathyroid hormone (PTH(1-34)) on metaphyseal bone healing. A wedge-shaped defect was created in the lateral aspect of the distal tibia in female New Zealand white rabbits (n=64) and was filled with tricalcium phosphate (TCP). Animals were assigned to four groups: 1) BMP-7 and PTH; 2) BMP-7; 3) PTH; and 4) control (TCP alone). In groups 1 and 2, 200 microg rhBMP-7 was incorporated into the TCP. Animals received daily subcutaneous injections of 10 microg/kg PTH(1-34) (groups 1 and 3) or saline (groups 2 and 4). Healing at 4 weeks was assessed using micro-computed tomography, histology, immunohistochemistry, and mechanical testing. Combined treatment with rhBMP-7 and PTH resulted in increased callus total volume (TV), mineralized volume (BV), average cross-sectional area, and bone mineral content (BMC) as compared to the control group (p

Over the past several years, there has been an increasing interest in the biology of bone repair and potential technologies for enhancing fracture-healing. Part of this interest is derived from the growing age of the population and the recognition that increased age carries an increased risk of complications after fracture. Although use of locally implanted or injected growth factors has received the most attention, systemic treatments for the enhancement of bone repair, especially for situations in which bone repair may be diminished or delayed, are now under investigation. Since the approval of parathyroid hormone (PTH) as an anabolic treatment for osteoporosis, there has been an increasing interest in other potential clinical uses for this compound in musculoskeletal conditions. It is now widely recognized that PTH administration is an effective therapy to increase bone mineral density and prevent fractures in patients with osteoporosis. More recently, a growing body of evidence has supported the conclusion that PTH will also be an effective anabolic therapy for the enhancement of bone repair after fracture. This review focuses on the recent research demonstrating the potential of PTH in the management of bone repair in a number of fracture models and also highlights the ongoing studies into the mechanisms of PTH actions on endochondral bone repair.

The hypoxia-inducible factor-1α (HIF-1α) pathway is the central regulator of adaptive responses to low oxygen availability and is required for normal skeletal development. Here, we demonstrate that the HIF-1α pathway is activated during bone repair and can be manipulated genetically and pharmacologically to improve skeletal healing. Mice lacking pVHL in osteoblasts with constitutive HIF-1α activation in osteoblasts had markedly increased vascularity and produced more bone in response to distraction osteogenesis, whereas mice lacking HIF-1 α in osteoblasts had impaired angiogenesis and bone healing. The increased vascularity and bone regeneration in the pVHL mutants were VEGF dependent and eliminated by concomitant administration of VEGF receptor antibodies. Small-molecule inhibitors of HIF prolyl hydroxylation stabilized HIF/VEGF production and increased angiogenesis in vitro . One of these molecules (DFO) administered in vivo into the distraction gap increased angiogenesis and markedly improved bone regeneration. These results identify the HIF-1α pathway as a critical mediator of neoangiogenesis required for skeletal regeneration and suggest the application of HIF activators as therapies to improve bone healing.

While developmental processes usually terminate when animals reach maturity, morphogenetic processes may be reinitiated in specific tissues as a consequence of injury. Fracture healing and bone repair are among the unique processes of postnatal tissue regeneration that are believed to mirror the ontological events that take place during embryological development of the skeleton. Many of the developmental processes and genes that are expressed preferentially in embryonic stem cells during the initiation and activation of the morphogenetic pathways of skeletal development are also expressed in fracture callus tissues. It is generally believed that it is the recapitulation of these ontological processes during fracture healing that allows bone to be repaired without the development of scar tissue, and ultimately leads to the regeneration of damaged tissue to its preinjury structure. This is in contrast to the repair of soft tissue that heals typically with some degree of fibroblastic scar formation. The interplay of a number of different tissues (vascular, hematopoietic, skeletal, and neural) is essential for the unimpeded regeneration of bone. Appropriate passage through this regenerative cascade is dependent on the proper orchestration of paracrine, autocrine, and systemic signaling pathways with the appropriate complement of stem cells needed to regenerate a skeletal organ. In context of the bone repair process both innate and adaptive immune cells and regulatory factors contribute to the initiation of the repair process, recruitment of skeletal stem cells, control of skeletal cell differentiation, and the remodeling of the regenerated tissues to the injured skeletal element’s original form and function.

Previous studies have demonstrated the ability of direct adenoviral BMP-2 (Ad.BMP-2) gene delivery to enhance bone repair. Nevertheless, in studies using a rat segmental defect model, it has not proved possible to achieve reliably full osseous union in all animals. To address this issue, we evaluated the effect of vector dose on healing. Critical-size defects were created in the right femora of 27 Sprague-Dawley rats. The defects received a single, intralesional, percutaneous injection of 2.7 x 10(7) (low dose), 2.7 x 10(8) (medium dose), or 2.7 x 10(9) (high dose) plaque-forming units of Ad.BMP-2. After 8 weeks, femora were evaluated by X-ray, dual-energy X-ray absorptiometry, microcomputed tomography (microCT), and histology. The high dose of vector bridged 100%, the medium dose 11%, and the low dose 25% of the defects, as evaluated by X-ray and microCT imaging. Bone mineral content and bone volume of the defects receiving the high dose of vector were significantly higher than those of both groups receiving lower doses. Histologically, defects treated with the high dose were filled by trabecular bone and small amounts of cartilage, whereas large areas of fibrous tissue and cartilage remained in the defects receiving lower doses. However, the newly formed bone lacked the structural organization of native bone, suggesting that further maturation is necessary.

The purpose of this study was to establish a reliable model of metaphyseal bone healing and to use this model to investigate the effect of recombinant human osteogenic protein 1 (rhOP-1; BMP-7) and parathyroid hormone fragment (PTH 1-34) on healing. A wedge-shaped osteotomy was created in the distal tibia of 16-week-old female New Zealand White rabbits (n = 20) and was bridged with a custom-made external fixator. Five experimental groups of four animals each were investigated. In groups 1-4 the osteotomy gap was filled with tricalcium phosphate (TCP), and the gap was left unfilled in group 5 ("normal healing"). In group 1, 200 microg OP-1 was mixed in with the TCP. Groups 2 and 3 received daily subcutaneous injections of 10 and 40 microg/kg PTH, respectively, beginning on postoperative day 1. Radiographs were taken weekly. Following sacrifice on postoperative day 28, peripheral quantitative computed tomography (pQCT), histology, and mechanical testing (axial compression and torsion) were performed. Only one animal failed to complete the full 4-week time course, and no infections were encountered. Bone healing occurred in all animals. OP-1 stimulated bone formation locally, while the lower dose of PTH enhanced bone formation systemically (p < 0.05). Tibiae treated with OP-1 exhibited higher torsional strength (p = 0.04) than those in the normal healing group. These results indicate that a reliable and reproducible surgical model of metaphyseal healing has been established. In addition, differences in systemic versus local effects of PTH and OP-1 in accelerating metaphyseal fracture healing were found.

Studies have shown that systemic PTH treatment enhanced the rate of bone repair in rodent models. However, the mechanisms through which PTH affects bone repair have not been elucidated. In these studies we show that PTH primarily enhanced the earliest stages of endochondral bone repair by increasing chondrocyte recruitment and rate of differentiation. In coordination with these cellular events, we observed an increased level of canonical Wnt-signaling in PTH-treated bones at multiple time-points across the time- course of fracture repair, supporting the conclusion that PTH responses are at least in part mediated through Wnt signaling. Introduction: Since FDA approval of PTH (PTH(1-34); Forteo) as a treatment for osteoporosis, there has been interest in its use in other musculoskeletal conditions. Fracture repair is one area in which PTH may have a significant clinical impact. Multiple animal studies have shown that systemic PTH treatment of healing fractures increased both callus volume and return of mechanical competence in models of fracture healing. Whereas the potential for PTH has been established, the mechanism(s) by which PTH produces these effects remain elusive. Materials and Methods: Closed femoral fractures were generated in 8-wk-old male C57Bl/6 mice followed by daily systemic injections of either saline (control) or 30 g/kg PTH(1-34) for 14 days after fracture. Bones were harvested at days 2, 3, 5, 7, 10, 14, 21, and 28 after fracture and analyzed at the tissue level by radiography and histomorphometry and at the molecular and biochemical levels level by RNase protection assay (RPA), real-time PCR, and Western blot analysis. Results: Quantitative CT analysis showed that PTH treatment induced a larger callus cross-sectional area, length, and total volume compared with controls. Molecular analysis of the expression of extracellular matrix genes associated with chondrogenesis and osteogenesis showed that PTH treated fractures displayed a 3-fold greater increase in chondrogenesis relative to osteogenesis over the course of the repair process. In addition, chondrocyte hypertrophy occurred earlier in the PTH-treated callus tissues. Analysis of the expression of potential mediators of PTH actions showed that PTH treatment significantly induced the expression of Wnts 4, 5a, 5b, and 10b and increased levels of unphosphorylated, nuclear localized -catenin protein, a central feature of canonical Wnt signaling. Conclusions: These results showed that the PTH-mediated enhancement of fracture repair is primarily asso- ciated with an amplification of chondrocyte recruitment and maturation in the early fracture callus. Associated with these cellular effects, we observed an increase in canonical Wnt signaling supporting the conclusion that PTH effects on bone repair are mediated at least in part through the activation of Wnt-signaling pathways. J Bone Miner Res 2007;22:1903-1912. Published online on August 6, 2007; doi: 10.1359/JBMR.070724

Skeletal development and turnover occur in close spatial and temporal association with angiogenesis. Osteoblasts are ideally situated in bone to sense oxygen tension and respond to hypoxia by activating the hypoxia-inducible factor alpha (HIF alpha) pathway. Here we provide evidence that HIF alpha promotes angiogenesis and osteogenesis by elevating VEGF levels in osteoblasts. Mice overexpressing HIF alpha in osteoblasts through selective deletion of the von Hippel-Lindau gene (Vhl) expressed high levels of Vegf and developed extremely dense, heavily vascularized long bones. By contrast, mice lacking Hif1a in osteoblasts had the reverse skeletal phenotype of that of the Vhl mutants: long bones were significantly thinner and less vascularized than those of controls. Loss of Vhl in osteoblasts increased endothelial sprouting from the embryonic metatarsals in vitro but had little effect on osteoblast function in the absence of blood vessels. Mice lacking both Vhl and Hif1a had a bone phenotype intermediate between those of the single mutants, suggesting overlapping functions of HIFs in bone. These studies suggest that activation of the HIF alpha pathway in developing bone increases bone modeling events through cell-nonautonomous mechanisms to coordinate the timing, direction, and degree of new blood vessel formation in bone.

Lysyl oxidases are required for collagen and elastin cross-linking and extracellular matrix maturation including in bone. The lysyl oxidase family consists of lysyl oxidase (LOX) and 4 isoforms (LOXL1-4). Here we investigate whether deletion of LOXL1, which has been linked primarily to elastin maturation, leads to skeletal abnormalities. Left femurs (n = 8), L5 vertebrae (n = 8), and tibiae (n = 8) were analyzed by micro-computed tomography in 13-week-old wild-type (WT) and LOXL1-/- male and female mice. Right femurs (n = 8) were subjected to immunohistochemistry for LOXL1, and histochemical/histology analyses of osteoclasts and growth plates. Sera from all mice were analyzed for bone turnover markers. Results indicate strong expression of LOXL1 in wild-type growth plates in femurs. Significant deterioration of trabecular bone structure in long bones and vertebrae from female was observed but not from male, mutant mice compared with WT. Decreases in BV/TV, Conn.D, trabecular thickness, and number in the femoral distal metaphysis were observed in female, but not in male, mutant mice. Trabecular spacing was increased significantly in femurs of female mutant mice. Findings were similar in trabeculae of L5 vertebrae from female mutant mice. The number of TRAP positive osteoclasts at the trabecular bone surface was increased in female mutant mice compared with WT females, consistent with increased serum RANKL and decreased OPG levels. Analysis of bone turnover markers confirmed increased bone resorption as indicated by significantly elevated CTX-1 in the serum of female LOXL1-/- mice compared to their wild-type counterparts, as well as decreased bone formation as measured by decreased serum levels of PINP. Picrosirius red staining revealed a loss of heterogeneity in collagen organization in female LOXL1-/- mice only, with little to no yellow and orange birefringence. Organization was also impaired in chondrocyte columns in both female and male LOXL1-/- mice, but to a greater extent in females. Data indicate that LOXL1-/- mutant mice develop appendicular and axial skeletal phenotypes characterized by decreased bone volume fraction and compromised trabecular microstructure, predominantly in females.

Uncovering the mechanisms of the sensitivity of bone healing to mechanical factors is critical for understanding the basic biology and mechanobiology of the skeleton, as well as for enhancing clinical treatment of bone injuries. This study refined an experimental method of measuring the strain microenvironment at the site of a bone injury during bone healing. This method used a rat model in which a well-controlled bending motion was applied to an osteotomy to induce the formation of pseudarthrosis that is composed of a range of skeletal tissues, including woven bone, cartilage, fibrocartilage, fibrous tissue, and clot tissue. The goal of this study was to identify both the features of the strain microenvironment associated with formation of these different tissues and the expression of proteins frequently implicated in sensing and transducing mechanical cues. By pairing the strain measurements with histological analyses that identified the regions in which each tissue type formed, we found that formation of the different tissue types occurs in distinct strain microenvironments and that the type of tissue formed is correlated most strongly to the local magnitudes of extensional and shear strains. Weaker correlations were found for dilatation. Immunohistochemical analyses of focal adhesion kinase and rho family proteins RhoA and CDC42 revealed differences within the cartilaginous tissues in the calluses from the pseudarthrosis model as compared to fracture calluses undergoing normal endochondral bone repair. These findings suggest the involvement of these proteins in the way by which mechanical stimuli modulate the process of cartilage formation during bone healing.

Background: Heterotopic bone formation has been observed in patients with traumatic brain injury; however, an association between such an injury and enhanced fracture-healing remains unclear. To test the hypothesis that traumatic brain injury causes a systemic response that enhances fracture-healing, we established a reproducible model of traumatic brain injury in association with a standard closed fracture and measured the osteogenic response with an in vitro cell assay and assessed bone-healing with biomechanical testing. Methods: A standard closed femoral fracture was produced in forty-three Sprague-Dawley rats. Twenty-three of the rats were subjected to additional closed head trauma that produced diffuse axonal injury similar to that observed in patients with a traumatic brain injury. Twenty-one days after the procedure, all animals were killed and fracture-healing was assessed by measuring callus size and by mechanical testing. Sera from the animals were used in subsequent in vitro experiments to measure mitogenic effects on established cell lines of committed osteoblasts, fibroblasts, and mesenchymal stem cells. Results: Biomechanical assessment demonstrated that the brain-injury group had increased stiffness (p = 0.02) compared with the fracture-only group. There was no significant difference in torsional strength between the two groups. Cell culture studies showed a significant increase in the proliferative response of mesenchymal stem cells after exposure to sera from the brain-injury group compared with the response after exposure to sera from the fracture-only group (p = 0.0002). This effect was not observed in fibroblasts or committed osteoblasts. Conclusions: These results support data from previous studies that have suggested an increased osteogenic potential and an enhancement of fracture-healing secondary to traumatic brain injury. Our results further suggest that the mechanism for this enhancement is related to the presence of factors in the serum that have a mitogenic effect on undifferentiated mesenchymal stem cells. Clinical Relevance: Fracture-healing may be enhanced by an associated traumatic brain injury. Further understanding of this systemic response could lead to important insights about systemic therapeutic strategies for the enhancement of skeletal repair.

Recombinant human parathyroid hormone (PTH [1-34]; teriparatide) is a new treatment for postmenopausal osteoporosis that can be systemically administered for the primary purpose of increasing bone formation. Because several studies have described the enhancement of fracture-healing and osteointegration in animals after use of PTH, we sought to critically analyze this skeletal effect.Two hundred and seventy male Sprague-Dawley rats underwent standard, closed femoral fractures and were divided into three groups that were administered daily subcutaneous injections of 5 or 30 mug/kg of PTH (1-34) or vehicle (control). The dosing was administered for up to thirty-five days. Groups were further divided into three subgroups and were killed on day 21, 35, or 84 after the fracture. The bones were subjected to mechanical torsion testing, histomorphometric analysis, or microquantitative computed tomography.By day 21, calluses from the group treated with 30 mug of PTH showed significant increases over the controls with respect to torsional strength, stiffness, bone mineral content, bone mineral density, and cartilage volume. By day 35, both groups treated with PTH showed significant increases in bone mineral content and density and total osseous tissue volume, and they demonstrated significant decreases in void space and cartilage volume (p0.05). Torsional strength was significantly increased at this time-point in the group treated with 30 mug of PTH (p0.05). While dosing was discontinued on day 35, analyses performed after eighty-four days in the group treated with 30 mug of PTH showed sustained increases over the controls with respect to torsional strength and bone mineral density. No change was noted in osteoclast density at the time-points measured, suggesting that treatment with PTH enhanced bone formation but did not induce bone resorption.These data show that daily systemic administration of PTH (1-34) enhances fracture-healing by increasing bone mineral content and density and strength, and it produces a sustained anabolic effect throughout the remodeling phase of fracture-healing.

Pleiotrophin (Ptn) is an extracellular matrix protein that regulates hippocampal synaptic plasticity and learning behavior in vivo. Since the overexpression of Ptn in transgenic mice leads to increased bone formation, we analyzed whether a deficiency in Ptn expression would have a negative effect on bone remodeling. Bones from Ptn-deficient mice and wild-type littermates were analyzed using radiography, muCT imaging and undecalcified histology. Biomechanical stability was determined in a three-point-bending assay. Cellular activities were assessed using dynamic histomorphometry and the determination of urinary collagen degradation products. Skeletons of Ptn-deficient mice have no gross abnormalities, displayed a normal size, and showed no differences in growth plate organization compared to wild-type littermates. There were no obvious differences in bone mass as determined by radiographic and muCT imaging. The absence of a bone remodeling phenotype in Ptn-deficient mice was further confirmed using static histomorphometry and biomechanical testing. Finally, the number, morphology, and function of osteoclasts, osteoblasts, and osteocytes were not altered in Ptn-deficient mice compared to wild-type littermates. The complete skeletal analysis of Ptn-deficient mice presented here demonstrates that the lack of Ptn in mice does not affect bone formation in vivo. Therefore, Ptn does not play a significant role in normal bone physiology.

Ischemic injury to the immature femoral head produces epiphyseal cartilage damage and cessation of endochondral ossification. This study suggests that VEGF facilitates the repair of the necrotic epiphyseal cartilage, which is essential for restoration of endochondral ossification and re-establishment of the growth of the immature femoral head after ischemic necrosis. Introduction: Legg-Calve-Perthes disease (LCPD) is a childhood form of osteonecrosis that produces growth arrest of the secondary center of ossification. The cessation of growth is caused by ischemic damage to the hypertrophic zone of the epiphyseal cartilage where endochondral ossification normally occurs. The role of vascular endothelial growth factor (VEGF) in restoring endochondral ossification in the epiphyseal cartilage after ischemic necrosis was investigated in a piglet model of LCPD because the resumption of normal growth is important for maintaining the spherical shape of the femoral head. Materials and Methods: Piglet femoral heads were assessed 24 h to 8 weeks after the surgical induction of ischemia. Western blot analysis, ribonuclease protection assay (RPA), immunohistochemistry, and in situ hybridization were performed. Results: Western blot analysis and RPA showed increased VEGF protein and mRNA expression, respectively, in the epiphyseal cartilage of the infarcted heads compared with the contralateral normal heads. In the normal femoral heads, VEGF-immunoreactivity (VEGF-IR) and transcripts were observed in the hypertrophic zone of the epiphyseal cartilage. In the infarcted heads, VEGF-IR and transcripts were no longer observed in the hypertrophic zone because of diffuse cell death in that zone from ischemia. However, VEGF-IR and transcripts were observed in the proliferative zone above the necrotic hypertrophic zone. At 8 weeks, vascular granulation tissue invasion of the necrotic hypertrophic zone was observed with active resorption of the necrotic cartilage. In some areas where the necrotic cartilage was completely resorbed, restoration of endochondral ossification was observed. In these areas, VEGF transcripts were observed in the newly formed hypertrophic zone. Conclusions: VEGF expression was increased, and its spatial expression was altered in the epiphyseal cartilage after ischemic necrosis of the immature femoral head. VEGF upregulation in the proliferative zone after ischemic damage may play a role in stimulating vascular invasion and granulation tissue formation in the necrotic hypertrophic zone of the epiphyseal cartilage. This may be an important step toward facilitating the resorption of the necrotic cartilage and restoration of endochondral ossification leading to further growth and development of the femoral head.

Distraction osteogenesis (DO) is one of the most dramatic in vivo applications of mechanical stimulation as a means of inducing bone regeneration. A simple and reproducible murine model of tibia distraction osteogenesis was developed using a monolateral fixator. Bone formation was assessed histologically over a 35-day time course. The steady state expression of a broad family of angiogenesis-associated genes was assessed by microarray hybridization analyses over the same time course, while the immediate gene response that was induced during each cycle of distraction was assessed at 30 min and 8 h after the first and last rounds of activation of the fixator. Distraction osteogenesis promoted new bone formation primarily through an intramembranous process with maximal osteogenesis during the active distraction period. Histological analysis also showed that dense cortical bone continued to be formed, during the consolidation phase, for 2 weeks after distraction ended. The analysis of steady state mRNA expression levels over the time course of DO showed that VEGF-A and neuropilin, an alternate receptor for VEGF-A, both angiopoietin (Ang) 1 and 2 factors, and the Ang receptor Tie2 were the critical angiogenic factors during DO. A key transcriptional regulator of many of the angiogenic factors, hypoxia-induced factor1alpha (Hif-1a), the FGF binding protein pleiotropin/OSF1, and multiple MMP(s), were also induced during the active distraction period. Examination of the expression of angiogenic factors that were induced after each cycle of activation, demonstrated that Hif-1a, Nrp1, and VEGF-A were all cyclically induced after each increment of distraction. These results suggest that these factors are early mediators that are produced by distraction and contribute toward the processes that promote bone formation. These experiments represent the first step in defining the molecular mechanisms that regulate skeletal regeneration and the functional relationship between angiogenesis and osteogenesis during distraction osteogenesis.

The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation. An interesting phenomenon was that BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media. In summary, the murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media. Our preliminary results suggest that the induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism.

Distraction osteogenesis has proven to be of great value for the treatment of a variety of musculoskeletal problems. Little is still known, however, about the phenotypic changes in the cells participating in the bone formation process, induced by the procedure. Recent findings of the expression of a contractile muscle actin isoform, α-smooth muscle actin (SMA), in musculoskeletal connective tissue cells prompted this immunohistochemical study of the expression of SMA in cells participating in distraction osteogenesis in a rat model. The tissues within and adjacent to the distraction site could be distinguished histologically on the basis of cell morphology, density, and extracellular matrix make-up. The percentage of SMA-containing cells within each tissue zone was graded from 0 to 4. The majority of the cells in each of the zones stained positive for SMA within five days of the distraction period. The SMA-containing cells included those with elongated morphology in the center of the distraction site and the active osteoblasts on the surfaces of the newly forming bone. These finding warrant further investigation of the role of this contractile actin isoform in distraction osteogenesis and investigation of the effects of modulation of this actin isoform on the procedure.

The role of alpha-smooth muscle actin (SMA)-expressing fibroblasts in the contraction of skin wounds has been known for three decades. Recent studies have demonstrated that osteoblasts can also express the gene for this contractile muscle actin isoform and can contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. These findings provided rationale for the hypothesis that SMA-expressing cells contribute to fracture healing by drawing the bone ends together. To begin to test this hypothesis, immunohistochemistry was employed to evaluate the distribution of connective tissue cells expressing SMA in a mouse model of successful fracture healing. The results demonstrated that the majority of the cells comprising the mesenchymal tissue interposed between the fracture ends contained SMA after 7 and 21 days, supporting the working hypothesis. Most of the osteoblasts lining the surfaces of newly forming bone and the chondrocytes comprising the cartilaginous callus also expressed this contractile actin isoform. The maximal SMA expression extended from 7 to 21 days postfracture. The finding of high levels of SMA expression in connective tissue cells participating in fracture healing suggests that SMA-enabled contraction may be playing a role in the healing process. These results warrant further study of the specific SMA-dependent cell behavior.

During endochondral bone development cartilage formation always precedes that of bone, leading to the hypothesis that chondrocytes provide inductive signals for osteogenesis. To test this hypothesis, C3H10T1/2 mesenchymal stem cells were cocultured in membrane separated trans-well culture chambers with nonhypertrophic chondrocytes, hypertrophic chondrocytes, calvaria osteoblasts, or tendon fibroblasts derived from embryonic chickens to assess if individual cell types would selectively promote osteogenic differentiation. Then, differentiation of C3H10T1/2 mesenchymal stem cells in coculture were compared with that induced by bone morphogenetic protein 7 or osteogenic protein-1 (BMP-7; OP-1) treatment. Osteogenesis, as determined by the expression of Cbfa1 and osteocalcin (OC) messenger RNAs (mRNAs), was induced strongly in C3H10T1/2 cells cocultured with both chondrocyte cell populations but was not induced by coculture with either osteoblasts or skin fibroblasts. Interestingly, treatment of C3H10T1/2 cells with BMP-7 induced both chondrogenesis and osteogenesis, and only osteogenic differentiation was observed in the C3H10T1/2 cells cocultured with chondrocytes. No alterations in the expression of mRNAs for BMP-1 to -8 were observed in the C3H10T1/2 cells under any of the coculture conditions. This shows that the induction of endogenous BMPs by coculture does not regulate osteogenesis in an autocrine manner. These results show that chondrocytes express soluble morphogenetic factors that selectively promote osteogenesis, and this selective effect is not mimicked by an exogenously added BMP.

Fractures are the most common large-organ, traumatic injuries to humans. The repair of bone fractures is a postnatal regenerative process that recapitulates many of the ontological events of embryonic skeletal development. Although fracture repair usually restores the damaged skeletal organ to its pre-injury cellular composition, structure and biomechanical function, about 10% of fractures will not heal normally. This article reviews the developmental progression of fracture healing at the tissue, cellular and molecular levels. Innate and adaptive immune processes are discussed as a component of the injury response, as are environmental factors, such as the extent of injury to the bone and surrounding tissue, fixation and the contribution of vascular tissues. We also present strategies for fracture treatment that have been tested in animal models and in clinical trials or case series. The biophysical and biological basis of the molecular actions of various therapeutic approaches, including recombinant human bone morphogenetic proteins and parathyroid hormone therapy, are also discussed.

The expression of neuropilin-1 (NRP1), a recently described VEGF and semaphorin receptor expressed by endothelial cells (EC) but some non-EC types as well, was analyzed in osteoblasts in vitro and in vivo. Cultured MC3T3-E1 osteoblasts expressed NRP1 mRNA and bound VEGF165 but not VEGF121, characteristic of the VEGF isoform-specific binding of NRP1. These cells did not express VEGFR-1 or VEGFR-2 so that VEGF binding to osteoblasts was strictly NRP1-dependent. In a chick osteocyte differentiation system, NRP1 was expressed by osteoblasts but its expression was absent as the cells matured into osteocytes. Immunohistochemical localization of NRP1 within the developing bones of 36-day-old mice and embryonic Day 17 chicks demonstrated that NRP1 was expressed by osteoblasts migrating alongside invading blood vessels within the metaphysis of the growth plate, as well as by osteoblasts at the developing edge of trabeculae within the marrow cavity. On the other hand, NRP1 was not expressed by osteocytes in either species, consistent with the in vitro results. In addition to osteogenic cells, NRP1 expression by EC was observed throughout the bone. Together these results suggest that NRP1 might have a dual function in bone by mediating osteoblast function directly as well as angiogenesis. J. Cell. Biochem. 81:82–92, 2001. © 2001 Wiley-Liss, Inc.