重组人骨形态发生蛋白-2诱导脂肪成体干细胞成异位软骨的研究

目的探索重组人骨形态发生蛋白-2（rhBMP-2）诱导脂肪成体干细胞异位软骨的生成能力。方法将获取的兔脂肪组织机械分割，通过Ⅰ型胶原酶消化后得到脂肪成体干细胞。经rhBMP-2诱导培养14d的脂肪成体干细胞微球种植在BALB／C裸鼠肌袋内。术后4、8周各处死2只动物，取材固定、脱钙、包埋后切片染色。镜下观察。结果经过rhBMP-2连续诱导培养14d的脂肪成体干细胞已具有软骨细胞的表型（细胞基质中富含蛋白多糖和Ⅱ型胶原），并不向成骨方向分化（Von kossa染色阴性）。术后4、8周有软骨细胞形成。结论rhBMP-2具有诱导脂肪成体干细胞向软骨细胞分化的潜能。     

Induction of CD-RAP mRNA during periosteal chondrogenesis.

Induction of chondrogenesis and maintenance of the chondrocyte phenotype are critical events for autologous periosteal transplantation, which is a viable approach for cartilage repair. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a recently discovered protein that is mainly produced in cartilage. During development, CD-RAP expression starts at the beginning of chondrogenesis and continues throughout cartilage maturation. In order to investigate the involvement of CD-RAP during periosteal chondrogenesis we have determined the nucleotide sequence of the rabbit CD-RAP mRNA and utilized this information to evaluate the temporal and spatial expression pattern of CD-RAP at the mRNA level during chondrogenesis. When the periosteal explants were cultured under chondrogenic conditions, the expression of CD-RAP was induced, as shown by a 40-fold increase in CD-RAP mRNA between days 7 and 10. The temporal expression pattern of CD-RAP closely mimicked that of collagen type IIB mRNA. Also, the CD-RAP mRNA was localized to the matrix forming chondrocytes in the cambium layer of the periosteum by in situ hybridization as indicated by colocalization with collagen type II mRNA and positive safranin O staining. These data suggest a regulatory role of CD-RAP in periosteal chondrogenesis, which is potentially important for both cartilage repair and fracture healing via callus formation.     

Repair of bone allograft fracture using bone morphogenetic protein-2

Long-term clinical data have shown that reconstruction using bone allografts provide adequate function after extensive tumor surgery. Complications such as nonunion of allograft-host interface, infection, and allograft fracture often require major revision surgeries. Allograft fractures usually do not induce the same repair process that is seen in normal fracture healing. The authors did an experimental study to test whether bone morphogenetic protein-2 can induce and achieve osseous repair in an allograft osteotomy model. Recombinant human bone morphogenetic protein-2 was applied at femoral intercalary allograft osteotomy sites in 20 rats. Forty additional rats served as controls (carrier alone and sham). Specimens in all groups were examined histologically and radiographically at 4 and 8 weeks. Specimens in the control groups showed only fibrosis by 8 weeks. In contrast, none of 10 specimens in the experimental group showed radiographic union at 8 weeks. New bone formation and integration with underlying allografts were seen in the experimental group as early as 4 weeks. These data suggest that fracture repair in the allograft bone can be triggered by a biologic regulator that is expressed during normal fracture healing.     

FGF-2 enhances TGF-beta1-induced periosteal chondrogenesis.

The use of periosteum as a cell source for the in vitro engineering of grafts for articular cartilage repair requires the development of methods to obtain high viable cell numbers in the early stages of culture. In this study, we demonstrate that the addition of a mitogen, fibroblast growth factor-2 (FGF-2), during the early stage of the in vitro culture of periosteum in the presence of transforming growth factor-beta1 (TGF-beta1), significantly enhances cell proliferation, which results in increased neo-cartilage formation at later stages. Periosteal explants were cultured in vitro within alginate or agarose based gels in the presence of either FGF-2 for the first week, TGF-beta1 for the first 2 weeks, FGF-2 and TGF-beta1 for the first week and first 2 weeks respectively, or no added factors. Consistent with previous studies, periosteum derived neo-chondrogenesis occurred only in the presence of TGF-beta1. The neo-cartilage was found to contain cartilage specific proteoglycans and Type-II collagen as determined by safranin-O and immunohistochemical staining respectively. Further medium supplementation with FGF-2 stimulated early cell proliferation (>3 fold higher total DNA content per explant at day 10). This resulted in a marked increase in the size of the cultured explants and in the total area of the explant staining positive for safranin-O (from around 50% to 85%, (p<0.05)) after 6 weeks culture. The ability to generate significant quantities of neo-cartilage within a biocompatible and biodegradable matrix such as alginate, which lacks the immunogenicity of agarose, could open new pathways to utilizing such constructs in articular cartilage tissue engineering applications.     

Colocalization of noggin and bone morphogenetic protein-4 during fracture healing.

The regulation of callus formation during fracture repair involves the coordinate expression of growth factors and their receptors. This article describes the temporal and spatial expression of noggin gene, an antagonist to bone morphogenetic protein (BMP), during the fracture repair process. Noggin expression was examined by means of Northern blotting and in situ hybridization and compared with the expression pattern of BMP-4 in a model of fracture repair in adult mice. Expression levels of noggin messenger RNA (mRNA) were enhanced in the early phase of fracture callus formation. The localization of the noggin mRNA was similar to that of BMP-4 mRNA. Distinct noggin mRNA signals were located predominantly in cells lining the periosteum and the cortical endosteum near the fracture site at 2 days after fracture. At 5, 10, and 21 days after fracture, noggin mRNA was detected in the chondrocytes and osteoblasts in the newly formed callus. The pattern of localization was indistinguishable from that of BMP-4. These results suggest that the noggin/BMP-4 balance could be an important factor in the regulation of callus formation during fracture healing.     

Serum-free media for periosteal chondrogenesis in vitro.

Organ culture studies involving whole explants of periosteum have been useful for studying chondrogenesis, but to date the standard culture model for these explants has required the addition of fetal bovine serum to the media. Numerous investigators have succeeded in culturing chondrocytes and embryonic cells in serum-free conditions but there have been no studies focused on achieving a defined, serum-free media for culturing periosteal explants. The purpose of the present investigation was to determine if whole periosteal explants can be grown and produce cartilage in serum-free conditions, and to define the minimum media supplements that would be conducive to chondrogenesis. 321 periosteal explants were obtained from the medial proximal tibiae of 31 two month-old NZ white rabbits and cultured using a published agarose suspension organ culture model and DMEM for six weeks. The explants were cultured with and without fetal bovine serum or bovine serum albumin and exposed to transforming growth factor beta alone, a combination of growth factors we call ChondroMix (10 ng/ml transforming growth factor beta, 50 ng/ml basic fibroblast growth factor, and 5 microg/ml growth hormone), and/or ITS+ (2.08 microg/ml each of insulin, transferrin, and selenious acid, plus 1.78 microg/ml linoleic acid and 0.42 mg/ml BSA). Maximal chondrogenic stimulation in this study was observed with the combination of ChondroMix and ITS+. However, the minimal requirement to match or exceed the level of chondrogenic stimulation seen in the standard model (TGF-1 in 10% FBS) was achieved simply by the addition of 2.0 microg/ml insulin in 0.1% BSA-containing medium (p < 0.05). Therefore, based on our results, it would be reasonable to assume that insulin is the component in ITS+ responsible for the observed increase in total cartilage growth. Lower concentrations of insulin were not effective, suggesting that the observed effect of insulin requires activation of the IGF-1 receptor.     

The effect of bone morphogenetic protein-2 expression on the early fate of skeletal muscle-derived cells

The identification of bone morphogenetic proteins (BMPs) has stimulated intense interest in BMP delivery approaches. Ex vivo BMP-2 gene delivery has recently been described using skeletal muscle-derived cells. Skeletal muscle-derived cells, because of proven efficient transgene delivery and osteocompetence, represent an attractive cell population on which to base ex vivo BMP-2 gene delivery. However, the early in vivo fate of BMP-2-expressing muscle-derived cells is unknown. This study investigates the in vivo effects of BMP-2 secretion on skeletal muscle-derived cells in terms of cell survival and cell differentiation. The first experiment compared survival of BMP-2-expressing cells with control cells during the first 48 h after in vivo implantation. The results demonstrate that BMP-2 secretion did not adversely affect cell survival 8, 24, or 48 h after intramuscular implantation. The second experiment histologically compared the fate of BMP-2-expressing muscle-derived cells to the same cells not expressing BMP-2. The results show that BMP-2 expression prevented in vivo myogenic differentiation and promoted osteogenic differentiation of the transduced cells. This study further supports the existence of osteoprogenitor cells residing within skeletal muscle. Moreover, it is demonstrated that BMP-2 secretion does not adversely affect early cell survival of muscle-derived cells. These data are important for future investigations into BMP-2 gene delivery approaches to the musculoskeletal system.     

Clinical evaluation of recombinant human bone morphogenetic protein-2

Recombinant human bone morphogenetic protein-2 is an osteoinductive protein that plays a pivotal role in bone growth and regeneration. Several hundred studies were conducted in the past 7 years in numerous animal models to establish unequivocally the efficacy, safety, mechanism of action, pharmacokinetics, and surgical handling properties of recombinant human bone morphogenetic protein-2, building a solid foundation for clinical development programs. Pilot clinical trials have shown the feasibility and safety of recombinant human bone morphogenetic protein-2 treatment, and defined the effective dose for its use in open long bone fractures and for augmentation or preservation of the alveolar bone in the dental ridge. Prospective observational clinical studies helped define clinical efficacy end points, identify significant variables, and estimate appropriate population sample size for pivotal clinical trials. Pivotal clinical trials of recombinant human bone morphogenetic protein-2 are underway in patients with open tibial shaft fractures and in patients with a deficiency of the alveolar ridge.     

基因治疗对兔下颌骨牵引过程中骨形成蛋白-2表达的影响

目的 探索电穿孔介导的基因治疗对兔下颌骨牵引成骨过程中骨形成蛋白-2(bone morphogenetic protein-2,BMP2)表达的影响.方法 新西兰大白兔双侧下颌骨截骨,术后3d开始牵引,连续牵引7d后,将实验动物分为A、B、C、D、E5组,A、B、C组分别在牵引区注射2 μg(0.1 μg/μl)重组质粒pIRES-hVEGF165-hBMP2、pIRES-hBMP2、pIRES-hVEGF165.D组与E组分别注射相同剂量的空质粒(pIRES)和生理盐水(NS).各组分别于固定期第7、14、28天处死动物,取材行免疫组织化学检测BMP2的表达情况,并利用病理图像分析系统进行分析.结果 BMP2主要在肉芽组织中的炎细胞及新生幼稚骨小梁表面的细胞组织中表达.固定7d时表达最强烈,各时点基因治疗组明显强于对照组.结论 电脉冲介导的基因治疗能使BMP2在牵引区的表达增强和表达时限延长并促进细胞的分裂增殖与分化,促进牵引区细胞基质的形成和新骨生成.     

Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone

The need to improve bone healing permeates the discipline of orthopedic surgery. Bone morphogenetic proteins (BMPs) are capable of inducing ectopic and orthotopic bone formation. However, the ideal approach with which to deliver BMPs remains unknown. Gene therapy to deliver BMPs offers several theoretical advantages over implantation of a recombinant BMP protein, including persistent BMP delivery and eliminating the need for a foreign body carrier. A replication defective adenoviral vector was constructed to carry the rhBMP-2 gene (AdBMP-2). The direct in vivo gene therapy approach was applied in both immunodeficient and immunocompetent animals to produce intramuscular bone as early as 2 weeks following injection. Radiographic and histologic analysis revealed radiodense bone containing mature bone marrow elements. Adenovirus-mediated delivery of a marker gene (β-galactosidase) into control animals produced no bone but indicated the cells transduced with the AdBMP-2 vector. Furthermore, comparisons between immunodeficient and immunocompetent animals illustrated the magnitude and significance of the immune response. Gene therapy to deliver BMP-2 has innumerable potential clinical applications from bone defect healing to joint replacement prosthesis stabilization. This study is the first to establish the feasibility of in vivo gene therapy to deliver active BMP-2 and produce bone.     

Recombinant human bone morphogenetic protein-2 enhances osteotomy healing in glucocorticoid-treated rabbits.

The objectives of this study were to evaluate the effect of chronic prednisolone treatment on osteotomy healing in rabbits and to determine whether recombinant human bone morphogenetic protein-2 (rhBMP-2) would enhance healing in the presence of chronic glucocorticoid therapy. Forty-nine skeletally mature, male rabbits were injected with either prednisolone (n = 26; 0.35 mg/kg per day, three times a week) or saline (n = 23). After a 6-week pretreatment period, bilateral ulnar osteotomies were created surgically. One osteotomy was treated with rhBMP-2 (0.2 mg/ml of rhBMP-2, 40 microg of rhBMP-2 total) delivered on an absorbable collage sponge (ACS), whereas the contralateral osteotomy remained untreated. Prednisolone or saline treatment was continued until the rabbits were killed either 6 weeks or 8 weeks after creation of the osteotomy. Osteotomy healing was evaluated by radiography, peripheral quantitative computed tomography (pQCT), torsional biomechanics, and undecalcified histology. Because we observed similar responses to both prednisolone and rhBMP-2/ACS treatment in the 6-week and 8-week cohorts, the results from these time points were combined. Serum osteocalcin and vertebral trabecular bone density were lower in the prednisolone-treated rabbits. Prednisolone treatment dramatically inhibited osteotomy healing. In the untreated ulnas, callus area and torsional strength were 25% and 55% less, respectively, in the prednisolone-treated rabbits than in the saline group (p < 0.001 for both). rhBMP-2/ACS enhanced healing in both the prednisolone- and the saline-treated groups, although the effect was larger in the prednisolone-treated rabbits. In the prednisolone-treated rabbits, callus area and torsional strength were 40% and 165% greater (p < 0.001 for both), respectively, in osteotomies treated with rhBMP-2/ACS compared with the contralateral, untreated osteotomies. Histological evaluation confirmed that osteotomy healing was inhibited by prednisolone and accelerated by rhBMP-2/ACS. In summary, a single application of rhBMP-2/ACS counteracted the inhibition of osteotomy healing caused by prednisolone exposure. These results suggest that rhBMP-2/ACS may be a useful treatment for enhancing fracture healing in patients who are undergoing chronic glucocorticoid therapy.     

The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are potential therapeutic factors for degenerative discs, and BMP-12 does not have the osteogenic potential of BMP-2, making it better suited for intradiscal injection. However, no reports have compared the actions of BMP-2 and -12 on human annulus fibrosus (AF) and nucleus pulposus (NP) cells nor evaluated adenoviral-mediated gene therapy in human AF cells. PURPOSE: To evaluate and compare the effects of recombinant human (rh) BMP-2, rhBMP-12, and adenoviral BMP-12 (Ad-BMP-12) on nucleus pulposus and annulus fibrosis cell matrix protein synthesis. STUDY DESIGN: In vitro study using rhBMP-2 and -12 and adenoviral BMP-12 with human intervertebral disc (IVD) cells. METHODS: Human NP and AF IVD cells were isolated, maintained in monolayer, and incubated with BMP-2 or -12 for 2 days. AF and NP cells were transduced with Ad-BMP-12, pellets formed, and incubated for 6 days. Growth factor-treated cells were labelled with either 35-S or 3H-proline to assay matrix protein synthesis. RESULTS: rhBMP-2 increased NP proteoglycan, collagen, and noncollagen protein synthesis to 355%, 388%, and 234% of control. RhBMP-12 increased the same NP matrix proteins' synthesis to 140%, 143%, and 160% of control. Effects on AF matrix protein synthesis were minimal. Ad-BMP-12 significantly increased matrix protein synthesis and DNA content of AF and NP cells in pellet culture. NP synthesis of all matrix proteins and AF synthesis of proteoglycans was increased when the data were normalized to pellet DNA. AF synthesis of noncollagen protein and collagen was not modulated by Ad-BMP-12 if the data are normalized to pellet DNA content. CONCLUSIONS: Both rhBMP-2 and -12 increase human NP cell matrix protein synthesis while having minimal effects on AF cells. However, Ad-BMP-12 did increase matrix protein synthesis in both NP and AF cells, making it a potential therapy for enhancing matrix production in the IVD. These responses plus the proliferative action of Ad-BMP-12 seen in the current studies, and the lack of an osteogenic action noted in other studies justifies future studies to determine if gene therapy with BMP-12 could provide protective and/or reparative actions in degenerating discs.     

Use of recombinant human bone morphogenetic protein-2 in spine surgery

Bone morphogenetic proteins are osteoinductive factors which have gained popularity in orthopaedicsurgery and especially in spine surgery. The use of recombinant human bone morphogenetic protein-2 has been officially approved by the United States Food and Drug Administration only for single level anterior lumbar interbody fusion, nevertheless it is widely used by many surgeons with off-label indications. Despite advantages in bone formation, its use still remains a controversial issue and several complications have been described by authors who oppose their wide use.     

激素性股骨头坏死病程中骨形态发生蛋白-2的改变及其意义

目的　观察激素性股骨头坏死病程中骨形态发生蛋白 2 (BMP2 )的变化。方法　用醋酸氢化泼尼松诱发早期股骨头坏死动物模型 ,实验动物根据醋酸氢化泼尼松用量分为A组 (对照组 )、B组 (4mg/kg体重 )、C组 (8mg/kg体重 )、D组 (16mg/kg体重 ) ,肌注给药 ,每周 1次。取股(肱 )骨头标本行免疫组织化学染色。采用图像分析技术 ,测得平均染色面积百分比和平均吸光度。结果　A、B、C、D组阳性染色区染色强度依次减弱 ,平均吸光度值依次降低 ,且A组和D组间差异有非常显著性 (P <0 .0 1)。A组平均染色面积百分比 (股骨头 2 5 .5 8± 7.76、肱骨头 2 4.98± 8.2 3)明显高于D组 (股骨头 12 .30± 6 .6 0、肱骨头 12 .5 0± 8.0 3,P <0 .0 1)。各实验组平均吸光度和平均染色面积百分比随用药时间延长而降低。结论　在激素性股骨头坏死病程中 ,BMP2表达受抑制 ,抑制程度与用药时间和用药剂量正相关。