GDF-5 can act as a context-dependent BMP-2 antagonist.

BACKGROUND: Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-ß family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5-as evident from its alternative name, cartilage derived morphogenetic protein 1-plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a "BMP-2 mimic" with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor. RESULTS: Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites. CONCLUSIONS: Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5's signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs.

Histomorphologic characteristics of bisphosphonate-related osteonecrosis of the jaw.

BACKGROUND: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a known side effect of the therapy with bisphosphonates. No specific pathologic aspects or histological features are included in the most current definition. This study investigates characteristics of BRONJ with a special emphasis on histomorphologic aspects, evaluating the role of Actinomyces spp. as well as other disease-promoting factors in a formal pathogenetic context. METHODS: We investigated 23 patients (14 female, nine male; mean age: 66 ± 11.8 years) who received bisphosphonates with a gender- /age-matched control group. Tissue specimens were treated according to local standards and analyzed histologically. RESULTS: In 18 (78.3%) BRONJ cases, we found Actinomyces spp. colonies. Bone remodeling could be found in three specimens (13%). Eight specimens (34.8%) showed signs of epithelial proliferation. Analysis of dental treatment before the onset of BRONJ did not reveal significant differences (P > 0.20). In 10 patients (83%; P > 0.05) of the reported cases a relationship between dental treatment and the occurrence of a purulent bone necrosis could be observed. Statistically significant differences in thickness of trabeculae were detected between the two study groups (P = 0.04). CONCLUSIONS: This study demonstrates the important influence of the osteoblast-osteoclast balance in a histomorphologic analysis. Together with cofactors, which are able to trigger the onset of BRONJ, a new pathogenesis model was developed.

Role of oxidative and nitrosative stress in autogenous bone grafts to the mandible using guided bone regeneration and a deproteinized bovine bone material.

UNLABELLED: The aim of this study was to evaluate the role of oxidative and nitrosative stress in autogenous bone grafts to the mandible based on immunohistochemical analysis. MATERIAL AND METHODS: Using a well-established sheep model autogenous bone grafts were harvested form the iliac bone. A combination of a Collagen Membrane (CM) and Deproteinized Bovine Bone Material (DBBM) was used to cover the bone graft (Experiment 2). This modification was compared with simple onlay bone grafts (Experiment 1). Immunohistochemically, the expression of specific stable degradation products of oxidative and nitrosative stress was compared between the two experimental groups. RESULTS: Specific markers for oxidative and nitrosative stress showed statistically significant differences in expression in the different experimental groups. The influence of oxidative and nitrosative stress on osteoblasts (OB), osteoclasts (OC), and osteocytes (OCy) was analysed. Experiment 2 showed increased expression of markers in OB and decreased expression in OC. CONCLUSIONS: Taking the result of this study and reports from the literature into consideration grafts in Experiment 2 showed less resorption and atrophy, higher activity of OB and inhibition of OC, and less expression of Reactive Oxygen and Nitrogen Species (RONS) as markers of oxidative stress within the graft. These data illustrate the improved remodelling processes in grafts using CM and DBBM.

Immunohistochemial study on the expression of von Willebrand factor (vWF) after onlay autogenous iliac grafts for lateral alveolar ridge augmentation.

INTRODUCTION: The main problems of autogenous bone transplants are their unpredictable atrophy and their loss of structure. One key factor lies in the poor revascularization of simple onlay grafts. The the aim of this study was to evaluate the revascularization processes in autogenous bone grafts from the iliac crest to the alveolar ridge. METHODS: In a sheep model, autogenous bone grafts were harvested from the iliac crest. A combination of a resorbable collagen membrane (CM) and deproteinized bovine bone material (DBBM) was used to modify the bone graft (experiment 2). This was compared with a simple onlay bone graft (control group, experiment 1). The amount of vessels in bone and connective tissue (CT), and the amount of CT were analyzed. The expression of von Willebrand factor (vWF) was compared between the two experimental groups using immunohistochemical analysis. RESULTS: The ratio of the amount of vessels in bone and CT changed over time, and more vessels could be detected in bone at 12-16 weeks of graft healing. The number of vessels were significantly higher in experiment 2 than in experiment 1. More CT was found in experiment 1, whereas the amount of CT in both experiments decreased over time. CONCLUSION: This study shows a more intensive and extensive revascularization in experiment 2, as significantly more vessels were detected. The decreased amount of CT in experiment 2 clarifies its clinical superiority.

Tissue engineering of human oral mucosa on different scaffolds: in vitro experiments as a basis for clinical applications.

OBJECTIVES: The lack of oral mucosa in oral and maxillofacial surgery for intraoral grafting after trauma or tumor resection can be balanced by tissue-engineered oral mucosa. The aim of this study was to generate a tissue-engineered oral mucosa equivalent (OME). STUDY DESIGN: First, primary oral fibroblasts were cultured for 7 days on different materials: Tissufoil E (TFE), dermal regeneration template (DRT), and Vicryl. Then, cocultures were established by seeding of primary oral keratinocytes and culturing for another 7-14 days. Immunohistochemical staining for CD90, cytokeratin 14 and collagen IV as well as gene expression analysis using reverse-transcription quantitative polymerase chain reaction were used to get information about cell architecture and basal membrane formation. RESULTS: Vicryl showed good mechanical stability but mixed cell growth. TFE provided the best cell growth with good cell architecture and basal membrane formation but showed degradation. The best results for the above-mentioned criteria were seen with DRT. CONCLUSIONS: It was possible to create OMEs on all 3 scaffolds. The arrangement of the cells strongly depends on the texture of the material.

Cryopreservation of autologous bone grafts: an experimental study on a sheep animal model.

A sheep animal model was used to investigate the clinical behavior of autologous bone transplants after cryopreservation. The aim of the present study was to compare fresh, cryopreserved and deep-frozen bone transplants in terms of their osseointegration. We used a serum-free cryopreservation protocol with DMSO as cryoprotectant for the bone transplants, which were harvested from the iliac crest of the sheep. The bicortical iliac bone grafts were either cryopreserved or immediately frozen to -80 degrees C for 4 weeks. Four, 8, 12 and 16 weeks after the autologous transplantation of the cryopreserved, fresh or deep-frozen bone transplants to the contralateral iliac crest, the animals were sacrificed and the bone specimens were evaluated clinically, by staining for hematoxylin/eosin and for tartrate-resistant acid phosphatase, by quantified computed tomography, immunohistochemistry (Ki67) and polychrome sequential labeling. The best results were obtained for the fresh specimens with 83% bone healing compared with 75% (cryopreserved bone) and 50% (deep frozen bone). All parameters indicate that bone formation and remodeling processes take place in fresh and cryopreserved transplants. The deep-frozen specimens displayed no fluorochrome uptake in the sequential labeling. These findings indicate that osseointegration of the fresh transplants was the most successful and that osteogenic effects in fresh and cryopreserved transplants are located in the surface area, whereas only the osteoconductive effects are important in the center of the transplants. Thus, cryopreservation is a useful method for the clinical routine because it keeps the osteogenic cells viable, making it superior to deep freezing of abundant bone.

Influence of host periosteum and recipient bed perforation on the healing of onlay mandibular bone graft: an experimental pilot study in the sheep.

PURPOSE: The aim of this study was to evaluate the role of host periosteum and recipient bed perforation on the healing of an onlay graft to the mandible based on histologic and immunohistochemical analysis. MATERIALS AND METHODS: Each of the 12 adult sheep used in the study received four iliac corticocancellous onlay bone grafts on the lateral surface of the mandible. In experiment 1, the block graft was placed in direct contact with the recipient bed and fixed with micro-screws, and in experiment 2, the recipient cortical bed was perforated before graft placement. The host periosteum around the graft was excised before flap replacement in experiment 3, and in experiment 4, a sheet of silicone membrane was placed between the graft and the recipient bed. The animals were euthanised at 4, 8, 12 and 16 weeks after surgery and the findings were analysed by routine microscopy (haematoxylin and eosin and tartrate-resistant acid phosphatase [TRAP] stains) and immunohistochemistry for proliferation and apoptotic markers (Ki67, caspase-3 and TUNEL stains). RESULTS: After 8 weeks, full graft-host integration was seen in all the experimental groups except experiment 4. After 16 weeks, pronounced graft resorption and volume reduction was observed in experiments 1 and 2, whilst experiment 3 was characterised with extensive connective tissue infiltration and severe resorption of the graft. The number of osteoclasts expressed peaked at 4 weeks in experiments 1 and 2 and at 16 weeks in experiment 3. Immunoreactivity for Ki67 by osteoblasts lining the trabecular bone of the graft's spongiosa expressed moderate level of Ki67 at 8 weeks, and thereafter declined markedly. The strongest expression of caspase-3 on the bone surface was observed after 16 weeks. DISCUSSION AND CONCLUSION: Recipient cortical bed perforation offered no advantage over non-perforated bed regarding healing and integration of a bone graft. Excision of the host overlying the periosteum was accompanied with rapid absorption of the grafts and partial or complete replacement with fibrous connective tissue. This study also demonstrated that cell death by apoptosis is a fundamental component of osteoblastic phenotypic differentiation during healing of corticocancellous bone graft.