Adipose tissue grafting for management of persistent anastomotic leak after low anterior resection.

BACKGROUND: An anastomotic leak is the most dreaded complication after low anterior resection. Adipose tissue grafting may induce healing in a persistent anastomotic defect. The aim of the present study was to report retrospectively reviewed outcomes for a series of patients who were managed with heterotopic grafted adipose tissue to facilitate anastomotic healing. METHODS: Patients with anastomotic leakage after low anterior resection sequentially treated with grafting of adipose tissue were included in the study. All patients had pelvic radiation during treatment and had a diverting ileostomy in situ. The cohort had a persistent defect despite being treated with available modalities such as suture repair, fibrin glue, Endo-Sponge and surgical debridement. The outcomes were reviewed and reported. RESULTS: There were 11 patients (8 males and 3 females) with a median age of 54 years (range 33-72 years). Five patients experienced complete healing of the anastomotic defect with successful reversal of the diverting ileostomy. The anastomotic defect of one other patient in the series appeared to have healed and hence his diverting ileostomy was reversed. However, he presented with a recurrent leak, which ultimately necessitated an abdominoperineal resection. Another patient had a persistent defect after an attempt at adipose tissue grafting and opted to proceed with a takedown of the anastomosis. In the remaining four patients, the outcome after adipose tissue grafting remains unknown, as two patients succumbed to metastatic disease, one was lost to follow-up and the remaining patient developed a recurrence which required pelvic exenteration. Procedural associated morbidity occurred in one patient who developed fat embolism, which was treated expectantly. CONCLUSIONS: Adipose tissue grafting is safe and feasible, though its effectiveness remains uncertain. It may be useful selectively in the management of persistent anastomotic leak after radiation and low anterior resection.

Post-reconstruction dermatitis of the breast.

BACKGROUND: Approximately one-third of women diagnosed with breast cancer undergo mastectomy with subsequent implant-based or autogenous tissue-based reconstruction. Potential complications include infection, capsular contracture, and leak or rupture of implants with necessity for explantation. Skin rashes are infrequently described complications of patients who undergo mastectomy with or without reconstruction. METHODS: A retrospective analysis of breast cancer patients referred to the Dermatology Service for diagnosis and management of a rash post-mastectomy and expander or implant placement or transverse rectus abdominis myocutaneous (TRAM) flap reconstruction was performed. Parameters studied included reconstruction types, time to onset, clinical presentation, associated symptoms, results of microbiologic studies, management, and outcome. RESULTS: We describe 21 patients who developed a rash on the skin overlying a breast reconstruction. Average time to onset was 25.7 months after expander placement or TRAM flap reconstruction. Clinical presentations included macules and papules or scaly, erythematous patches and plaques. Five patients had cultures of the rash, which were all negative. Skin biopsy was relatively contraindicated in areas of skin tension, and was reserved for non-responding eruptions. Treatments included topical corticosteroids and topical antibiotics, which resulted in complete or partial responses in all patients with documented follow-ups. CONCLUSION: Our findings suggest that tension and post-surgical factors play a causal role in this hitherto undescribed entity: "post-reconstruction dermatitis of the breast." This is a manageable condition that develops weeks to years following breast reconstruction. Topical corticosteroids and antibiotics result in restoration of skin barrier integrity and decreased secondary infection.

Obesity but not high-fat diet impairs lymphatic function.

BACKGROUND/OBJECTIVES: High-fat diet (HFD)-induced obesity has significant negative effects on lymphatic function, but it remains unclear whether this is a direct effect of HFD or secondary to adipose tissue deposition. METHODS: We compared the effects of HFD on obesity-prone and obesity-resistant mice and analyzed lymphatic function in vivo and in vitro. RESULTS: Only obesity-prone mice had impaired lymphatic function, increased perilymphatic inflammation and accumulation of lipid droplets surrounding their lymphatic endothelial cells (LECs). LECs isolated from obesity-prone mice, in contrast to obesity-resistant animals, had decreased expression of VEGFR-3 and Prox1. Exposure of LECs to a long-chain free fatty acid increased cellular apoptosis and decreased VEGFR-3 expression, while inhibition of intracellular inhibitors of VEGFR-3 signaling pathways increased cellular viability. CONCLUSIONS: Collectively, our studies suggest that HFD-induced obesity decreases lymphatic function by increasing perilymphatic inflammation and altering LEC gene expression. Reversal of diminished VEGFR-3 signaling may rescue this phenotype and improve lymphatic function.

Repair of a critical size defect in the rat mandible using allogenic type I collagen.

Mandibular fractures, resulting from either trauma or reconstructive surgery, can be challenging craniofacial problems. The morbidity of failed fracture healing is significant and may require bone grafting. Donor site morbidity and finite amounts of autogenous bone are major drawbacks of autogenous bone grafting. Similarly, the use of allografts and xenografts may be associated with an increased risk of rejection, infection, and nonunion. To circumvent the limitations of bone grafting, research efforts have focused on formulating a suitable bone substitute. The purpose of our study was to evaluate the efficacy of type I collagen implants in repairing critical sized mandibular defects in rats. Twelve male Sprague-Dawley rats (200-300g) were divided equally into control and experimental groups. Full thickness, round, four millimeter in diameter defects were created in the ramus of the right mandible of all rats using an electrical burr at low speed. The defects were irrigated of all bone chips, and either filled with a precisely fitted disk of allogenic collagen type I gel (experimental animals) or left empty (control animals). Animals were killed 6 weeks after surgery and healing of the bone defects was assessed in a blinded fashion using radiologic and histologic analysis. Radiologic analysis of the control group revealed a clear circular right mandibular defect in all animals, whereas the collagen disk implant group revealed an indistinct to nonexistent right mandibular defect in all animals. Densitometric analysis revealed a significant difference between these groups (* P = 0.01). Similarly, gross analysis of control mandibles revealed a 4mm round, soft-tissue filled defect, while implanted defects demonstrated gross bone spanning the defect. Finally, histologic analysis of all control mandibles revealed clearly demarcated bony edges at the defect border with connective tissue spanning the defect. In contrast, histological analysis of all implanted mandibles revealed indistinct bony edges at the defect border with a thin layer of osteoblasts and viable bone spanning the defects. We have demonstrated the ability of type I collagen to promote healing of a membranous bony defect that would not otherwise heal at 6 weeks. The suitability of type I collagen as a carrier matrix provides ample opportunity for tissue-engineered approaches to further facilitate bony defect healing. Promoting bone formation through tissue engineering matrices offers great promise for skeletal healing and reconstruction.

Quantitative assessment of cranial defect healing and correlation with the expression of TGF-beta.

Circular parietal defects from 3 to 12 mm in diameter were made in 45 6-month old skeletally mature guinea pigs, and animals were sacrificed after survival periods of 3 days to 12 weeks. The original defect was harvested in continuity with a rim of surrounding bone and the adjacent dura and pericranium. After 12 weeks, all 3 and 5 mm defects were completely covered by a bridge of bone, while residual defects were noted within the 8 and 12 mm wounds. Percentage of new bone formation was significantly higher within 3 mm defects, than in all larger defects at each time interval from 1 week on (P < .05), reaching a mean of 93% in 3 mm defects and remaining below a mean of 31% in the remaining defect sizes. Immunolocalization demonstrated an osteogenic front in which the osteoblasts stained strongly for all isoforms of TGF-beta, with the intensity decreasing after the majority of the defects had reossified; this front was located at the advancing bone edge of the defect as well as the endocranial side adjacent to the dura. In conclusion, isoforms of TGF-beta are upregulated during a limited "window" of time corresponding to the period of calvarial reossification, and are localized to osteoblasts within an osteogenic front at the periphery and dural surfaces of the defects.

Distraction osteogenesis of the craniofacial skeleton.

Distraction osteogenesis is becoming the treatment of choice for the surgical correction of hypoplasias of the craniofacial skeleton. Its principle is based on the studies of Ilizarov, who showed that osteogenesis can be induced if bone is expanded (distracted) along its long axis at the rate of 1 mm per day. This process induces new bone formation along the vector of pull without requiring the use of a bone graft. The technique also provides the added benefit of expanding the overlying soft tissues, which are frequently deficient in these patients. This article reviews the authors' 11-year clinical and research experience with mandibular distraction osteogenesis. It highlights the indications and contraindications of the technique and emphasizes the critical role that basic science research has played in its evolution.

Hypoxia regulates osteoblast gene expression.

Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO(2) = 35-40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-beta1, -beta2, and -beta3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-beta1 gene expression within 3 h of hypoxia. Although neither TGF-beta2 nor TGF-beta3 expression was affected by hypoxia, the TGF-beta type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators.

Rat mandibular distraction osteogenesis: latency, rate, and rhythm determine the adaptive response.

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.

Expression of bone morphogenetic proteins during membranous bone healing.

For the reconstructive plastic surgeon, knowledge of the molecular biology underlying membranous fracture healing is becoming increasingly vital. Understanding the complex patterns of gene expression manifested during the course of membranous fracture repair will be crucial to designing therapies that augment poor fracture healing or that expedite normal osseous repair by strategic manipulation of the normal course of gene expression. In the current study, we present a rat model of membranous bone repair. This model has great utility because of its technical simplicity, reproducibility, and relatively low cost. Furthermore, it is a powerful tool for analysis of the molecular regulation of membranous bone repair by immunolocalization and/or in situ hybridization techniques. In this study, an osteotomy was made within the caudal half of the hemimandible, thus producing a stable bone defect without the need for external or internal fixation. The healing process was then catalogued histologically in 28 Sprague-Dawley rats that were serially killed at 1, 2, 3, 4, 5, 6, and 8 weeks after operation. Furthermore, using this novel model, we analyzed, within the context of membranous bone healing, the temporal and spatial expression patterns of several members of the bone morphogenetic protein (BMP) family, known to be critical regulators of cells of osteoblast lineage. Our data suggest that BMP-2/-4 and BMP-7, also known as osteogenic protein-1 (OP-1), are expressed by osteoblasts, osteoclasts, and other more primitive mesenchymal cells within the fracture callus during the early stages of membranous fracture healing. These proteins continue to be expressed during the process of bone remodeling, albeit less prominently. The return of BMP-2/-4 and OP-1 immunostaining to baseline intensity coincides with the histological appearance of mature lamellar bone. Taken together, these data underscore the potentially important regulatory role played by the bone morphogenetic proteins in the process of membranous bone repair.

In vivo modulation of FGF biological activity alters cranial suture fate.

Gain-of-function mutations in fibroblast growth factor receptors have been identified in numerous syndromes associated with premature cranial suture fusion. Murine models in which the posterior frontal suture undergoes programmed fusion after birth while all other sutures remain patent provide an ideal model to study the biomolecular mechanisms that govern cranial suture fusion. Using adenoviral vectors and targeted in utero injections in rats, we demonstrate that physiological posterior frontal suture fusion is inhibited using a dominant-negative fibroblast growth factor receptor-1 construct, whereas the normally patent coronal suture fuses when infected with a construct that increases basic fibroblast growth factor biological activity. Our data may facilitate the development of novel, less invasive treatment options for children with craniosynostosis.

Rat mandibular distraction osteogenesis: part III. Gradual distraction versus acute lengthening.

Distraction osteogenesis is a well-established method of endogenous tissue engineering. This technique has significantly augmented our armamentarium of reconstructive craniofacial procedures. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been extensively described, the molecular mechanisms governing successful membranous distraction remain unknown. Using an established rat model, the molecular differences between successful (i.e., osseous union with gradual distraction) and ineffective (i.e., fibrous union with acute lengthening) membranous bone lengthening was analyzed. Herein, the first insight into the molecular mechanisms of successful membranous bone distraction is provided. In addition, these data provide the foundation for future targeted therapeutic manipulations designed to improve osseous regeneration. Vertical mandibular osteotomies were created in 52 adult male Sprague-Dawley rats, and the animals were fitted with customized distraction devices. Twenty-six animals underwent immediate acute lengthening (3 mm; a length previously shown to result in fibrous union) and 26 animals were gradually distracted (after a 3-day latency period, animals were distracted 0.25 mm twice daily for 6 days; total = 3 mm). Four mandibular regenerates were harvested from each group for RNA analysis on 5, 7, 9, 23, and 37 days postoperatively (n = 40). Two mandibular regenerates were also harvested from each group and prepared for immunohistochemistry on postoperative days 5, 7, and 37 (n = 12). In addition to the 52 experimental animals, 4 control rats underwent sham operations (skin incision only) and mandibular RNA was immediately collected. Control and experimental specimens were analyzed for collagen I, osteocalcin, tissue inhibitor of metalloproteinase-1, and vascular endothelial growth factor mRNA and protein expression. In this study, marked elevation of critical extracellular matrix molecules (osteocalcin and collagen I) during the consolidation phase of gradual distraction compared with acute lengthening is demonstrated. In addition, the expression of an inhibitor of extracellular matrix turnover, tissue inhibitor of metalloproteinase-1, remained strikingly elevated in gradually distracted animals. Finally, this study demonstrated that neither gradual distraction nor acute lengthening appreciably alters vascular endothelial growth factor expression. These results suggest that gradual distraction osteogenesis promotes successful osseous bone repair by regulating the expression of bone-specific extracellular matrix molecules. In contrast, decreased production or increased turnover of bone scaffolding proteins (i.e., collagen) or regulators of mineralization (i.e., osteocalcin) may lead to fibrous union during acute lengthening.

Osteoblast expression of vascular endothelial growth factor is modulated by the extracellular microenvironment.

Angiogenesis, the formation of new blood vessels, is crucial to the process of fracture healing. Vascular disruption after osseous injury results in an acidic, hypoxic wound environment. We have previously shown that osteoblasts can produce vascular endothelial growth factor (VEGF) in response to a variety of stimuli. In this study we examined pH and lactate concentration, two components of the putative fracture extracellular microenvironment, and determined their relative contribution to regulation of rat calvarial osteoblast VEGF production under both normoxic and hypoxic conditions. Our results demonstrate that pH and lactate concentration do independently affect osteoblast VEGF mRNA and protein production. Acidic pH (7.0) significantly decreased VEGF production, under normoxic and hypoxic conditions (P < 0.05), compared with neutral pH (7.4). This decrease was primarily transcriptionally regulated, because the rate of VEGF mRNA degradation was unchanged at pH 7.0 vs. 7.4. Similarly, an elevated lactate concentration (22 mM) also depressed osteoblast elaboration of VEGF at both neutral and acidic pH (P < 0.001). Furthermore, the effects of increasing acidity and elevated lactate appeared to be additive.

Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: implications for suture fusion and patency.

Despite its prevalence, the etiopathogenesis of craniosynostosis is poorly understood. To better understand the biomolecular events that occur when normal craniofacial growth development goes awry, we must first investigate the mechanisms of normal suture fusion. Murine models in which the posterior frontal (PF) suture undergoes programmed sutural fusion shortly after birth provide an ideal model to study these mechanisms. In previous studies, our group and others have shown that sutural fate (i.e., fusion vs. patency) is regulated by the dura mater (DM) directly underlying a cranial suture. These studies have led to the hypothesis that calvarial DM is regionally differentiated and that this differentiation guides the development of the overlying suture. To test this hypothesis, we evaluated the messenger RNA (mRNA) expression of osteogenic cytokines (transforming growth factor beta1 [TGF-beta1] and TGF-beta3) and bone-associated extracellular matrix (ECM) molecules (collagen I, collagen III, osteocalcin, and alkaline phosphatase) in freshly isolated, rat dural tissues associated with the PF (programmed to fuse) or sagittal (SAG; remains patent) sutures before histological evidence of sutural fusion (postnatal day 6 [N6]). In addition, osteocalcin protein expression and cellular proliferation were localized using immunohistochemical staining and 5-bromo-2'deoxyuridine (BrdU) incorporation, respectively. We showed that the expression of osteogenic cytokines and bone-associated ECM molecules is potently up-regulated in the DM associated with the PF suture. In addition, we showed that cellular proliferation in the DM associated with the fusing PF suture is significantly less than that found in the patent SAG suture just before the initiation of sutural fusion N6. Interestingly, no differences in cellular proliferation rates were noted in younger animals (embryonic day 18 [E18] and N2). To further analyze regional differentiation of cranial suture-associated dural cells, we established dural cell cultures from fusing and patent rat cranial sutures in N6 rats and evaluated the expression of osteogenic cytokines (TGF-beta1 and fibroblast growth factor 2 [FGF-2]) and collagen I. In addition, we analyzed cellular production of proliferating cell nuclear antigen (PCNA). These studies confirmed our in vivo findings and showed that dural cell cultures derived from the fusing PF suture expressed significantly greater amounts of TGF-beta1, FGF-2, and collagen I. In addition, similar to our in vivo findings, we showed that PF suture-derived dural cells produced significantly less PCNA than SAG suture-derived dural cells. Finally, coculture of dural cells with fetal rat calvarial osteoblastic cells (FRCs) revealed a statistically significant increase in proliferation (*p < 0.001) in FRCs cocultured with SAG suture-derived dural cells as compared with FRCs cocultured alone or with PF suture-derived dural cells. Taken together, these data strongly support the hypothesis that the calvarial DM is regionally differentiated resulting in the up-regulation of osteogenic cytokines and bone ECM molecules in the dural tissues underlying fusing but not patent cranial sutures. Alterations in cytokine expression may govern osteoblastic differentiation and ECM molecule deposition, thus regulating sutural fate. Elucidation of the biomolecular events that occur before normal cranial suture fusion in the rat may increase our understanding of the events that lead to premature cranial suture fusion.

Arteriovenous malformation of a persistent median artery with a bifurcated median nerve.

A patient with arteriovenous malformations of the volar forearm and hand arising from a persistent median artery with an associated bifid median nerve is presented. Surgeons should be aware of high median nerve bifurcations, particularly when a persistent median artery is identified, and should remember that additional structures that can lead to nerve compression may be present in the carpal tunnel. Specifically, more than one median nerve may need to be identified and protected in such cases.

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.

A molecular analysis of the isolated rat posterior frontal and sagittal sutures: differences in gene expression.

Although it is one of the most commonly occurring craniofacial congenital disabilities, craniosynostosis (the premature fusion of cranial sutures) is nearly impossible to prevent because the molecular mechanisms that regulate the process of cranial suture fusion remain largely unknown. Recent studies have implicated the dura mater in determining the fate of the overlying cranial suture; however, the molecular biology within the suture itself has not been sufficiently investigated. In the murine model of cranial suture fusion, the posterior frontal suture is programmed to begin fusing by postnatal day 12 in rats (day 25 in mice), reliably completing bony union by postnatal day 22 (day 45 in mice). In contrast, the sagittal suture remains patent throughout the life of the animal. Using this model, this study sought to examine for the first time what differences in gene expression--if any--exist between the two sutures with opposite fates. For each series of experiments, 35 to 40 posterior frontal and sagittal suture complexes were isolated from 6-day-old Sprague-Dawley rat pups. Suture-derived cell cultures were established, and ribonuicleic acid was derived from snap-frozen, isolated suture tissue. Results demonstrated that molecular differences between the posterior frontal and sagittal suture complexes were readily identified in vivo, although these distinctions were lost once the cells comprising the suture complex were cultured in vitro. Hypothetically, this change in gene expression resulted from the loss of the influence of the underlying dura mater. Significant differences in the expression of genes encoding extracellular matrix proteins existed in vivo between the posterior frontal and sagittal sutures. However, the production of the critical, regulatory cytokine transforming growth factor beta-1 was equal between the two suture complexes, lending further support to the hypothesis that dura mater regulates the fate of the overlying cranial suture.

Osteogenesis in cranial defects: reassessment of the concept of critical size and the expression of TGF-beta isoforms.

Transforming growth factor-betas (TGF-beta) have been demontstrated to be upregulated during osteoblast function in vitro and during cranial suture fusion in vivo. The authors hypothesized that spontaneous reossification of calvarial defects was also associated with upregulation of TGF-beta. The present study was designed to (1) evaluate the concept of a critical-size defect within the calvaria in an adult guinea pig model and (2) investigate the association between the ossification of calvarial defects and TGF-beta upregulation. Paired circular parietal defects with diameters of 3 and 5 mm and single parietal defects with diameters of 8 or 12 mm were made in 45 six-month-old skeletally mature guinea pigs. Three animals per defect size were killed after survival periods of 3 days, 1 week, 4 weeks, 8 weeks, or 12 weeks. New bone ingrowth was evaluated by assessing for linear closure by a traditional linear method and by a modified cross-sectional area method using an image analysis system in which the thickness of new bone was taken into account. Immunohistochemistry was performed using rabbit polyclonal antibodies to localize TGF-beta1, -beta2, and -beta3. All specimens were photographed, and the intensity of immunostaining was graded based on subjective photographic assessment by three independent reviewers. No defect demonstrated any measurable bone replacement after a survival period of 3 days. All 3- and 5-mm defects were completely reossified after 12 weeks based on the linear analysis of new bone, indicating these defects to be less than critical size. However, new bone formation in the 5-mm defects never exceeded a mean of 40 percent by cross-sectional area of new bone. Percent of new bone formation by cross-sectional area was significantly higher within 3-mm defects than in all larger defects 4 weeks after the craniotomy, reaching a mean of 89 percent new bone by 12 weeks. Persistent gaps were noted on linear analysis of the 8- and 12-mm wounds by 12 weeks, and mean percent new bone by cross-sectional area remained below 30 percent. Immunolocalization demonstrated osteogenic fronts at the advancing bone edge and the endocranial side, in which the osteoblasts stained strongly for all isoforms of TGF-beta. The intensity of osteoblast expression waned considerably after the majority of the defect had reossified. These data indicate that histometric analysis based on cross-sectional area more accurately reflects the osteogenic potential of a cranial defect than does linear inspection of defect closure. Although the interpretation of immunolocalization studies is highly subjective, independent assessment by three reviewers indicates that isoforms of TGF-beta were upregulated during a limited "window" of time corresponding to the period of active calvarial reossification, and expression of TGF-beta corresponded to osteoblast activity within osteogenic fronts.

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.