Expression and role of laminin-1 in mouse pancreatic organogenesis.

Previous studies have suggested that basement membrane alone may induce ductal differentiation and morphogenesis in the undifferentiated embryonic pancreas. The mechanism by which this induction occurs has not been investigated. Studies of other organ systems such as the lungs and mammary glands, where differentiation has been shown to be induced by basement membrane, have suggested a major role for laminin as a mediator of ductal or tubular morphogenesis and differentiation. We first defined the ontogeny of laminin-1 in the developing mouse pancreas. To determine the specific role of basement membrane laminin in pancreatic ductal morphogenesis and differentiation, we microdissected 11-day mouse embryonic pancreatic epithelium free from its surrounding mesenchyme and then suspended the explants in a 3-dimensional organ culture to allow us to assay cell differentiation and morphogenesis. When the pancreatic epithelium buds off the foregut endoderm, the pancreatic mesenchyme diffusely expresses laminin-1. This laminin subsequently organizes to the interface between the epithelium and the mesenchyme by E12.5. As gestation progresses, epithelial cells in direct contact with laminin-1 seem to differentiate into ducts and acini, whereas those spared intimate contact with laminin-1 appeared to organize into islets. Although basement membrane gel could induce pancreatic ductal morphogenesis of embryonic pancreatic epithelium, this induction was blocked when we added neutralizing antibodies against any of the following: 1) laminin (specifically laminin-1), 2) the "cross-region" of laminin-1, and 3) the alpha6 moiety of the integrin receptor, which is known to bind laminins. Immunohistochemistry, however, showed that pancreatic duct cell-specific differentiation (carbonic anhydrase II) without ductal morphogenesis was still present, despite the blockage of duct morphogenesis by the anti-laminin-1 neutralizing antibodies. Interestingly, there appeared to be a decrease in carbonic anhydrase II expression over time when the epithelia were grown in a collagen gel, rather than in a basement membrane gel. The pattern of laminin-1 expression in the embryonic pancreas supports the conclusion that laminin-1 is important in the induction of exocrine (ducts and acini) differentiation in the pancreas. Furthermore, our data demonstrate that 1) pancreatic ductal morphogenesis appears to require basement membrane laminin-1 and an alpha6-containing integrin receptor; 2) the cross-region of basement membrane laminin is a biologically active locus of the laminin molecule necessary for pancreatic ductal morphogenesis; 3) duct-specific cytodifferentiation, in the form of carbonic anhydrase II expression, is not necessarily coupled to duct morphogenesis; and 4) the basement membrane gel may contain components (e.g., growth factors) other than laminin-1 that can sustain both carbonic anhydrase II expression and, possibly, the capacity to form ducts, despite the absence of duct structures.

Adenovirus-mediated gene therapy of osteoblasts in vitro and in vivo.

Modulation of biological pathways governing osteogenesis may accelerate osseous regeneration and reduce the incidence of complications associated with fracture healing. Transforming growth factor beta1 (TGF-beta1) is a potent growth factor implicated in the regulation of osteogenesis and fracture repair. The use of recombinant proteins, however, has significant disadvantages and has limited the clinical utility of these molecules. Targeted gene therapy using adenovirus vectors is a technique that may circumvent difficulties associated with growth factor delivery. In this study, we investigate the efficacy of replication-deficient adenoviruses containing the human TGF-beta1 and the bacterial lacZ genes in transfecting osteoblasts in vitro and osseous tissues in vivo. We demonstrate that adenovirus-mediated gene therapy efficiently transfects osteoblasts in vitro with the TGF-beta1 virus causing a marked up-regulation in TGF-beta1 mRNA expression even 7 days after transfection. Increased TGF-beta1 mRNA expression was efficiently translated into protein production and resulted in approximately a 46-fold increase in TGF-beta1 synthesis as compared with control cells (vehicle- or B-galactosidase-transfected). Moreover, virally produced TGF-beta1 was functionally active and regulated the expression of collagen IalphaI (5-fold increase) and the vascular endothelial growth factor (2.5-fold increase). Using an adenovirus vector encoding the Escherichia coli LacZ gene, we demonstrated that adenovirus-mediated gene transfer efficiently transfects osteoblasts and osteocytes in vivo and that transfection can be performed by a simple percutaneous injection. Finally, we show that delivery of the hTGF-beta1 gene to osseous tissues in vivo results in significant changes in the epiphyseal plate primarily as a result of increased thickness of the provisional calcification zone.

Mechanisms of fibroblast growth factor-2 modulation of vascular endothelial growth factor expression by osteoblastic cells.

Normal bone growth and repair is dependent on angiogenesis. Fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGFbeta) have all been implicated in the related processes of angiogenesis, growth, development, and repair. The purpose of this study was to investigate the relationships between FGF-2 and both VEGF and TGFbeta in nonimmortalized and clonal osteoblastic cells. Northern blot analysis revealed 6-fold peak increases in VEGF mRNA at 6 h in fetal rat calvarial cells and MC3T3-E1 osteoblastic cells after stimulation with FGF-2. Actinomycin D inhibited these increases in VEGF mRNA, whereas cycloheximide did not. The stability ofVEGF mRNA was not increased after FGF-2 treatment. Furthermore, FGF-2 induced dose-dependent increases in VEGF protein levels (P < 0.01). Although in MC3T3-E1 cells, TGFbeta1 stimulates a 6-fold peak increase in VEGF mRNA after 3 h of stimulation, we found that both TGFbeta2 and TGFbeta3 yielded 2- to 3-fold peak increases in VEGF mRNA levels noted after 6 h of stimulation. Similarly, both TGFbeta2 and TGFbeta3 dose dependently increased VEGF protein production. To determine whether FGF-2-induced increases in VEGF mRNA may have occurred independently of TGFbeta, we disrupted TGFbeta signal transduction (using adenovirus encoding a truncated form of TGFbeta receptor II), which attenuated TGFbeta1 induction of VEGF mRNA, but did not impede FGF-2 induction ofVEGF mRNA. In summary, FGF-2-induced VEGF expression by osteoblastic cells is a dose-dependent event that may be independent of concomitant FGF-2-induced modulation of TGFbeta activity.

Minimally invasive repair of pectus excavatum: a single institution's experience.

BACKGROUND: The Nuss repair of pectus excavatum is a relatively new, minimally invasive surgical (MIS) alternative to the traditional open "Ravitch-type" operation. We have one of the larger single-center experiences to date, and we conducted this clinical study to evaluate our early experience, emphasizing initial outcome and technical modifications designed to minimize complications. METHODS: A retrospective chart review was performed on 112 patients who underwent 116 pectus excavatum repairs between January 1995 and January 2001. The Nuss procedure was performed in 80 patients, and open repair was performed in 32 patients. Information about demographics, deformity, operative course, complications, and early outcome was recorded. RESULTS: Operative duration was 143 minutes for the open group and 53 minutes for the Nuss MIS group (P <.001). Blood loss was 6 mL/kg for the open group and 0.5 mL/kg for the MIS group (P <.001). Postoperative hospitalization was 3.2 days for the open group versus 3.7 days for the MIS group (P<.05). CONCLUSIONS: The MIS pectus repair can be performed safely with minimal blood loss and reduced operative time. Short-term analysis of the quality of repair, including absence of preoperative symptoms, patient satisfaction, and cosmetic appearance are encouraging.

Glans approximation procedure urethroplasty for the wide, deep meatus.

OBJECTIVES: To assess the reliability, cosmesis, and complication rate of the glans approximation procedure (GAP). METHODS: We reviewed 37 consecutive GAP urethroplasties performed at the Children's Mercy Hospital in Kansas City, Missouri over a 5-year period, performed by three different pediatric surgeons. All patients selected had a large, deep ventral groove, typically with a wide open urethral meatus. RESULTS: The mean age was 18 months, with a mean follow-up of 28 months. Of the 37, there was one urethrocutaneous fistula that has since been easily repaired with good results. The parents have been very pleased with the results in all cases, with a straight and strong urinary stream. There was one episode of transient erythema, possibly representing an infection, which resolved after 3 days of oral antibiotics. CONCLUSIONS: The GAP is technically easy, reliable, and offers a relatively low complication rate for the repair of a very select group of patients with hypospadias with a deep ventral glanular groove and wide-mouthed meatus.

Basement membrane exposure defines a critical window of competence for pancreatic duct differentiation from undifferentiated pancreatic precursor cells.

We previously showed that the undifferentiated pancreatic epithelium can differentiate into islets, ducts, or acini depending on its milieu and that laminin is necessary for pancreatic duct formation. Therefore we wanted to study the plasticity of laminin-induced duct differentiation the better to understand mechanisms of pancreatic duct lineage selection induced by basement membrane. Mouse embryonic pancreases were dissected at gestational day 11 (E11.5), and epithelium was isolated from its surrounding mesenchyme. Some epithelia were cultured in a collagen gel devoid of laminin. These epithelia were "rescued" at days 1-7 of culture by transferring them to a laminin-rich matrix (Matrigel) for 7 additional days. Other epithelia were instead first cultured in Matrigel, and then placed into collagen. Immunohistochemistry was performed for insulin, amylase, and carbonic anhydrase II. Pancreatic epithelia rescued from collagen into laminin during days 1-4 after harvest were still able to form ducts, whereas epithelia deprived of laminin for longer than this 4-day window were not. Pancreatic epithelia exposed to laminin for as little as 1 day, and then placed into collagen, still retained the ability to make ducts. Thus there is a clear cut-off in the development of the pancreatic epithelium at E11.5, after which laminin appears necessary to induce duct formation. We believe that such "windows of competence" in embryonic development imply that developmental programs in the embryo allow some flexibility.

Ontogeny of activin B and follistatin in developing embryonic mouse pancreas: implications for lineage selection.

Activin, a member of the transforming growth factor-beta superfamily, has been shown to be a critical regulator in exocrine and endocrine pancreas formation. The purpose of our study was to describe the ontogeny of activin B and its inhibitor, follistatin, in developing pancreas and to elucidate potential mechanisms for exocrine and endocrine lineage selection. Mouse embryonic pancreata were dissected at various ages (day 10 [E10.5] to birth [E18.5]), sectioned, and immunostained for activin B (one of two existing isomers, A and B), follistatin, insulin, and glucagon. In addition, reverse transcriptase-polymerase chain reaction was employed to determine the messenger RNA expression of follistatin in isolated pancreatic epithelia and mesenchyme of various ages. Activin B was first detected at E12.5 in epithelial cells coexpressing glucagon. At E16.5 these coexpressors appeared as clusters in close proximity to early ducts. By E18.5 activin B was localized to forming islets where cells coexpressed glucagon and were arranged in the mantle formation characteristic of mature alpha cells. Follistatin was found to be ubiquitous in pancreatic mesenchyme at early ages by immunohistochemical analysis, disappearing sometime after E12.5. Follistatin reappeared in E18.5 islets and remains expressed in adult islets. Follistatin messenger RNA was first detected in epithelium at E11.5, preceding its protein expression in islets later in gestation. We propose that mesenchyme-derived follistatin inhibits epithelium-derived activin at early embryonic ages allowing for unopposed exocrine differentiation and relative suppression of endocrine differentiation. At later ages the decrease in the amount of mesenchyme relative to epithelium and the subsequent drop in follistatin levels liberates epithelial activin to allow differentiation of endocrine cells to form mature islets by the time of birth.

Initiation of gastrin expression during the development of the mouse pancreas.

Gastrin expression occurs in the pancreas in only two situations: (1) in cases of gastrinoma and (2) in the embryonic/fetal pancreas. The initiation of gastrin expression in the embryonic pancreas may be recapitulated during gastrinoma tumorigenesis. For this reason, we have tried to identify the point of onset of gastrin expression in the developing pancreas. Previously, determining the point of onset for genes in embryonic tissues has been difficult because of low expression levels and small tissue samples. Using the sensitive polymerase chain reaction assay, we were able to determine, with a sensitivity of 10 molecules of mRNA, the earliest expression of gastrin in the developing pancreas. This expression occurred at 30 somites, or at a gestation of 9.5 to 10 days.

Improvement in survival of mice with proximal small bowel obstruction treated with octreotide.

Small bowel obstruction is a common disorder in surgical practice. The major morbidity of bowel obstruction relates to intestinal distension and ischemia. We hypothesized that octreotide, a potent inhibitor of gut secretion, would reduce mortality in a mouse model of lethal small bowel obstruction. C57 mice were anesthetized with urethane and prepared with either proximal jejunal or distal ileal obstruction. After 8 hours, surviving mice were randomized to receive either octreotide (100 micrograms/kg) or saline subcutaneously every 8 hours. Octreotide significantly improved survival in mice with proximal obstruction by life table analysis. Mean survival increased from 31 +/- 3 to 41 +/- 4 hours. In distal obstruction, octreotide treatment resulted in a trend towards improved survival; however, this trend failed to reach statistical significance by life table analysis. The improvement in survival in this mouse model suggests that octreotide may be a valuable adjunct in the treatment of patients with small bowel obstruction.

Molecular approaches to understanding organogenesis.

The elucidation of the molecular mechanisms of mammalian organogenesis is the foundation on which we can build an improved understanding of organ pathology and pathophysiology. This paper uses the lung and the pancreas as paradigms to demonstrate how advances in basic molecular developmental biology research has translated into new appreciation of, and even novel potential treatment strategies for, congenital anomalies and mature diseases of these organs.

Fetal wound repair: where do we go from here?

In contrast to adult wound healing, early-gestation fetal skin wound healing occurs rapidly, in a regenerative fashion, and without scar formation. The accelerated rate of healing, relative lack of an acute inflammatory response, and an absence of neovascularization distinguishes fetal from adult wound healing. However, this remarkable ability of the fetus to heal without scarring still remains poorly understood. The uncertainties include the role of cytokines, extracellular matrix components, homeobox genes, and certain cell types in the scarless wound repair process. Nevertheless, some strides have been made within the last two decades. This report, discusses the current knowledge of the mechanisms and characteristics of scarless fetal wound healing. Furthermore, to shy away from being just another all inclusive review, the authors point out deficiencies in the knowledge base on this important topic. Last, the future direction of research is discussed that may elucidate the mechanisms regulating the scarless repair phenomena.

Scarless healing. The fetal wound.

Fetal wounds heal without a scar early in gestation, and may hold the key to scarless repair. Several important concepts central to the fetal wound-healing response have been determined. The fetal fibroblast modulates the wound-healing response through collagen deposition, extracellular matrix deposition, and growth factor secretion. Fetal repair is both gestational-age and wound-size dependent, with a transition from scarless to scarring repair occurring during fetal life. Fetal fibroblasts manifest a decreased ability to induce dermal appendage formation from fetal epithelium at the same time that scarring in the fetus begins, suggesting that epithelial-mesenchymal interactions play an important role in scarless fetal repair. The fetal immune response during wound healing differs from the adult response, with a primarily mononuclear cell infiltrate and decreased activity and presence of polymorphonuclear leukocytes, whereas the cytokine profile of the fetal wound differs markedly from that of the adult wound. Patterning genes (homeobox genes) involved in organogenesis may prove integral to fetal healing, and are emerging as an active area of research. Once the biology of fetal wound healing is fully determined, attempts to manipulate the adult wound undoubtedly will progress rapidly, and scarless repair may become a clinical reality in children and adults.

Immunolocalization of basic fibroblast growth factor and fibroblast growth factor receptor-1 and receptor-2 in rat cranial sutures.

Craniosynostosis is a common disorder with an unknown etiology. Recent genetic mapping studies have demonstrated a strong linkage between several familial craniosynostotic syndromes and mutations in fibroblast growth factor receptor 1 (FGF-R1) and 2 (FGF-R2). The purpose of this experiment was to investigate by immunohistochemistry the protein production of these receptors as well as of their most prevalent ligand, basic fibroblast growth factor (bFGF), before, during, and after sutural fusion in rat cranial sutures. The posterior frontal (normally fuses between postnatal days 12 and 22) and sagittal (remains patent) sutures of embryonic day 20 and neonatal days 6, 12, 17, 22, and 62 (n = 3 per group) were harvested, fixed, and decalcified. Five-micrometer sections were stained with polyclonal antibodies against bFGF, FGF-R1, and FGF-R2, and patterns of immunohistochemical staining were assessed by independent reviewers. Our results indicate that increased bFGF production correlates temporally with suture fusion, with increased staining of the dura underneath the fusing suture prior to fusion followed by increased staining within osteoblasts and sutural cells during fusion. FGF-R1 and, to a lesser extent FGF-R2 immunostaining revealed a different pattern of localization with increased immunostaining within the patent sagittal suture at these time points. These results implicate bFGF in the regulation of sutural fusion and may imply autoregulatory mechanisms in fibroblast growth factor receptor expression.

Rat mandibular distraction osteogenesis: Part I. Histologic and radiographic analysis.

The application of distraction osteogenesis to craniofacial surgery has altered the approach and treatment of congenital and acquired craniofacial defects. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been described extensively, relatively little is known about the molecular regulation of this process. The elucidation of the molecular mechanisms of distraction osteogenesis has important clinical implications because it may facilitate the use of recombinant proteins or gene therapy to accelerate bone regeneration. Molecular analysis of distraction osteogenesis has been hindered by the use of large animal models in which only limited genetic information is available. In this study, a rat model of mandibular distraction osteogenesis is described. This report includes a pilot study (n = 50) to develop an appropriate distraction device and to determine the optimal placement of the osteotomy. The study subsequently included 80 animals, 35 of which were distracted at a rate of 0.25 mm per day for 6 days (1.5 mm total) and 35 that were distracted at a rate of 0.25 mm twice per day (3.0 mm total). These animals were killed at various time points (after latency and during the distraction and consolidation periods) and displayed histologic and radiographic findings of membranous bone distraction osteogenesis that were consistent with those in large ,animal and clinical models. In addition, five animals each were acutely lengthened 1.5 mm and 3.0 mm and demonstrated a fibrous nonunion. Furthermore, the utility of this model is demonstrated in the analysis of the molecular mechanisms of distraction osteogenesis by applying the polymerase chain reaction to total cellular RNA isolated from normal and distracted rat mandibles. In conclusion, it is believed that the rat model of distraction osteogenesis has significant advantages over traditional models, including decreased costs and facilitation of molecular analysis.

Gene expression of TGF-beta, TGF-beta receptor, and extracellular matrix proteins during membranous bone healing in rats.

Poorly healing mandibular fractures and osteotomies can be troublesome complications of craniomaxillofacial trauma and reconstructive surgery. Gene therapy may offer ways of enhancing bone formation by altering the expression of desired growth factors and extracellular matrix molecules. The elucidation of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth factors during normal (i.e., successful) fracture repair. Transforming growth factor beta1 (TGF-beta1), its receptor (Tbeta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thought to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 24 adult rats underwent mandibular osteotomy with rigid external fixation. In addition, four identically treated rats that underwent sham operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 37 days after the procedure) to examine gene expression of TGF-beta1 and Tbeta-RII, osteocalcin, and type I collagen. Northern blot analysis was used to compare gene expression of these molecules in experimental animals with that in control animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta1 and T-RII proteins were immunolocalized in an additional group of nine animals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta1 expression 7 days postoperatively; TGF-beta1 expression returned thereafter to near baseline levels. Tbeta-RII mRNA expression was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mRNA expression was dramatically downregulated shortly after osteotomy and remained low during the early phases of fracture repair. Osteocalcin expression trended slowly upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type IalphaI mRNA expression was acutely downregulated shortly after osteotomy, peaked on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-beta1 staining was predominantly localized to the osteotomized bone edges, periosteum, surrounding soft tissues, and residual inflammatory cells. By postoperative day 37, complete bony healing was observed, and TGF-beta1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, Tbeta-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, Tbeta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the osteotomized bone edges. On postoperative day 37, Tbeta-RII protein was seen in osteocytes, osteoblasts, and the newly formed periosteum in the remodeling bone. These observations agree with those of previous in vivo studies of endochondral bone formation, growth, and healing. In addition, these results implicate TGF-beta1 biological activity in the regulation of osteoblast migration, differentiation, and proliferation during mandibular fracture repair. Furthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical cases.

Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro.

Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.

The effects of ionizing radiation on osteoblast-like cells in vitro.

The well-described detrimental effects of ionizing radiation on the regeneration of bone within a fracture site include decreased osteocyte number, suppressed osteoblast activity, and diminished vascularity. However, the biologic mechanisms underlying osteoradionecrosis and the impaired fracture healing of irradiated bone remain undefined. Ionizing radiation may decrease successful osseous repair by altering cytokine expression profiles resulting from or leading to a change in the osteoblastic differentiation state. These changes may, in turn, cause alterations in osteoblast proliferation and extracellular matrix formation. The purpose of this study was to investigate the effects of ionizing radiation on the proliferation, maturation, and cytokine production of MC3T3-E1 osteoblast-like cells in vitro. Specifically, the authors examined the effects of varying doses of ionizing radiation (0, 40, 400, and 800 cGy) on the expression of transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and alkaline phosphatase. In addition, the authors studied the effects of ionizing radiation on MC3T3-E1 cellular proliferation and the ability of conditioned media obtained from control and irradiated cells to regulate the proliferation of bovine aortic endothelial cells. Finally, the authors evaluated the effects of adenovirus-mediated TGF-beta1 gene therapy in an effort to "rescue" irradiated osteoblasts. The exposure of osteoblast-like cells to ionizing radiation resulted in dose-dependent decreases in cellular proliferation and promoted cellular differentiation (i.e., increased alkaline phosphatase production). Additionally, ionizing radiation caused dose-dependent decreases in total TGF-beta1 and VEGF protein production. Decreases in total TGF-beta1 production were due to a decrease in TGF-beta1 production per cell. In contrast, decreased total VEGF production was secondary to decreases in cellular proliferation, because the cellular production of VEGF by irradiated osteoblasts was moderately increased when VEGF production was corrected for cell number. Additionally, in contrast to control cells (i.e., nonirradiated), conditioned media obtained from irradiated osteoblasts failed to stimulate the proliferation of bovine aortic endothelial cells. Finally, transfection of control and irradiated cells with a replication-deficient TGF-beta1 adenovirus before irradiation resulted in an increase in cellular production of TGF-beta1 protein and VEGF. Interestingly, this intervention did not alter the effects of irradiation on cellular proliferation, which implies that alterations in TGF-beta1 expression do not underlie the deficiencies noted in cellular proliferation. The authors hypothesize that ionizing radiation-induced alterations in the cytokine profiles and differentiation states of osteoblasts may provide insights into the cellular mechanisms underlying osteoradionecrosis and impaired fracture healing.

Immature versus mature dura mater: II. Differential expression of genes important to calvarial reossification.

The ability of immature animals and newborns to orchestrate successful calvarial reossification is well described. This capacity is markedly attenuated in mature animals and in humans greater than 2 years of age. Previous studies have implicated the dura mater as critical to successful calvarial reossification. The authors have previously reported that immature, but not mature, dural tissues are capable of elaborating a high expression of osteogenic growth factors and extracellular matrix molecules. These findings led to the hypothesis that a differential expression of osteogenic growth factors and extracellular matrix molecules by immature and mature dural tissues may be responsible for the clinically observed phenotypes (i.e., immature animals reossify calvarial defects; mature animals do not). This study continues to explore the hypothesis through an analysis of transforming growth factor (TGF)-beta3, collagen type III, and alkaline phosphatase mRNA expression. Northern blot analysis of total RNA isolated from freshly harvested immature (n = 60) and mature (n = 10) dural tissues demonstrated a greater than three-fold, 18-fold, and nine-fold increase in TGF-beta3, collagen type III, and alkaline phosphatase mRNA expression, respectively, in immature dural tissues as compared with mature dural tissues. Additionally, dural cell cultures derived from immature (n = 60) and mature dura mater (n = 10) were stained for alkaline phosphatase activity to identify the presence of osteoblast-like cells. Alkaline phosphatase staining of immature dural cells revealed a significant increase in the number of alkaline phosphatase-positive cells as compared with mature dural tissues (p < 0.001). In addition to providing osteogenic humoral factors (i.e., growth factors and extracellular matrix molecules), this finding suggests that immature, but not mature, dura mater may provide cellular elements (i.e., osteoblasts) that augment successful calvarial reossification. These studies support the hypothesis that elaboration of osteogenic growth factors (i.e., TGF-beta33) and extracellular matrix molecules (i.e., collagen type III and alkaline phosphatase) by immature, but not mature, dural tissues may be critical for successful calvarial reossification. In addition, these studies suggest for the first time that immature dural tissues may provide cellular elements (i.e., osteoblasts) to augment this process.

Differential expression of receptor tyrosine kinases and Shc in fetal and adult rat fibroblasts: toward defining scarless versus scarring fibroblast phenotypes.

The remarkable ability of the fetus to heal early gestation skin wounds without scarring remains poorly understood. Taking advantage of recent advances in signal transduction, the tyrosine phosphorylation patterns of fetal rat fibroblasts, representing the scarless cutaneous repair phenotype, and adult rat fibroblasts, representing scarforming phenotype, were examined whether there were inherent differences in cellular signaling. Specifically, correlation of the phosphorylation patterns with the expression levels of the signaling molecules that transmit information from the plasma membrane receptor to the nucleus was sought. By using three different cell lines of explanted fibroblasts from gestational day 13 fetal rat skin (n = 24) and 1-month-old postnatal adult rat skin (n = 3), immunoblotting was performed to compare tyrosine phosphorylation patterns. The results revealed five major protein bands of interest in fetal rat fibroblasts, but not in the adult rat fibroblasts. These phosphorylated protein bands are of interest because of their possible role in wound repair and may have the potential to regulate cellular responses to the extracellular matrix and their secondary signaling molecules. It was hypothesized that these bands represented receptor tyrosine kinases, epidermal growth factor receptor, and discoidin domain receptor 1, and their downstream adaptor protein Shc that binds receptor tyrosine kinases to transduce signals intracellularly. Furthermore, elevated expression of platelet-derived growth factor receptor-beta in adult compared with fetal fibroblasts was demonstrated, suggesting that decreased expression of certain growth factors may also be important for the scarless phenomenon to occur.

Transforming growth factor beta superfamily members: role in cartilage modeling.

Normal and abnormal extracellular matrix turnover is thought to result, in part, from the balance in the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). The clinical manifestations of an imbalance in these relationships are evident in a variety of pathologic states, including osteoarthritis, deficient long-bone growth, rheumatoid arthritis, tumor invasion, and inadequate cartilage repair. Articular cartilage defects commonly heal as fibrocartilage, which is structurally inferior to the normal hyaline architecture of articular cartilage. Transforming growth factor-beta 1 (TGF-beta1), a cytokine central to growth, repair, and inflammation, has been shown to upregulate TIMP-1 expression in human and bovine articular cartilage. Additionally, members of the TGF-beta superfamily are thought to play key roles in chondrocyte growth and differentiation. Bone morphogenetic protein-2 (BMP-2), a member of this superfamily, has been shown to regulate chondrocyte differentiation states and extracellular matrix composition. It was proposed that, by optimizing extracellular matrix composition, BMP-2 would enhance articular cartilage healing. After determining the release kinetics of BMP-2 from a collagen type I implant (Long-Evans male rats; two implants/rat, n = 14), it was found that, in a tissue engineering application, BMP-2 induced a hyaline-like repair of New Zealand White rabbit knee articular cartilage defects (3-mm full-thickness defects in the femoral trochlea; 2 defects/rabbit, n = 36). The quality of cartilage repair with BMP-2 (with or without chondrocytes) was significantly better than defects treated with BMP-2, as assessed by a quantitative scoring scale. Immunohistochemical staining revealed TIMP-1 production in the cartilage defects treated with BMP-2. When studied in vitro, it was found that BMP-2 markedly increased TIMP-1 mRNA by both bovine articular and human rib chondrocytes. Additionally, increased TIMP-1 mRNA was translated into increased TIMP-1 protein production by bovine chondrocytes. Taken together, these data suggest that BMP-2 may be a useful cytokine to improve healing of cartilaginous defects. Furthermore, these data suggest that the beneficial effects of BMP-2 may be, in part, related to alterations in extracellular matrix turnover.