Accelerating Bone Generation and Bone Mineralization in the Interparietal Sutures of Rats Using an rhBMP-2/ACS Composite after Rapid Expansion

This study aims to investigate the effects of rhBMP-2/ACS composite on bone regeneration and mineralization during expansion of the interparietal suture in rats. Forty 10-week-old Sprague-Dawley rats were divided into four groups (n=10). The first group (intact group) did not receive any intervention. The second group (expansion control group) received an expansion force of 60 g. The remaining two groups received an expansion force of 60 g and were implanted with an atelo-type I absorbable collagen sponge and rhBMP-2/ACS composite positioned on the suture beneath the periosteum. The relapse, relapse ratio, relevant bone remodelling, and calcium and osteocalcin contents were evaluated. Bone regeneration in the interparietal suture was estimated by the histological method. The osteocalcin content was measured by radioimmunoassay, and the calcium content was measured by atomic absorption spectrophotometry. Bone regeneration was more active in the suture after application of the expansion force compared with that of the suture without any intervention. Bone bridges formed in the rhBMP-2/collagen composite group. Both osteocalcin and calcium content were higher in the rhBMP-2/collagen composite group than in the other three groups (P<0.01). The relapse ratio in the rhBMP-2/collagen group was much lower than that in the other two expansion groups (P<0.01). RhBMP-2/ACS composite can promote bone regeneration and bone mineralization in the expanded suture and decrease the relapse ratio. Thus, the rhBMP-2/ACS composite may be therapeutically beneficial to the inhibition of relapse and shortening of the retention period during rapid expansion.     

Reconstruction of radial bone defect using gelatin sponge and a BMP-2 combination graft

Many bioactive molecules like recombinant human bone morphogenetic protein 2 (rhBMP-2) have been developed for mineralized bone grafts, for which proper scaffolds are necessary to successfully apply the bioactive molecules. In this study, we tested the osteogenic efficacy of rhBMP-2 produced in-house in combination with gelatin sponge as the scaffold carrier in a rabbit radial defect model. The efficacy of the rhBMP-2 was determined by alkaline phosphatase activity assay of C2C12 cells. Two groups of ten rabbits each were treated with rhBMP-2/gelatin sponge, or gelatin sponge only. At 4 weeks, rhBMP-2/gelatin sponge grafts showed more bone regeneration than gelatin sponge grafts, as determined by X-ray radiography, micro-computed tomography, and histological analyses. At 8 weeks, rhBMP-2/gelatin sponge grafts exerted much stronger osteogenic effects. The study demonstrates the improved osteogenic efficacy of the rhBMP-2/gelatin sponge grafts in a rabbit radial bone defect model acting as a bone-inductive material. [BMB Reports 2013; 46(6): 328-333]     

Evaluation of the bony repair in rat cranial defect using near infrared reflectance spectroscopy and discriminant analysis

We set out to determine whether near infrared reflectance spectroscopy (NIRS) combined with principal component analysis–linear discriminant analysis (LDA) or, variable selection techniques employing successive projection algorithm or genetic algorithm (GA) could evaluate the bone repair in cranial critical‐size (5 mm) defect after stimulation with collagen sponge scaffold and/or infrared low‐level laser therapy directly on the local. Forty‐five Winstar rats were divided into nine groups of five each, namely: group H – healthy, n = 5 (without treatment and without cranial critical‐size defect), (GI positive control – n = 5, 21 days or n = 5, 30 days) without treatment and with cranial critical‐size defect; (GII‐n = 5, 21 days or n = 5, 30 days) cranial critical‐size defect filled with collagen sponge scaffold; (GIII–n = 5, 21 days or n = 5, 30 days) cranial critical‐size defect submitted to low‐level laser therapy; (GIV–n = 5, 21 days or n = 5, 30 days) cranial critical‐size defect submitted to combined collagen sponge scaffold + low‐level laser therapy treatment. In relation to the histological analysis, the collagen sponge scaffold + low‐level laser therapy treatment group (GIV) 30 days showed the best result with the presence of secondary bone, immature bone (osteoid) and newly formed connective tissue (periosteum). GA–LDA model also successfully classified control class of the others classes. Thus, the results provided by the good‐quality classification model revealed the feasibility of NIRS for application to evaluation of the wound healing in rat cranial defect, thanks to the short analysis time of a few seconds and nondestructive advantages of NIRS as an alternative approach for bone repair purposes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1160–1168, 2017     

Effects of rhBMP-2 and osteopromotive membranes on experimental bone grafting.

A problem with using autogeneic bone for onlay grafting is its degree of incorporation and volumetric persistence. This study explored whether improved graft volumetric maintenance can be achieved by recombinant human bone morphogenetic protein-2 (rhBMP-2) and whether a combination of rhBMP-2 and osteopromotive membranes is advantageous in this respect. Unicortico-cancellous bone grafts were positioned below the temporal muscle bilaterally in 48 adult Lewis rats. The recipient bone was ground, and the grafted area was randomly treated according to one of eight different alternatives. Two doses of rhBMP-2 (4 microg/80 microl or 32 microg/80 microl buffer) in a collagenous carrier were tested, with or without coverage with expanded polytetrafluoroethylene membranes. As controls, membrane and carrier, alone or in combination, and onlay grafts only were used. The results were evaluated after 4 and 20 weeks by routine histologic examination and immunohistochemical labeling for various bone and cartilage matrix proteins. After 4 weeks, rhBMP-2-treated grafts showed complete integration, whereas for controls, only the membrane-alone group attained incorporation after 20 weeks. The combined treatment with high-dose rhBMP-2 and membrane demonstrated, as compared with the remaining rhBMP-2 groups, pronounced bone formation and less graft resorption, resulting in maintained or increased graft size. This was observed after 4 weeks, and the result remained at 20 weeks. Treatment with high-dose rhBMP-2 without membrane placement, in contrast, resulted in extensive resorption and graft size reduction at 20 weeks. Control groups showed less success in graft size persistence. It was concluded that a combination of high-dose rhBMP-2 and osteopromotive membranes had a synergistic effect, leading to rapid, complete graft integration and size maintenance. In the absence of a membrane, the protein seemed to accelerate the remodeling of the graft.     

Reinforced bioartificial dermis constructed with collagen threads

In this work, a novel type of composite scaffold was designed, which has the suitability of both high biocompatibility and strong mechanical properties, for use in bioartificial dermis applications. The reinforced scaffold consisted of a lyophilized collagen sponge formed around a cross-linked collagen meshwork with an average thread diameter of approximately 55 μm. Fibroblasts were cultured in the reinforced collagen sponge for 7 days, during which time the pores in the sponge became filled with cells that secreted extracellular matrix (ECM) to form a bioartificial dermis. Results of ultimate tensile strength (UTS) measurements and compression tests indicated that the bioartificial dermis formed around the reinforced collagen sponge showed about ten times the strength of the bioartificial dermis formed around a typical collagen sponge (1.5 ± 0.05 vs. 0.15 ± 0.05 and 2.5 ± 0.1 vs. 0.2 ± 0.08 MPa, respectively). As a result, reinforced collagen mesh improved mechanical properties and this technique will be possible to make stronger scaffolds, not only for artificial skin applications but also various artificial tissues, such as synthetic cartilage, bone, and blood vessels.     

Effects of bone morphogenetic protein-7 stimulation on osteoblasts cultured on different biomaterials

The objective of the present study was to investigate the effects of an in vitro stimulation of human osteoblasts by recombinant human bone morphogenetic protein-7 (rhBMP-7) on the collagen types and the quantity of the collagen cross-links synthesized in a three-dimensional culture on various biomaterials for bone replacement. Trabecular bone chips were harvested from human iliac crests, and cell cultures were established at standard conditions. One hundred and fifty nanograms per milliliter of rhBMP-7 was added. For the second passage a cell scraper was used to bring the cells into suspension, and 100 microl osteoblasts (at a density of 3.3 x 10(5)) were transferred onto nine blocks of either Bio-Oss, Tutoplast, or PepGen p-15. Blocks incubated with cells that were not treated with rhBMP-7 served as controls. Cell colonization of the biomaterials was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after a period of 2, 4, and 6 weeks. Throughout the experiment medium, supernatants were collected and collagen was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the collagen cross-link residues hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were quantified by HPLC. Within 4 weeks the cells became confluent on all of the studied biomaterials. All samples synthesized bone specific LP and collagen type I. However, in rhBMP-7-stimulated samples, the amount of HP and LP found was increased by 45% compared to non-stimulated samples. Cell proliferation and collagen synthesis was similar on the different biomaterials, but was consistently reduced in specimen not stimulated with rhBMP-7. In vitro stimulation of osteoblasts on Bio-Oss, Tutoplast, or PepGen p-15 with rhBMP-7 and subsequent transplantation of the constructs might lead to an enhanced osseointegration of the biomaterials in vivo.     

Ectopic bone formation in titanium mesh loaded with bone morphogenetic protein and coated with calcium phosphate

The osteoinductive properties of porous titanium fiber mesh, with or without a calcium phosphate coating and loaded with recombinant human bone morphogenic protein-2 (rhBMP-2) or rhBMP-2 and native bovine BMP (S-300) were investigated in a rat ectopic assay model. A total of 112 calcium phosphate-coated and 112 noncoated porous titanium implants, either loaded with rhBMP-2 and S-300 or loaded with rhBMP-2 alone, were subcutaneously placed in 56 Wistar-King rats. The rats were killed 5, 10, 20, and 40 days postoperatively, and the implants were retrieved.Histologic analysis demonstrated that all growth factor and carrier combinations induced ectopic cartilage and bone formation at 5 and 10 days, respectively. At 20 days, bone formation increased and was characterized by trabecular bone and bone marrow-like tissue. At 40 days, more lamellar bone and hemopoietic bone marrow-like tissue were present. At both times, more bone had been formed in calcium phosphate-coated implants than in noncoated samples. Further, in rhBMP-2 and S-300-loaded specimens, bone formation was higher than in rhBMP-2 only-loaded specimens. In rhBMP-2 only-loaded specimens, bone formation was mainly localized inside the mesh material, whereas in specimens loaded with both rhBMP-2 and S-300, the bone was localized inside and surrounding the titanium mesh. The histological findings were confirmed by calcium content and alkaline phosphatase activity measurements. In addition, all specimens showed osteocalcin expression as early as 5 days postoperatively.Our results show that the combination of titanium mesh with BMPs can induce ectopic bone formation and that this bone formation seems to be similar to "enchondral" ossification. In addition, a thin calcium phosphate coating can have a beneficial effect on the bone-inducing properties of a scaffold material. Finally, rhBMP-2 and native BMP act synergistically in ectopic bone induction.     

Effect of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2

FK506 is an immunosuppressant that is used widely in organ transplantation, and it has recently been recognized as effective for promoting the growth of bone grafts [J. Bone Miner. Res. 15 (2000) 1147]. In this study, we evaluated the influence of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2) using atelopeptide type I collagen as a carrier. We administered FK506 (1 mg/kg/day intramuscularly) on days -2 to 0, -2 to 7, and -2 to sacrifice. rhBMP-2 was implanted into the calf muscle of Wistar rats (thirty per group) and the implant was sampled on days 7, 14, and 21. Radiographic evaluation, histological examination, and biochemical analysis were performed. It was found that FK506 promoted the early stage of osteoinduction after short-term administration. However, long-term administration of this agent accelerated both bone formation and bone resorption. In order to use FK506 effectively for promoting bone growth, we must further examine the appropriate dose, method, and period of administration.     

Injectable Biocomposites for Bone Healing in Rabbit Femoral Condyle Defects

A novel biomimetic bone scaffold was successfully prepared in this study, which was composed of calcium sulfate hemihydrate (CSH), collagen and nano-hydroxyapatite (nHAC). CSH/nHAC was prepared and observed with scanning electron microscope and rhBMP-2 was introduced into CSH/nHAC. The released protein content from the scaffold was detected using high performance liquid chromatography at predetermined time interval. In vivo bone formation capacity was investigated by means of implanting the scaffolds with rhBMP-2 or without rhBMP-2 respectively into a critical size defect model in the femoral condyle of rabbit. The releasing character of rhBMP-2 was that an initial burst release (37.5%) was observed in the first day, followed by a sustained release and reached 100% at the end of day 20. The CSH/nHAC showed a gradual decrease in degradation with the content of nHAC increase. The results of X-rays, Micro CT and histological observation indicated that more new bone was formed in rhBMP-2 group. The results implied that this new injectable bone scaffold should be very promising for bone repair and has a great potential in bone tissue engineering.     

重组人骨形态发生蛋白-6的表达、纯化及其活性分析

利用RT-PCR从人胎盘组织中获取BMP-6成熟肽的cDNA 片段,并克隆到表达载体pET-15b中, 构建hBMP_6成熟肽的非融合蛋白表达质粒pET-BMP6,转化E.coli BL21（DE3）。IPTG 诱导4h后,工程菌高表达rhBMP-6成熟肽,在SDS-PAGE上出现预期的新蛋白带(≈15kD), 约占菌体总蛋白的10%,表达产物以包涵体形式存在。分离和纯化的包涵体溶解于8 mol/L尿素,在变性溶解状态下经阳离子交换层析,得到目的蛋白纯度达95%以上。再经稀释复性后,约80%的rhBMP-6形成同源二聚体。体外活性分析结果显示：rhBMP-6可以提高C3H10T1/2 细胞碱性磷酸酶活性及促进I型胶原、Osterix（Osx）和骨钙素（Osteocalcin）等成骨细胞表型转化标记基因mRNA的表达,证明制备的rhBMP_6具有诱导非骨源性细胞分化成为成骨细胞的作用。     

不同剂量rhBMP-2/CPC 体内骨诱导活性的比较研究*

目的:对比不同剂量rhBMP-2与多孔CPC复合后的诱导成骨效应,探讨与多孔CPC复合后的rhBMP-2的量效关系.方法:将0.5 mg/ml、1 mg/ml、2 mg/ml、3 mg/ml 4种不同剂量的rhBMP-2与多孔CPC材料复合后,植入36只小鼠双侧股部肌肉内,分别于术后1周、2周及4周取材,通过大体观察、组织学分析、形态计量学分析、荧光双标测定,观察4组诱导成骨情况.结果:植入1周,rhBMP-2与多孔CPC材料复合表现出了较明显的剂量依赖性,含有较多rhBMP-2的材料内诱导形成的骨组织也较多,但骨组织的增加并未随着rhBMP-2剂量的增加而连续递增,2 mg组和3 mg组新生骨组织含量无明显差异(P＞0.05).植入4周,新生骨组织向材料内部生长,但此时的新生骨组织面积较2周增加不显著(P＞0.05).0.5 mg组新生骨组织含量仍处于最低水平,而其它三组之间却无明显差异(P＞0.05).结论:在0.5 mg/ml-2.0 mg/ml剂量范围,与多孔CPC复合的rhBMP-2诱导成骨量与其剂量成正比,最佳剂量为2 mg/ml.     

Regulation of the binding of the BMP-2 growth factor with collagen by blood plasma fibronectin

The release kinetics of recombinant human bone morphogenic factor 2 (rhBMP-2) from collagen hydrogel in the presence of human blood plasma have been studied. The expulsion of rhBMP-2 from the collagen-BMP-2 complex by the competitive adhesion of collagen-binding proteins penetrating from plasma was firstly recognized. It was experimentally proven that that blood plasma fibronectin is the main collagen-binding protein, which is responsible for the controlled release of rhBMP-2. As a result, a new collagen hydrogel with the incorporation of fibronectin was created which retained rhBMP-2 for a twice longer period as compared to the ordinary collagen hydrogel. A distinctive feature of this new collagen-fibronectin matrix is the slow release of rhBMP-2 in the first three days which allows for the avoiding of adverse effects in clinics caused by the rapid release of large amounts of rhBMP-2 from collagen hydrogel.     

Prefabricated vascularized bone flap: a tissue transformation technique for bone reconstruction

In this study, an attempt was made to transform a muscle vascularized pedicle raised on host vessels into a vascularized bone flap, using recombinant human bone morphogenetic protein 2 (rhBMP-2). The purpose of this study was to produce new bone vascularized in nature to increase the survival rate of the subsequently grafted bone and to fabricate the newly formed bone into the desired shape. Silicone molds in the shape of a rat mandible were used to deliver rat bone matrix impregnated with or without rhBMP-2. A muscle pedicle the same size as the mold was raised on the saphenous vessels in the rat thigh and then sandwiched in the center of the silicone molds. The molds were sliced in half and each section was filled with rat bone matrix that was impregnated either with 25 microg of rhBMP-2 for the experimental group or with diluting material alone for the control group. The sandwiched flaps were then secured by tying them to the adjacent muscles and were harvested at 2 and 4 weeks after surgery. Three and six rats were used in the control and experimental groups at each time point, respectively. Bone formation was assessed in the ex vivo specimens by macroscopic, radiologic, and histologic evaluation. Macroscopically, the continuation of the vascular pedicle was clearly visible for both the control and experimental muscle flaps. However, no evidence of muscle-tissue transformation was observed in the control flaps, whereas all the flaps treated with rhBMP-2 produced new bone that replicated the shape of the mold exactly and had saphenous vessels supplying the newly formed bone. This study demonstrates that this experimental model has the potential to be therapeutically applied for effective bone reconstruction.     

Changes with age and the effect of recombinant human BMP-2 on proteoglycan and collagen gene expression in rabbit anulus fibrosus cells

In order to compare the difference between young and old intervertebral disc cells and theirresponsiveness to recombinant human bone morphogenetic protein-2 (rhBMP-2),disc cells were isolatedfrom the anulus fibrosus (AF) and transition zones of lumbar discs from eight old and eight young NewZealand white rabbits.Compared with the ceils from the young rabbits,cells from old rabbits respond less torhBMP-2 treatment with respect to sulfated-glycosaminoglycan (sGAG) synthesis and aggrecan geneexpression.But in collagen Ⅰ and collagen Ⅱ gene expressions,there are no significant differences betweenthe old and the young.When comparing sGAG content,aggrecan,and collagen Ⅱ gene expression of the oldAF cells after rhBMP-2 treatment with that of the young AF cells without rhBMP-2 treatment,the old AFcells with rhBMP-2 treatment have a greater capacity to synthesize sGAG bound in the cells and to releasesGAG in the media,as well as to express aggrecan and collagen Ⅱ gene.It can be concluded that old AF cellsafter rhBMP-2 treatment have a greater capacity to synthesize sGAG and express aggrecan and collagen Ⅱ ascompared to young AF cells without rhBMP-2 treatment.Thus rhBMP-2 can reverse the decline in theanabolic capacity of the disc cells with ageing.So it seems that rhBMP-2 has potential for use as an agent toretard a key component of disc degeneration and loss of disc matrix.     

Non-functional Fas ligand increases the formation of cartilage early in the endochondral bone induction by rhBMP-2

It has previously been shown that mice with a defect in Fas ligand-mediated apoptosis have an enhancement of ectopic bone formation. We investigated the expression of bone-related markers--alkaline phosphatase, collagen, bone sialoprotein, osteocalcin, osteopontin, and bone morphogenetic proteins (BMP) -2, -4, and -7; and cytokines interleukin-1alpha (IL-1), IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) in ectopic new bone induced by recombinant human (rh) BMP-2 in mice without functional Fas-ligand (gld mice). At day 6 after rhBMP-2 implantation, gld mice formed more cartilage and mesenchyme compared with their wild type littermates. At later stages, gld mice did not differ from the control mice in the volume of newly formed tissue, expressing higher level of BMP genes and lower levels of genes involved in osteoblast maturation--bone sialoprotein and osteopontin. Differences in the levels of expression of IL-1alpha and TNF-alpha were observed only at day 12 after rhBMP-2 implantation. These results suggest that gld mice have an increased recruitment of cells of mesenchymal origin and an abnormal pattern of differentiation and maturation of the newly formed mesenchymal tissues.     

Recombinant human bone morphogenetic protein-2 promotes Indian hedgehog-mediated osteo-chondrogenic differentiation of a human chondrocytic cell line in vivo and in vitro

We examined osteo-chondrogenic differentiation of a human chondrocytic cell line (USAC) by rhBMP-2 in vivo and in vitro. USAC was established from a transplanted tumor to athymic mouse derived from an osteogenic sarcoma of the mandible. USAC usually shows chondrocytic phenotypes in vivo and in vitro. rhBMP-2 up-regulated not only the mRNA expression of types II and X collagen, but also the mRNA expression of osteocalcin and Cbfa1 in USAC cells in vitro. In vivo experimental cartilaginous tissue formation was prominent in the chamber with rhBMP-2 when compared with the chamber without rhBMP-2. USAC cells implanted with rhBMP-2 often formed osteoid-like tissues surrounded by osteoblastic cells positive for type I collagen. rhBMP up-regulated Ihh, and the expression of Ihh was well correlated with osteo-chondrogenic cell differentiation. These results suggest that rhBMP-2 promotes chondrogenesis and also induces osteogenic differentiation of USAC cells in vivo and in vitro through up-regulation of Ihh.     

重组人骨形态发生蛋白-7与脱钙骨基质复合物双重骨诱导作用的生物学评价

将不同剂量的重组人骨形态发生蛋白-7(rhBMP-7)与脱钙骨基质(DBM)分别复合后,植入小鼠股部内侧肌间隙,三周后取材,通过组织学检查、碱性磷酸酶(ALP)及钙含量的测定比较各组的骨诱导活性。结果显示,三组复合物均有骨组织生成,中、高剂量组可见骨小梁、板层骨和原始骨髓腔,血管和骨髓丰富;低剂量组的成骨量明显少于中、高剂量组,且新骨的成熟度低于其他两组,DBM少部分吸收;单独植入rhBMP-7组有编织骨形成;而DBM组可见成骨细胞的聚集。rhBMP-7/DBM复合组在ALP和Ca含量水平上与同等剂量的两对照组相比均有显著性差异(P<0.01),而rhBMP-7三种剂量之间均有显著性差异(P<0.01)。这充分说明DBM作为rhBMP-7的合适载体,具有缓释作用,且二者复合可起到双重骨诱导活性;而且rhBMP-7的骨诱导活性具有一定的剂量依赖性。     

Bone morphogenetic protein-2 binds as multilayers to a collagen delivery matrix: an equilibrium thermodynamic analysis

Recombinant human bone morphogenetic protein-2 (rhBMP-2) promotes bone growth but must be retained at the delivery site for optimal efficacy in vivo. rhBMP-2 release from a collagen-based matrix has shown favorable pharmacokinetics. The present study assessed binding affinity and binding saturation of rhBMP-2 to a collagen matrix as a function of solution and rhBMP-2 isoform variables. Results indicate that rhBMP-2 binds to the collagen matrix with affinities on the order of 10(3) to 10(4) M(-1). Maximum binding, nu, was primarily a function of pH for heterogeneous rhBMP-2 and the extended (T(266)/T(266)) isoform. However, binding saturation of the 相似文献   

Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2

BACKGROUND: Adipose tissue has been demonstrated to contain a population of progenitor cells that can differentiate into bone and cartilage. Studies have suggested that adipose-derived adult stem (ADAS) cells can be induced to differentiate into chondrocytes by transforming growth factor-beta (TGF-beta). In this study, we examined whether bone morphogenetic protein-2 (BMP-2), as a member of the TGF-beta superfamily, could regulate ADAS cells to differentiate into a chondrolineage. METHODS: ADAS cells were isolated and induced by rhBMP-2. These cells were cultured in pellets for 2 weeks, and the chondrogenic phenotype was observed in vitro and in vivo. ADAS cells cultured without BMP-2 were used as controls. RESULTS: After 2 weeks of culture, the differentiated ADAS cells reacted positively to Alcian blue and collagen II, and the content of collagen II protein was obviously up-regulated at day 14. Glycosaminoglycan (GAG) content gradually increased from day 2 to day 14 (P < 0.05). However, H&E staining and collagen II expression were weak, and there was a little collagen II protein and GAG detected in the control group. Additionally, the pellets of ADAS cells induced by rhBMP-2 were transplanted into BALB/C nude mice and formed cartilage lacuna at week 8 in vivo. DISCUSSION: These data demonstrate that rhBMP-2 induce ADAS cells to differentiate into chondrocytes in vitro and in vivo. This is useful for basic and clinical studies aimed at repairing cartilage damage. But in a control group, ADAS cells tended towards differentiation into chondrocytes, which was affected by ITS. We will be exploring the mechanism further.     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.