The role of bone morphogenetic proteins in articular cartilage development, homeostasis and repair.

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of secreted ligands. BMPs regulate a diverse range of developmental processes during embryogenesis and postnatal development, and control the differentiation of several musculoskeletal tissues including bone, cartilage, tendon and ligaments. The ability of BMPs to modulate the phenotype of cells in these tissue lineages suggests that these factors could be valuable for musculoskeletal tissue regeneration. In fact, BMPs-2 and -7 are already in clinical use for bone regeneration. This review addresses the signaling mechanisms by which BMPs regulate cellular processes, the role of BMPs in articular cartilage development and joint formation, and the data that supports the use of BMPs for in vitro phenotypic support of articular chondrocyte cultures, chondrogenic differentiation of mesenchymal stem cells (MSCs) and articular cartilage repair. Given the documented importance of BMP activity for normal joint formation, articular cartilage development and maintenance, the chondrogenic activity of BMPs when applied to MSC cultures and the encouraging outcomes of several in vivo cartilage repair studies, BMP therapies hold considerable promise for effective cartilage repair and/or regeneration. Future advances in the control of BMP elution from biocompatible matrices and prolonged, dose-controlled BMP expression by genetically engineered cells should substantially improve cartilage repair strategies using BMPs and similar chondro-protective proteins.     

Bmp/Smad信号通路及其在哺乳动物卵巢发育中的作用

开展绵羊多羔性状基因的研究对于揭示其分子调控机制和提高绵羊繁殖力具有重要意义。骨形态发生蛋白(BMPs)为转化生长因子-β(TGF-β)超家族成员,与哺乳动物繁殖活动密切相关。研究表明,BMPs可促使原始卵泡向次级卵泡转化,对哺乳动物卵巢颗粒细胞增殖、生殖激素的合成和分泌以及卵母细胞成熟和排卵等方面起重要调节作用,而BMPs发挥功能主要依赖于经典的Bmp/Smad信号通路。本文就Bmp/Smad信号通路成员的表达、对早期胚胎发育的影响以及在哺乳动物卵巢发育中的作用等方面的研究进行总结,以期为进一步研究BMPs及其信号通路的调控机制提供参考。     

与绵羊多胎繁殖性状相关的BMP家族基因的研究进展

文章就BMP家族基因中与绵羊多胎繁殖性状相关的主效基因和候选基因进行了综述,主要包括BMP2、BMP4、BMP15、GDF9以及BMP的受体BMPRH、BMPR—IB等基因的最新研究进展,阐述了其对绵羊繁殖率的影响,供研究者参考。     

Recombinant Bone Morphogenetic Proteins: Novel Substances for Enhancing Bone Healing

Bone morphogenetic proteins (BMPs) are differentiative factors whose principal function is to induce transformation of undifferentiated mesenchymal cells into chondroblasts and osteoblasts in a dose-dependent manner. Bone morphogenetic proteins have been isolated postnatally in mammals from bone matrix, periosteal cells, mesenchymal cells of marrow stroma, tooth anlagen, and cells of osteosarcoma and chondrosarcoma. Distribution in additional embryonic tissues implies a broader organogenic function. Bone morphogenetic proteins are the only differentiative factors able to singularly induce de novo bone formation in vitro and in vivo. Recombinant DNA technology allows their production in large and highly purified quantities. The BMPs' osteoinductive ability has been shown with a variety of carriers including collagens and polymers at heterotopic and orthotopic sites in a wide range of species. They are presently being readied for clinical use as alternatives to bone grafts. Other potential applications include use as pulp capping agents, promoters of implant osteointegration and soft tissue reunion with bone, treatments for nonadaptive bone disease, and implants for use with mitotically expanded skeletal stem cell populations. Errors in the genetic coding of BMPs may manifest as clinical disease entities.     

Delivery of growth factors using gene therapy to enhance bone healing

OBJECTIVE: To review the delivery of growth factors using gene therapy for enhancing long-bone fracture healing. STUDY DESIGN: Literature review. METHODS: MEDLINE and CAB Abstracts literature search (1980-2004). RESULTS: Non-union and infected non-union are relatively common complications of long-bone fractures in human and veterinary patients. Growth factors are cytokines that regulate many cell functions important in fracture healing. Exogenous growth factors can be delivered to the fracture site as recombinant proteins or using gene therapy. Recombinant human bone morphogenetic protein-2 and -7 (rhBMP-2 and -7), in particular, enhance fracture healing in numerous experimental and clinical studies. Some limitations with use of recombinant proteins may be overcome by use of gene therapy. Gene therapy involves delivery of the growth factor gene to cells at the fracture site using a viral or non-viral vector. The gene is then expressed (protein synthesis) by cells at the fracture site. Delivery of the BMP gene to the fracture site using gene therapy has been evaluated in laboratory animal models of non-union, with favorable results and without complications. CONCLUSION: Delivery of growth factors, particularly members of BMP family, to the fracture site using gene therapy may be a method to enhance fracture healing. Use of this technology may improve the prognosis for patients with long-bone fractures. CLINICAL RELEVANCE: Clinical application of gene therapy could improve the prognosis for human and veterinary patients with long-bone fractures, but has not been evaluated clinically.     

Effect of transforming growth factor-beta1 on bone regeneration in critical-sized bone defects after irradiation of host tissues

OBJECTIVE: To determine whether sustained release of transforming growth factor (TGF)-beta1 from a gelatin hydrogel would enhance bone regeneration in critical-sized long-bone defects and overcome inhibitory effects of preoperative irradiation. ANIMALS: 24 adult New Zealand White rabbits. PROCEDURE: Rabbits were allocated to 2 groups. Twelve rabbits received localized megavoltage radiation to the right ulna by use of a cobalt 60 teletherapy unit, and 12 rabbits received no irradiation. Then, a 1.5-cm defect was aseptically created in the right ulna of each rabbit. Gelatin hydrogel that contained 5 microg of adsorbed recombinant-human (rh)TGF-beta1 was placed in the defect of 12 rabbits (6 irradiated and 6 nonirradiated), and the other 12 rabbits received hydrogel without rhTGF-beta1. Rabbits were euthanatized 10 weeks after surgery. New bone formation within the defect was analyzed by use of nondecalcified histomorphometric methods. A 1-way ANOVA was used to compare differences among groups. RESULTS: New bone formation within the defect was significantly greater in TGF-beta1-treated rabbits than in rabbits treated with hydrogel carrier alone. Local delivery of rhTGF-beta1 via a hydrogel carrier in irradiated defects resulted in amounts of bone formation similar to those for nonirradiated defects treated by use of rhTGF-beta1. CONCLUSIONS AND CLINICAL RELEVANCE: Local delivery of TGF-beta1 by use of a hydrogel carrier appears to have therapeutic potential for enhancing bone formation in animals after radiation treatments. IMPACT FOR HUMAN MEDICINE: This technique may be of value for treating human patients at risk for delayed bone healing because of prior radiation therapy.     

Differential expression of bone morphogenetic protein 4-6 (BMP-4, -5, and -6) and growth differentiation factor-9 (GDF-9) during ovarian development in neonatal pigs

Growth differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMPs), comprise the largest subgroups of ligands in the TGF-β superfamily, and have been shown to be involved in follicle development in mammals. However, whether these factors are involved in folliculogenesis in pigs is still unknown. The present study was performed to determine the relationships between early folliculogenesis and the expression of GDF-9 and BMP (BMP-4, -5 and -6) mRNAs in neonatal pigs. Ovaries were removed at 5, 16, 28 and 39 days after birth to examine the follicular population (the right ovary of each animal) and to detect mRNA expression (the left ovary of each animal). Primordial follicles accounted for >80% of the ovarian follicles from 5 days until 39 days after birth. A marked increase in primary follicles and the appearance of secondary follicles were observed in the ovaries at 28 days after birth. BMP-4, -5, and -6 and GDF-9 mRNAs were expressed by ovaries at 5-, 16-, 28- and 39-day-old pigs. The peak expression of BMP-4, -5, and -6 and GDF-9 mRNAs was observed in the ovaries at 5, 39, 28 and 16 days, respectively, after birth. These data demonstrate that folliculogenesis in piglets might be controlled by the interaction with these factors. We conclude that BMPs and GDF-9 may have distinct functions in several stages of follicle development in neonatal pig ovaries.     

Use of bone morphogenetic proteins for augmentation of bone regeneration

A large body of preclinical and clinical data now documents that recombinant BMPs can be used for skeletal regeneration in humans and animals. Recombinant human BMP-2 and BMP-7 have been approved for use in human patients with long-bone fractures and nonunions and in patients undergoing lumbar fusion or various maxillofacial and dental regenerative procedures. These products have also been made available for veterinary use.     

Effect of trehalose coating on basic fibroblast growth factor release from tailor-made bone implants

Artificial bone implants are often incorporated with osteoinductive factors to facilitate early bone regeneration. Calcium phosphate, the main component in artificial bone implants, strongly binds these factors, and in a few cases, the incorporated proteins are not released from the implant under conditions of physiological pH, thereby leading to reduction in their osteoinductivity. In this study, we coated tailor-made bone implants with trehalose to facilitate the release of basic fibroblast growth factor (bFGF). In an in vitro study, mouse osteoblastic cells were separately cultured for 48 hr in a medium with a untreated implant (T-), trehalose-coated implant (T+), bFGF-incorporated implant (FT-), and bFGF-incorporated implant with trehalose coating (FT+). In the FT+ group, cell viability was significantly higher than that in the other groups (P<0.05). Scanning electron microscopy (SEM) and X-ray diffraction (XRD) revealed that trehalose effectively covered the surface of the artificial bone implant without affecting the crystallinity or the mechanical strength of the artificial bone implant. These results suggest that coating artificial bone implants with trehalose could limit the binding of bFGF to calcium phosphate.     

Mammary gland secretory concretions contain non-collagenous bone matrix proteins

Secretory concretions in mammary gland alveoli are commonly of microscopical size. However, some concretions reach clinically palpable dimensions and may occlude teat canals and obstruct milk flow. We studied secretory concretions in sheep, goat and cow mammary glands, using routine histological staining methods, conventional histochemistry and electron microscopy. As concretions frequently mineralize, immunostaining for keratan sulphate and calcium-binding non-collagenous bone matrix proteins (bone sialoprotein, osteocalcin, osteonectin and osteopontin) was performed. Concretions consisted of organic matrix (condensed secretions) with calcium precipitates. Mineralized deposits mostly show concentric organization, bound haematoxylin, and were readily identified in H&E-stained sections. Mineral components of concretions reacted for calcium carbonate and phosphate, organic matrix was found to contain sialoglycan material. Immunohistochemistry revealed bone sialoprotein, osteonectin and keratan sulphate in cow and goat concretions. Osteocalcin was detected in sheep, cow and goat concretions, whilst osteopontin was not identified in any of the specimens studied. Our results indicate the presence of non-collagenous bone matrix proteins (except osteopontin) in mammary gland concretions. These glycoproteins are commonly thought to govern mineralization of organic matrix and are assumed also to promote mineral deposition in mammary gland secretory concretions. Besides caseins, these particular glycoproteins have to be considered as calcium-binding milk proteins.     

Total body and regional measurements of bone mineral content and bone mineral density in pigs by dual energy X-ray absorptiometry.

Dual energy X-ray absorptiometry (DXA) was used to make total body and regional measurements of bone mineral content, bone mineral density, and bone area during the growth of pigs from 3 to 138 kg. In all, 1,053 total body scans were performed on 587 live pigs. Regional measurements consisted of the front legs, trunk, and back legs. In addition, bone mineral density readings were recorded for the head, pelvis, spine, and ribs. From about 5 to 75 kg, a greater percentage of the total body bone mineral content (BMC) was located in the trunk region. However, the percentage of BMC in the front and back legs continued to increase linearly whereas the percentage of BMC in the trunk region peaked at about 25 kg and then decreased logarithmically. Allometric analysis revealed that up to about 30 kg the BMC increased more rapidly in the trunk region compared to the front or back leg regions (P > 0.05), but after 30 kg the increase in BMC was more rapid in the leg regions (P < 0.05). Overall, the rate of increase in BMC in the back legs was slightly more than in the front legs (P > 0.05). Positive allometric growth of BMC was observed when compared with the increase in bone area for the same region. By far, the highest measured level of bone mineral density (BMD) was in the head region (P < 0.05), followed in order by the front legs, spine, back legs, pelvis, and ribs. Over the entire range of growth from 3 to 138 kg, the highest relative growth coefficient for the increase in BMD occurred in the pelvic and back leg regions and the lowest was in the ribs (P < 0.05). For pigs < 30 kg, the highest growth coefficient for BMD relative to BW was in the spine (P > 0.05). The growth coefficients for BMD in the back legs and total body increased in pigs > 30 kg and those of the front legs and trunk regions decreased.     

Oocyte-somatic cell-endocrine interactions in pigs

Oocyte-somatic cell communication is bi-directional and essential for both oocyte and follicular granulosa and theca cell function and development. We have shown that the oocyte secretes factors that stimulate porcine granulosa cell proliferation in serum-free culture, and suppress progesterone production, thereby preventing premature luteinisation. Possible candidates for mediating some of these effects are the bone morphogenetic proteins (BMPs) that belong to the transforming growth factor beta family. They are emerging as a family of proteins critical for fertility and ovulation rate in several mammals, and they are expressed in various cell types in the ovary. We have evidence for a functional BMP system in the porcine ovary and BMP receptors are present in the egg nests in the fetal ovary and in the granulosa cells, oocytes and occasional theca cells throughout subsequent development. In addition to paracrine interactions in the ovary, the porcine oocyte and its developmental potential can also be influenced by nutritional manipulation in vivo. We have demonstrated that feeding a high plane of nutrition to gilts for 19 days prior to ovulation increased oocyte quality compared to control animals fed a maintenance diet, as determined by oocyte maturation in vitro. This was associated with a number of changes in circulating reproductive and metabolic hormones and also in the follicular fluid in which the oocyte is nurtured. Further studies showed a similar increase in prenatal survival on Day 30 of gestation, demonstrating a direct link between oocyte quality/maturation and embryo survival. Collectively, these studies emphasise the importance of the interactions that occur between the oocyte and somatic cells and also with endocrine hormones for ovarian development, and ultimately for the production of oocytes with optimal developmental potential.     

Basic avian bone growth and healing.

There have been few studies on the process of fracture repair in avian species. Most of the information shows similarities between avian and mammalian bone growth and fracture repair, but there are differences. The main finding confirms that fractures must be reduced properly, stabilized, and immobilized with an adequate blood supply to the bone fragments for optimal healing. The return to function of extremities, particularly the legs and wings, is an important consideration when internal fixation methods are used. Causing little or no collateral damage to soft tissue and joint when implanting internal hardware is ideal and reduces the likelihood of impaired function. Whether internal or external fixation methods are used for fracture reduction, the knowledge of avian bone growth and fracture repair is essential for veterinarian understanding and when discussing the healing process with clients.     

Appositional bone formation rates in the Beagle

Measurements of haversian appositional bone formation rates were carried out on 400 haversian systems in 28-month-old standardized research colony-raised Beagles, using the midshaft of the 11th rib. The appositional bone formation rate was gradual and regular when the circumference of the outer tetracycline ring was more than 0.35 mm, but the regularity of the measured rate disappeared when the outer tetracycline ring circumference was less than 0.35 mm. Erroneous conclusions could be drawn from measurements of appositional bone formation rate in these animals if this fact were not taken into consideration, and we concluded that in pre- and postexperimental biopsy sections in the ribs of these animals, haversian systems with tetracycline ring circumferences above 0.35 mm must be measured in both situations if the differences in appositional bone formation rate are to be construed as being truly significant.     

Long-term bone marrow culture systems: normal and cyclic hematopoietic dogs.

A continuous long-term liquid culture in both a micro and macro system that incorporates bone marrow cells from normal and cyclic hematopoietic dogs is described. An adherent layer composed of fibroblasts, endothelial cells, mononuclear phagocytic cells, and fat-containing cells is essential for continuous hematopoiesis. Hematopoiesis was measured by the recovery of the nonadherent cells and the generation of committed granulocyte-monocyte progenitor cells for a period of seven weeks. Optimum growth factors include the use of horse serum, fetal bovine serum, dog serum, hydrocortisone, a 33 degrees C incubation temperature and feeding twice a week. As is true for both human and murine marrow liquid cultures, horse serum and hydrocortisone are essential for development and maintenance of fat-containing cells in the described systems. Both factors are important in hematopoiesis but their respective roles have not been defined. Normal and cyclic hematopoietic dogs bone marrow cells are comparable in their ability to establish long-term cultures. The micro-method (Linbro-well culture) gave similar results in maintaining hematopoiesis as did a macromethod (flask culture).     

BMP-2 and -6 modulate porcine theca cell function alone and co-cultured with granulosa cells

Bone morphogenetic proteins (BMPs) are emerging as a family of proteins crucial in the regulation of fertility and ovulation rate. We have shown that porcine theca cells express BMP receptors, however, there is a paucity of information regarding the effect(s) of BMPs on theca cell function. The purpose of this study was to investigate the effects of BMP-2 and -6 on theca cells cultured under serum-free conditions in terms of steroidogenesis, cAMP release and proliferation. The study was further extended to determine whether BMP responses in theca cells are affected by the addition of granulosa cells to the culture system. Both BMPs suppressed progesterone and androstenedione synthesis by theca cells (P < 0.05) after 144 h in culture. Oestradiol synthesis was suppressed (P < 0.05) by BMP-2, but not BMP-6, and theca cell proliferation was stimulated (P < 0.05) by BMP-6, but not BMP-2, after 144 h in culture. Both BMP-2 and -6 inhibited cAMP release (P < 0.05) by theca cells. Furthermore, progesterone and androstenedione synthesis by co-cultured theca and granulosa cells were suppressed (P < 0.05) whereas cell proliferation was stimulated (P < 0.05). These results provide strong evidence for a functional BMP system in the porcine ovary and that theca cells are responsive to BMPs in terms of steroidogenesis and proliferation. BMP-2 and -6 may have a role as luteinisation inhibitors in this polyovular species.     

Thermostable antigens from the bone marrow of several animal species.

Studies on bone marrow extracts from ten animal species have revealed the presence of thermostable antigens. Similar antigens were found in other organs as shown by immunodiffusion and cross-reactivity was demonstrated between the thermostable proteins from horse and dog and between cow and sheep proteins. Using an immunoenzymatic reaction with glucose oxidase as the marker, the thermostable antigens were localized in the blood and bone marrow polymorphonuclear leukocytes and the myeloid cell line.     

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.     

Evidence for a functional bone morphogenetic protein (BMP) system in the porcine ovary

Bone morphogenetic proteins (BMPs) play important roles in controlling fertility and ovulation rate. There is however, little information on the BMP system in the ovary of a large polyovular species. The aims of the present study were to investigate BMP-2 and -6 protein expression in the porcine ovary, their effects on granulosa cells in culture and their mechanism of action. Cells and oocytes were recovered from healthy antral follicles 2–6 mm in diameter. When assessed by Western blotting, oocytes and follicular fluid contained BMP-2 and -6. In addition, BMP-2 and -6 were observed in granulosa cells and BMP-2 was also found in theca cells. Granulosa cells were cultured in a serum-free system for 144 h in the presence of increasing doses (0, 3, 30 and 100 ng/ml) of BMP-2 or BMP-6. Both BMPs suppressed progesterone production in a dose-dependent manner after 48 h (P < 0.001) and 144 h (P < 0.05). Only BMP-6 stimulated cell proliferation at 100 ng/ml (P < 0.05). Investigation into the mechanism of action found that BMP-2 and -6 decreased cyclic adenosine monophosphate (cAMP) production (P < 0.01), expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) protein (P < 0.001) and steroidogenic acute regulatory protein (StAR) (BMP-6 only; P < 0.05). This supports the hypothesis that BMP-2 and -6 act as luteinization inhibitors. In conclusion, these findings provide evidence for the presence of a complex signalling mechanism in the porcine ovary and suggest that both BMP-2 and -6 may act in a paracrine manner to control granulosa cell function in this large polyovulatory species.     

Evaluation of bone toxicity in various bones of aged rats

The aim of the present study was to provide a method for evaluating bone toxicity induced by drugs in various bones in aged rats. Male Crl:CD (SD) rats at 46 weeks of age were administered 15 mg/m(2) body surface area of doxorubicin, which effects the growth plate in weanling rats, weekly for 9 weeks by intravenous injection, and the femur, sternum, humerus and tibia were examined histopathologically. In the doxorubicin-treated group, thinning of the growth plate was remarkably observed in the proximal tibia and humerus; however, these changes were not observed in other regions. In addition, the osteoclast number per bone perimeter in the proximal tibia was significantly higher than others in control aged rat. Thus, recognizing the various histological reactions related to the time of epiphyseal closure is important for evaluating bone toxicity in aged rats.