Cellular compatibility of improved scaffold material with deproteinized heterogeneous bone

Objective： To study cellular compatibility of improved scaffold material with deproteinized heterogeneous bone and provide experimental basis on choosing the scaffold material in bone tissue engineering. Methods： Bone marrow stromal cells （BMSC） were co-cultured with heterogeneous deproteinized bone in vitro. The contrast phase microscope, scanning electron microscope, MTT assay, flow cytometry were performed and the BGP content and ALP activities were detected in order to observe the cell growth, adhesion in the material, cell cycle and cell viability. Results. The scaffold material of deproteinized heterogeneous bone had no inhibitory effect on cellular proliferation, differentiation and secretion function of BMSCs. Conclusions ： The established heterogeneous deproteinized bone has good biocompatibility with BMSCs and is a potentially ideal scaffold material for bone tissue engineering.     

软骨细胞外基质源性取向支架与骨髓基质干细胞体外软骨组织工程的初步研究

Objective To fabricate cartilage extracellular matrix (ECM) oriented scaffolds and investigate the attachment, proliferation, distribution and orientation of bone marrow mesenchymal stem cells (BMSCs) cultured within the scaffolds in vitro. Methods Cartilage slices were shattered in sterile phosphate-buffered saline (PBS) and the suspension were differentially centrifugated untill the micro- fiber of the cartilage extracellular matrix was disassociated from the residue cartilage fragments. At last the supernatant were centrifugated, the precipitation were collected and were made into 2%-3% suspension. Using unidirectional solidification as a freezing process and freeze-dried method, the cartilage extracellular matrix derived oriented scaffolds was fabricated. The scaffolds were then cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution. By light microscope and scan electron microscope (SEM) observation, histological staining, and biomechanical test, the traits of scaffolds were studied. After being labelled with PKH26 fluorescent dye, rabbit BMSCs were seeded onto the scaffolds. The attachment, proliferation and differentiation of the cells were analyzed using inverted fluorescent microscope. Results The histological staining showed that toluidine blue, safranin O, alcian blue and anti-collagen Ⅱ immunohistochemistry staining of the scaffolds were positive. A perpendicular pore-channel structures which has a diameter of 100 μm were verified by light microscope and SEM analysis. The cell-free scaffolds showed the compression moduli were (2.02±0.02) MPa in the mechanical testing. Inverted fluorescent microscope showed that most of the cells attached to the scaffold. Cells were found to be widely distributed within the scaffold, which acted as a columnar arrangement. The formation of a surface cells layer was found on the surface of the scaffolds which resembled natural cartilage. Coclusion The cartilage extracellular matrix derived oriented scaffolds have promising biological, structural, and mechanical properties.     

骨髓源性肝干细胞的筛选、扩增和分化

Objective To explore the feasibility of direct separation, selective proliferation and differentiation of BDLSC from hone marrow cells with culture systems containing cholestatic serum in vitro.Methods Whole bone marrow cells of rats cultured in routine medium were replaced with con-ditioning selection media containing eholestatic sera of different concentrations after they attached to the plates.The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures.Then the selected BDLSC were induced to proliferating culture system and dif-ferentiating culture system.Each passage of the proliferated stem cells was subjected to flow cytome-try for detection of stem cell markers.The morphology and phenotypie markers of BDLSC were char-acterized using immunohistochemistry, RT-PCR and electron microscopy.The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.Results The 50 ml/L eholestatie serum was the optimal concentration for the selection of BDLSC at which BDLSC could sur-vive while the other populations of the bone marrow cells could not.The purified BDLSC could prolif-erate for 6 passages and maintained stable markers in our proliferating system.When the culture sys-tem changed to differentiating system, hepatocyte-like colony-forming units (HCFU) were formed.HCFU expressed markers of embryonic hepatoeytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and eytoehrome P450-2b1), and hepatocyte nuclear factors (HNF-1α and HNF-3β).They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.Conclusion BDLSC can be selected directly from whole bone marrow cells and pure BDLSC can also proliferate for 6 passages.The differentiated cells have hepatocyte-like phenotype and function.BDLSC will he a new way to provide a readily avail-able alternate source of cells for clinical hepatocyte therapy.     

骨髓源性肝干细胞的筛选、扩增和分化

Objective To explore the feasibility of direct separation, selective proliferation and differentiation of BDLSC from hone marrow cells with culture systems containing cholestatic serum in vitro.Methods Whole bone marrow cells of rats cultured in routine medium were replaced with con-ditioning selection media containing eholestatic sera of different concentrations after they attached to the plates.The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures.Then the selected BDLSC were induced to proliferating culture system and dif-ferentiating culture system.Each passage of the proliferated stem cells was subjected to flow cytome-try for detection of stem cell markers.The morphology and phenotypie markers of BDLSC were char-acterized using immunohistochemistry, RT-PCR and electron microscopy.The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.Results The 50 ml/L eholestatie serum was the optimal concentration for the selection of BDLSC at which BDLSC could sur-vive while the other populations of the bone marrow cells could not.The purified BDLSC could prolif-erate for 6 passages and maintained stable markers in our proliferating system.When the culture sys-tem changed to differentiating system, hepatocyte-like colony-forming units (HCFU) were formed.HCFU expressed markers of embryonic hepatoeytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and eytoehrome P450-2b1), and hepatocyte nuclear factors (HNF-1α and HNF-3β).They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.Conclusion BDLSC can be selected directly from whole bone marrow cells and pure BDLSC can also proliferate for 6 passages.The differentiated cells have hepatocyte-like phenotype and function.BDLSC will he a new way to provide a readily avail-able alternate source of cells for clinical hepatocyte therapy.     

骨髓源性肝干细胞的筛选、扩增和分化

Objective To explore the feasibility of direct separation, selective proliferation and differentiation of BDLSC from hone marrow cells with culture systems containing cholestatic serum in vitro.Methods Whole bone marrow cells of rats cultured in routine medium were replaced with con-ditioning selection media containing eholestatic sera of different concentrations after they attached to the plates.The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures.Then the selected BDLSC were induced to proliferating culture system and dif-ferentiating culture system.Each passage of the proliferated stem cells was subjected to flow cytome-try for detection of stem cell markers.The morphology and phenotypie markers of BDLSC were char-acterized using immunohistochemistry, RT-PCR and electron microscopy.The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.Results The 50 ml/L eholestatie serum was the optimal concentration for the selection of BDLSC at which BDLSC could sur-vive while the other populations of the bone marrow cells could not.The purified BDLSC could prolif-erate for 6 passages and maintained stable markers in our proliferating system.When the culture sys-tem changed to differentiating system, hepatocyte-like colony-forming units (HCFU) were formed.HCFU expressed markers of embryonic hepatoeytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and eytoehrome P450-2b1), and hepatocyte nuclear factors (HNF-1α and HNF-3β).They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.Conclusion BDLSC can be selected directly from whole bone marrow cells and pure BDLSC can also proliferate for 6 passages.The differentiated cells have hepatocyte-like phenotype and function.BDLSC will he a new way to provide a readily avail-able alternate source of cells for clinical hepatocyte therapy.     

骨髓源性肝干细胞的筛选、扩增和分化

Objective To explore the feasibility of direct separation, selective proliferation and differentiation of BDLSC from hone marrow cells with culture systems containing cholestatic serum in vitro.Methods Whole bone marrow cells of rats cultured in routine medium were replaced with con-ditioning selection media containing eholestatic sera of different concentrations after they attached to the plates.The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures.Then the selected BDLSC were induced to proliferating culture system and dif-ferentiating culture system.Each passage of the proliferated stem cells was subjected to flow cytome-try for detection of stem cell markers.The morphology and phenotypie markers of BDLSC were char-acterized using immunohistochemistry, RT-PCR and electron microscopy.The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.Results The 50 ml/L eholestatie serum was the optimal concentration for the selection of BDLSC at which BDLSC could sur-vive while the other populations of the bone marrow cells could not.The purified BDLSC could prolif-erate for 6 passages and maintained stable markers in our proliferating system.When the culture sys-tem changed to differentiating system, hepatocyte-like colony-forming units (HCFU) were formed.HCFU expressed markers of embryonic hepatoeytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and eytoehrome P450-2b1), and hepatocyte nuclear factors (HNF-1α and HNF-3β).They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.Conclusion BDLSC can be selected directly from whole bone marrow cells and pure BDLSC can also proliferate for 6 passages.The differentiated cells have hepatocyte-like phenotype and function.BDLSC will he a new way to provide a readily avail-able alternate source of cells for clinical hepatocyte therapy.     

骨髓源性肝干细胞的筛选、扩增和分化

Objective To explore the feasibility of direct separation, selective proliferation and differentiation of BDLSC from hone marrow cells with culture systems containing cholestatic serum in vitro.Methods Whole bone marrow cells of rats cultured in routine medium were replaced with con-ditioning selection media containing eholestatic sera of different concentrations after they attached to the plates.The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures.Then the selected BDLSC were induced to proliferating culture system and dif-ferentiating culture system.Each passage of the proliferated stem cells was subjected to flow cytome-try for detection of stem cell markers.The morphology and phenotypie markers of BDLSC were char-acterized using immunohistochemistry, RT-PCR and electron microscopy.The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.Results The 50 ml/L eholestatie serum was the optimal concentration for the selection of BDLSC at which BDLSC could sur-vive while the other populations of the bone marrow cells could not.The purified BDLSC could prolif-erate for 6 passages and maintained stable markers in our proliferating system.When the culture sys-tem changed to differentiating system, hepatocyte-like colony-forming units (HCFU) were formed.HCFU expressed markers of embryonic hepatoeytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and eytoehrome P450-2b1), and hepatocyte nuclear factors (HNF-1α and HNF-3β).They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.Conclusion BDLSC can be selected directly from whole bone marrow cells and pure BDLSC can also proliferate for 6 passages.The differentiated cells have hepatocyte-like phenotype and function.BDLSC will he a new way to provide a readily avail-able alternate source of cells for clinical hepatocyte therapy.     

骨髓源性肝干细胞的筛选、扩增和分化

Objective To explore the feasibility of direct separation, selective proliferation and differentiation of BDLSC from hone marrow cells with culture systems containing cholestatic serum in vitro.Methods Whole bone marrow cells of rats cultured in routine medium were replaced with con-ditioning selection media containing eholestatic sera of different concentrations after they attached to the plates.The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures.Then the selected BDLSC were induced to proliferating culture system and dif-ferentiating culture system.Each passage of the proliferated stem cells was subjected to flow cytome-try for detection of stem cell markers.The morphology and phenotypie markers of BDLSC were char-acterized using immunohistochemistry, RT-PCR and electron microscopy.The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.Results The 50 ml/L eholestatie serum was the optimal concentration for the selection of BDLSC at which BDLSC could sur-vive while the other populations of the bone marrow cells could not.The purified BDLSC could prolif-erate for 6 passages and maintained stable markers in our proliferating system.When the culture sys-tem changed to differentiating system, hepatocyte-like colony-forming units (HCFU) were formed.HCFU expressed markers of embryonic hepatoeytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and eytoehrome P450-2b1), and hepatocyte nuclear factors (HNF-1α and HNF-3β).They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.Conclusion BDLSC can be selected directly from whole bone marrow cells and pure BDLSC can also proliferate for 6 passages.The differentiated cells have hepatocyte-like phenotype and function.BDLSC will he a new way to provide a readily avail-able alternate source of cells for clinical hepatocyte therapy.     

骨髓源性肝干细胞的筛选、扩增和分化

Objective To explore the feasibility of direct separation, selective proliferation and differentiation of BDLSC from hone marrow cells with culture systems containing cholestatic serum in vitro.Methods Whole bone marrow cells of rats cultured in routine medium were replaced with con-ditioning selection media containing eholestatic sera of different concentrations after they attached to the plates.The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures.Then the selected BDLSC were induced to proliferating culture system and dif-ferentiating culture system.Each passage of the proliferated stem cells was subjected to flow cytome-try for detection of stem cell markers.The morphology and phenotypie markers of BDLSC were char-acterized using immunohistochemistry, RT-PCR and electron microscopy.The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.Results The 50 ml/L eholestatie serum was the optimal concentration for the selection of BDLSC at which BDLSC could sur-vive while the other populations of the bone marrow cells could not.The purified BDLSC could prolif-erate for 6 passages and maintained stable markers in our proliferating system.When the culture sys-tem changed to differentiating system, hepatocyte-like colony-forming units (HCFU) were formed.HCFU expressed markers of embryonic hepatoeytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and eytoehrome P450-2b1), and hepatocyte nuclear factors (HNF-1α and HNF-3β).They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.Conclusion BDLSC can be selected directly from whole bone marrow cells and pure BDLSC can also proliferate for 6 passages.The differentiated cells have hepatocyte-like phenotype and function.BDLSC will he a new way to provide a readily avail-able alternate source of cells for clinical hepatocyte therapy.     

Osteogenic potential of rabbit dermal fibroblasts cultured in vitro：a scanning electron microscopic study

ＴＳｈａｎｇｈａｉＩｎｓｔｉｔｕｔｅｏｆＴｒａｕｍａｔｏｌｏｇｙａｎｄＯｒｔｈｏｐａｅｄｉｃｓ,Ｓｈａｎｇｈａｉ２０００２５,Ｃｈｉｎａ（ＣｈａｉＢＦａｎｄＬｉＨ）ＳｈａｎｇｈａｉＮｏ．２ＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ（ＴａｎｇＸＭ）ｈｅｆｉｂｒｏｂｌａｓｔｓｔａｋｅａｐｉｖｏｔａｌｒｏｌｅｉｎｔｈｅｒｅｐａｉｒｐｒｏｃｅｓｓｅｓｏｆｖａｒｉｏｕｓｗｏｕｎｄｅｄｔｉｓｓｕｅｓ．Ｔｈｉｓｈｏｌｄｓｔｒｕｅｉｎｈｅａｌｉｎｇｏｆｆｒａｃｔｕｒｅｄｂｏｎｅｔｏｏ．Ｃｏｎｖｅｎｔｉｏｎａｌｈｉｓｔｏ…     

A comparative histologic analysis of tissue-engineered bone using platelet-rich plasma and platelet-enriched fibrin glue.

OBJECTIVE: The aim of this study was to compare the effects of platelet-rich plasma (PRP) and platelet-enriched fibrin glue on bone formation in bone tissue engineering. STUDY DESIGN: PRP was mixed with bone marrow mesenchymal stem cells and bone morphogenetic protein-2 (BMP-2), and the composites were injected into the subcutaneous space on the dorsum of nude mice. On the contralateral side of the dorsum, platelet-enriched fibrin glue/bone marrow mesenchymal stem cells/BMP-2 composites were injected. Bone formation was evaluated after 12 weeks. RESULTS: The volumes of subcutaneous nodules formed in nude mice were 55 +/- 18 microL at the PRP/bone marrow mesenchymal stem cells/BMP-2 sites and 135 +/- 27 microL at the platelet-enriched fibrin glue/bone marrow mesenchymal stem cells/BMP-2 sites. Histomorphometric analysis demonstrated that the nodules contained 14.9 +/- 4.1% newly formed bone when using PRP and 19.8 +/- 3.6% newly formed bone when using platelet-enriched fibrin glue. CONCLUSION: The results indicated that the osteogenic characteristics of platelet-enriched fibrin glue are superior to PRP in bone tissue engineering.     

体外诱导成纤维细胞成骨活性表达的研究

目的探讨成纤维细胞(fibroblast,FB)体外成骨表达的诱导因素,为其能否成为骨组织工程的种子细胞提供理论依据.方法分离和纯化新西兰兔肉芽组织FB,按1×105/ml分别接种于含纤维结合蛋白(fibronectin,FN)10、20、40、60、80μg/ml条件培养液中(实验1～5组),对照组为不含FN的无血清培养液.于培养14 d后,行细胞组织学观察及钙化结节形成率、趋骨性四环素荧光标记、3H-TdR标记、骨钙素测定及3H脯氨酸标记,检测FB成骨表型表达的标志.结果组织学观察实验组发现FB由梭形逐渐趋向多突形和圆形,细胞核偏向一侧,细胞重叠成多层结构;其表面有钙盐堆积,逐渐呈云雾状物质,经不断融合和扩大形成不透光的结节.干预培养14 d钙化结节形成率:对照组15.35%±3.45%,实验1～5组分别为53.73%±9.49%、75.21%±9.80%、98.34%±15.20%、61.83%±10.04%、45.11%±8.70%,实验组与对照组比较,差异有统计学意义(P＜0.05);实验3组与其它各实验组比较,差异有统计学意义(P＜0.05).趋骨性四环素特异性标记为新生骨组织;FB增殖活性,实验3、4、5组7 d时,实验2、3、4组14 d时与对照组比较,差异有统计学意义(P＜0.05).实验1～5组FB分泌骨钙素,实验2～5组3H-脯氨酸掺入量高于对照组,7、14d时与对照组比较差异有统计学意义(P＜0.05).结论适量的FN可以促进FB增殖、胶原蛋白的合成及骨钙素分泌,FN可以诱导FB成骨表达,适宜浓度为40～60μg/ml.     

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).     

Mineralization in in vitro cultures of rabbit marrow stromal cells

Localized regions of mineralization were found in confluent cultures of rabbit marrow fibroblastic cells. The mineralized tissue developed within clusters of giant fat cells in the spaces between the cells. Investigations with light and electron microscopy demonstrated that in these sites there was some differentiation of the fibroblastic cells in an osteogenic direction, shown by changes to more polygonal shapes, and the synthesis of well-banded collagen similar to that found in bone tissue. Differentiation may be due, in part, to increased cell density in a confined space. Growth of the mineralized tissue was observed in the living cultures with a fluorescence microscope. Electron probe microanalysis confirmed that the mineral formed was hydroxyapatite. Initiating sites of mineralization included membranous vesicular bodies, lipid, and products of cellular degeneration. Once initiated, mineralization appeared to spread rapidly into adjacent collagenous and other structures, suggesting the appearance of a mixture of skeletal-type and dystrophic mineralization.     

A comparative qualitative histological analysis of tissue-engineered bone using bone marrow mesenchymal stem cells, alveolar bone cells, and periosteal cells.

For tissue-engineered bone formation, autogenous osteogenic cells are of paramount importance for successful bone formation. In order to investigate the donor cell-related differences in tissue-engineered bone, cultured bone marrow mesenchymal stem cells, cultured alveolar bone cells, and cultured periosteal cells were examined for their in vivo potential to form bone. These cells were isolated from dogs, expanded in vitro, mixed with autologous fibrin glue and BMP-2, and then injected into the subcutaneous space on the dorsum of nude mice. Bone formation was evaluated at 12 weeks. Histomorphometric analysis demonstrated that the subcutaneous nodules formed in nude mice contained 26.9% newly formed bone when using the bone marrow mesenchymal stem cells, 41.1% newly formed bone when using the alveolar bone cells, and 58.2% newly formed bone when using the periosteal cells. The results suggest that periosteal cells are the best choice for enhancing bone formation in tissue engineering of bone regeneration.     

增强型绿色荧光蛋白标记技术对骨折后骨髓间充质干细胞的迁移示踪

目的采用增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)标记技术,对外源性骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs)迁移至骨折区域进行示踪,为骨修复中干细胞作用机制的研究创造条件。方法EGFP标记日本大耳白兔MSCs,部分标记细胞经成骨诱导培养后检测其细胞表型。另取日本大耳白兔18只制成双侧尺骨骨折模型,随机分为实验组与对照组,每组9只,分别于术前24h、术后1和24h,将扩增的标记MSCs和未标记MSCs以1×107个/kg剂量分别注入实验组和对照组的耳缘静脉;术后48h处死动物,取左侧尺骨断端组织行冰冻切片、荧光显微镜观察,右侧样本组织固定后行石蜡切片、HE染色、光镜观察。结果MSCs培养后分化及表型良好,标记MSCs仍具有较快的增殖能力,经成骨诱导培养后,可表达碱性磷酸酶,并具有钙结节形成能力;兔体内实验术后48h动物均存活,HE染色见骨折断端为血肿组织。术前24h、术后1和24h静脉注入标记细胞后均可在骨断端组织中观察到EGFP阳性细胞,而对照组则未见EGFP阳性细胞。结论兔MSCs经EGFP标记后仍然具有成骨细胞诱导能力;EGFP示踪技术提示,静脉途径给予标记的MSCs可以迁移聚集到骨折区域的血肿组织内。     

pcDNA_(3.1)-OGP基因转染兔骨髓基质干细胞的表达及生物学效应的观察

[目的]观察成骨生长肽(osteogenic growth peptid,OGP)基因转染兔骨髓基质干细胞后的表达及表达产物对骨髓基质干细胞向成骨细胞分化的影响.[方法]构建重组真核表达载体pcDNA_(3.1)-OGP,并在脂质体介导下,将其导入兔骨髓基质干细胞.通过G418筛选获得阳性克隆.用RT-PCR方法榆测OGP基因在骨髓基质干细胞内的表达.Ⅰ型胶原和碱性磷酸酶的检测观察转染pcDNA_(3.1)-OGP后骨髓基质干细胞向成骨细胞分化情况.[结果]成功构建真核表达载体pcDNA_(3.1)-OGP,用RT-PCR方法及碱性磷酸酶和Ⅰ型胶原检测证实OGP基因能在骨髓基质干细胞中表达并促进其向成骨细胞分化.[结论]经pcDNA_(3.1)-OGP转染的兔骨髓基质干细胞不仅可以表达OGP,而且具有向成骨细胞系分化的特性.     

Osteogenesis by canine and rabbit bone marrow in diffusion chambers

Summary Osteogenic activity of canine and rabbit bone marrow and marrow stromal fibroblasts (MSF) derived from marrow culturedin vitro was evaluated using diffusion chambers. Marrow from young dogs and rabbits grown in cell culture produced confluent layers of MSF. Diffusion chambers containing 0.18–7.6×10⁶ allogeneic MSF were inserted into the peritoneal cavities of 5 dogs and 6 rabbits. Chambers recovered from the dogs (15/16) contained only loose fibrous tissue while chambers from rabbits (9/13) contained membranous bone and cartilage. Diffusion chambers implanted with 1.0–32.4×10⁷ fresh allogeneic marrow cells suspended in cell culture medium were inserted into the peritoneal cavities of 11 dogs and 9 rabbits for 3–8 weeks, and after recovery examined histologically. Membranous bone was formed in 4/40 chambers containing canine marrow while bone and hyaline cartilage was formed in 21/27 chambers containing rabbit marrow. This apparent species difference in incidence of bone marrow osteogenesis may relate to a lower concentration of osteogenic precursor cells in canine marrow, a failure of osteogenic precursor cells to differentiate to osteoblasts in a somewhat artificial environmentin vivo (viz diffusion chambers), a lack of cell-matrix interaction to stimulate cell differentiation, inappropriately short diffusion chamber implantation times post grafting, or a difference in ontogenetic stage of development of marrow donors with rabbit cells being physiologically younger.     

补肾活血方调控 microRNA-210促进 BMSCs 成骨分化的研究

目的研究补肾活血方促进骨髓间充质干细胞(Bone Mesenehymal Stem Cells,BMSCs)成骨分化的分子机制。方法贴壁培养法分离纯化大鼠BMSCs,将培养成功的BMSCs分为空白组、成骨诱导组和中药组,培养7d、14d行矿化结节茜素红染色(Alizarin Red S,ARS),倒置显微镜观察矿化骨结节形成情况,采用real-time PCR检测miR-210的表达变化。结果与空白组比较,成骨诱导组和中药组BMSCs钙化结节数量明显增多,差异有非常显著的统计学意义(P0.01)。在成骨诱导剂、补肾活血方水提液的作用下,与空白组比较,miR-210表达明显降低,且中药组较成骨诱导组更明显降低miR-210表达。结论补肾活血方通过降低miR-210表达促进BMSCs成骨分化。