脂联素通过调节BMP信号通路促进骨髓干细胞成骨分化

目的 研究脂联素（adiponectin, ApN）对骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）成骨分化的作用,并探讨其可能的机制。方法 体外培养BMSCs,构建ApN过表达质粒及干扰质粒,转染至BMSCs中。将BMSCs随机分为5组：对照组、过表达组、过表达空载组、干扰组和干扰空载组。茜素红染色观察各组细胞钙化沉积。碱性磷酸酶（alkaline phosphatase, ALP）染色观察各组细胞成骨分化能力。qRT-PCR检测ApN受体、骨形态发生蛋白（bone morphogenetic protein, BMP）信号通路及成骨相关基因表达情况。结果 与对照组相比,过表达组BMSCs中钙化沉积和ALP阳性表达增多,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著升高（P<0.05）;干扰组BMSCs中钙化沉积和ALP阳性表达减少,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著降低（P<0.05）。结论 ApN可能通过上调BMP信号通路促进BMSCs成骨分化。     

Exogenous collagen-enhanced recruitment of mesenchymal stem cells during rabbit articular cartilage repair

Background Despite the well-known effect of type-I collagen in promoting cartilage repair, the mechanism still remains unknown. In this study we investigated this mechanism using a rabbit model of cartilage defects.

Animals and methods 5-mm-diameter full-thickness defects were created on both patellar grooves of 53 Japanese white rabbits (approximately 13 weeks old). The left defect was filled with collagen gel and the right defect was left empty. The rabbits were killed and examined morphometrically until the twenty-fourth postoperative week, by (1) evaluation of matrix production, (2) enumeration of the total number of cells engaged in cartilage repair, (3) enumeration of the proliferating cells, (4) localization of mesenchymal stem cells, and (v) localization of apoptotic cells.

Results We found that type-I collagen enhances cell recruitment, and thereby increases the number of proliferating cells. A considerable proportion of the proliferating cells were identified as bone marrow-derived mesenchymal stem cells. However, type-I collagen does not prevent the chondrocyte precursors from undergoing apoptotic disengagement from the chondrogenic lineage.

Interpretation Type-I collagen promotes cartilage repair by enhancing recruitment of bone marrowderived mesenchymal stem cells. Additional use of agent(s) that sustain mesenchymal stem cells along the chondrogenic path of differentiation may constitute an appropriate environment for cartilage repair.     

Tartrate-resistant acid phosphatase 5b (TRAP 5b) as a marker of osteoclast activity in the early phase after cementless total hip replacement

Background After total hip replacement, increased bone metabolism is seen. A local periprosthetic osteopenia can be measured by dual-energy X-ray absorptiometry (DXA), but it is still unkown whether biochemical markers can be used to monitor the local remodeling at an earlier stage.

Patients and methods In this prospective study we compared the biochemical markers tartrate-resistant acid phosphatase 5b (TRAP 5b), bone ALP, osteocalcin and CrossLaps with periprosthetic DXA in 17 consecutive patients after uncemented total hip replacement.

Results We found a highly significant early increase in TRAP 5b after 2 weeks and 6 weeks, which was followed by a densitometrically detectable decrease in bone mineral density after 26 weeks, especially in periprosthetic section Gruen zone 7. Bone ALP and osteocalcin levels as markers of osteoblast activity, and also Cross-Laps as a further marker of osteoclast activity, did not appear to allow any significant prediction of local bone remodeling.

Discussion Our findings show that TRAP 5b is a sensitive parameter for monitoring of osteoclast activity after cementless total hip replacement, and may predict local osteopenia.     

Effect of bone marrow grafting on the titanium porous-coated implant in bilateral total knee arthroplasty

Background and purpose Poor bone ingrowth into the porous coating of tibial components has been reported. We hypothesized that iliac marrow grafting might be useful to enhance bone ingrowth into a porous-coated implant. The first part of this study was to examine the presence of fibroblast colony-forming units (CFUF) containing osteogenic precursor cells in tibial bone marrow and iliac bone marrow. The second aim was to compare the clinical and radiographic results after bilateral total knee arthroplasty (TKA) with and without autologous bone marrow transplantation to the bone-implant interface.

Methods Simultaneous bilateral TKA was performed in 21 patients with osteoarthritis. Aspirated iliac bone marrow was transplanted to the interface of one randomly selected porous-coated tibial component in each patient, and contralateral knees served as controls. All of the 21 patients were followed for 5 years.

Results The average number of CFU-F was significantly lower in tibial marrow than in iliac marrow (p = 0.008). The final fluoroscopically-guided radiographs revealed a decrease in the number of knees with radiolucent lines after marrow grafting compared to those without grafting (p = 0.004).

Interpretation Iliac bone marrow is useful as a bone grafting material to enhance the biological fixation in porous-coated implants.     

微管仿生人工骨对犬骨髓基质细胞生物学行为的影响

[目的]研究不同微管结构仿生人T骨对犬骨髓基质细胞（BMSCs）增殖和分化的影响。[方法]仿生人工骨分为正交结构（A组）和同心结构（B组），以磷酸钙骨水泥（CPC）／重组人骨形成蛋白2（rhBMP-2）（C组）、单纯CPC（D组）为对照组：梯度密度离心法获取犬骨髓单个核细胞，取第3代与人工骨复合培养。通过荧光显微镜、扫描电镜观察细胞在人工骨表面附壁和生长情况；以MTT实验检测各组细胞增殖能力；测定碱性磷酸酶（ALP）检测BMSCs的成骨活性：[结果]细胞在各组人工骨表面黏附、生长，以A、B组数量较多；MTT法检测显示A、B、C组吸光度（A）值大于D组（P〈0．05）、A、B、C组A值相似（P〉0．05）；ALP测定显示ALP活性A、B组〉C组〉D组（P〈0．05），A、B组间差异无统计学意义（P〉0．05）。[结论]微管结构仿生人工骨与犬BMSCs有良好的生物相容性，是BMP的良好缓释载体，可以促进犬骨髓基质细胞向成骨方向分化.     

重组人骨形成蛋白2与骨诱导剂对大鼠骨髓间充质干细胞增殖分化的影响

目的观察重组人骨形成蛋白2(recombinant human bone morphogenetic protein2,rhBMP-2)与骨诱导剂对SD大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)的增殖与成骨作用。方法体外培养大鼠MSCs,分为实验组与对照组。对照组:不加rhBMP-2与骨诱导剂。实验组:骨诱导剂单独作用于SD大鼠MSCs(A组);rhBMP-2分别以浓度为10(B组)、50(C组)、100(D组)、200μg/L(E组)单独作用于SD大鼠MSCs;rhBMP-2分别以浓度为10(F组)、50(G组)、100(H组)、200μg/L(I组)联合骨诱导剂作用于SD大鼠MSCs。测定第3、6、9、12天的增殖状况(MTT法)、碱性磷酸酶(alkaline phosphatase,ALP)活性和骨钙素(osteocalcin,OC)水平。结果倒置相差显微镜观察SD大鼠MSCs原代培养时,细胞接种12h后即可贴壁;48h细胞成梭形,形似成纤维细胞;4d时细胞为多角形、纺锤形;6d时,成纤维细胞散在分布,少量呈集落样生长,为漩涡状、放射状排列;10d左右,细胞基本铺满瓶底,融合成片。传代细胞5~7d即可长满瓶底。各时间点A~I组均能显著促进MSCs成骨活性(ALP和OC)的表达。B~E组同时具有促进MSCs增殖作用,并呈浓度依赖性;F~I组增殖及ALP、OC含量均高于A~E组,比较差异有统计学意义(P<0.05)。结论rhBMP-2与骨诱导剂联合作用可在促进MSCs增殖的同时提高其成骨活性。     

Characterization of growth and osteogenic differentiation of rabbit bone marrow stromal cells

BACKGROUND: The rabbit is recognized as an excellent model to study the repair of bony defects with tissue engineered constructs. However, the use of rabbit bone marrow stromal cells (RBMSCs) has been limited despite the proven benefits of autologous BMSCs in the formation of bone. The purpose of this study was to characterize the growth and differentiation pattern of RBMSCs and their response to growth factors. MATERIAL AND METHODS: BMSCs were isolated from New Zealand White rabbits and cultured. Serial cell counts of parallel cultures were taken daily to determine cell growth. Response of RBMSCs to varying doses of recombinant human BMP-2 (rhBMP-2) and their time course was gauged by alkaline phosphatase (ALP) activity. The osteoblastic differentiation potential of RBMSCs in response to rhBMP-2 treatment was determined by evaluating the expression pattern of various genes involved with osteogensis using northern analysis. Von Kossa staining was performed to determine the effect of rhBMP-2 on the mineralization capabilities of RBMSCs. RESULTS: The growth rate of RBMSCs severely declined after first passage and this rate was further suppressed by TGF-beta1. The optimal dose response of rhBMP-2 was determined to be 50 ng/ml and its time course displayed increasing alkaline phosphatase activity over time. Two osteogenic markers, collagen I and osteopontin, were up regulated by rhBMP-2 treatment. Finally, the mineralization capability of RBMSCs was determined to be enhanced by rhBMP-2 treatment. CONCLUSION: Our work indicates that RBMSCs possess strong osteogenic potential and can be successfully applied toward bone tissue engineering in a rabbit model.     

No positive effects of OP-1 device on the incorporation of impacted graft materials after 8 weeks: a bone chamber study in goats

Background Bone morphogenetic proteins (BMPs) have the potential to improve clinical outcome after hip revision surgery by improving graft incorporation and implant fixation. However, impaction of cancellous bone grafts and TCP/HA bone substitute mixed with OP-1 device in a bone chamber in goats in a previous study led to reduced fibrous tissue ingrowth after 4 weeks. New bone formation was not promoted by OP-1. In the current study we examined whether this reduction represented a final loss of ingrowth or was just a delay, and whether the reduction can be overcome and ultimately results in a better late ingrowth.

Methods Bone chambers with impacted allografts and impacted TCP/HA granules mixed with 2 doses of OP-1 device were implanted in proximal medial goat tibias. Impacted allografts and TCP/HA not treated with OP-1 served as controls. After 8 weeks, the incorporation was evaluated using histology and histomorphometry.

Results Histology revealed evidence of bone graft incorporation, which proceeded in a similar way in both allografts and TCP/HA, with and without the addition of OP-1. After 8 weeks, no difference in bone ingrowth was found between the OP-1 groups and their controls. It was only in the allografts that the addition of OP-1 resulted in more fibrous tissue ingrowth.

Interpretation We conclude that the previously observed delay in fibrous tissue ingrowth can be only partially overcome.     

Wnt3a联合骨形态发生蛋白2对骨髓间充质干细胞增殖及成骨分化的影响

目的 观察经典Wnt/β-catenin以及骨形态发生蛋白(BMP)信号通路对大鼠骨髓来源的间充质干细胞(BMSCs)体外增殖以及向成骨方向分化的影响.方法 应用密度梯度离心联合贴壁筛选法分离培养骨髓间充质干细胞并分4组:对照组、Wnt组、BMP组、Wnt3a和BMP-2联合组,在不同时间点用细胞计数试剂盒-8(CCK-8)法检测细胞增殖活性,碱性磷酸酶(ALP)活性定量测定、Yon Kossa染色观察细胞向成骨分化和基质矿化程度.逆转录-聚合酶链反应(RT-PCR)检测成骨特异性标志物表达.结果 培养第7天,CCK-8测吸光度值联合组为0.845,Wnt组为0.738,明显高于对照组0.409(P＜0.05).培养第6天和第12天,ALP活性吸光度值联合组为63.8和144.3,BMP-2组为40.8和104.1,均明显高于对照组7.3和18.9(P ＜0.05).培养14 d后,联合组骨钙素mRNA表达量高于对照组,而Runx2及Osterix表达量与其他组比较增高不显著.培养3周后,Yon Kossa染色显示联合组钙结节数量及大小均高于对照组.结论 Wnt3a因子和骨形态发生蛋白-2联合诱导能有效地促进骨髓间充质干细胞增殖及向成骨方向分化.     

两种细胞因子联合应用对骨髓基质干细胞增殖和成骨活性的影响

目的 研究骨形态发生蛋白融合基因-4/7(BMP-4/7)和碱性成纤维细胞生长因子(bFGF)联合应用对体外培养兔骨髓基质干细胞(BMSCs)增殖和成骨活性的影响.方法 体外培养BMSCs,在第3代细胞培养液中加入不同浓度的BMP-4/7和bFGF,依据加入不同基因浓度组合的不同分为5个实验组(A组:80 ng/mL BMP-4/7,B组:80 ng/mL bFGF,C组:30 ng/mL BMP-4/7+30ng/mL bFGF,D组:50 ng/mL BMP-4/7+50 ng/mL bFGF,E组:80 ng/mL BMP-4/7+80 ng/mL bFGF)和对照组(不加任何因子),采用绘制生长曲线,甲基噻唑基四唑(MTT)比色法检测细胞增殖活力,碱性磷酸酶(ALP)和降钙素(OC)活性检测法比较各组间差异,观察不同浓度的BMP-4/7和bFGF联合应用对兔BMSCs增殖和成骨活性的影响. 结果 传代后第5天对照组个别单核细胞贴壁,呈长梭形;A组细胞增殖,呈旋涡状排列;B组细胞较为密集,部分融合成片;C组细胞呈集落式生长,生长旺盛;D组细胞生长密集,可见明显的钙结节;E组细胞密集,可见细胞性钙结节形成.各组OD值、ALP含量、OC含量随着作用时间的延长而增加,各组不同培养时间的OD值差异均有统计学意义(P<0.01);且C、D、E组均高于A、B组,差异均有统计学意义(P<0.05).C、D、E组内随着作用浓度的增加,细胞增殖及成骨活性增强,呈浓度依赖关系,差异均有统计学意义(P<0.05). 结论 合理的联合应用BMP-4/7和bFGF可促进BMSCs细胞增殖,促进成骨活性,两者对BMSCs有明显的协同增强效应.     

Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells.

Efficacious bone regeneration could revolutionize the clinical management of many bone and musculoskeletal disorders. Bone morphogenetic proteins (BMPs) can regulate the differentiation of mesenchymal stem cells into cartilage, bone, tendon/ligament, and fat lineages. Early data documented the osteogenic potential of rhBMP2 and rhBMP7/OP-1. However, prior to this work that summarized several of our recent studies, no comprehensive analysis had been undertaken to characterize relative osteogenic activity of all BMPs. Using recombinant adenoviruses expressing 14 BMPs, we have demonstrated that, besides BMP2 and BMP7, BMP6 and BMP9 exhibit the highest osteogenic activity both in vitro and in vivo. We further demonstrated that several BMPs may exert synergistic effect on osteogenic differentiation, and that osteogenic BMPs produce a distinct set of molecular fingerprints during osteogenic differentiation. The reported work should expand our current understanding of BMP functions during osteogenic differentiation. It is conceivable that osteogenic BMPs (i.e., BMP2, 4, 6, 7, and 9) may be used to formulate synergistic pairs among themselves and/or with other less osteogenic BMPs for efficacious bone regeneration in clinical settings.     

Autogenous regulation of a network of bone morphogenetic proteins (BMPs) mediates the osteogenic differentiation in murine marrow stromal cells

The expression patterns of (bone morphogenetic proteins) BMPs during fracture repair and pre-natal bone development suggest that these processes are regulated through the coordinated actions of multiple BMPs. Murine bone marrow stromal cells (MSCs) in culture provide a well recognized ex vivo system of mesenchymal stem cell differentiation in which the effects of BMPs can be examined. Studies were performed to determine if MSC differentiation is dependent on the endogenous expression of multiple BMPs and to characterize their interactions. MSCs were harvested from the bone marrow of tibiae and femora of 8 to 10-week-old male C57/B6 mice and prepared by standard methods. Osteogenic differentiation was assessed by histological assays, alkaline phosphatase enzyme activity and assays for the expression of multiple mRNAs for BMPs and osteogenic development. The role of autogenously expressed BMPs in controlling the osteogenic differentiation of marrow stromal cells in vitro was assessed in both gain-of-function and loss-of-function experiments. Gain of function experiments were carried out in the presence of exogenously added BMP-2 or -7 and loss-of-function experiments were carried out by BMP antagonism with noggin and BMP-2 antibody blockade. Osteogenic differentiation was concurrent with and proportional to increases in the expression of BMPs-2, -3, -4, -5, -6 and -8A. BMP antagonism with either noggin or BMP-2 antibody blockade inhibited osteogenic differentiation by 50% to 80%, respectively, and reduced the expression of endogenous levels of BMPs-2, -3, -5 and -8A. In contrast, antagonism induced the expression of BMP-4 and -6. The addition of rhBMP-2 or -7 enhanced osteogenic differentiation and produced a reciprocal expression profile in the endogenous BMPs expression as compared to BMP antagonism. BMP antagonism could be rescued through the competitive addition of rhBMP-2. These studies demonstrated that osteogenic differentiation was regulated by a complex network of multiple BMPs that showed selective increased and decreased expression during differentiation. They further demonstrated that BMP-2 was a central regulator in this network.     

改良复合磷酸钙骨水泥诱导分化成骨作用的体外研究

目的 通过体外实验观察兔骨髓基质干细胞(BMSCs)与改良复合磷酸钙骨水泥(BCPC)共培养的生物相容性及诱导成骨活性,探讨其可能的机制.方法 配制相同大小规格的BCPC及普通磷酸钙骨水泥(CPC)块,实验分为4组:空白对照组(不加载体)、普通CPC组、BCPC组、加BMP组(加BMP的BCPC).将各组材料与兔BMSCs复合培养,倒置显微镜下观察细胞生长情况,进行细胞计数,碱性磷酸酶(ALP)试剂盒检测细胞合成ALP活性变化,扫描电镜观察细胞在材料表面的生长及长入情况,real-time PCR检测成骨及成软骨相关基因mRNA表达情况并比较表达差别.结果 复合培养后倒置显微镜观察各组细胞与载体边缘接触良好,各组细胞增殖差异无统计学意义(P＞0.05);扫描电镜观察细胞在BCPC表面生长增殖良好,逐渐伸出伪足并长入材料孔隙;复合培养7 d后,BCPC组及加BMP组比CPC组和空白对照组表达更高的ALP活性,差异有统计学意义(P＜0.05);复合培养第10天,BCPC组及加BMP组分别与CPC组及空白对照组比较,其成骨及成软骨相关基因mRNA表达差异有统计学意义(P＜0.05).结论 配制的BCPC具有良好的生物相容性和一定的成骨诱导能力,其疏松的结构及更大的孔径适宜细胞长入材料内部.     

富血小板血浆与地塞米松对人骨髓基质干细胞成骨分化作用的对比研究

目的 通过系统评价富血小板血浆(PRP)与地塞米松(DEX)对人骨髓基质干细胞(BMSCs)成骨分化的影响,为PRP临床骨组织修复应用提供更为可靠的实验依据.方法 将体外培养的BMSCs分为单纯血清培养组(FCS组)、PRP诱导组和DEX组,通过相差显微镜观察碱性磷酸酶(ALP)染色、钙盐沉积染色,RT-PCR法检测碱性磷酸酶(ALP)、骨钙素(OC)、Ⅰ型胶原(Coll-Ⅰ)、骨连接蛋白(ON)、中心结合因子(Cbfα1)mRNA表达系统评价PRP的成骨分化能力. 结果 PRP抑制了BMSCs向三角形、多角形细胞转变,DEX则诱导BMSCs向三角形、多角形细胞转变;PRP抑制了ALP分泌,钙盐沉积;DEX增加了ALP分泌,促进钙化结节形成.与FCS组相比,DEX促进了ALP、OCmRNA表达,PRP抑制了ALP、OC mRNA表达;PRP、DEX对Coll-Ⅰ、ON、Cbfα1 mRNA表达均无影响.结论 在本实验条件下,PRP对人BMSCs体外成骨分化的直接作用是抑制效应;在体外PRP并不能代替DEX作为人BMSCs成骨分化的诱导因子.     

多孔双相陶瓷复合重组人骨形态发生蛋白-2和碱性成纤维细胞生长因子缓释微球的生物相容性

目的 观察复合重组人骨形态发生蛋白-2(rhBMP-2)和碱性成纤维细胞生长因子(bFGF)缓释微球的多孔双相陶瓷(BCP)组织相容性.方法 A组(rhBMP-2+bFGF缓释微球/BCP)、B组(单纯BCP)、C组(bFGF缓释微球/BCP)、D组(rhBMP-2缓释微球/BCP)和E组(不含BCP).其中bFGF、dlBMP的浓度各为5、15μg,材料均为5 mm×5 mm×1mm.采用体外细胞培养的方法测定细胞黏附能力、浸提液对于兔骨髓基质细胞(BMSCs)增殖和成骨分化的影响及体外诱导BMSCs成骨分化的能力.结果 细胞在材料上黏附、生长良好.各组噻唑蓝(MTT)吸光度值、碱性磷酸酶(ALP)值均随培养时间的延长而增大.A组与B、D和E组比较对MSC有显著的促增殖作用,但低于C组(P<0.05).A组ALP活性显著高于B、C和E组,但低于D组(P<0.05).结论 复合rhBMP-2和bFGF缓释微球的BCP具有良好的生物相容性.     

联合rhBMP-2与成骨细胞诱导骨髓基质细胞成骨分化的实验研究

目的探讨rhBMP-2和成骨细胞联合诱导骨髓基质细胞(BMSCs)的成骨分化能力。方法应用全骨髓贴壁法分离培养4周龄SD大鼠的BMSCs,通过改进酶消化法结合反复贴壁法培养1日龄SD乳鼠成骨细胞。实验分为4组:rhBMP-2诱导组、成骨细胞诱导组、rhBMP-2+成骨细胞诱导组、单纯培养液组。倒置相差显微镜及扫描电镜观察细胞生长情况;分别收集第3、6、9天细胞培养液上清液定性及定量测定碱性磷酸酶并进行统计学分析。结果 rhBMP-2诱导组、成骨细胞诱导组、rhBMP-2+成骨细胞诱导组均检测出成骨细胞生成,其中,rhBMP-2+成骨细胞诱导组碱性磷酸酶含量明显大于前两组,差异有统计学意义(P0.05)。结论rhBMP-2、成骨细胞、rhBMP-2联合成骨细胞均能提供成骨微环境,并在体外诱导BMSCs向成骨细胞分化,rhBMP-2联合成骨细胞对BMSCs的成骨分化具有更优的诱导能力。     

Implantation of perichondrium-derived chondrocytes in physeal defects of rabbit tibiae

Background Various cells and scaffolds have been used experimentally as interposition materials after physeal bar resection to treat partial physeal growth arrest. However, results of these cell therapies are unpredictable.

Methods We investigated the chondrogenic differentiation of rabbit perichondrial cells in vitro. We also investigated radiographic and histological changes after implantation of the perichondrium-derived chondrocytes, embedded in fibrin beads, into defects created in the medial aspect of the proximal tibial physis of 26 8-week-old New Zealand White rabbits. Physeal defects of the contralateral tibiae were left intact without any treatment, or were treated with fibrin beads not containing cells.

Results The perichondrial cells cultured in the alginate- fibrin beads were positive in immunohistochemical staining for S-100 protein and the extracellular area was stained with Safranin-O. RNase protection assay showed that the expression of type II collagen and aggrecan continued In the cell and bead-treated tibiae, varus deformity was reduced significantly and tibial length was maintained longer than that of the contralateral tibia. Histologically, cartilage clusters of varying size with intervening micro-bony trabeculae were found in the cell and bead-treated tibiae. The lateral, remaining physis in the untreated tibiae showed altered histology.

Interpretation Implantation of fibrin beads containing perichondrium-derived chondrocytes into the physeal defect helps to prevent angular deformation and shortening of long bone, but not completely. Physeal bony bar formation adversely affects the growth of the remaining physis from early stages onward.