分离培羊人体脂肪基质细胞，经过软骨定向诱导后复合藻酸盐凝胶，研究基异位软骨生成能力。方法将脂肪抽吸术获取的人体脂肪机械分割，通过I型胶原酶消化后得到脂肪10ug/LTCF-β，50mg/L维生素C的DMEM/F12培羊基中诱导培羊14d，获取的细胞按1×1010/L的细胞密度与质量分数为1.2%的藻酸钠复合、加入102mmol/LCaCl，充分泥匀，使之交联，将细胞-藻酸盐凝胶1ml注入每只体重为25g的BALB/C裸小隶鼠背部皮下（共4办），同时在裸小鼠臀部两侧皮下各注入相同量的无细胞藻酸盐及单纯细胞作为自岙对照、术后4、8周各处列2只动物，取材固定、脱钙、包埋后切自染色。镜下观察。结果经连续导培羊14d的脂肪基质细胞已具明显成骨表型（细胞基质中富含硫酸软骨素及II型胶原）。4、8周时，注射细胞-藻酸盐胶标本均显示成明显的软骨形成。肥大的软骨细非金属位于富含硫在术手3-4周已完全吸收。结论脂肪基质细胞过导培羊具有向软骨细胞表型分化的潜能。

目的:分离人体脂肪基质细胞并诱导培养其成骨表型。方法:将脂肪抽吸术获取的人体脂肪进行机械分割，通过Ⅰ型胶原酶消化后得到脂肪基质细胞，在BGJb培养基中原代培养10d，消化传代后用含体积分数10%FBS及10-8mol/L地塞米松，50mg/L左旋抗坏血酸，10nmol/L维生素D3，10mmol/Lβ2磷酸甘油钠的DMEM/F12培养基中诱导培养14d，观察细胞形态，绘制细胞生长曲线并对其成骨表型进行鉴定。结果：脂肪基质细胞呈成纤维细胞样贴壁生长，其中的成体干细胞经诱导培养后体积明显增大，胞核大面圆，胞浆丰富，群体倍增时间为66h。Gomori萘酚磷酸酯法染色显示其胞浆内富含碱性磷酸酶颗粒，vonKossa染色表明聚集的细胞团能形成矿化结节。结论：脂肪基质细胞中的成体干细胞经过矿化诱导培养后可向成骨细胞分化，并具有明显的成骨表型。

Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferatoractivated receptor (PPAR-γ 2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.

Functional engineering of musculoskeletal tissues generally involves rapid expansion of progenitor cells in vitro while retaining their potential for further differentiation and then induction in specific culture conditions. The autologous adipose-derived stromal cells (ASCS) are considered to contain pluripotent mesenchymal stem cells. Imaging with expression of green fluorescent protein (GFP) facilitates the detailed research on ASCs physiological behavior during differentiation into a variety of cell lineages both in vitro and in vivo. In this study, we aimed to confirm the trans-germ plasticity of homogeneously marked ASCs from GFP transgenic mice. Simultaneously, the term and intensity of GFP expression in ASCs were also focused on during variant inductions, when cells were incubated with multiple growth factors and adjuvant. ASCs were harvested from inguinal fat pads of transgenic nude mice, passaged 3 times in monolayer cultures, and then transferred to osteogenic, adipogenic, neurogenic, and myogenic medium. The morphological characterization of inductive cells was observed using phase-contrast microscopy and histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation. The expression of marker genes or proteins was measured using RT-PCR and immunocytochemical analysis. Collagen type I, osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, peroxisome proliferator-activated receptor(PPAR)-γ 2 and lipoprotein lipase (LPL) were positive in adipogenic ones, glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) were positive in neurogenic ones, and α-smooth muscle actin (α-SMA) was positive in myogenic ones. Moreover, the results of fluorescence microscopic imaging suggested that there was no significant decline of GFP expression during ASCs differentiation and the level of GFP maintained stable till differentiated ASCs showed apoptotic phenotype. So the endogenous GFP and multilineage potential of transgenic ASCs had no influences on each other. Since the population of GFP ASCs can be easily identified, it is proposed that they may be promising candidate seed cells for further studieson ASCs tissue engineering, especially the study on engineered tissues formed in vivo. (Mol Cell Biochem xxx: 1–10, 2005)

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo. When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo, the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT-PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage-specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo. These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo, however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.