Rumio Taga

Bone morphogenetic proteins: from structure to clinical use

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor ss superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

Down-regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin-10 knockout mice

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappaB ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.

Rat subcutaneous tissue response to macrogranular porous anorganic bovine bone graft

The ideal bone graft must present biocompatibility, osteoconductive and osteoinductive properties, resistance and plasticity. Xenogenic grafts of bovine cancellous bone origin are particularly interesting due to their biologically designed porous structure that enhance both cellular and vascular invasion. The purpose of this study was to evaluate the tissue response induced by bovine macrogranular porous anorganic bone implanted in rat subcutaneous tissue. Forty rats were assigned to 2 groups, as follows: the control group received empty collagen capsules and the test group received subcutaneous implants of the test material. Samples were collected after 10, 20, 30 and 60 days and processed histologically. Histological analysis showed at 10 days a granulomatous inflammatory infiltrate, rich in multinucleated giant cells and free of lymphocytes or plasma cells, similarly to mineralized allograft implanted in rat subcutaneous. In later periods, there was a significant decrease in the inflammatory infiltrate and an increase in fibrosis around graft particles. In conclusion, the test material induced a foreign body-type granuloma with subsequent fibrosis around the graft particles implanted in rat subcutaneous and did not elicit any immune response, thus being considered biocompatible.

Radiographic and histological study of perennial bone defect repair in rat calvaria after treatment with blocks of porous bovine organic graft material

Over the last few years, various bone graft materials of bovine origin to be used in oromaxillofacial surgeries have entered the market. In the present study, we determined the capacity of a block organic bone graft material (Gen-ox, Baumer SA, Brazil) prepared from bovine cancellous bone to promote the repair of critical size bone injuries in rat calvaria. A transosseous defect measuring approximately 8mm in diameter was performed with a surgical trephine in the parietal bone of 25 rats. In 15 animals, the defects were filled with a block of graft material measuring 8mm in diameter and soaked in the animals own blood, and in the other 10 animals the defects were only filled with blood clots. The calvariae of rats receiving the material were collected 1, 3 and 6 months after surgery, and those of animals receiving the blood clots were collected immediately and 6 months after surgery. During surgery, the graft material was found to be of easy handling and to adapt perfectly to the receptor bed after soaking in blood. The results showed that, in most animals treated, the material was slowly resorbed and served as a space filling and maintenance material, favoring angiogenesis, cell migration and adhesion, and bone neoformation from the borders of the lesion. However, a foreign body-type granulomatous reaction, with the presence of numerous giant cells preventing local bone neoformation, was observed in two animals of the 1-month subgroup and in one animal of the 3-month subgroup. These cases were interpreted as resulting from the absence of demineralization and the lack of removal of potential antigen factors during production of the biomaterial. We conclude that, with improvement in the quality control of the material production, block organic bone matrix will become a good alternative for bone defect repair in the oromaxillofacial region due to its high osteoconductive capacity.

Relationship between lower-lip fistulae and cleft lip and/or palate in Van der Woude syndrome

OBJECTIVE This study was conducted to evaluate the relationship between fistulae of the lower lip and cleft lip and/or palate in patients with Van der Woude syndrome. METHODS The medical records of 11,000 patients with cleft lip and/or palate registered at the Cleft Lip-Palate Research and Rehabilitation Hospital, University of São Paulo, Bauru were reviewed. Of these patients, 133 (1.2%) presented with Van der Woude syndrome. RESULTS Of the 133 patients, 88 (66.2%) exhibited full clefts, 22 (16.5%) only cleft lip, and 23 (17.3%) only cleft palate. The lower-lip fistulae observed in these 133 patients were bilateral symmetric in 66 (49.7%), bilateral asymmetric in 42 (31.6%), microform in 19 (14.3%), median in 5 (3.8%), and unilateral in 1 (0.7%). CONCLUSION This population sample appears to exhibit the previously published tendency for bilateral, unilateral, or mixed-type congenital fistulae to be associated with cleft lip with or without cleft palate, while so-called microforms or conic elevations are almost exclusively associated with cleft palate.

Tissue response to a membrane of demineralized bovine cortical bone implanted in the subcutaneous tissue of rats

The treatment of persistent bone defects has encouraged the search for proper techniques or bone substitutes. In Dentistry, a common problem in the treatment of periodontal bone defects is the growth of tissues within the lesion, such as the junctional epithelium, which impair regeneration of these tissues. Guided tissue regeneration (GTR), based on the separation of the tissues by means of membranes or barriers, was developed in an attempt to improve periodontal regeneration. The aim of this study was to histologically evaluate the tissue response to a membrane of demineralized bovine cortical bone implanted in the subcutaneous tissue of rats. The study periods were 1, 3, 7, 15, 30 and 60 days after implantation. Analysis of the histological sections demonstrated a moderate to intense inflammatory response at 1 and 3 days, moderate at 7 and 15 days, and almost absent at 30 and 60 days. Resorption of the membrane began 15 days after implantation, and at 60 days only remnants could be detected in some animals. We concluded that the demineralized bovine cortical bone membrane was well tolerated by the tissues and is completely resorbed after 30-60 days by mononuclear cells and multinucleated giant cells, which disappear upon completion of the process.

Bone repair and augmentation using block of sintered bovine‐derived anorganic bone graft in cranial bone defect model

OBJECTIVE To histomorphometrically investigate the repair of critical size defects (CSDs) and bone augmentation in cranial walls using block of sintered bovine-derived anorganic bone (sBDAB) graft. MATERIAL AND METHODS Forty guinea-pigs were divided into test (n=20) and CSD control (n=20) groups. In each animal, a full-thickness bone defect with 9.5 mm diameter was made in the frontal bone. The defects were filled with an sBDAB block soaked in blood in the test group and with blood clot in the CSD control group. The skulls were collected at 0 h (n=2) and 30, 90 and 180 days (n=6/group and period) postoperatively. The volume density and total volume of newly formed bone, sBDAB, blood vessels and connective tissue, vertical thickness of removed bone plug, sBDAB block and graft area were evaluated. RESULTS The vertical thickness of the adapted sBDAB block was 3.8 times higher than that of the removed bone plug and did not show significant difference between periods, filling in average 29.8% of the total graft region. The sBDAB block exhibited complete osseointegration with the borders of the defect at 90 days. At 90 and 180 days, the vertical thickness of the graft was 279% in the average, and the total volume of bone augmentation was, respectively, 78.8% and 148.5% higher compared with the removed bone plug. The defects of the CDS control group showed limited osteogenesis and filling by connective tissue plus tegument. CONCLUSION The sBDAB block can be used to promote repair of CSDs and bone augmentation in the craniomaxillofacial region, due to its good osteoconductive and slow resorptive properties.

Lack of repair of rat skull critical size defect treated with bovine morphometric protein bound to microgranular bioabsorbable hydroxyapatite

The ability of a pool of bovine bone morphogenetic proteins bound to synthetic microgranular hydroxyapatite (BMPb-HA) to stimulate bone repair was determined in rat critical size defects. An 8-mm diameter defect was created in the calvaria of 25 rats. In 15 rats, the defects were filled with BMPb-HA homogenized with blood (experimental group), and in 10 rats the defects were filled only with blood clots (control). The calvariae of experimental rats were collected 1, 3 and 6 months after surgery and of the control rats at the end of surgery and 6 months thereafter. The morphometric results obtained in the radiographs showed an absence of new bone formation at 1 and 3 months post-surgery and, histologically, the defects were filled with fibrous connective tissue and numerous foci of a foreign body-type granulomatous reaction around hydroxyapatite agglomerates. At the end of 6 months, the number and size of the granulomatous foci decreased and the area of the defects was reduced by 22% compared to the 0-hour control due to the formation of new bone at their borders, although the mean area was similar to the 6-month control. We conclude that the use of BMPb-HA in the treatment of critical size bone defects of the rat skull leads to the formation of a foreign body-type granulomatous reaction that markedly inhibits new bone formation, suggesting that synthetic microgranular hydroxyapatite does not represent a good carrier for BMP-induced bone formation.

Tibial segmental bone defect treated with bone plate and cage filled with either xenogeneic composite or autologous cortical bone graft - An experimental study in sheep

Tibia segmental defect healing in sheep were clinically, radiographically and histologically evaluated. Twelve young sheep aged four to five months were divided into two groups, G1 and G2. A 3.5 cm long segmental defect was created in the right tibial diaphysis with maintenance of the periosteum. The bone defects in both groups were stabilized with a bone plate combined with a titanium cage. In G1 the cage was filled with pieces of autologous cortical bone graft. In G2 it was filled with a composite biomaterial which consisted of inorganic bovine bone, demineralized bovine bone, a pool of bovine bone morphogenetic proteins bound to absorbable ultra-thin powdered hydroxyapatiteand bone-derived denaturized collagen. Except for one G1 animal, all of them showed normal limb function 60 days after surgery. Radiographic examination showed initial formation of periosteal callus in both groups at osteo-tomy sites, over the plate or cage 15 days postoperatively. At 60 and 90 days callus remodeling occurred. Histological and morphometric analysis at 90 days after surgery showed that the quantity of implanted materials in G1 and G2 were similar, and the quantity of new bone formation was less (p = 0.0048) and more immature in G1 than G2, occupying 51 +/- 3.46% and 62 +/- 6.26% of the cage space, respectively. These results suggest that the composite biomaterial tested was a good alternative to autologous cortical bone graft in this experimental ovine tibial defect. However, additional evaluation is warranted prior to its clinical usage.

Expression of metalloproteinase 2 in the cell response to porous demineralized bovine bone matrix.

SummaryThe purpose of the study was to analyze the involvement of metalloproteinase 2 (MMP-2) and macrophages in the tissue and cell response to the organic graft material produced from bovine cancellous bone. Thirty adult male white Wistar rats (Rattus norvegicus) received implants of blocks of demineralized bovine bone matrix between the fasciae of the quadriceps muscle. The specimens collected at 3, 7, 14, 21 and 28 days after implantation (n = 6/period). Sections of 6 μm thick were stained with hematoxylin and eosin and immunolabeled with anti-MMP-2 and anti-CD68 using standard avidin–biotin–peroxidase method. The tissue response to the material was initially mediated by polymorphonuclear neutrophils, evolving to a mononuclear inflammatory infiltrate with macrophages and few lymphocytes and plasma cells and presence of inflammatory multinucleated giant cells (GC) in contact with the material that exhibited signs of resorption. The number of cells immunolabeled to MMP-2 was highest at day 7 (103.2 ± 39.1), but significantly decreased (F = 3.67; p = 0.044) until day 28 (45.9 ± 13.1). CD68 immunostaining also significantly decreased (F = 6.75; p = 0.007) from day 7 (49.5 ± 10.4) to day 28 (19.5 ± 8.9). A positive and statistically significant correlation was observed between the evolutions of these two variables. The material had been almost completely resorbed at day 28. Among cells present at the granuloma, anti-MMP-2 immunostaining was predominant and more intense in macrophages, yet lightly immunolabeled multinucleated giant cells were found in close contact with the material. Thus, considering the experimental limitations of this study, we concluded that MMP-2 produced by macrophages participates in the resorption of demineralized bovine bone.