Faculty Bibliography

Neoangiogenesis is essential for successful wound repair. Platelets are among the earliest cells recruited to a site of skeletal injury and are thought to provide numerous factors critical to successful repair. The release of platelet-derived growth factor (PDGF) after skeletal injury increases osteoblast proliferation, chemotaxis, and collagen synthesis; however, its angiogenic effect on osteoblast biology remains unknown. The purpose of this study was to investigate the effect of recombinant human (rh)PDGF-BB on the synthesis of vascular endothelial growth factor (VEGF) by primary neonatal rat calvarial osteoblasts. Furthermore, the authors investigated whether PDGF works in concert with hypoxia, another component of the fracture microenvironment, to additively or synergistically induce VEGF production. Osteoblast cultures were stimulated with varying concentrations of rhPDGF-BB (1, 10, 50, and 100 ng/ml) in normoxic and hypoxic (<1% oxygen) conditions for 0, 3, 6, 12, and 24 hours, and VEGF gene expression was analyzed by Northern blot analysis. To determine whether rhPDGF-BB-induced VEGF messenger RNA (mRNA) expression was transcriptionally mediated or required de novo protein synthesis, transcription, and translation, studies were performed using actinomycin D and cycloheximide, respectively. Treatment with 50 ng/ml rhPDGF-BB resulted in a 2.4-fold increase in VEGF mRNA expression after 3 hours. Interestingly, rhPDGF-BB and hypoxia seemed to have an additive effect, resulting in a 3.7-fold increase in VEGF mRNA expression after 6 hours in primary neonatal rat calvarial osteoblasts. Furthermore, by using actinomycin D and cycloheximide, the authors demonstrated that the rhPDGF-BB-induced VEGF mRNA expression was transcriptionally mediate and not dependent on de novo protein synthesis. These data demonstrate that rhPDGF-BB transcriptionally increases osteoblasts VEGF mRNA expression in vitro. Furthermore, the semiquantitative results suggest that rhPDGF-BB and hypoxia act additively to increase VEGF mRNA expression. It is postulated that similar mechanisms may occur in vivo, at a site of skeletal injury, to induce neoangiogenesis and promote fracture repair

Distraction osteogenesis has become a mainstay in bone tissue engineering and has significantly improved our armamentarium for reconstructive craniomaxillofacial procedures. However, although the biomechanical, histological, and ultrastructural changes associated with distraction osteogenesis have been widely described, the molecular mechanisms governing the formation of new bone in the interfragmental gap of gradually distracted bone segments remain largely unclear. Recently, a rat model of mandibular distraction was described that provides an excellent environment for deciphering the molecular mechanisms that mediate distraction osteogenesis. This article presents the hypotheses and current research that have furthered knowledge of the molecular mechanisms that govern distraction osteogenesis.Recent studies have implicated a growing number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (transforming growth factor-beta1, -beta2, -beta3, bone morphogenetic proteins, insulin-like growth factor-1, fibroblast growth factor-2) and extracellular matrix proteins (osteonectin, osteopontin) during distraction osteogenesis have been best characterized and are discussed in this article. It is believed that understanding the biomolecular mechanisms that mediate membranous distraction osteogenesis may guide the development of targeted strategies designed to improve distraction osteogenesis and accelerate bone healing

The endothelium is a metabolically active secretory tissue, capable of responding to a wide array of environmental stimuli. Hypoxia and vascular endothelial growth factor (VEGF) are two components of the putative fracture microenvironment. This study investigated the role of hypoxia and VEGF on endothelial cell activation as it relates to the bone repair process. It was hypothesized that endothelial cells may have an important osteogenic role in fracture healing through the production of bone morphogenetic protein-2 (BMP-2), an osteogenic cytokine at the fracture site. Therefore, BMP-2 mRNA and protein expression in endothelial cells under hypoxia and/or VEGF treatment was studied. The authors observed a 2-fold to 3-fold up-regulation of BMP-2 mRNA expression in bovine capillary endothelial cells and human microvascular endothelial cells stimulated with hypoxia or rhVEGF. Furthermore, the combined effects of hypoxia and rhVEGF appeared to be additive on BMP-2 mRNA expression in bovine capillary endothelial cells. Actinomycin D and cycloheximide studies suggested that the increased mRNA expression was transcriptionally regulated. BMP-2 protein expression was up-regulated after 24 and 48 hours of treatment with either hypoxia or rhVEGF in bovine capillary endothelial cells. Surprisingly, the data suggest that endothelial cells may play not only an angiogenic role but also an osteogenic role by a direct stimulation of the osteoblasts, through the enhanced expression of a potent osteogenic factor, BMP-2, at the fracture site

Popularized by Gavril Ilizarov in the 1960s, monofocal distraction osteogenesis has become a well-established method of endogenous bone engineering. This revolutionary surgical technique has significantly augmented the available reconstructive orthopedic and craniomaxillofacial procedures. Bifocal distraction osteogenesis, or bone transportation, is a modification of monofocal distraction that involves moving a free segment of living bone to fill an intercalary bone defect. Bifocal distraction has been applied successfully to reconstruct complex mandibular and long bone defects. Because traumatic or postsurgical calvarial defects do not spontaneously heal in humans older than 18 to 24 months of age, we hypothesized that bifocal distraction osteogenesis could be applied to the skull to close critical size calvarial defects. Critical size (15 x 15 mm) calvarial defects were created in eight New Zealand White rabbits. Next, a 15-mm x 10-mm calvarial box osteotomy was created just anterior to the skull defect. This osteotomy created a free bone segment that could be transported. A custom-made transport distraction device was fixed into place and the skin incision was closed. After a 4-day latency period, the distraction device was activated (0.5 mm once daily for 30 days) in seven animals; the distraction device in one animal was not activated and served as a control. All animals underwent 30 days of consolidation and were then killed. Radiographs and computed tomographic scans were performed at the following time points: end of latency period (postoperative day 4), mid-distraction (postoperative day 19), and end of consolidation period (postoperative day 64). Gross and histologic analysis was performed to evaluate the quality of the bony regenerate. The control animal healed with a fibrous union. Complete closure of the skull defects was observed in five of seven rabbits at the end of the consolidation period. One animal was removed from the study because of an early loosening of the distraction device, and one was removed because of device failure. Of the remaining five animals that completed the distraction protocol, radiographs and computerized tomographic scans showed successful ossification in all five rabbits at the end of the consolidation period. This study suggests that transport distraction osteogenesis is a promising technique that may be applied to a variety of commonly encountered craniofacial problems such as nonhealing calvarial defects

For decades surgeons have exploited the ability of infants to reossify large calvarial defects. To demonstrate the role of dura mater-osteoblast communication during the process of calvarial reossification, the authors used a novel in vitro system that recapitulates the in vivo anatomic relationship of these cell populations. Primary cultures of osteoblast cells from 2-day-old Sprague-Dawley rat pups were grown on six-well plates, and cultures of immature, non-suture-associated dura mater cells from 6-day-old Sprague-Dawley rat pups were grown on Transwell inserts. When the osteoblast and dura mater cell cultures reached confluence, they were combined. This Transwell co-culture system permitted the two cell populations to grow together in the same well, but it prevented direct cell-to-cell contact. Therefore, the authors were able to determine, for the first time, whether paracrine signaling from immature, non-suture-associated dura mater could influence the biologic activity of osteoblasts.Osteoblasts co-cultured with dural cells proliferated significantly faster after 2 days (2.1 x 10(5) +/- 2.4 x 10(4) versus 1.4 x 10(5) +/- 2.2 x 10(4), p < or = 0.05) and 4 days (3.1 x 10(5) +/- 5 x 10(4) versus 2.2 x 10(5) +/- 4.0 x 10(4), p < or = 0.01) than did osteoblasts cultured alone. After 20 days, co-cultured osteoblasts expressed greater amounts of mRNA for several markers of osteoblast differentiation, including collagen I alpha I (4-fold), alkaline phosphatase (2.5-fold), osteopontin (3-fold), and osteocalcin (4-fold), than did osteoblasts cultured alone. After 30 days, co-cultured osteoblasts produced bone nodules that were significantly greater both in number (324 +/- 29 nodules versus 252 +/- 29 nodules per well, p , < or = 0.04) and total area of nodules (65 +/- 11 mm(2) versus 24 +/- 1.6 mm(2), p < or = 0.003) than osteoblasts cultured alone.To begin to understand how dural cells effect changes in osteoblast gene expression, the authors compared the expression of candidate genes, transforming growth factor beta 1 and fibroblast growth factor 2, in dural cells and osteoblasts before and after 5 days of culture. Interestingly, the dura mater produced marked amounts of these osteogenic cytokines compared with osteoblasts.The described co-culture system demonstrated that co-cultured osteoblasts proliferated more rapidly and experienced an increased rate and degree of cellular maturation than did osteoblasts cultured alone. The authors hypothesize that this effect was due to paracrine signaling (e.g., transforming growth factor beta 1 and fibroblast growth factor 2) from the dura mater, and they are investigating those mechanisms in ongoing experiments. Collectively these data verify that immature, non-suture-associated dura mater can influence the biologic activity of osteoblasts. Moreover, the production of cytokines derived from the dura mater (e.g., transforming growth factor beta 1 and fibroblast growth factor 2), and they may begin to explain why immature animals and infants with intact dura mater can reossify large calvarial defects

The transforming growth factor beta (TGF-beta) superfamily encompasses a number of important growth factors including several TGF-beta isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-beta 1, -beta 2, and -beta 3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-beta isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-beta isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-beta 1 and type I collagen mRNA transcription. To assess the effects of extended TGF-beta treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-beta isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-beta isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-beta isoform treatment increased osteopontin expression in primary osteoblasts after 4 and 10 days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-beta isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-beta isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation

Distraction osteogenesis is a well-established technique of endogenous tissue engineering. The biomechanical factors thought to affect the quality of the distraction regenerate include the latency, rate, rhythm, and consolidation period. In an effort to understand the impact of these parameters on regenerate bone formation, this study was designed to decipher the most adaptive response in a rat model of mandibular distraction osteogenesis. Ninety-six adult Sprague-Dawley rats were divided into 16 subgroups (n = 6 per subgroup) based on variations in the distraction parameters (i.e., latency, rate, and rhythm). After a 28-day consolidation period, the mandibles were harvested, decalcified, and sectioned. A standardized histologic ranking system was used to evaluate the effect of each protocol on the adaptive response of the regenerate bone. In this study, we have demonstrated that the latency period dramatically affects the success of distraction osteogenesis. Furthermore, distraction rates up to 0.50 mm per day stimulated excellent regenerate bone formation, whereas greater distraction rates produced a fibrous union. Finally, higher frequency distraction (i.e., increased rhythm) appeared to accelerate regenerate bone formation. We believe that defining the critical parameters of this model will improve future analysis of gene expression during rat mandibular distraction osteogenesis and may facilitate the development of biologically based strategies designed to enhance regenerate bone formation

The purpose of this study was to establish a novel mouse model of membranous osteotomy healing. By applying this model to transgenic mice or using in situ hybridization techniques, we can subsequently investigate candidate genes that are believed to be important in membranous osteotomy healing. In the current study, 20 adult male CD-1 mice underwent a full-thickness osteotomy between the second and third molars of the right hemimandible using a 3-mm diamond disc and copious irrigation. Compo-Post pins were secured into the mandible, 2 mm anterior and posterior to the osteotomy. After the soft tissues were reapproximated and the skin was closed, an acrylic external fixator was attached to the exposed posts for stabilization. The animals were killed on postoperative day number 7, 10, 14, and 28 (n=5 animals per time point). The right hemimandibles were decalcified and embedded in paraffin for histologic evaluation or immunohistochemistry localizing osteocalcin. At 7 days after the osteotomy, early intramembranous bone formation could be seen extending from either edge of the osteotomized bone. By 10 days, an increasing number of small blood vessels could be seen within and around the osteotomy. At 14 days, the bone edges were in close approximation, and by 28 days the callus had been replaced by actively remodeling woven bone in all specimens examined. Immunohistochemistry demonstrated that osteocalcin expression correlated temporally with the transition from a soft to a hard callus. Furthermore, osteocalcin was spatially confined to osteoblasts actively laying down new osteoid or remodeling bone. This study describes a novel mouse model of membranous osteotomy healing that can be used as a paradigm for future osteotomy healing studies investigating candidate genes critical for osteogenesis and successful bone repair

It is unclear whether cleft palate formation is attributable to intrinsic biomolecular defects in the embryonic elevating palatal shelves or to an inability of the shelves to overcome a mechanical obstruction (such as the tongue in Pierre Robin sequence) to normal fusion. Regardless of the specific mechanism, presumably embryonic palatal shelves are ultimately unable to bridge a critical distance and remain unapproximated, resulting in a clefting defect at birth. We propose to use a palate organ culture system to determine the critical distance beyond which embryonic palatal shelves fail to fuse (i.e., the minimal critical intershelf distance). In doing so, we hope to establish an in vitro cleft palate model that could then be used to investigate the contributions of various signaling pathways to cleft formation and to study novel in utero treatment strategies.Palatal shelves from CD-1 mouse embryos were microdissected on day 13.5 of gestation (E13.5; term = 19.5 days), before fusion. Using a standardized microscope ocular grid, paired palatal shelves were placed on a filter insert at precisely graded distances ranging from 0 (in contact) to 1.9 mm (0, 0.095, 0.19, 0.26, 0.38, 0.48, 0.57, 0.76, 0.95, and 1.9 mm). A total of 68 paired palatal shelves were placed in serum-free organ culture for 96 hours (n = 68). Sample sizes of 10 were used for each intershelf distance up to and including 0.48 mm (n = 60). For intershelf distances of 0.57 mm and greater, two-paired palatal shelves were cultured (n = 8). All specimens were assessed grossly and histologically for palatal fusion.Palatal fusion occurred in our model only when intershelf distances were 0.38 mm or less. At 0.38 mm, eight of 10 palates appeared grossly adherent, whereas six of 10 demonstrated clear fusion histologically with resolution of the medial epithelial seam and continuity of the palatal mesenchyme. None of the 18 palates fused when placed at intershelf distances of 0.48 mm or greater.Using our selected intershelf distances as a guideline, we have established an approximate minimal critical intershelf distance (0.48 mm) at which we can reliably expect no palatal fusion. Culturing palatal shelves at intershelf distances of 0.48 mm or greater results in nonfusion or clefting in vitro. This model will allow us to study biomolecular characteristics of unfused or cleft palatal shelves in comparison with fused shelves. Furthermore, we plan to study the efficacy of grafting with exogenous embryonic mesenchyme or candidate factors to overcome clefting in vitro as a first step toward future in utero treatment strategies