体外诱导人肾间质成纤维细胞成骨分化

 目的: 观察在体外培养条件下,使用成骨诱导培养液或不同浓度钙离子诱导人肾间质成纤维细胞发生成骨分化的效果,初步探讨肾脏Randall斑形成可能的细胞机制。方法: 体外培养人肾间质成纤维细胞,实验分为5组:成骨诱导组(加成骨诱导液)、CaⅠ组(加0.5 mmol/L Ca²⁺液)、CaⅡ组(加1.5 mmol/L Ca²⁺液)、Ca Ⅲ 组(加2.5 mmol/L Ca²⁺液)和对照组(加PBS)。各组细胞分别培养至第1、3、6、9天时,采用MTT法检测细胞活力;诱导至第9天时,用细胞钙茜素红染色液和钙钴法磷酸酶染色液对各组细胞进行染色,观察钙结节形成和碱性磷酸酶的表达情况。另外,分别采用real-time PCR和Western blot检测各组细胞不同时间点Runt相关转录因子2(Runx2)的 mRNA和蛋白表达水平。 结果: 在1.5 mmol/L 和2.5 mmol/L Ca²⁺浓度条件下细胞的活力明显受抑制。细胞染色结果证实成骨诱导液组和钙离子组均可以见到典型的红色钙结节和黑色块状的硫化钴沉淀物。成骨诱导组细胞Runx2的mRNA和蛋白相对表达量(0~9 d)逐渐升高(P<0.05);诱导至第9天时,3个钙离子组细胞Runx2的mRNA和蛋白表达呈浓度依赖性升高(P<0.05)。结论: 在体外培养环境下,人肾成纤维细胞可以在成骨诱导培养液的刺激作用下发生成骨分化;另外,在高钙离子环境,人肾成纤维细胞也发生了类似的成骨分化。肾乳头组织中的成纤维细胞在高钙离子等因素的作用下发生成骨分化,这可能是肾脏Randall斑形成的细胞学基础。     

Gene expression of markers of osteogenic differentiation of human mesenchymal cells on collagen I-modified microrough titanium surfaces

Microrough, doubly acid etched titanium surfaces (Ti) were further modified by amination and covalent coupling of fibrillar collagen type I (ColTi). Human Mesenchymal Cells (HMC) adhesion and growth, and relevant osteogenic differentiation in nonosteogenic (basal) medium were evaluated by fluorescence microscopy, scanning electron microscopy, and RT-PCR for a three-week period. Results show strongly enhanced HMC adhesion and cell density at short experimental time on ColTi, together with complete spreading of the cell body over the microrough surface topography. RT-PCR analysis of several genes involved in osteogenesis indicate, since the first week of culturing, significant progression of HMC on ColTi along the osteogenic pathway. These results indicate that the adopted process of surface immobilization of collagen, mandatory to impart collagenase resistance in implant sites, does not impair biospecific interactions between HMC and collagen. Thus, it is possible to upgrade properties arising from the control of Ti surfaces topography by surface-chemistry driven enhanced recruitment of precursor osteogenic cells and pro-osteogenic stimula.     

Increased expression of integrin alphavbeta5 induces the myofibroblastic differentiation of dermal fibroblasts

The biological effect of cytokines is mainly determined by the cytokine-receptor interaction, which is modulated by the concentration and the activity of cytokines and/or their receptors. Because alphav-containing integrins can bind to and/or activate latent TGF-beta, these integrins have been thought to be involved in the pathogenesis of fibrotic disorders. Our recent observations that alphavbeta5 is up-regulated in scleroderma fibroblasts and that the transient overexpression of alphavbeta5 increases the human alpha2(I) collagen gene expression in normal fibroblasts suggest the involvement of alphavbeta5 in the self-activation system in scleroderma fibroblasts. In this study, we established stable transfectants with alphavbeta5 using normal dermal fibroblasts and demonstrated that such cells differentiated into myofibroblasts by the stimulation of autocrine TGF-beta. This observation is explained by 1) alphavbeta5 recruiting latent TGF-beta1 on the cell surface, 2) endogenous active TGF-beta localizing on the cell surface, and 3) alphavbeta5 interacting with TGF-beta receptors. Furthermore, blockade of alphavbeta5 reversed the myofibroblastic phenotype in scleroderma fibroblasts. These data identify a novel mechanism for the establishment of autocrine TGF-beta signaling in dermal fibroblasts by the up-regulation of alphavbeta5 and suggest the possibility of regulating fibrotic disorders, especially scleroderma, by targeting this integrin.     

The effect of heparin-functionalized PEG hydrogels on three-dimensional human mesenchymal stem cell osteogenic differentiation

Poly(ethylene glycol) (PEG) hydrogels functionalized with heparin were utilized as a three-dimensional culture system for human mesenchymal stem cells (hMSCs). Heparin-functionalized hydrogels supported hMSC viability, as quantified through live/dead imaging, and induced osteogenic differentiation, as measured by increased alkaline phosphatase (ALP) production and osteopontin (OPN) and collagen I (COL I) gene expression over the 5-week study. Further exploration of the potential mechanism of heparin-induced osteogenic differentiation was performed. Specifically, the availability of bone morphogenetic protein 2 (BMP2) and fibronectin (FN) in the culture system was controlled and hMSC osteogenic differentiation was evaluated as a function of the microenvironment. BMP2 availability increased both ALP production and OPN gene expression, while FN increased ALP production, but not OPN gene expression. Furthermore, immunostaining of integrin expression revealed that viability and differentiation were differentially affected by integrin production, where both alpha5beta1 and alphavbeta3 integrin-ligand interactions supported viability, while only the alpha5beta1 integrin played a role in hMSC osteogenic differentiation.     

微渠多孔羟基磷灰石支架诱导骨髓间充质干细胞成骨分化的miRNA表达谱分析

文题释义： 羟基磷灰石：是目前最为理想的生物活性材料,具有生物相容性、骨传导性和骨诱导性,植入人体后对组织无刺激和排斥作用,能与骨形成很强的化学结合,用作骨缺损的充填材料,能为新骨的形成提供支架,发挥骨传导作用,是理想的硬组织替代材料。 MicroRNA(miRNA)：是一类内生的、长度为20-24个核苷酸的单链小分子RNA,由具有发夹结构的70-90个碱基大小的单链RNA前体经过Dicer酶加工后生成,其可以通过几个miRNAs的组合在转录后水平精细调控基因的表达。 背景：多孔羟基磷灰石支架具有良好的体内外成骨效能,但其所涉及的miRNAs复杂调控机制相关研究较少。 目的：探讨多孔羟基磷灰石支架材料介导大鼠骨髓间充质干细胞成骨矿化过程中相关miRNA表达谱的变化。 方法：体外分离、培养和鉴定大鼠骨髓间充质干细胞,将骨髓间充质干细胞与多孔羟基磷灰石支架共培养为实验组,骨髓间充质干细胞单独培养为空白对照组,分别进行成骨诱导7 d,运用miRNA高通量测序技术分析两组骨髓间充质干细胞成骨矿化过程中相关miRNA表达谱的变化并进行GO分析,筛选出两组中表达差异明显的miRNA分子并进行qRT-PCR验证。 结果与结论：①与空白对照组比较,成骨诱导7 d时实验组BMP2、ALP、Runx2 mRNA表达上调,其中BMP2上调明显(P < 0.05);②microRNA高通量测序结果显示miR-210-3p、miR-146a-5p等13个miRNAs明显上调;let-7c-3p、let-3615等17个miRNAs明显下调;③GO分析上调的miRNA靶基因主要参与生物学调节、细胞基因表达、基因表达调节等,包括NF-κB、Toll样受体9、细胞间黏附、白细胞介素1调节、血管生成、Hippo等信号通路;④实时荧光定量qPCR验证结果显示miRNA-210在实验组上调15倍,miR-146a-5p在实验组上调10倍(P < 0.05);⑤结果表明,新型微渠多孔羟基磷灰石支架可以通过上调骨髓间充质干细胞miRNA-210-3p和miR-146a表达,促进骨髓间充质干细胞的成骨分化。 ORCID: 0000-0002-8722-1548(郑佳俊) 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     

Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold

Functional engineering of musculoskeletal tissues generally involves the use of differentiated or progenitor cells seeded with specific growth factors in biomaterial scaffolds. Ideally, the scaffold should be a functional and structural biomimetic of the native extracellular matrix and support multiple tissue morphogenesis. We have previously shown that electrospun, three-dimensional nanofibrous scaffolds that morphologically resemble collagen fibrils are capable of promoting favorable biological responses from seeded cells, indicative of their potential application for tissue engineering. In this study, we tested a three-dimensional nanofibrous scaffold fabricated from poly(epsilon-caprolactone) (PCL) for its ability to support and maintain multilineage differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in vitro. hMSCs were seeded onto pre-fabricated nanofibrous scaffolds, and were induced to differentiate along adipogenic, chondrogenic, or osteogenic lineages by culturing in specific differentiation media. Histological and scanning electron microscopy observations, gene expression analysis, and immunohistochemical detection of lineage-specific marker molecules confirmed the formation of three-dimensional constructs containing cells differentiated into the specified cell types. These results suggest that the PCL-based nanofibrous scaffold is a promising candidate scaffold for cell-based, multiphasic tissue engineering.     

Behavior of human dermal fibroblasts in three-dimensional fibrin clots: dependence on fibrinogen and thrombin concentration

Fibrin sealant products are used in hemostasis and tissue sealing, and potentially as a cell delivery vehicle. In this study, fibrin sealant was evaluated as a delivery vehicle for human dermal fibroblasts. Fibroblast proliferation and migration were assessed in various dilutions of fibrin sealant by changing the fibrinogen and thrombin concentration. Fibroblasts proliferated well within three-dimensional (3-D) fibrin clots consisting of fibrinogen (5-17 mg/mL) and thrombin (1-167 U/mL). These fibroblasts also retained good morphology and growth characteristics after migrating out of the 3-D fibrin clots. Furthermore, using Western blot and fluorescence-activated cell-sorting analysis, we found that the expression of growth factors and interleukins in the entire fibroblast-fibrin construct was dependent on the fibrin sealant formulation. For example, in a formulation in which fibroblasts showed modest proliferation and migration, interleukin 8 was secreted to a lesser extent than in a formulation that supported robust proliferation and migration. To our knowledge, this is the first time that it has been shown that modifying the concentration of fibrinogen and thrombin affects fibroblast behavior within formed 3-D fibrin clots. In addition, some of these formulations present an ideal delivery vehicle for fibroblasts that could be used for the treatment of chronic wounds.     

Microarray-based identification of age-dependent differences in gene expression of human dermal fibroblasts

Senescence is thought to play an important role in the progressive age-related decline in tissue integrity and concomitant diseases, but not much is known about the complex interplay between upstream regulators and downstream effectors. We profiled whole genome gene expression of non-stressed and rotenone-stressed human fibroblast strains from young and oldest old subjects, and measured senescence associated β-gal activity. Microarray results identified gene sets involved in carbohydrate metabolism, Wnt/β-catenin signaling, the cell cycle, glutamate signaling, RNA-processing and mitochondrial function as being differentially regulated with chronological age. The most significantly differentially regulated mRNA corresponded to the p16 gene. p16 was then investigated using qPCR, Western blotting and immunocytochemistry. In conclusion, we have identified cellular pathways that are differentially expressed between fibroblast strains from young and old subjects.     

Altered expression of connective tissue genes in postnatal chondroinduced human dermal fibroblasts

In a novel model for postnatal chondroinduction, normal human dermal fibroblasts (hDFs) cultured with demineralized bone powder (DBP) express chondrocyte features after 7 days. Representational difference analysis (RDA) prior to overt chondroblastogenesis (3 days) revealed altered expression of connective tissue genes (collagens, collagen receptors, and post-translational enzymes). Northern or RT-PCR analysis at 3, 7, 14, or 21 d showed different expression patterns for those genes. COL3A1 was transiently upregulated, whereas upregulation of COL11A1, integrin alpha-11, lysyl oxidase, and lysyl hydroxylase 2 mRNAs persisted for 7 days. Downregulation of decorin was sustained for 21 d. The expression of the post-translational enzymes induced by DBP was unique when compared with human skin and human articular chondrocytes. Thus, the data suggest an "induced chondroblast" stage with a unique connective tissue gene expression profile that may result in a matrix supportive of chondrogenesis by postnatal cells.     

胶原蛋白支架对载杜仲叶提取物处理的人牙周膜干细胞增殖和成骨分化的作用

文题释义：杜仲：是中国特有的杜仲科植物,其活性成分具有促进某些间充质干细胞的增殖,调节骨代谢和促进骨生成,同时抑制骨吸收,并加速骨痂改建的药理作用。临床上,杜仲主要用于预防和治疗骨质疏松症、骨关节炎和骨折等疾病。 人牙周膜干细胞：一种来自牙周韧带的多能间充质干细胞,具有良好的自我更新和多向分化潜能。通过不同的体外诱导条件刺激,它可以分化成骨样细胞、软骨样细胞、牙周韧带细胞、脂肪样细胞和神经元样细胞,并且就细胞来源而言,它是用于牙周组织再生的优选种子细胞。 背景：胶原蛋白支架是一种良好的组织工程材料,但目前其对于载杜仲叶提取物的人牙周膜干细胞的生物相容性、增殖及成骨活性的影响尚未有报道。 目的：通过对载杜仲叶提取物的人牙周膜干细胞与胶原蛋白支架的体外共培养,研究人牙周膜干细胞在支架材料上的形态特征、黏附情况、增殖及成骨分化功能。 方法：将载杜仲叶提取物的人牙周膜干细胞与胶原蛋白支架体外共培养作为实验组,以人牙周膜干细胞与胶原蛋白支架的共培养作为阳性对照组,无支架的人牙周膜干细胞常规培养作为空白对照组。 结果与结论：①扫描电镜下可见,实验组支架上有大量人牙周膜干细胞附着,且细胞突触与支架紧密相连,生长状态良好,细胞间相互接触、紧密相连;②共培养4,8和12 h后,实验组和阳性对照组人牙周膜干细胞的黏附率均呈上升趋势,实验组黏附率高于阳性对照组(P < 0.05);③MTT结果显示,复合培养3,5和7 d后,实验组的细胞吸光度值高于阳性对照组(P < 0.05);④成骨诱导7,14 d后,实验组碱性磷酸酶活性高于阳性对照组(P < 0.05);⑤RT-PCR检测结果显示,成骨刺激作用21 d后,实验组和阳性对照组中成骨相关基因Runx2、OPN和OCN的表达显著高于空白对照组(P < 0.01),实验组上述基因的表达均高于阳性对照组(P < 0.05);⑥上述数据说明,胶原蛋白支架对于载杜仲叶提取物的人牙周膜干细胞具有较好的生物相容性,复合培养后可保持细胞形态、促进其增殖及成骨分化功能。 ORCID: 0000-0002-6784-9979(刘焱) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

17beta-estradiol regulates the secretion of TGF-beta by cultured human dermal fibroblasts

Estrogen plays an important role in skin homeostasis, as demonstrated by the changes seen in the skin of post-menopausal women, changes reversed by HRT. Estrogen also has a role in wound healing, since estrogen deficiency as occurs post-menopausally and in ovariectomised animals, is associated with a reduced rate of wound healing. Estrogen appears to modulate all phases of wound healing with effects on inflammatory cells, epithelialization, angiogenesis, extracellular matrix deposition and tissue remodelling. This study was designed to investigate the effects of 17beta-estradiol on cultured human dermal fibroblasts using an in vitro wound-healing assay. The end points investigated were cell migration, proliferation, total collagen secretion and active TGF-beta1 secretion. 17beta-estradiol significantly increased the migration and proliferation of cultured dermal fibroblasts following mechanical wounding, although the secretion of total soluble collagen was not altered. An increase in TGF-beta1 was demonstrated by unwounded confluent dermal fibroblast monolayers in response to 17beta-estradiol, but paradoxically, a decrease in the secretion of TGF-beta1 was demonstrated in the mechanically wounded dermal fibroblasts. These results identify human dermal fibroblasts as estrogen target cells and provide further evidence for a role by which estrogen regulates this particular cell type as part of the wound-healing process. However, the paradoxical nature of the effect of estrogen on TGF-beta1 secretion following mechanical wounding suggests that the cellular mechanism of action is complex. A greater understanding of the cell-specific action of estrogen may help to develop therapies that will improve cutaneous wound healing in the future.     

Physiological variables affecting collagen lattice contraction by human dermal fibroblasts

Normal human dermal fibroblasts cultured in collagen lattices can compact that matrix by the process known as lattice contraction. That process is a model of the pathological one of scar contracture or wound contraction and is affected by several factors. Lattice contraction is promoted by the addition of adequate amounts of fetal bovine serum to the medium (maximum contraction with 10% serum). The process requires energy, of which glucose and pyruvate have been shown to be adequate sources. When glucose is used as the substrate, the major pathway of energy generation appears to be anaerobic metabolism. When pyruvate is the only substrate, aerobic metabolism may be crucial. The synthesis of DNA is not required for lattice contraction, while protein synthesis is, although the identities of the specific proteins are unknown. Impairment of calcium ion transport inhibits lattice contraction, and the specific inhibition of calmodulin-calcium interactions by W-7 blocks contraction. W-7 at a concentration of 6 x 10(-6) M blocks lattice contraction completely, while it has no effect at any lower concentration. Impairing dynamic microtubule activity impairs contraction. Disrupting microfilaments by cytochalasin B completely blocks lattice contraction. Microfilament function and calcium-calmodulin may be linked by a mechanism involving myosin-ATPase. The process of cell-mediated lattice contraction requires the production of energy, protein synthesis, and a functional cytoskeleton.     

The influence of three-dimensional nanofibrous scaffolds on the osteogenic differentiation of embryonic stem cells

Embryonic stem cells represent a potentially unlimited cell source for tissue engineering applications. However, in order to be used for such applications, embryonic stem cells' differentiation must be controlled to only the desired lineages. In this study, we examine the effects of nanofibrous architecture and biochemical cues on the osteogenic differentiation of embryonic stem cells compared to the more traditional architecture without the nanofibrous features in two dimensions (thin matrix or flat films) and three dimensions (scaffolds) in vitro. After three weeks of culture the nanofibrous thin matrices were capable of supporting mRNA expression of osteogenic differentiation markers in embryonic stem cells without osteogenic supplements, while solid films required osteogenic supplements and growth factors to achieve mRNA expression of osteogenic differentiation markers. Nanofibrous scaffolds substantially enhanced mRNA expression of osteogenic differentiation markers compared to solid-walled scaffolds, nanofibrous thin matrices or solid films. After 4 weeks of culture, nanofibrous scaffolds were found to contain 3 times more calcium and stronger osteocalcin stain throughout the scaffolds than the solid-walled scaffolds. Overall, the nanofibrous architecture enhanced the osteogenic differentiation and mineralization of embryonic stem cells compared to the solid-walled architecture in both two and three-dimensional cultures.     

Enhancement of PDGF-BB mitogenic activity on human dermal fibroblasts by biospecific dextran derivatives

Dextran derivatives are biosynthetic polyanionic polymers which exert some of the heparin properties such as regulating the activity of several heparin-binding growth factors. Based on a reproducible synthetic procedure, we have synthesized a new generation of dextran derivatives named dextran methylcarboxylate benzylamide sulfate (DMCBSu). Here we investigated the ability of a library of well-characterized DMCBSu to interact with platelet-derived growth factor-BB (PDGF-BB), which has essential roles during wound healing. Using gel mobility shift assay, our results indicate that benzylamide and sulfate groups act synergically to bind to PDGF-BB. Furthermore we show that depending on their chemical composition, functionalized dextrans are able to potentiate the mitogenic activity of PDGF-BB on human dermal fibroblasts. This enhancing effect is accompanied with changes in PDGF-BB-induced signaling events, as determined by the use of specific inhibitors and by western blot. Our results suggest that the use of such biopolymers combined with a local administration of the growth factor could increase the efficiency of the biomolecule activity in future therapeutic strategies.     

Effects of 17beta-estradiol on matrix metalloproteinase-1 synthesis by human dermal fibroblasts

BACKGROUND: Hormone replacement therapy (HRT) has been used in treatment of various menopausal disorders. It has been well documented that HRT increases the amount of dermal collagen and skin thickness in vivo. However little is known about the effects of female sex hormones on dermal fibroblasts in vitro. OBJECTIVE: The aim of this study is to determine whether or not 17beta-estradiol affects mRNA expression and production of type I collagen, matrix metalloproteinases-1 (MMP-1), tissue inhibitor metalloproteinases-1 (TIMP-1) or transforming growth factor-beta1 (TGF-beta1) by human dermal fibroblasts. METHODS: Fibroblasts were cultured with and without 17beta-estradiol for 6h. We evaluated the changes of mRNA expressions and protein production of type I collagen, MMP-1, TIMP-1 and TGF-beta1. RESULTS: The mRNA expressions of collagen alpha1(I), MMP-1, TIMP-1, TGF-beta1 were not changed by 17beta-estradiol stimulations at a concentration of 10(-12) to 10(-8) M. However, 17beta-estradiol at concentrations of 10(-12) and 10(-10) M exhibited inhibitory effects on proMMP-1, but not type I collagen or TIMP-1 synthesis. The synthesis of TGF-beta1 by fibroblasts stimulated with 10(-8) M of estradiol was significantly increased as compared with the control. However, the level of TGF-beta type II receptor phosphorylation was not elevated under the same conditions. CONCLUSION: Suppressed synthesis of MMP-1 at a low concentration of 17beta-estradiol may be partly involved in the dermal tissue remodeling to inhibit the degradative change.     

Influence of culture method on the proliferation and osteogenic differentiation of human adipo-stromal cells in nonwoven fabrics

The initial attachment, proliferation, and osteogenic differentiation of stromal cells from human fat tissue were investigated in three-dimensional nonwoven fabrics prepared from polyethylene terephthalate (PET) fiber with different diameters. The largest number of cells initially attached was observed in the nonwoven fabrics prepared from PET fiber with a diameter of 22.0 microm, irrespective of fabric porosity. The number of cells attached was larger and the cells were distributed more homogeneously in the fabrics by the agitated seeding method than by the static seeding method. The culture method depended on the time profile of cell proliferation. Cell proliferation improved in the following order: stirred (spinner flask) culture method > agitated culture method > static culture method. In addition, cells proliferated homogeneously in fabrics by the stirred culture method. When evaluated as a measurement of cell osteogenic differentiation, the activity of alkaline phosphatase (ALP) was not influenced by the diameter of fabrics. The static culture method tended to enable cells to enhance ALP activity, in contrast with the stirred and agitated culture methods. It is concluded that fabric fiber diameter and culture method greatly affected the proliferation and differentiation of cells in nonwoven fabrics.