Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle.

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the 'P-36 protocol' for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT(2) receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus-oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.

Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis

Aims: The purpose of this study was to evaluate the expression of proteins that participate in the osteoinduction stage (VEGF, BMP2 and CBFA1) of the process of bone regeneration of defects created in rat calvariae and filled with autogenous bone block grafts. Materials and methods: 10 adult male rats (Rattus norvegicus albinus, Wistar) were used, who received two bone defects measuring 5 mm each in the calvariae. The bone defects constituted two experimental groups (n = 10): Control Group (CONT) (defects filled with a coagulum); Graft Group (GR) (defects filled with autogenous bone removed from the contralateral defect). The animals were submitted to euthanasia at 7 and 30 days post-operatively. Results: Quantitative analysis demonstrated significantly greater bone formation in Group GR, but the presence of the studied proteins was significantly greater in the CONT Group in both time intervals of observation. Conclusion: It was not possible in this study in cortical bone block groups to detect the osteoinductive proteins in a significant amount during the repair process. © 2013 European Association for Cranio-Maxillo-Facial Surgery.

Horizontal ridge augmentation of the atrophic anterior maxilla using rhBMP-2/ACS or autogenous bone grafts: A proof-of-concept randomized clinical trial

Aim To compare the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge carrier (ACS) with autogenous bone graft for augmentation of the edentulous atrophic anterior maxilla. Methods Twenty-four subjects were enrolled in a randomized, controlled, parallel-group, open-label clinical trial. Subjects either received rhBMP-2/ACS (1.5 mg/ml) or particulated autogenous bone harvested from the mandibular retromolar region. A titanium-mesh was used to provide space and wound stability. A guide was used to standardize clinical recordings using an analogue caliper. Alveolar ridge width was also assessed using cone-beam computed tomography. Results rhBMP-2/ACS yielded significantly greater radiographic horizontal bone gain compared with autogenous bone graft at immediate subcrestal levels (1.5 ± 0.7 versus 0.5 ± 0.9 mm; p = 0.01); non-significant differences were observed at mid- (2.9 ± 0.8 versus 2.9 ± 0.9 mm; p = 0.98) and apical (1.7 ± 0.9 versus 1.8 ± 1.1 mm; p = 0.85) crestal levels. No significant differences in clinical horizontal bone gain were observed at 6 months between rhBMP-2/ACS and autogenous bone graft (3.2 ± 0.9 mm versus 3.7 ± 1.4 mm; p = 0.31). Sixty-two implants were placed after 6 month of healing with no significant differences between groups for number of implants, implant size, primary stability and survival. Conclusions rhBMP-2/ACS appears a realistic alternative for augmentation of the edentulous atrophic anterior maxilla. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Regulação da expressão de fatores secretados pelo oócito (FSOs) e seus receptores durante a maturação in vitro (MIV) bovina e ações no controle da expressão de cumulus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bone Morphogenetic Proteins: Structure, biological function and therapeutic applications

Bone Morphogenetic Proteins (BMPs) are multifunctional secreted cytokines, which belong to the TGF-beta superfamily. These glycoproteins act as a disulfide-linked homo- or heterodimers, being potent regulators of bone and cartilage formation and repair, cell proliferation during embryonic development and bone homeostasis in the adult. BMPs are promising molecules for tissue engineering and bone therapy. The present review discusses this family of proteins, their structure and biological function, their therapeutic applications and drawbacks, their effects on mesenchymal stem cells differentiation, and the cell signaling pathways involved in this process. (C) 2014 Published by Elsevier Inc.

Caracterização de osteoblastos diferenciados de células-tronco mesenquimais da medula óssea de ratos espontâneamente hipertensos (SHR)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Impact of Bone Harvesting Techniques on Cell Viability and the Release of Growth Factors of Autografts

Background: Autogenous bone grafts obtained by different harvesting techniques behave differently during the process of graft consolidation; the underlying reasons are however not fully understood. One theory is that harvesting techniques have an impact on the number and activity of the transplanted cells which contribute to the process of graft consolidation. Materials and Methods: To test this assumption, porcine bone grafts were harvested with four different surgical procedures: bone mill, piezosurgery, bone drilling (bone slurry), and bone scraper. After determining cell viability, the release of molecules affecting bone formation and resorption was assessed by reverse transcription polymerase chain reaction and immunoassay. The mitogenic and osteogenic activity of the conditioned media was evaluated in a bioassay with isolated bone cells. Results: Cell viability and the release of molecules affecting bone formation were higher in samples harvested by bone mill and bone scraper when compared with samples prepared by bone drilling and piezosurgery. The harvesting procedure also affected gene expression, for example, bone mill and bone scraper samples revealed significantly higher expression of growth factors such as bone morphogenetic protein-2 and vascular endothelial growth factor compared with the two other modalities. Receptor activator of nuclear factor kappa B ligand expression was lowest in bone scraper samples. Conclusion: These data can provide a scientific basis to better understand the impact of harvesting techniques on the number and activity of transplanted cells, which might contribute to the therapeutic outcome of the augmentation procedure.

Functionalization of deproteinized bovine bone with a coating-incorporated depot of BMP-2 renders the material efficiently osteoinductive and suppresses foreign-body reactivity

The repair of critical-sized bony defects remains a challenge in the fields of implantology, maxillofacial surgery and orthopaedics. As an alternative bone-defect filler to autologous bone grafts, deproteinized bovine bone (DBB) is highly osteoconductive and clinically now widely used. However, this product suffers from the disadvantage of not being intrinsically osteoinductive. In the present study, this property was conferred by coating DBB with a layer of calcium phosphate into which bone morphogenetic protein 2 (BMP-2) was incorporated. Granules of DBB bearing a coating-incorporated depot of BMP-2--together with the appropriate controls (DBB bearing a coating but no BMP-2; uncoated DBB bearing adsorbed BMP-2; uncoated DBB bearing no BMP-2)--were implanted subcutaneously in rats. Five weeks later, the implants were withdrawn for a histomorphometric analysis of the volume densities of (i) bone, (ii) bone marrow, (iii) foreign-body giant cells and (iv) fibrous capsular tissue. Parameters (i) and (ii) were highest, whilst parameters (iii) and (iv) were lowest in association with DBB bearing a coating-incorporated depot of BMP-2. Hence, this mode of functionalization not only confers DBB with the property of osteoinductivity but also improves its biocompatibility--thus dually enhancing its clinical potential in the repair of bony defects.

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects

Introduction Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM). Materials and Methods The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5). Results PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups. Conclusion Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus.

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo

The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for this seems to be an unbalanced colonization of the scaffolds with cells, due to insufficient pore size.

Mandibular reconstruction using a combination graft of rhBMP-2 with bone marrow cells expanded in vitro

BACKGROUND: The aim of this study was to evaluate the efficacy of a combination graft, using recombinant human bone morphogenetic protein-2 (rhBMP-2) and culture-expanded cells derived from bone marrow, for bone regeneration in a nonhuman primate mandible. METHODS: Five Japanese monkeys were used. Three milliliters of bone marrow was obtained from the tibia and plated into culture flasks. Adherent cells were cultured until near confluence; then, the proliferated cells were transferred to a three-dimensional culture system using collagen beads as the cell carrier. The medium was supplemented with ascorbic acid, beta-glycerophosphate, and dexamethasone to promote osteoblastic differentiation. After further proliferation on beads, the cells were mixed with a collagen sponge that was impregnated with rhBMP-2 and grafted into surgically created segmental bone defects of the mandibles. Three animals received this treatment, and either culture-expanded cells alone or collagen beads without cells were implanted into the remaining two monkeys as controls. The animals were killed 24 weeks after surgery, and the results were assessed by radiographic and histologic evaluation. RESULTS: The combination graft of culture-expanded bone marrow cells with rhBMP-2 in a collagen sponge regenerated the mandibular bone completely. By contrast, the graft of culture-expanded cells alone resulted in only a small amount of bone formation, and the implantation of collagen beads alone led to no bone formation. CONCLUSION: The combination graft of rhBMP-2 and culture-expanded cells, which requires only a small amount of bone marrow, is a reliable method for the reconstruction of segmental bone defects of the mandible.

Effects of Delta12-prostaglandin J2 on bone regeneration and growth factor expression in rats

OBJECTIVES: Cyclopentenone prostaglandins have been shown to promote osteoblast differentiation in vitro. The aim of this study was to examine in a rat model the effects of local delivery of Delta(12)-prostaglandin J(2) (Delta(12)-PGJ(2)) on new bone formation and growth factor expression in (i) cortical defects and (ii) around titanium implants. MATERIAL AND METHODS: Standardized transcortical defects were prepared bilaterally in the femur of 28 male Wistar rats. Ten microliters of Delta(12)-PGJ(2) at 4 concentrations (10(-9), 10(-7), 10(-5) and 10(-3) mol/l) in a collagen vehicle were delivered inside a half-cylindrical titanium chamber fixed over the defect. Contralateral defects served as vehicle controls. Ten days after surgery, the amount of new bone formation in the cortical defect area was determined by histomorphometry and expression of platelet-derived growth factor (PDGF)-A and -B, insulin-like growth factor (IGF)-I/II, bone morphogenetic protein (BMP)-2 and -6 was examined by immunohistochemistry. In an additional six rats, 24 titanium implants were inserted into the femur. Five microliters of carboxymethylcellulose alone (control) or with Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) were delivered into surgically prepared beds prior to implant installation. RESULTS: Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) significantly enhanced new bone formation (33%, P<0.05) compared with control cortical defects. Delivery of Delta(12)-PGJ(2) at 10(-3) mol/l significantly increased PDGF-A and -B and BMP-2 and -6 protein expression (P<0.05) compared with control defects. No significant difference was found in IGF-I/II expression compared with controls. Administration of Delta(12)-PGJ(2) also significantly increased endosteal new bone formation around implants compared with controls. CONCLUSION: Local delivery of Delta(12)-PGJ(2) promoted new bone formation in the cortical defect area and around titanium implants. Enhanced expression of BMP-2 and -6 as well as PDGF-A and -B may be involved in Delta(12)-PGJ(2)-induced new bone formation.

Chondrogenic differentiation of bovine synovium: bone morphogenetic proteins 2 and 7 and transforming growth factor beta1 induce the formation of different types of cartilaginous tissue

OBJECTIVE: To compare the potential of bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7) and transforming growth factor beta1 (TGFbeta1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed. METHODS: Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP-2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP-2, BMP-7, and TGFbeta1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level. RESULTS: BMP-2 induced the chondrogenic differentiation of synovial explants in a dose- and time-dependent manner under serum- and dexamethasone-free conditions. The expression levels of cartilage-related genes increased in a time-dependent manner. BMP-7 was more potent than BMP-2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFbeta1 differed in terms of both cell phenotype and gene expression profiles. CONCLUSION: The 3 tested members of the TGFbeta superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

Astrocytes from the contused spinal cord inhibit oligodendrocyte differentiation of adult oligodendrocyte precursor cells by increasing the expression of bone morphogenetic proteins.

Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.

Enhancement of bone healing using non-glycosylated rhBMP-2 released from a fibrin matrix in dogs and cats

OBJECTIVES To test a non-glycosylated recombinant human bone morphogenetic protein-2 (ngly-rhBMP-2)/fibrin composite, which has been shown experimentally to enhance healing of bone defects in rodents, in a clinical case series of dogs and cats undergoing treatment for fracture non-unions and arthrodesis. METHODS A ngly-rhBMP-2/fibrin composite was applied in 41 sites in 38 dogs and cats for which a cancellous bone autograft was indicated, replacing the graft. RESULTS Bridging of the bone defect with functional bone healing was achieved in 90 per cent of the arthrodesis and fracture nonunions treated in this manner. CLINICAL SIGNIFICANCE This prospective clinical study demonstrates the beneficial effects of ngly-rhBMP-2 in a specially designed fibrin matrix on the treatment of bone defects, and validates the use of this composite as an alternative to bone autografts in dogs and cats.