骨组织工程中人骨髓间质干细胞动态种植和静态种植的比较研究

分别采用动态种植(旋转烧瓶法)和静态种植方法种植人骨髓间质干细胞,于种植后的两周内测定细胞-载体构件中DNA含量;利用组织学光镜和扫描电镜观察细胞的分布情况;运用荧光标记RT-PCR技术测定相关成骨基因的表达。构件中DNA含量测定表明,对于静态种植,当初始种植密度为每载体400×103(8.9×104/mm3)时,DNA的含量达到最高;在此基础上提高初始种植密度,并不能进一步提高构件DNA含量。光镜和扫描电镜观察可见动态种植后人骨髓间质干细胞在载体中的分布相对均匀,静态种植后细胞在载体中出现聚集现象;荧光标记RT-PCR证明,体外构件培养两周后,动态种植后的细胞-载体构件中有较多的成骨基因表达。提示人骨髓间质干细胞的静态种植效率较低;动态种植是一种优于静态种植的可行方法。     

In Vivo Ectopic Implantation Model to Assess Human Mesenchymal Progenitor Cell Potential

Clinical interest on human mesenchymal progenitor cells (hMPC) relies on their potential applicability in cell-based therapies. An in vitro characterization is usually performed in order to define MPC potency. However, in vitro predictions not always correlate with in vivo results and thus there is no consensus in how to really assess cell potency. Our goal was to provide an in vivo testing method to define cell behavior before therapeutic usage, especially for bone tissue engineering applications. In this context, we wondered whether bone marrow stromal cells (hBMSC) would proceed in an osteogenic microenvironment. Based on previous approaches, we developed a fibrin/ceramic/BMP-2/hBMSCs compound. We implanted the compound during only 2 weeks in NOD-SCID mice, either orthotopically to assess its osteoinductive property or subcutaneously to analyze its adequacy as a cell potency testing method. Using fluorescent cell labeling and immunohistochemistry techniques, we could ascertain cell differentiation to bone, bone marrow, cartilage, adipocyte and fibrous tissue. We observed differences in cell potential among different batches of hBMSCs, which did not strictly correlate with in vitro analyses. Our data indicate that the method we have developed is reliable, rapid and reproducible to define cell potency, and may be useful for testing cells destined to bone tissue engineering purposes. Additionally, results obtained with hMPCs from other sources indicate that our method is suitable for testing any potentially implantable mesenchymal cell. Finally, we propose that this model could successfully be employed for bone marrow niche and bone tumor studies.     

Matrix-mediated retention of in vitro osteogenic differentiation potential and in vivo bone-forming capacity by human adult bone marrow-derived mesenchymal stem cells during ex vivo expansion

Mesenchymal stem cells (MSCs) represent an attractive cell source for tissue engineering applications, since they are readily isolated from adult bone marrow and have the ability to differentiate along multiple mesenchymal lineages, including osteogenic. Currently, utilization of MSCs for bone tissue engineering is limited because of the attenuation of their osteogenic differentiation potential and in vivo bone-forming capacity following ex vivo expansion on conventional tissue culture plastic (TCP). Previously, we demonstrated that a denatured type I collagen (DC) matrix promotes the maintenance of MSC in vitro osteogenic differentiation potential during ex vivo expansion in contrast to TCP. In this study, we further demonstrate that the maintenance of MSC osteogenic differentiation potential is primarily due to the ability of DC matrix to influence the retention of early passage osteogenic functions in late passage (LP) cells during ex vivo expansion, in contrast to solely enhancing attenuated LP cellular functions during osteogenic differentiation. Serum-associated factors played a significant role in influencing the retention of MSC osteogenic differentiation potential during expansion on the DC matrix. Significantly, the results show that although LP cells expanded ex vivo on TCP highly attentuate their in vivo bone-forming capacity, the expansion of MSCs on DC matrix preserves this ability as determined by histological, histomorphometric, and bone mineral density evaluations of MSC-seeded hydroxyapatite/tricalcium phosphate scaffolds following an 8-week implantation period within a heterotopic muscle pouch model. These findings provide further insight into the importance of matrix-mediated effects on MSC function and selective factors important in this process.     

Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells

Mesenchymal stem cells (MSCs) from human adult bone marrow (haMSCs) represent a promising source for bone tissue engineering. However, their low frequencies and limited proliferation restrict their clinical utility. Alternative postnatal, perinatal, and fetal sources of MSCs appear to have different osteogenic capacities, but have not been systematically compared with haMSCs. We investigated the proliferative and osteogenic potential of MSCs from human fetal bone marrow (hfMSCs), human umbilical cord (hUCMSCs), and human adult adipose tissue (hATMSCs), and haMSCs, both in monolayer cultures and after loading into three-dimensional polycaprolactone-tricalcium-phosphate scaffolds.Although all MSCs had comparable immunophenotypes, only hfMSCs and hUCMSCs were positive for the embryonic pluripotency markers Oct-4 and Nanog. hfMSCs expressed the lowest HLA-I level (55% versus 95%-99%) and the highest Stro-1 level (51% versus 10%-27%), and had the greatest colony-forming unit-fibroblast capacity (1.6x-2.0x; p < .01) and fastest doubling time (32 versus 54-111 hours; p < .01). hfMSCs had the greatest osteogenic capacity, as assessed by von-Kossa staining, alkaline phosphatase activity (5.1x-12.4x; p < .01), calcium deposition (1.6x-2.7x in monolayer and 1.6x-5.0x in scaffold culture; p < .01), calcium visualized on micro-computed tomography (3.9x17.6x; p < .01) and scanning electron microscopy, and osteogenic gene induction. Two months after implantation of cellular scaffolds in immunodeficient mice, hfMSCs resulted in the most robust mineralization (1.8x-13.3x; p < .01).The ontological and anatomical origins of MSCs have profound influences on the proliferative and osteogenic capacity of MSCs. hfMSCs had the most proliferative and osteogenic capacity of the MSC sources, as well as being the least immunogenic, suggesting they are superior candidates for bone tissue engineering.     

Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation

To engineer bone tissue, mechanosensitive cells are needed that are able to perform bone cell-specific functions, such as (re)modeling of bone tissue. In vivo, local bone mass and architecture are affected by mechanical loading, which is thought to provoke a cellular response via loading-induced flow of interstitial fluid. Adipose tissue is an easily accessible source of mesenchymal stem cells for bone tissue engineering, and is available in abundant amounts compared with bone marrow. We studied whether adipose tissue-derived mesenchymal stem cells (AT-MSCs) are responsive to mechanical loading by pulsating fluid flow (PFF) on osteogenic stimulation in vitro. We found that ATMSCs show a bone cell-like response to fluid shear stress as a result of PFF after the stimulation of osteogenic differentiation by 1,25-dihydroxyvitamin D3. PFF increased nitric oxide production, as well as upregulated cyclooxygenase-2, but not cyclooxygenase-1, gene expression in osteogenically stimulated AT-MSCs. These data suggest that AT-MSCs acquire bone cell-like responsiveness to pulsating fluid shear stress on 1,25-dihydroxyvitamin D3-induced osteogenic differentiation. ATMSCs might be able to perform bone cell-specific functions during bone (re)modeling in vivo and, therefore, provide a promising new tool for bone tissue engineering.     

Matrix-mediated retention of osteogenic differentiation potential by human adult bone marrow stromal cells during ex vivo expansion

During prolonged cultivation ex vivo, adult bone marrow stromal stem cells (BMSCs) undergo two probably interdependent processes, replicative aging and a decline in differentiation potential. Recently, our results with primary human fibroblasts indicated that growth on denatured collagen (DC) matrix results in the reduction of the rate of cellular aging. The present study has been undertaken to test whether the growth of human BMSCs under the same conditions would translate into preservation of cellular aging-attenuated functions, such as the ability to express HSP70 in response to stress as well as of osteogenic differentiation potential. We report here that growth of BMSCs on a DC matrix versus tissue culture polystyrene significantly reduced one of the main manifestations of cellular aging, the attenuation of the ability to express a major protective stress response component, HSP70, increased the proliferation capacity of ex vivo expanded BMSCs, reduced the rate of morphological changes, and resulted in a dramatic increase in the retention of the potential to express osteogenic-specific functions and markers upon treatment with osteogenic stimulants. BMSCs are a promising and increasingly important cell source for tissue engineering as well as cell and gene therapeutic strategies. For use of BMSCs in these applications, ex vivo expansion is necessary to obtain a sufficient, therapeutically useful, number of cells; however, this results in the loss of differentiation potential. This problem is especially acute in older patients where more extensive in vitro expansion of smaller number of stem/progenitor cells is needed. The finding that growth on certain biomaterials preserves aging-attenuated functions, enhances proliferation capacity, and maintains differentiation potential of BMSCs indicates a promising approach to address this problem.     

Genetic Modification of Stem Cells to Enhance Bone Repair

Orthopaedic surgeons are often faced with difficult bone loss problems. Conventional bone grafting is usually accomplished with autogenous iliac crest bone graft that provides osteogenic cells, osteoinductive growth factors, and an osteoconductive matrix. Cadaveric bone allograft and bone graft substitutes are inferior to autogenous bone graft because they fail to supply osteogenic cells or a significant amount of osteoinductive growth factors. Recombinant growth factors such as bone morphogenetic protein-2 and osteogenic protein-1 are currently in clinical use but these proteins require supraphysiologic dosing and considerable expense while failing to provide a sustained osteoinductive signal at the implantation site. Mesenchymal stem cells capable of differentiating into mesodermal tissues have been isolated and expanded in culture from several different sources including bone marrow, adipose tissue, and muscle. In the presence of appropriate growth factors these cells can differentiate into osteoblast lineage cells that will form bone in vitro and in vivo. Recent attention has focused on genetic modification of mesenchymal stem cells to both produce and respond to osteogenic growth factors with the goal of developing a tissue engineering strategy for bone repair. This review examines the current potential and limitations of these cellular systems for bone repair.     

Ectopic osteogenesis of hBMP-2 gene-transduced human bone mesenchymal stem cells/BCB

We determined the feasibility of using scaffolds of adenoviral human BMP2 gene (AdBMP2)-modified human bone marrow mesenchymal stem cells (hBMSCs) and antigen-free bovine cancellous bone (BCB) to construct bone tissue. hMSCs were infected with AdBMP-2. Expression of BMP-2 and alkaline phosphatase confirmed successful secretion of active BMP-2. The osteogenic capability of a composite of AdBMP2-modified hMSCs with BCB was evaluated in athymic mice (group A). BCB (group B), hMSCs/BCB (group C), adenoviral β‐galactosidase genes (Adβgal)-transfected hMSCs/BCB (group D) were controls. Formation of bone tissue was assessed by histological methods 4 weeks and 8 weeks after implantation. Implanted cells were identified by human Y-chromosome-specific fluorescence in-situ hybridization (FISH). hMSCs differentiated into osteogenic cells, and bone formation was observed. Obvious bone formation was not noted at any time point in control groups. We hypothesize that the described method is a promising method for bone regeneration.     

In vitro concurrent endothelial and osteogenic commitment of adipose-derived stem cells and their genomical analyses through comparative genomic hybridization array: novel strategies to increase the successful engraftment of tissue-engineered bone grafts

In the field of tissue engineering, adult stem cells are increasingly recognized as an important tool for in vitro reconstructed tissue-engineered grafts. In the world of cell therapies, undoubtedly, mesenchymal stem cells from bone marrow or adipose tissue are the most promising progenitors for tissue engineering applications. In this setting, adipose-derived stem cells (ASCs) are generally similar to those derived from bone marrow and are most conveniently extracted from tissue removed by elective cosmetic liposuction procedures; they also show a great potential for endothelization. The aim of the present work was to investigate how the cocommitment into a vascular and bone phenotype of ASCs could be a useful tool for improving the in vitro and in vivo reconstruction of a vascularized bone graft. Human ASCs obtained from abdominoplasty procedures were loaded in a hydroxyapatite clinical-grade scaffold, codifferentiated, and tested for proliferation, cell distribution, and osteogenic and vasculogenic gene expression. The chromosomal stability of the cultures was investigated using the comparative genomic hybridization array for 3D cultures. ASC adhesion, distribution, proliferation, and gene expression not only demonstrated a full osteogenic and vasculogenic commitment in vitro and in vivo, but also showed that endothelization strongly improves their osteogenic commitment. In the end, genetic analyses confirmed that no genomical alteration in long-term in vitro culture of ASCs in 3D scaffolds occurs.     

Multilineage cells from human adipose tissue: implications for cell-based therapies

Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.     

The derivation of mesenchymal stem cells from human embryonic stem cells

Human embryonic stem cells (hESCs) hold promise for tissue regeneration therapies by providing a potentially unlimited source of cells capable of undergoing differentiation into specified cell types. Several preclinical studies and a few clinical studies use human bone marrow stromal cells (hBMSCs) to treat skeletal diseases and repair damaged tissue. However, hBMSCs have limited proliferation and differentiation capacity, suggesting that an alternate cell source is desirable, and hESCs may serve this purpose. Here we describe a protocol for the reproducible derivation of mesenchymal stem cells from hESCs (hES-MSCs). The hES-MSCs have a similar immunophenotype to hBMSCs, specifically they are CD73+, STRO-1+ and CD45-, and are karyotypically stable. The derived hES-MSCs are also capable of differentiating into osteoblasts and adipocytes. When the hES-MSCs were genetically modified with the lineage-specific Col2.3-GFP lentivirus and cultured in osteogenic medium, increased GFP expression was detected over time, indicating the hES-MSCs have the capacity to differentiate down the osteogenic lineage and had progressed toward a mature osteoblast phenotype.     

Review: gene- and stem cell-based therapeutics for bone regeneration and repair

Many clinical conditions require regeneration or implantation of bone. This is one focus shared by neurosurgery and orthopedics. Current therapeutic options (bone grafting and protein-based therapy) do not provide satisfying solutions to the problem of massive bone defects. In the past few years, gene- and stem cell-based therapy has been extensively studied to achieve a viable alternative to current solutions offered by modern medicine for bone-loss repair. The use of adult stem cells for bone regeneration has gained much focus. This unique population of multipotential cells has been isolated from various sources, including bone marrow, adipose, and muscle tissues. Genetic engineering of adult stem cells with potent osteogenic genes has led to fracture repair and rapid bone formation in vivo. It is hypothesized that these genetically modified cells exert both an autocrine and a paracrine effects on host stem cells, leading to an enhanced osteogenic effect. The use of direct gene delivery has also shown much promise for in vivo bone repair. Several viral and nonviral methods have been used to achieve substantial bone tissue formation in various sites in animal models. To advance these platforms to the clinical setting, it will be mandatory to overcome specific hurdles, such as control over transgene expression, viral vector toxicity, and prolonged culture periods of therapeutic stem cells. This review covers a prospect of cell and gene therapy for bone repair as well as some very recent advancements in stem cell isolation, genetic engineering, and exogenous control of transgene expression.     

In vitro differentiation of human mesenchymal stem cells on Ti6Al4V surfaces

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.     

Spatial immobilization of bone morphogenetic protein-4 in a collagen-PLGA hybrid scaffold for enhanced osteoinductivity

The introduction of bioactive molecules into three-dimensional porous scaffolds to mimic the in vivo microenvironment is a promising strategy for tissue engineering and stem cell research. In this study, bone morphogenetic protein-4 (BMP4) was spatially immobilized in a collagen-PLGA hybrid scaffold with a fusion BMP4 composed of an additional collagen-binding domain derived from fibronectin (CBD-BMP4). CBD-BMP4 bound to the collagen-PLGA hybrid scaffold and the BMP4-immobilized hybrid scaffold supported cell adhesion and proliferation. The osteogenic induction effect of the immobilized CBD-BMP4 was investigated with three-dimensional culture of human bone marrow-derived mesenchymal stem cells in the BMP4-immobilized collagen-PLGA hybrid scaffold. The in vivo implantation experiment demonstrated that the immobilized CBD-BMP4 maintained its osteoinductive activity, being capable of up-regulating osteogenic gene expression and biomineralization. The strong osteoinductivity of the BMP4-immobilized scaffold suggests it should be useful for bone tissue engineering, stem cell function manipulation and bone substitutes.     

人Muse细胞体外诱导为神经前体细胞的研究

探讨成人骨髓来源的Muse细胞体外诱导为神经前体细胞的技术方法。从健康成人骨髓中分离出骨髓基质细胞（hBMSCs）,利用流式细胞分选技术从中筛选出Muse细胞,采取免疫荧光细胞化学染色和qPCR技术鉴定Muse细胞的多能干细胞特性;通过神经诱导培养基体外诱导Muse细胞分化为神经前体细胞（Muse-NPCs）,采用免疫荧光细胞化学染色和qPCR技术鉴定其神经干细胞标志物表达情况。从hBMSCs中分选出约0.58%的Muse细胞,Muse细胞表达多能干细胞标志物SSEA-3、Nanog和Oct4,其mRNA表达水平均高于hBMSCs（P＜0.01）;诱导后的Muse-NPCs表达神经干细胞标志物Nestin、βIII-tubulin,其mRNA表达水平均高于hBMSCs及Muse 细胞（P＜0.01）。从成人骨髓中成功分离出具有多能干细胞特性的Muse细胞,用体外诱导形成Muse-NPCs,可为今后细胞治疗修复神经损伤提供新的种子细胞。     

Stem cell technology and bioceramics: from cell to gene engineering

Mesenchymal stem cells reside in bone marrow and, when these cells are incorporated into porous ceramics, the composites exhibit osteo-chondrogenic phenotypic expression in ectopic (subcutaneous and intramuscular) or orthotopic sites. The expressional cascade is dependent upon the material properties of the delivery vehicle. Bioactive ceramics provide a suitable substrate for the attachment of the cells. This is followed by osteogenic differentiation directly on the surface of the ceramic, which results in bone bonding. Nonbioactive materials show neither surface-dependent cell differentiation nor bone bonding. The number of mesenchymal stem cells in fresh adult bone marrow is small, about one per one-hundred-thousand nucleated cells, and decreases with donor age. In vitro cell culture technology can be used to mitotically expand these cells without the loss of their developmental potency regardless of donor age. The implanted composite of porous ceramic and culture-expanded mesenchymal stem cells exhibits in vivo osteo-chondrogenic differentiation. In certain culture conditions, these stem cells differentiate into osteoblasts, which make bone matrix on the ceramic surface. Such in vitro prefabricated bone within the ceramic provides immediate new bone-forming capability after in vivo implantation. Prior to loading of the cultured, marrow-derived mesenchymal stem cells into the porous ceramics, exogenous genes can be introduced into these cells in culture. Combining in vitro manipulated mesenchymal stem cells with porous ceramics can be expected to effect sufficient new bone-forming capability, which can thereby provide tissue engineering approaches to patients with skeletal defects in order to regenerate skeletal tissues.     

人骨髓间充质干细胞的体外培养、鉴定及成骨分化

背景：国内外对骨髓间充质干细胞的体外成骨诱导分化研究手段、测定指标均不够全面。 目的：建立并完善一整套人骨髓间充质干细胞的分离培养及鉴定方法,探讨其体外成骨分化能力。 方法：采用密度梯度离心法分离培养人骨髓间充质干细胞,流式细胞仪鉴定细胞表面表型。传至第3代时更换成骨诱导培养基进行成骨分化诱导。 结果与结论：人骨髓间充质干细胞生长旺盛,传代后增殖旺盛,第3代骨髓间充质干细胞表面表型CD44、CD73、CD90表达阳性,CD34表达阴性。诱导后的成骨细胞碱性磷酸酶活性增加,Gomori、Von kossa、茜素红染色均阳性。RT-PCR检测诱导后细胞有Ⅰ型胶原、碱性磷酸酶、骨钙素、骨唾液酸蛋白、骨桥蛋白及骨连接蛋白基因的表达,证明了人骨髓间充质干细胞成功向成骨方向分化。表明实验建立了一整套稳定、成熟的骨髓间充质干细胞分离、培养、扩增方案。     

骨髓基质干细胞治疗骨不连的新进展

背景：骨髓基质干细胞体外培养增殖力强、易于向成骨细胞及软骨细胞方向分化且成骨性能稳定等特点,成为骨组织工程中合适的种子细胞。 目的：总结分析采用骨髓基质干细胞作为种子细胞,分析其直接移植于骨不连部位或复合支架或转基因治疗骨不连所具有的优劣势。 方法：检索1992/2011西文生物医学期刊文献数据及CNKI 数据库有关骨不连研究,骨髓基质干细胞分离、培养,在骨不连方面的应用,骨组织工程细胞支架方面的文献,英文检索词为“bone marrow stromal stem cells,nonunions, repairing,tissue engineering”,中文检索词为“骨髓基质干细胞,骨修复,骨不连,组织工程”。排除重复性研究,保留23篇进一步归纳总结。 结果与结论：利用骨髓基质干细胞作为种子细胞,直接植入骨不连部位,或与适当的支架材料结合,或用骨髓基质干细胞作为靶细胞,导入外源目的基因诱导成骨的基因治疗来修复骨缺损的方法,给骨缺损的治疗带来光明的前景。但同时也存在骨髓基质干细胞增殖、分化合适条件难以准确确定,经皮移植自体骨髓基质干细胞植入体内后容易流失,不能在植入部位形成有效的细胞浓度,支架材料尚不能完全符合临床要求,以及如何将骨组织工程与基因治疗的方法结合起来等问题,需要进一步的研究。     

骨髓间充质细胞复合胶原-壳聚糖材料联合骨搬移修复胫骨缺损：随机对照实验方案

背景：骨髓间充质干细胞发挥成骨作用需要支架材料的辅助,一方面支架材料不仅可将细胞运载至骨缺损区域,另一方面还可作为新骨生长的框架结构。胶原-壳聚糖复合材料是骨组织工程较为理想的支架材料之一,同时其具有骨诱导性,比常规支架材料更优越的成骨能力。骨搬移技术在临床上在修复长段骨缺损方面已得到广泛应用,但也存在成骨慢、外固定时间长、骨不连等缺憾。如何进一步加快骨形成速度,减少并发症发生,已成当前亟待解决的问题。实验假设：骨髓间充质干细胞复合胶原-壳聚糖支架移植能提高胫骨缺损骨搬移修复效果。 方法/设计：随机对照动物实验。分为体外和体内实验两部分。体外实验中取月龄一两个月的新西兰大白兔股骨骨髓,提取骨髓间充质干细胞,培养至第3代,将细胞悬液滴于胶原-壳聚糖支架材料,构建骨髓间充质干细胞复合胶原-壳聚糖支架。体内实验选用24只三四月龄新西兰大白兔,被随机分配接受如下干预：骨搬移、支架植入、骨搬移联合支架植入。研究的主要观察指标为植入材料与骨缺损界面的生长情况、X射线检测的缺损区骨修复情况、苏木精-伊红染色及扫描电镜观察缺损区成骨情况、免疫组织化学染色检测成骨区Ⅰ型胶原蛋白的表达情况、扫描电子显微镜观察移植材料与宿主骨的界面键合情况、超微结构及新骨的生成。 讨论：实验结果将有助于确定对骨缺损进行骨搬移治疗过程中,应用骨髓间充质干细胞复合胶原-壳聚糖支架移植促进骨缺损再生修复效果的可行性。 实验方案获基金支持情况：获辽宁省科学技术计划项目资助(2012225019)。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程