骨组织工程的种子细胞--骨髓基质细胞的研究进展

骨髓基质细胞作为骨组织工程的种子细胞具有广阔前景.许多实验证实骨髓基质细胞具有间充质干细胞特性,表现为较强的增殖能力和向多种间充质细胞分化的潜能.目前已建立了体外培养骨髓基质干细胞的方法,而且正在摸索进一步纯化的方法和诱导分化的条件.已有利用其成骨特性体内移植实验,表明在适当的条件下,接种在组织工程材料上的骨髓基质细胞可以形成新骨.     

Graft materials and bone marrow stromal cells in bone tissue engineering

Bone augmentation procedures rely on osteogenic/osteoconductive properties of bone graft material (BGM). A further improvement is represented by use of autologous bone marrow stromal cells (BMSC), expanded in vitro and seeded on BGM before implantation in the bone defect. The effect of different BGMs on BMSC osteogenic differentiation was evaluated. BMSC were cultured in vitro in the presence of different BGM (natural, synthetic, and mixed origins). Cellular morphology was analyzed with scanning electron microscopy. The capability of BMSC to differentiate was determined in vitro by alkaline phosphatase gene expression and enzyme activity at different time points (7, 14, and 28 days) and in vivo by ectopic bone formation of implanted tissue constructs in an immunodeficient murine model. BGM supports the cell adhesion and osteogenic differentiation of BMSC developing a useful tool in the bone tissue engineering.     

骨髓间充质干细胞构建组织工程化小口径血管

目的采用在动物体外构建初级组织工程化血管、体内强化的方法,探讨构建小口径组织工程化血管的可能性.方法体外培养骨髓间充质干细胞(BMSC),用含全反式维甲酸(AT-RA)、双丁酰环磷酸腺苷(db-cAMP)的DMEM-LG培养液和含血管内皮细胞生长因子(VEGF)的培养液诱导BMSC分别向血管平滑肌样细胞和血管内皮样细胞分化.免疫荧光观察平滑肌样细胞β肌动蛋白的表达和内皮样细胞vWF的表达.电镜观察超微结构的改变.诱导的血管平滑肌样细胞和血管内皮样细胞,分层种植于胶原包埋聚乙醇酸(PGA)的复合支架表面,将细胞和支架复合体种植于动物皮下,于植入后第4、8周再次麻醉动物,取出植入皮下的组织工程化血管,行组织学检查、压力实验及免疫荧光检查.结果诱导14 d后,BMSC能够分化为血管平滑肌样细胞和血管内皮样细胞:β肌动蛋白和vWF呈阳性表达,电镜证实细胞出现了相应的形态学改变.人工血管组织学观察见管壁结构清晰.单纯支架组可承受100～150 mm Hg(1mm Hg=0.133 kPa)的血管腔内压力,实验组则均可承受200mm Hg的血管腔内压力不破裂.实验组皮下培养8周Brdu标记细胞的免疫荧光结果显示部分细胞核呈现明亮的黄绿色荧光.结论以动物皮下为生物反应器可构建出组织工程化血管,其大体结构和天然血管相似.     

In vitro control of human bone marrow stromal cells for bone tissue engineering

For the clinical application of cultured human mesenchymal stem cells (MSCs), cells must have minimal contact with fetal calf serum (FCS) because it might be a potential vector for contamination by adventitious agents. The use of human plasma and serum for clinical applications also continues to give rise to considerable concerns with respect to the transmission of known and unknown human infectious agents. With the objective of clinical applications of cultured human MSCs, we tested the ability of autologous plasma, AB human serum, FCS, and artificial serum substitutes containing animal-derived proteins (Ultroser G) or vegetable-derived proteins (Prolifix S6) to permit their growth and differentiation in vitro. To conserve as much autologous plasma as possible, we attempted to mix it at decreasing concentrations with the serum substitute containing vegetable-derived mitogenic factors. Under control conditions, by day 10 all the fibroblast colony-forming units (CFU-Fs) were alkaline phosphatase (ALP) positive. However, their number and size were highly variable among donors. Better CFU-F formation was obtained with Ultroser G, and with human AB serum and autologous plasma mixed at, respectively, 5 and 1% with Prolifix S6. The effects of these mixtures on CFU-F formation demonstrate synergy, with the human serum or plasma supplying the factors that favor differentiation of MSCs while Prolifix S6 supplies the mitogenic factors. Finally, we demonstrated the possibility of controlling human MSC growth and differentiation in vitro. Notably, by means of a minimal quantity of human serum or human plasma mixed with a new serum substitute containing vegetable-derived proteins, we displayed growth and differentiation of human MSCs comparable to that obtained with FCS or serum substitutes containing animal-derived proteins. These results will have crucial significance for future applications of cultured human MSCs in bone tissue engineering.     

Lapine and canine bone marrow stromal cells contain smooth muscle actin and contract a collagen-glycosaminoglycan matrix.

Lapine and canine marrow stromal cells were found to contain a contractile actin isoform, alpha-smooth muscle actin (SMA), by immunohistochemistry and Western blot analysis. The SMA was found to be incorporated into stress fibers that were prominently displayed by the cells in monolayer culture. The cell content of this actin isoform increased with passage number. The contractility of SMA-expressing stromal cells was demonstrated by their contraction of collagen-glycosaminoglycan analogs of extracellular matrix into which they were seeded. The demonstration that marrow-derived stromal cells express the SMA gene may explain recent findings of this expression in musculoskeletal connective tissue cells including osteoblasts, chondrocytes, and fibrochondrocytes that may be derived from this mesenchymal stem cell. The implications of these findings for tissue engineering strategies employing marrow stromal cells are also discussed.     

Effects of pH on human bone marrow stromal cells in vitro: implications for tissue engineering of bone

The objective of this study was to address the hypothesis that changes in extracellular pH alter collagen gene expression, collagen synthesis, and alkaline phosphatase activity in bone marrow stromal cells (BMSCs). Potential effects of pH on cell function are of particular importance for tissue engineering because considerable effort is being placed on engineering biodegradable polymers that may generate a local acidic microenvironment on degradation. Human and murine single-cell marrow suspensions were plated at a density of 2 x 10(4) cells/cm(2). After 7 days in culture, the pH of the culture medium was adjusted to one of six ranges: > or = 7.8, 7.5.-7.7, 7.2-7.4, 6.9-7.1, 6.6-6.8, or < or = 6.5. After 48 h of exposure to an altered pH, alkaline phosphatase activity and collagen synthesis decreased significantly with decreasing pH. This decrease was two-to threefold as pH decreased from 7.5 to 6.6. In contrast, alpha1(I) procollagen mRNA levels increased two- to threefold as pH was decreased. The trend in osteocalcin mRNA expression was opposite to that of collagen. Small shifts in extracellular pH led to significant changes in the ability of BMSCs to express markers of the osteoblast phenotype. These pH effects potentially relate to the microenvironment supplied by a tissue-engineering scaffold and suggest that degrading polymer scaffolds may influence the biologic activity of the cells in the immediate environment.     

Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold

To investigate the potential application of bone marrow stromal cells (BMSCs) and an injectable sodium alginate/gelatin scaffold for bone tissue engineering (BTE). The phenotype of osteogenic BMSCs was examined by mineralized nodules formation and type I collagen expression. Cell proliferation was evaluated by MTT assay. The biocompatibility of scaffold and osteogenic cells were examined by hematoxylin and eosin (H&E) staining. Ectopic bone formation as well as closure of rabbit calvarial critical-sized defects following scaffold-cell implantation were analyzed by histological examination and computed tomography (CT) scanning. Spindle-shaped osteogenic cells of high purity were derived from BMSCs. The osteogenic cells and sodium alginate/gelatin (2:3) scaffold presented fine biocompatibility following cross-linking with 0.6% of CaCl(2). After implantation, the scaffold-cell construct promoted both ectopic bone formation and bone healing in the rabbit calvarial critical-sized defect model. Our data demonstrated that the sodium alginate/gelatin scaffold could be a suitable biomaterial for bone engineering, and the scaffold-osteogenic cells construct is a promising alternative approach for the bone healing process.     

Human bone marrow stromal cells: In vitro expansion and differentiation for bone engineering

Stromal cells from marrow hold a great promise for bone regeneration. Even if they are already being exploited in many clinical settings, the biological basis for the source and maintenance of their proliferation/differentiation potential after in vitro isolation and expansion needs further investigation.

Most studies on osteogenic differentiation of marrow stromal cells (MSC) have been performed using bone marrow from the iliac crest. In this study, MSC were derived from spare femoral bone marrow obtained during hip replacement surgery from 20 adult donors. After in vitro isolation the cells were grown in osteogenic medium, and their proliferation and differentiation analysed during in vitro expansion. We found that MSC isolated from the femur of adult patients consistently maintain an osteogenic potential. Using biochemical signals, these cells turn to fully differentiated osteoblasts with a predictable set of molecular and phenotypic events of in vitro bone deposition. When seeded on polycaprolactone-based scaffold or surfaces, the proliferation and mineralization of femur-derived MSC were modulated by the surface chemistry/topography. Despite remarkable differences between individual colony-forming ability, alkaline phosphatase production, and mineralization ability, these cells are a potential source for bone engineering, either by direct autologous reimplantation or by ex vivo expansion and reimplantation combined to a proper scaffold.     

Comparing various off-the-shelf methods for bone tissue engineering in a large-animal ectopic implantation model: bone marrow, allogeneic bone marrow stromal cells, and platelet gel

Construction of bone grafts for regenerative medicine would highly benefit from off-the-shelf components, such as allogeneic bone marrow stromal cells (BMSCs) and blood-derived growth factors from platelet concentrate. Although allogeneic BMSCs are considered immunosuppressive, their use in transplantation studies is still cautioned. In this study, we used off-the-shelf goat allogeneic BMSCs, per-operatively aspirated bone marrow (BM) and platelet gel (PLG). Ten goats received six different hybrid constructs consisting of biphasic calcium phosphate scaffolds seeded with PLG or plasma that were mixed with BM, allogeneic BMSCs or left without cells. All constructs were implanted in the paraspinal muscles for 9 weeks. Fluorochromes were administered at 2, 3, and 5 weeks to assess onset of bone formation. Analysis revealed that the scaffolds without cells yielded small amounts of bone. Allogeneic BMSCs had a positive effect on the amount and early onset of bone formation. Fresh BM did not enhance ectopic bone formation. The PLG, which contained higher levels of transforming growth factor beta than plasma, did not result in more bone either. Fluorochrome incorporation results indicate that the presence of seeded cells in the constructs accelerates bone formation. This study shows a potential role of allogeneic BMSCs in bone tissue-engineering research.     

大鼠骨髓来源的平滑肌祖细胞培养及鉴定

目的培养和鉴定从鼠骨髓中分离的平滑肌祖细胞,观察其在体外增殖、分化过程中相关细胞表型的变化。方法用密度梯度离心法获得鼠骨髓单个核细胞,用条件培养基进行诱导分化,免疫荧光双标技术（α-SMA、CD14）鉴定平滑肌祖细胞（SPC）。Western blot检测α-SMA蛋白,Real-time PCR法检测α-SMA mRNA表达。结果鼠骨髓单个核细胞诱导培养4d时开始贴壁,7d变为梭形,14d呈典型的“峰”“谷”样形态;4d时开始出现α-SMA-CD14双荧光阳性细胞;培养1d时无α-SMA蛋白表达,4d后增强,10～14d达高峰,21d仍持续高水平;培养1d的细胞α-SMA mRNA表达低（P〈0.01）,4d后增强,14d达高峰（P〈0.01）,21d后仍持续高水平。结论本试验成功地从鼠骨髓单个核细胞中分离培养出SPC,并在体外扩增分化成平滑肌样细胞。     

动脉内皮损伤后平滑肌细胞维甲酸受体的表达及维甲酸的作用

目的 研究动脉内皮损伤后ＶＳＭＣ中ＲＡＲ表达的规律及其与内膜增生的关系,方法 放射配基法测量用维甲酸与未用药两组内皮损伤后不同时期的受体最大结合容量和平衡解离常数抽时用形态学方法测量血管内膜面积。结果 动脉内皮损伤后早期ＶＳＭＣ中ＲＡＲ数量显著低于正常（Ｐ〈０．０１）,于第４周恢复正常,同时血管膜面积增加,用维甲酸后,ＲＡＲ显著增加,于术后２周即接近正常,血管内膜面积显著少于未用药组,结论 动脉损     

Systematic neuronal and muscle induction systems in bone marrow stromal cells: the potential for tissue reconstruction in neurodegenerative and muscle degenerative diseases

Because bone marrow stromal cells (MSCs) are easily accessible from both healthy donors and patients and can be expanded on a therapeutic scale, they have attracted attention for cell-based therapy. Benefits of MSCs have been discussed mainly from two aspects: one is their tissue protective and immunomodulatory effects, and the other is their capability under specific manipulations to differentiate into various cell types. In this review, their differentiation into functional neural and muscle cell lineages is the focus, and their potential to the application for tissue reconstruction in neurodegenerative and muscle degenerative diseases is discussed.     

The expression of cross-linked elastin by rabbit blood vessel smooth muscle cells cultured in polyhydroxyalkanoate scaffolds

In order to evaluate the possibility for making artificial blood vessels, a series of microbial copolyesters poly(3-hydroxybutyrate-co-4-hydroxybutyrate)s (P3HB4HB)s containing 0-40 mol% 4-hydroxybutyrate (4HB) were studied for growth and formation of elastin of rabbit blood vessel smooth muscle cells (RaSMCs) incubated on the solvent-casting polyester films. Porous scaffolds of all P3HB4HBs except P(HB-40 mol% 4HB) were used to compare their potentials as materials for tissue engineering blood vessel (TEBV). The polyesters were studied using static contact angles, differential scanning calorimetry (DSC), cell count kit-8 (CCK-8) and Fastin elastin assays. Simultaneously, SEM, H&E and DAPI staining were employed to study cell morphology and cell growth in the polyester scaffolds. Viability of rabbit blood vessel smooth muscle cells (RaSMCs) grown on P(HB-7 mol% 4HB) films was the strongest among all other tested polyester films during the whole growth process. H&E and DAPI staining clearly suggested good cells' growth and even confluent growth in the P3HB4HB scaffolds. Fastin elastin assay demonstrated that 4HB component in the P3HB4HB copolymer benefited the elastin formation and accumulation. P(HB-20 mol% 4HB) showed a 160% more elastin production compared with that on the well-studied poly(3-hydroxybutyrate-co-12 mol% 3-hydroxyhexanoate) (PHBHHx) incubated under the same conditions. Since the P3HB4HB has adjustable elasticity and strength, combined with its ability to induce elastin formation, it can be regarded as a useful material for developing into a material for artificial blood vessels.     

In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering

Mineralized and partially or fully demineralized biomaterials derived from bovine bone matrix were evaluated for their ability to support human bone marrow stromal cell (BMSC) osteogenic differentiation in vitro and bone-forming capacity in vivo in order to assess their potential use in clinical tissue-engineering strategies. BMSCs were either seeded on bone-derived scaffolds and cocultured in direct cell-to-scaffold contact, allowing for the exposure of soluble and insoluble matrix-incorporated factors, or cocultured with the scaffold preparations in a transwell system, exposing them to soluble matrix-incorporated factors alone. Osteoblast-related markers, alkaline phosphatase (ALP) activity and bone sialoprotein (BSP) and osteopontin (OP) mRNA expression were evaluated in BMSCs following 14 days of cocultivation in both systems. The data demonstrate that BMSCs from some donors express significantly higher levels of all osteoblast-related markers following cocultivation in direct cell-to-scaffold contact with mineralized scaffolds in comparison to fully demineralized preparations, while BMSCs from other donors display no significant differences in response to various scaffold preparations. In contrast, BMSCs cocultured independently with soluble matrix-incorporated factors derived from each scaffold preparation displayed significantly lower levels of ALP activity and BSP mRNA expression in comparison to untreated controls, while no significant differences were observed in marker levels between cells cocultured similarly with different biomaterial preparations. In addition, BMSCs were seeded directly on mineralized and partially or fully demineralized biomaterials and implanted in subcutaneous sites of athymic mice for 8 weeks to evaluate their in vivo bone-forming capacity. The ex vivo incorporation of BMSCs into all bone-derived scaffold preparations substantially increased the mean extent and frequency of samples containing de novo bone formation over similar nonseeded controls, as determined by histological and histomorphometrical analysis. No statistically significant differences were observed in the extent or frequency of bone formation between various scaffold preparations seeded with BMSCs from different donors. These results demonstrate that the in vivo osteoinductivity of bone-derived scaffolds can be modulated by ex vivo incorporated BMSCs and the extent of scaffold demineralization plays a significant role in influencing in vitro osteogenic differentiation of BMSCs depending on the coculture system and BMSC donor.     

Meniscal regeneration using tissue engineering with a scaffold derived from a rat meniscus and mesenchymal stromal cells derived from rat bone marrow

The purpose of this study was to regenerate a meniscus using a scaffold from a normal meniscus and mesenchymal stromal cells derived from bone marrow (BM-MSCs). Thirty Sprague-Dawley rat menisci were excised and freeze-thawed three times with liquid nitrogen to kill the original meniscal cells. Bone marrow was aspirated from enhanced green fluorescent protein transgenic Sprague-Dawley rats. BM-MSCs were isolated, cultured for 2 weeks, and 2 x 10(5) cells were then seeded onto the meniscal scaffolds. Using a fluorescent microscope and immunohistochemical staining, repopulation of enhanced green fluorescent protein positive cells was observed in the superficial zone of the scaffold after 1 week of culture, and then in the deep zone after 2 weeks. At 4 weeks, expression of extracellular matrices was detected histologically and expression of mRNA for aggrecan and type X collagen was detected. Stiffness of the cultured tissue, assessed by the indentation stiffness test, had increased significantly after 2 weeks in culture, and approximated the stiffness of a normal meniscus. From this study, we conclude that a scaffold derived from a normal meniscus seeded with BM-MSCs can form a meniscus approximating a normal meniscus.     

Engineered bone from bone marrow stromal cells: a structural study by an advanced x-ray microdiffraction technique

The mechanism of mineralized matrix deposition was studied in a tissue engineering approach in which bone tissue is formed when porous ceramic constructs are loaded with bone marrow stromal cells and implanted in vivo. We investigated the local interaction between the mineral crystals of the engineered bone and the biomaterial by means of microdiffraction, using a set-up based on an x-ray waveguide. We demonstrated that the newly formed bone is well organized inside the scaffold pore, following the growth model of natural bone. Combining wide angle (WAXS) and small angle (SAXS) x-ray scattering with high spatial resolution, we were able to determine the orientation of the crystallographic c-axis inside the bone crystals, and the orientation of the mineral crystals and collagen micro-fibrils with respect to the scaffold. In this work we analysed six samples and for each of them two pores were studied in detail. Similar results were obtained in all cases but we report here only the most significant sample.