Refining retinoic acid stimulation for osteogenic differentiation of murine adipose-derived adult stromal cells.

Murine adipose-derived adult stromal cells (ADAS) seeded onto appropriate scaffolds and pre-incubated with retinoic acid have been shown to generate in vivo bone rapidly. Prompt resorption ensues, however, as a result of osteoclastogenesis, likely secondary to retinoic acid carryover. In this study, we determined the effects of abbreviated retinoic acid exposure on ADAS osteogenic differentiation. Histological staining and gene expression analysis revealed that longer retinoic acid exposure resulted in better in vitro bone differentiation. However, significant osteogenesis was observed in ADAS after just 15 days of retinoic acid supplementation, suggesting that continual culture with retinoic acid is unnecessary for initiation of the osteogenic program. This was confirmed using ADAS pre-incubated in monolayer with an abbreviated 15 days of retinoic acid exposure before implantation into critical-sized calvarial defects. Similar rates of regeneration were observed between ADAS exposed to for 15 days or for a full 25-day course of retinoic acid before defect repair. Furthermore, by limiting retinoic acid exposure to ADAS in monolayer without scaffold, accelerated bone formation was observed without concomitant osteoclastic resorption. These data suggest that skeletal regeneration may be improved by modulating retinoic acid exposure before implantation, markedly accelerating the repair of bone defects using ADAS.

Differential gene expression between juvenile and adult dura mater: a window into what genes play a role in the regeneration of membranous bone.

BACKGROUND: Although reossification of large calvarial defects is possible in children, adults lack this tissue engineering capacity. In this study, the authors compared the differences in gene expression between juvenile and adult dura mater using a mouse cDNA microarray with 42,000 unique elements. METHODS: Non-suture-associated parietal bone was harvested from 6-day-old and 60-day-old mice. The dura mater was carefully dissected from the calvarial disk and snap-frozen. RNA was extracted from pooled dura mater for microarray analysis. The 25 most differentially expressed genes were listed, as were selected bone-related genes. In addition, quantitative real-time reverse-transcriptase polymerase chain reaction confirmation of selected genes-BMP-2, BMP-4, and BMP-7; and osteopontin (OP), osteocalcin (OC), and FGFR-1-was performed. RESULTS: Juvenile dura mater expressed significantly greater amounts of BMP-2 and OP. Minimal difference in OC expression was observed between juvenile and adult dura mater. Extracellular matrix proteins (Col3a1, 5a1, 6a1, and fibronectin 1), osteoblast differentiation markers (Runx2/Cbfa1, Itm2a, and FGFR-1), and the growth factor Ptn were among other genes with greater expression in juvenile dura mater. Markers of osteoclasts (Acp5, MMP9, Ctsk) and the multiple candidate gene Ntrk2 were also expressed at higher levels in the juvenile dura mater. CONCLUSIONS: These findings suggest a more differentiated osteoprogenitor population to exist along with a greater presence of osteoclasts in the juvenile dura mater relative to adults. In addition to establishing a baseline difference in gene expression between juvenile and adult dura mater, new genes potentially critical to the regenerative potential of juvenile calvaria were identified.

Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling.

Although the multilineage potential of human adipose-derived adult stromal cells (ADAS) has been well described, few published studies have investigated the biological and molecular mechanisms underlying osteogenic differentiation of mouse ADAS. We report here that significant osteogenesis, as determined by gene expression and histological analysis, is induced only when mouse ADAS are cultured in the presence of retinoic acid with or without recombinant human bone morphogenetic protein (BMP)-2 supplementation. Furthermore, a dynamic expression profile for the BMP receptor (BMPR) isoform IB was observed, with dramatic up-regulation during osteogenesis. Western blot analysis revealed that retinoic acid enhanced levels of BMPR-IB protein during the first 7 days of osteogenic differentiation and that RNAi-mediated suppression of BMPR-IB dramatically impaired the ability of ADAS to form bone in vitro. In contrast, absence of BMPR-IA did not significantly diminish ADAS osteogenesis. Our data therefore demonstrate that the osteogenic commitment of multipotent mouse ADAS requires retinoic acid, which enhances expression of the critical BMPR-IB isoform.

Connective tissue growth factor-specific monoclonal antibody therapy inhibits pancreatic tumor growth and metastasis.

Pancreatic cancer is highly aggressive and refractory to most existing therapies. Past studies have shown that connective tissue growth factor (CTGF) expression is elevated in human pancreatic adenocarcinomas and some pancreatic cancer cell lines. To address whether and how CTGF influences tumor growth, we generated pancreatic tumor cell lines that overexpress different levels of human CTGF. The effect of CTGF overexpression on cell proliferation was measured in vitro in monolayer culture, suspension culture, or soft agar, and in vivo in tumor xenografts. Although there was no effect of CTGF expression on proliferation in two-dimensional cultures, anchorage-independent growth (AIG) was enhanced. The capacity of CTGF to enhance AIG in vitro was linked to enhanced pancreatic tumor growth in vivo when these cells were implanted s.c. in nude mice. Administration of a neutralizing CTGF-specific monoclonal antibody, FG-3019, had no effect on monolayer cell proliferation, but blocked AIG in soft agar. Consistent with this observation, anti-CTGF treatment of mice bearing established CTGF-expressing tumors abrogated CTGF-dependent tumor growth and inhibited lymph node metastases without any toxicity observed in normal tissue. Together, these studies implicate CTGF as a new target in pancreatic cancer and suggest that inhibition of CTGF with a human monoclonal antibody may control primary and metastatic tumor growth.

Guided tissue regeneration enhances bone formation in a rat model of failed osteogenesis.

BACKGROUND: Guided tissue regeneration is a technique that uses barrier materials to enhance tissue regeneration. Although previously demonstrated to be an effective way of enhancing craniofacial osteogenesis in several animal models, the ability of guided tissue regeneration to augment bone formation in the context of distraction osteogenesis is unknown. In the current study, the authors applied the principle of guided tissue regeneration to their rat mandibular distraction osteogenesis model in an attempt to enhance bone regeneration. METHODS: Twelve (n = 6 per group) adult Sprague-Dawley rats underwent routine gradual distraction (5 days' latency, 4-mm distraction over 8 days, 4 to 6 weeks of consolidation) and acute distraction (immediate lengthening to 4 mm, 6 to 8 weeks of consolidation). An additional 10 animals underwent acute distraction followed by application of bioabsorbable Gore Resolut XT membranes (acute distraction plus guided tissue regeneration). Membranes were completely wrapped around the distraction gap. Animals were killed 6 and 8 weeks postoperatively and mandibles analyzed radiographically and histologically. RESULTS: Quantitative histomorphometric analyses were performed to compare relative bone formation between all three groups. Gradual distraction mandibles achieved bony union by 6 weeks with 86 percent bone formation, which increased to 98 percent by 8 weeks. Acute distraction mandibles healed with a fibrous nonunion and only 37 percent bone formation by 8 weeks. In contrast, acute distraction plus guided tissue regeneration-treated mandibles formed significantly more bone than acute distraction mandibles by 6 weeks (57 percent) and achieved bony bridging by 8 weeks, with 88 percent new bone formation. CONCLUSION: The authors' data demonstrate that guided tissue regeneration can significantly enhance bone formation in a fibrous nonunion model of mandibular distraction osteogenesis.

Craniofacial bone tissue engineering.

Repair and reconstruction of the craniofacial skeleton represents a significant biomedical burden, with thousands of procedures per-formed annually secondary to injuries and congenital malformations. Given the multitude of current approaches, the need for more effective strategies to repair these bone deficits is apparent. This article explores two major modalities for craniofacial bone tissue engineering: distraction osteogenesis and cellular based therapies. Current understanding of the guiding principles for each of these modalities is elaborated on along with the knowledge gained from clinical and investigative studies. By laying this foundation, future directions for craniofacial distraction and cell-based bone engineering have emerged with great promise for the advancement of clinical practice.

The osteogenic potential of adipose-derived mesenchymal cells is maintained with aging.

BACKGROUND: Adipose-derived mesenchymal cells are multipotent progenitor cells derived from the vascular-stromal compartment of adipose tissue. Although we have recently shown that these cells, from both juvenile and adult animals, are capable of forming bone in vivo, a detailed examination of the differences in the biology of these two populations (and in particular their ability to form bone) has not been performed. METHODS: Adipose-derived mesenchymal cells were harvested from juvenile (6-day-old) and adult (60-day-old) mice. Differences in cellular attachment, proliferation, and proliferating cell nuclear antigen production were assessed. The ability of cells to undergo adipogenic differentiation was determined by Oil Red O staining. Early osteogenic differentiation was determined with alkaline phosphatase staining, and terminal differentiation with von Kossa staining as well as determination of extracellular matrix calcium content. All experiments were performed in triplicate. RESULTS: Greater attachment, proliferation, and proliferating cell nuclear antigen production were seen in juvenile as compared with adult adipose-derived mesenchymal cells. The juvenile cells underwent significantly greater adipogenic differentiation than did adult cells (p < 0.001). Interestingly, the adult cells were capable of robust early and terminal osteogenic differentiation, with levels of all three osteo-genic assays being similar to those seen in juvenile cells. Differences were not statistically significant. CONCLUSIONS: Although biologic differences exist between adipose-derived mesenchymal cells from juveniles and adults, the osteogenic capacity of these cells appears to be minimally affected by donor age. This suggests that these cells may be a particularly useful cellular resource in the design of cell-based therapies for skeletal regeneration in an aging population.

Expression and possible mechanisms of regulation of BMP3 in rat cranial sutures.

BACKGROUND: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial sutures and the factors regulating its expression in vitro. METHODS: Microarray analysis was performed on rat posterior frontal and sagittal cranial sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonsuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: BMP3 expression in the posterior frontal suture decreased over the time course analyzed, whereas it increased in the sagittal suture. Notably, BMP3 expression was higher in the patent sagittal suture during the window of posterior frontal suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). CONCLUSIONS: BMP3 is expressed in rat cranial sutures in a temporal pattern suggesting involvement in cranial suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial suture biology.

Microarray analysis of mechanical shear effects on flexor tendon cells.

BACKGROUND: Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. The authors hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. METHODS: To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. RESULTS: Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, the authors also found up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with the authors' microarray data. CONCLUSIONS: The authors demonstrate an overall antifibrotic expression pattern in response to shear stress in tendon cells that may provide insight into the mechanisms by which early mobilization decreases adhesion formation without impaired tendon healing.

Differential transcriptional expression profiles of juvenile and adult calvarial bone.

BACKGROUND: It has widely been observed that young children are capable of reossifying large calvarial defects, while adults lack this endogenous tissue-engineering capacity. The ability of juvenile animals to regenerate calvarial defects has been investigated in multiple animal models, including mice. In this study, the authors used cDNA microarrays to investigate the expression of osteogenesis-associated genes upstream and downstream of Runx2 in juvenile and adult mouse calvaria. METHODS: Nonsuture-associated parietal bone discs were harvested from 6-day-old (n = 50) and 60-day-old (n = 35) male CD-1 mice. After separation of the underlying dura mater and overlying pericranium, the calvarial discs were snap-frozen and RNA was extracted from pooled samples of calvaria for microarray analysis. Genes analyzed included cytokines, receptors, and cell-surface and matrix proteins both upstream and downstream of Runx2. RESULTS: Genes associated with the Runx2 pathway had notably higher levels in the juvenile versus adult calvaria. All genes except for osteocalcin were expressed at least twofold higher in the juvenile calvaria. This pattern was validated with quantitative real-time polymerase chain reaction. In addition, mRNA for potent osteoinductive growth factors was present at higher levels in the juvenile compared with the adult calvaria. CONCLUSIONS: These findings reflect a genomic environment of active osteoblast differentiation and ossification in the juvenile calvaria compared with the adult "quiescent" calvarial tissue. These data suggest that a decreased osteogenic potential of adult calvarial osteoblasts may, in part, explain the inability of adult animals to heal calvarial defects.

Angiogenesis is required for successful bone induction during distraction osteogenesis.

UNLABELLED: The role of angiogenesis during mechanically induced bone formation is incompletely understood. The relationship between the mechanical environment, angiogenesis, and bone formation was determined in a rat distraction osteogenesis model. Disruption of either the mechanical environment or endothelial cell proliferation blocked angiogenesis and bone formation. This study further defines the role of the mechanical environment and angiogenesis during distraction osteogenesis. INTRODUCTION: Whereas successful fracture repair requires a coordinated and complex transcriptional program that integrates mechanotransductive signaling, angiogenesis, and osteogenesis, the interdependence of these processes is not fully understood. In this study, we use a system of bony regeneration known as mandibular distraction osteogenesis (DO) in which a controlled mechanical stimulus promotes bone induction after an osteotomy and gradual separation of the osteotomy edges to examine the relationship between the mechanical environment, angiogenesis, and osteogenesis. MATERIALS AND METHODS: Adult Sprague-Dawley rats were treated with gradual distraction, gradual distraction plus the angiogenic inhibitor TNP-470, or acute distraction (a model of failed bony regeneration). Animals were killed at the end of distraction (day 13) or at the end of consolidation (day 41) and examined with muCT, histology, and immunohistochemistry for angiogenesis and bone formation (n = 4 per time-point per group). An additional group of animals (n = 6 per time-point per group) was processed for microarray analysis at days 5, 9, 13, 21, and 41. RESULTS AND CONCLUSIONS: Either TNP-470 administration or disruption of the mechanical environment prevented normal osteogenesis and resulted in a fibrous nonunion. Subsequent analysis of the regenerate showed an absence of angiogenesis by gross histology and immunohistochemical localization of platelet endothelial cell adhesion molecule in the groups that failed to heal. Microarray analysis revealed distinct patterns of expression of genes associated with osteogenesis, angiogenesis, and hypoxia in each of the three groups. Our findings confirm the interdependence of the mechanical environment, angiogenesis, and osteogenesis during DO, and suggest that induction of proangiogenic genes and the proper mechanical environment are both necessary to support new vasculature for bone induction in DO.

Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover.

Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.

FGF-2 acts through an ERK1/2 intracellular pathway to affect osteoblast differentiation.

An abundance of genetic and experimental data have suggested that fibroblast growth factor (FGF) signaling plays a central role in physiological and pathological cranial suture fusion. Although alterations in the differentiation and proliferation of sutural osteoblasts may be a key mediator of this process, the mechanisms by which FGF signaling regulates osteoblast differentiation remain incompletely understood. In the current study, the authors show that recombinant human FGF-2 alters osteoblastic expression of bone morphogenetic protein-2 and Msx-2 in vitro to favor cellular differentiation and osteoinduction. The ERK1/2 intracellular signaling cascade was shown to be necessary for recombinant human FGF-2-mediated bone morphogenetic protein-2 transcriptional changes. Furthermore, the cellular production of an intermediate transcriptional modifier was found to be necessary for the recombinant human FGF-2-mediated gene expression changes in bone morphogenetic protein-2 and Msx-2. Together, these findings offer new insight into the mechanisms by which FGF-2 modulates osteoblast biology.

New developments in pediatric plastic surgery research.

Pediatric plastic surgery research is a rapidly expanding field. Unique in many ways, researchers in this field stand at the union of multiple scientific specialties, including biomedical engineering, tissue engineering, polymer science, molecular biology, developmental biology, and genetics. The goal of this scientific effort is to translate research advances into improved treatments for children with congenital and acquired defects. Although the last decade has seen a dramatic acceleration in research related to pediatric plastic surgery, the next 10 years will no doubt lead to novel treatment strategies with improved clinical outcomes.

Age-dependent residual tensile strains are present in the dura mater of rats.

The objectives of this study were to determine whether residual tensile strains exist in the dura mater of mammals in vivo, and whether the strains are age-dependent. We made incisions in the parietal dura mater of immature and mature rats, and measured the retraction of the dura mater from each incision. We then used a finite-element model to calculate the strain present in the parietal dura mater of each rat. We found that age-dependent residual tensile strains are present in the dura mater of rats. The mean average residual strain of the immature rats was significantly larger than that of the mature rats (4.96+/-1.54% (s.d.) versus 0.39+/-0.13%, p<0.0001), with the mean strain calculated in the mature rats of the order of the minimum measurement that could be made using our experimental approach. In addition, in the immature rats mean residual strain in the longitudinal direction was significantly larger than mean residual strain in the transverse direction (6.11+/-3.62% versus 3.82+/-2.64%, p=0.0218). Our findings show that age-dependent residual tensile strains exist in the dura mater of rats. We speculate that these strains may reflect the rate and direction of cranial growth and may also influence cranial healing.

High-dose retinoic acid modulates rat calvarial osteoblast biology.

Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 microM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 microM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis.

Quantitative transcriptional analysis of fusing and nonfusing cranial suture complexes in mice.

Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior frontal and sagittal suture complexes (including the associated dura mater, suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior frontal suture as compared with the sagittal suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior frontal suture in mice.

Applications of a mouse model of calvarial healing: differences in regenerative abilities of juveniles and adults.

Young children are capable of healing large calvarial defects, whereas adults lack this endogenous osseous tissue-engineering capacity. Despite the important clinical implications, little is known about the molecular and cell biology underlying this differential ability. Traditionally, guinea pig, rabbit, and rat models have been used to study the orchestration of calvarial healing. To harness the research potential of knockout and transgenic mice, the authors developed a mouse model for calvarial healing. Nonsuture-associated parietal defects 3, 4, and 5 mm in diameter were made in both juvenile (6-day-old, n = 15) and adult (60-day-old, n = 15) mice. Calvariae were harvested after 8 weeks and analyzed radiographically and histologically. Percentage of healing was quantified using Scion Image software analysis of calvarial radiographs. A significant difference in the ability to heal calvarial defects was seen between 6-day-old and 60-day-old mice when 3-, 4-, or 5-mm defects were created. The authors' analysis revealed that juvenile mice healed a significantly greater percentage of their calvarial defects than adult mice (juvenile mean percentage of healing: 3-mm defects, 59 percent; 4-mm defects, 65 percent; 5-mm defects, 44 percent; adult mean percentage of healing: <5 percent in all groups; p < 0.05). All three defect sizes were found to be critical in the adult, whereas significant healing was seen regardless of the size of the defect in juvenile mice. The establishment of this model will facilitate further, detailed evaluation of the molecular biology underlying the different regenerative abilities of juvenile versus adult mice and enhance research into membranous bone induction by making available powerful tools such as knockout and transgenic animals.

Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts.

Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.