Mesenchymal stem cells for tissue engineering of bone: 3D-cultivation and osteogenic differentiation on mineralized collagen

AIM: Due to their plasticity and high proliferation capacity in vitro, human mesenchymal stem cells (MSC) are promising candidates for substitution of mesenchymal tissues, such as bone. According to the tissue engineering concept, combinations of cells and three dimensional scaffolds are used to replace damaged tissue. Although various attempts have been made, the optimal combination of cells and artificial scaffold has not been found so far. METHODS: In this work, human MSC were isolated from bone marrow aspirates according to standard protocols and cultivated on mineralized collagen. Osteogenic differentiation was induced by medium containing dexamethasone, ascorbic acid and beta-glycerophosphate. Cell proliferation on the scaffold (WST-1 vitality assay, total protein measurement) and osteogenic differentiation (quantitative Real-Time-RT-PCR) were monitored for 24 days. RESULTS: Viable cells were found within the matrix throughout the cultivation period using histological and histochemical methods. Effective osteogenic differentiation could be demonstrated by the increase of expression of osteogenic marker genes (such as alkaline phosphatase) on a molecular level. CONCLUSION: Our results make the cell/matrix construct investigated in this work a promising candidate for tissue engineering of bone using mesenchymal stem cells. This has to be tested further by in vivo analysis.     

骨髓基质干细胞成骨的研究进展

组织工程学是生命科学中新兴学科，是生物医学工程领域的一个快速发展的新方向。骨组织工程学是骨外科学主要发展方向之一。骨髓基质干细胞有明确的成骨作用，其特点为来源丰富、提取方便、体外扩增迅速、易于成骨诱导、可导入标记性或治疗性基因，因此认为骨髓基质干细胞是骨组织工程学中理想的种子细胞。本文就骨髓基质干细胞成骨进展作一综述。     

骨髓基质干细胞及其在骨组织工程中的应用

骨髓基质干细胞（BMSCs）是一群存在于骨髓中的非造血干细胞,具有较强的自我更新能力和多向分化潜能,在体外不同的诱导条件下可分化为骨细胞、软骨细胞、肌腱细胞、脂肪细胞、神经细胞、平滑肌细胞等多种细胞.BMSCs的应用是目前国际上骨组织工程领域的重要研究内容,具有广泛的临床应用前景.近年来,许多实验室从分子、生化、物理等水平对其成骨分化调控进行了深入研究,取得了较大的进展.     

Osteogenic stem cells and the stromal system of bone and marrow

According to current hypothesis, cells of the osteogenic lineage, which includes both osteoblasts and chondroblasts, are derived from a stromal stem cell in the postnatal organism. That there exist osteogenic precursors in association with the soft, fibrous tissue of the marrow stroma is well established. An osteogenic tissue comprised of cartilage and bone is formed when marrow or marrow cell suspensions are cultured in vivo within diffusion chambers. Bone with a functional marrow organ is formed when marrow or marrow cell suspensions are transplanted heterotopically, e.g., under the renal capsule. Cultures of marrow stromal fibroblasts are readily established in vitro from single-cell bone marrow suspensions. Such cultures do not demonstrate overt differentiation in an osteogenic direction in vitro. When transplanted in vivo, however, they differentiate to form cartilage and bone in diffusion chambers and bone with a functional marrow organ when transplanted heterotopically. Single-cell bone marrow suspensions can be cultured in vitro under conditions that facilitate the formation of stromal fibroblast colonies. Circumstantial evidence supports the conclusion that each colony is derived from a single initiating cell termed a colony-forming unit-fibroblastic (CFU-F). A proportion of CFU-F demonstrates extensive proliferative potential both in vitro and in vivo. In vitro the extensive proliferative potential of a subset of CFU-F has been shown to be associated with a capacity for extensive self-renewal. On transplantation in vivo, the progeny of a proportion of CFU-F has been shown to be capable of proliferating and differentiating into all the stromal cell lines necessary for the formation of bone and reconstitution of the hematopoietic inductive microenvironment. These findings provide strong circumstantial evidence to support the hypothesis that there are stem cells present within the marrow stroma that are capable of giving rise to cells of a number of different lineages, including those of the osteogenic lineage (chondroblasts and osteoblasts).     

补肾中药对骨髓间充质干细胞成骨分化的作用研究

骨髓间充质干细胞作为骨损伤后,骨修复、重建的种子细胞,越来越成为骨组织工程学中的研究热点。骨修复、重建的关键核心是如何能够提高骨髓间充质干细胞的高效增殖和成骨定向分化。以"肾藏精,主骨,生髓"的中医理论为依据,同时实验和临床也证实,补肾中药可以有效地促进骨髓间充质干细胞增殖、以及定向成骨分化。为骨损伤之后的修复和重建提供新的治疗途径。笔者就近年补肾中药促进骨髓间充质干细胞成骨分化的研究作一综述。     

骨髓间充质干细胞成骨分化的研究与进展

超临界大面积或大段骨缺损仍是临床面临的难题,研究者们致力于研发人工骨材料,但为了解决人工骨材料成骨效应不良的问题,人们越来越关注骨髓间充质干细胞在骨组织工程中的应用。本综述以骨髓间充质干细胞、成骨分化、成骨细胞、临床应用"Bone marrow mesenchymal stem cells,osteogenesis differentiation,osteoblasts cell,clinical application"为检索词,应用计算机搜索CNKI数据库、万方数据库、维普数据库、PUBMED数据库,全面归纳总结骨髓间充质干细胞的分离培养、骨髓间充质干细胞鉴定方法、成骨诱导方法、成骨分化鉴定及其在临床中的应用,为证实其作为种子细胞治疗骨组织疾病提供理论依据。学者们初步研究了骨髓间充质干细胞结合移植、组织工程等技术来治疗骨和软骨缺损、骨关节炎、股骨头坏死等疾病,均取得了较好的临床疗效。但是骨髓间充质干细胞会有一定的优缺点,其临床试验及远期疗效的验证还需进一步深入研究。     

In vitro differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissues

Bone marrow stromal cells, progenitor cells involved in repair of bone and cartilage, can potentially provide a source for autologous skeletal tissue engineering. We investigated which factors were required to induce in vitro differentiation of avian bone marrow stromal cells into three-dimensional cartilaginous and bone-like tissues. Bone marrow stromal cells from embryonic chicks were expanded in monolayers, seeded onto biodegradable polyglycolic acid scaffolds, and cultured for 4 weeks in orbitally mixed Petri dishes. Cell-polymer constructs developed an organized extracellular matrix containing glycosaminoglycans and collagen, whereas control bone marrow stromal cell pellet cultures were smaller and consisted predominantly of fibrous tissue. Bone marrow stromal cells expanded with fibroblast growth factor-2 and seeded onto polymer scaffolds formed highly homogeneous three-dimensional tissues that contained cartilage-specific molecular markers and had biochemical compositions comparable with avian epiphyseal cartilage. When cell-polymer constructs were cultured in the presence of beta-glycerophosphate and dexamethasone, the extracellular matrix mineralized and bone-specific proteins were expressed. Our work shows that cell expansion in the presence of fibroblast growth factor-2 and cultivation on a three-dimensional polymer scaffold allows differentiation of chick bone marrow stromal cells into three-dimensional cartilaginous tissues. In the in vitro system studied, the same population could be selectively induced to regenerate either cartilaginous or bonelike tissue.     

Human maxillary tuberosity and jaw periosteum as sources of osteoprogenitor cells for tissue engineering.

OBJECTIVE: Bone tissue engineering is a promising approach for bone reconstruction in oral-maxillofacial surgery. This study investigates the suitability of oral skeletal tissues as convenient and accessible sources of osteogenic progenitors as an alternative to the iliac crest bone marrow. STUDY DESIGN: Samples of maxilla tuberosity (MT) and maxillary and mandibular periosteum (MP) were obtained during routine oral surgery, and donor site morbidity was assessed using a "split-mouth" approach. Cells isolated from MT (bone marrow stromal cells; MT-BMSCs) and from MP (periosteal cells; M-PCs), were analyzed for clonogenicity, phenotype, expression of osteogenic markers, and ability to form bone in vivo. RESULTS: Both MT-BMSCs and M-PCs included clonogenic cells, showed comparable phenotypic profiles, and expressed early osteogenic markers. Most importantly, both cell populations formed bone upon ectopic in vivo transplantation. CONCLUSION: MT-BMSCs and M-PCs behaved as osteoprogenitor cells in vitro and in vivo. MT and MP may be considered as suitable sources of cells for bone tissue engineering in humans.     

玻璃化冻存骨髓基质干细胞的实验研究

目的：骨髓基质干细胞（BMSCs）是构建组织工程化骨主要的种子细胞,实现BMSCs的深低温保存对骨组织工程具有重要意义。目前,玻璃化冻存是最有发展前景的冻存方法,因此希望通过改进玻璃化液和预处理条件来提高BMSCs的玻璃化冻存效果。方法：采用不同组成及浓度的玻璃化液在不同的预处理条件下对第2（P2）代成骨诱导的犬骨髓基质干细胞（cBMSCs）进行玻璃化冻存实验,通过复苏后的细胞存活率来选择一种理想的玻璃化液及适合的预处理条件,并在此基础上再与常规冻存的实验结果进行比较,同时考虑冻存过程对细胞成骨活性的影响。结果：将VS226作为玻璃化液,在0℃／5min的预处理条件下玻璃化冻存P2代成骨诱导的cBMSCs,与常规冻存后的细胞存活率相比无统计学差异,且冻存过程对此细胞的成骨能力没有影响。结论：采用VS226来玻璃化冻存BMSCs,可实现为骨组织工程提供大量种子细胞的目的。     

Editorial Commentary: Stem Cells. They Are in the Fat Tissue,Bone Marrow,and Even in the Synovial Fluid of the Knee Joint

Adult stem cells have been isolated in bone marrow and adipose tissue. These mesenchymal stromal cells (MSCs) have the ability to differentiate into osteogenic, chondrogenic, and adipogenic cell lines. The study by Branch et al. has identified MSCs in the synovial fluid of the knee in patients after anterior cruciate ligament injury and in patients with osteoarthritis of the knee. When mixing synovial fluid with whole blood and using a commercially available platelet-rich plasma–processing system, the total number of MSCs doubled in both groups when compared with the cell count in synovial fluid only. However, it is not clear whether the MSCs in the processed synovium–whole blood mix include synovial MSCs versus MSCs from only the blood. In addition, cell counts were substantially lower when compared with the typical concentrations of MSCs in bone marrow aspirate. The clinical application is yet to be defined.     

Multipotent stromal cells derived from the infrapatellar fat pad of the knee

Tissue engineering approaches for promoting the repair of skeletal tissues have focused on cell-based therapies involving multipotent stromal cells. Recent studies have identified such cells in several tissues in the adult human, including skin, muscle, bone marrow, and subcutaneous fat. This study examined the hypothesis that the infrapatellar fat pad of the adult knee contains progenitor cells that have the ability to differentiate into chondrocytes, osteoblasts, or adipocytes under appropriate culture conditions. Cells isolated from the fat pad stroma had a profile of cell-surface molecules similar but not identical to that of bone marrow-derived mesenchymal stem cells. Using defined culture conditions, fat pad-derived stromal cells were induced to differentiate cells with phenotypic characteristics of: (1) chondrocytes, synthesizing cartilage matrix molecules; (2) adipocytes, producing lipid vacuoles and leptin; or (3) osteoblasts, forming mineralized tissue. The culture conditions also modulated the expression of characteristic gene markers for each lineage. This study supports the hypothesis that multipotent stromal cells are present in many connective tissues in the adult human. Given its location and accessibility, the fat pad may prove to be a potential source of progenitor cells for musculoskeletal tissue engineering.     

What should be the characteristics of the ideal bone graft substitute? Combining scaffolds with growth factors and/or stem cells

Reconstruction of large bone defects or non-unions resulting from biochemical disorders, tumour resections or complicated fractures is still a challenge for orthopaedic and trauma surgery. On the one hand, autografts harbour most features of ideal bone graft substitutes but on the other hand, they have a lot insurmountable disadvantages. An ideal bone graft substitute should be biomechanically stable, able to degrade within an appropriate time frame, exhibit osteoconductive, osteogenic and osteoinductive properties and provide a favourable environment for invading blood vessels and bone forming cells. Whilst osteoconductivity of biomaterials for bone tissue engineering strategies can be directed by their composition, surface character and internal structure, osteoinductive and osteogenic features can be provided by growth factors originally participating in fracture healing and/or multipotent mesenchymal stromal/stem cells (MSC) capable of rebuilding bone and marrow structures. In this review, aspects of the clinical application of the most commonly used growth factors for bone repair, the bone morphogenetic proteins (BMPs), and the potential use of human MSC for clinical application will be discussed.     

In vitro osteogenic differentiation and in vivo bone-forming capacity of human isogenic jaw periosteal cells and bone marrow stromal cells

OBJECTIVE: To compare the in vitro osteogenic differentiation and in vivo ectopic bone forming capacity of human bone marrow stromal cells (BMSCs) and jaw periosteal cells (JPCs), and to identify molecular predictors of their osteogenic capacity. SUMMARY BACKGROUND DATA: JPC could be an appealing alternative to BMSC for the engineering of cell-based osteoinductive grafts because of the relatively easy access to tissue with minimal morbidity. However, the extent of osteogenic capacity of JPC has not yet been established or compared with that of BMSC. METHODS: BMSCs and JPCs from the same donors (N = 9), expanded for 2 passages, were cultured for 3 weeks in osteogenic medium either in monolayers (Model I) or within 3-dimensional porous ceramic scaffolds, following embedding in fibrin gel (Model II). Cell-fibrin-ceramic constructs were also implanted ectopically in nude mice for 8 weeks (Model III). Cell differentiation in vitro was assessed biochemically and by real-time RT-PCR. Bone formation in vivo was quantified by computerized histomorphometry. RESULTS: JPCs had lower alkaline phosphatase activity, deposited smaller amounts of calcium (Model I), and expressed lower mRNA levels of bone sialoprotein, osteopontin, and osterix (Models I and II) than BMSCs. JPCs produced ectopic bone tissue at lower frequency and amounts (Model III) than BMSCs. Bone sialoprotein, osteopontin, and osterix mRNA levels by BMSCs or JPCs in Model II were markedly higher than in Model I and significantly more expressed by cells that generated bone tissue in Model III. CONCLUSIONS: Our data indicate that JPCs, although displaying features of osteogenic cells, would not be as reliable as BMSCs for cell-based bone tissue engineering, and suggest that expression of osteoblast-related markers in vitro could be used to predict whether cells would be osteoinductive in vivo.     

骨髓基质细胞体内外成骨的实验研究

目的：探讨骨髓基质细胞（Bone marrow stromal cell,BMSc)向成骨细胞转化的条件,利用骨髓基质细胞构建组织工程化骨组织。方法：采用地塞米松、β-甘油磷酸钠诱导体外培养的兔骨髓基,上差异显微镜和碱性磷酸酶检测基向成骨细胞转化的能力。将骨髓基质细胞与生物活性玻璃陶瓷（Bioactive glass ceramic,BGC)复合后植入免自体肌袋内,观察成骨过程。结果：在适当诱导条件下,骨髓基质细胞可向成骨细胞分化,在体内外表现出明显的成骨能力,地塞米松、β-甘油磷酸钠起着重要的作用。结论：骨髓基质细胞是骨组织工程的良好细胞来源,利用组织工程化方法可构建新生骨组织。     

兔骨髓基质细胞的分离培养

目的 为骨组织工程寻找一种理想的种子细胞。方法 采取兔骨髓组织，应用梯度离心获取骨髓基质细胞，体外培养传代，通过光镜、透射电镜及成骨特性的鉴定，观察细胞的形态、生长特点及成骨特性。结果 培养的骨髓基质细胞形态多为三角形或梭形，生长增殖迅速，具有成骨能力，易于定向分化为成骨细胞。结论 自骨髓获得的骨髓基质细胞具有明显的增殖能力和成骨活性，可以做为骨组织工程中比较理想的种子细胞。     

成骨生长肽对大鼠骨髓基质细胞增殖和成骨分化的作用

目的：观察成骨生长肽诱导体外培养的大鼠骨髓基质细胞增殖及向成骨分化的作用。方法：取6周龄SD大鼠，贴壁法分离培养骨髓基质细胞，在不同浓度成骨生长肽的诱导下，观察细胞形态变化，绘制骨髓基质细胞生长曲线，碱性磷酸酶和钙结节组织化学染色。结果：成骨生长肽对骨髓基质细胞增殖和成骨分化的作用呈剂量依赖性：成骨生长肽浓度在10^-10及10^-11mol／L时促进骨髓基质细胞增殖，而在10^-8及10^-9mol／L时对骨髓基质细胞的增殖略有抑制作用；成骨生长肽浓度在10^-10及10^-11mol／L时骨髓基质细胞碱性磷酸酶染色基本呈阴性，与对照组相比差异无统计学意义，而在10^-8及10^-9mol／L浓度下可以显著提高骨髓基质细胞碱性磷酸酶染色的阳性率。结论：成骨生长肽可以明显促进大鼠骨髓基质细胞增殖及向成骨细胞分化，其促成骨活性具有显著的浓度依赖性。     

温阳补肾中药促进骨髓基质细胞分化的实验研究进展

骨髓基质细胞（BMSCs）是存在于骨髓中的一种干细胞,具有自我更新、高度增殖能力和多向分化的潜能,故在细胞替代治疗、基因治疗、组织工程等方面具有良好的应用前景。但由于骨髓中BMSCs含量极少,必须通过体外培养扩增、诱导分化才能满足临床需求。近年来,研究发现中药诱导BMSCs增殖、分化是非常有潜力的一条途径,基于＂肾主骨生髓＂和＂肾藏精、精生髓＂的中医理论,中医学者们就温阳补肾中药在促进BMSCs增殖和分化方面进行了探索,发现该类药物具有促进BMSCs增殖及向成骨、软骨、神经细胞分化的作用。本文就此方面的研究进行阐述和介绍。     

Simvastatin induces osteogenic differentiation of murine embryonic stem cells

Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast‐like cells, and embryonic stem cells have shown statins to have cholesterol‐independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self‐proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications. © 2010 American Society for Bone and Mineral Research.