控释神经营养因子与细胞移植减少损伤脊髓的胶质瘢痕

背景：前期研究发现控释胶质细胞源性神经营养因子与骨髓间充质干细胞源神经元样细胞联合移植可有效促进猕猴脊髓损伤后运动功能和感觉功能的恢复。 目的：观察控释胶质细胞源性神经营养因子联合骨髓间充质干细胞源神经元样细胞移植抑制猴脊髓损伤后胶质瘢痕形成的作用是否优于单纯细胞移植。 方法：取12只恒河猴,采用改良Allen氏法制作急性重度脊髓损伤模型,随机数字表法分为3组,实验组以控释胶质细胞源性神经营养因子联合自体骨髓间充质干细胞源神经元样细胞移植修复,对照组以自体骨髓间充质干细胞源神经元样细胞移植修复,空白对照组以磷酸盐缓冲液修复。修复后5个月,取出脊髓组织制成石蜡标本,应用免疫组织化学染色显示胶质瘢痕的形态特征、构成特点及瘢痕中神经纤维的再生情况,检测胶质瘢痕面积及胶质纤维酸性蛋白染色的平均吸光度值。 结果与结论：脊髓损伤部位胶质瘢痕由混合性增生的星形胶质细胞和组织细胞构成。空白对照组脊髓胶质瘢痕累及范围广,星形胶质细胞增生显著,神经丝蛋白免疫组织化学染色阴性,胶质瘢痕面积、胶质纤维酸性蛋白染色平均吸光度值高于实验组与对照组(P < 0.05);实验组、对照组脊髓胶质瘢痕累及范围较局限,神经丝蛋白免疫组织化学染色显示有少量神经纤维通过瘢痕区,并且实验组胶质瘢痕面积、胶质纤维酸性蛋白染色平均吸光度值低于对照组(P < 0.05)。结果表明,控释胶质细胞源性神经营养因子联合骨髓间充质干细胞源性神经元样细胞移植可更强抑制脊髓损伤后胶质瘢痕的形成。     

骨细胞形成过程中成纤维细胞生长因子受体3的调节作用及机制

文题释义： 失神经骨折：机体骨折合并有各类、各层面神经的损伤。随着社会经济的飞速发展,这类骨折的发生也逐年增多,在愈合过程中表现出较单纯骨折愈合加速且骨痂过量生长,甚至在肌肉中出现异位骨化,尤其是关节周围的骨折,严重影响关节功能和治疗效果,已成为修复重建和组织工程领域的研究热点。 创伤性神经损伤：包括创伤性颅脑损伤、脊髓损伤和周围神经损伤。这类损伤伴有骨折的患者表现出较单纯骨折愈合加速且骨痂过量生长,甚至在肌肉中出现异位骨化。 背景：在临床治疗中,创伤性神经损伤伴有骨折的患者表现出较单纯骨折愈合加速且骨痂过量生长,甚至在肌肉中出现异位骨化,严重影响这类骨折的治疗效果。对于影响失神经后骨折愈合加速的具体原因和机制,目前并不清楚。 目的：探索成纤维细胞生长因子受体3抑制剂在骨折愈合过程中的作用及表达变化规律。 方法：实验方案经兰州大学第二医院动物实验伦理委员会批准。选用60只雌性SD大鼠,制作坐骨神经损伤的胫骨横行骨折模型,随机分为实验组及对照组2组。实验组造模后腹腔注射成纤维细胞生长因子受体3阻滞剂;对照组造模后给等剂量生理盐水。分别于造模后4,7,10,14,21 d拍X射线片后取胫骨大体标本(每个时间点6只),取胫骨进行苏木精-伊红染色、Masson三色法染色组织学观察;计算大鼠胫骨骨细胞密度和骨小梁密度;测定胫骨组织纤维率。 结果与结论：①两组大鼠胫骨X射线观察差别不显著;②苏木精-伊红染色、Masson三色法染色结果显示实验组修复效果比对照组较好;③2组大鼠胫骨骨细胞密度、骨小梁密度及胫骨组织纤维率在7-14 d有明显差别(实验组>对照组);④抑制成纤维细胞生长因子受体3在周围神经失神经情况下能够加快骨折愈合,促进骨痂的塑形。成纤维细胞生长因子受体3在骨折后7-14 d时间段表达最为活跃。 ORCID: 0000-0002-7687-4383(许少策) 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     

骨髓间充质干细胞移植对结肠吻合口愈合的影响

背景:骨髓间充质干细胞具有很大的增殖能力、多向分化潜能、向损伤组织定位修复的功能、低免疫原性和免疫调节作用,其可以用来治疗传统治疗方案难以治愈的疾病,具有广阔临床应用价值,成为细胞移植治疗的新热点。目的:观察局部注射的骨髓间充质干细胞在缺血性结肠吻合模型大鼠肠黏膜的定植情况及对结肠吻合口愈合的影响。方法:将40只雌性SD大鼠随机分为4组:对照4 d组和对照7 d组,行左侧结肠缺血性吻合后肠黏膜下局部注射0.5 m L胎牛血清,骨髓间充质干细胞移植4 d组和骨髓间充质干细胞移植7 d组,行左侧结肠缺血性吻合后,取培养至第3代的骨髓间充质干细胞(0.5 m L,1×107个细胞)直接在肠黏膜下注射移植。分别测定各组大鼠吻合口爆破压,取吻合口处肠管行羟脯氨酸含量测定,苏木精-伊红染色及Masson染色行组织病理学评分,Y染色体原位杂交检测供鼠来源的细胞迁徙及定植情况。结果与结论:骨髓间充质干细胞移植组吻合口爆破压、羟脯氨酸含量较对照组明显增高,组织病理学评分提示除上皮形成及炎症指标外,黏膜坏死、胶原蛋白沉积量、成纤维细胞活性及新生血管形成指标骨髓间充质干细胞移植组优于对照组。结果表明移植同种异体大鼠骨髓间充质干细胞可在受体缺血性结肠吻合模型肠道内定植,并可加速结肠吻合口的愈合,其发挥疗效主要是通过旁分泌的形式促进机体内源性修复,而骨髓间充质干细胞直接分化为肠黏膜上皮细胞作用较小。     

骨髓间充质干细胞移植对大鼠心梗后细胞因子VEGF、SDF-1、TGF-β1表达及心脏功能的影响

目的 观察急性心肌梗死大鼠移植异体骨髓间充质干细胞后血管内皮生长因子（VEGF）、基质细胞源性因子－1（SDF-1）、转化生长因子β1（TGF-β1）及心脏功能的变化。方法 密度梯度离心法和贴壁筛选法获得SD大鼠骨髓间充质干细胞,实验动物随机分为2组：细胞移植组即实验组（n＝8）、无细胞移植组即对照组（n＝8）。于移植4周后处死动物,免疫组化检测VEGF、SDF-1、TGF-β1及Buxco 系统测大鼠的心功能。结果 实验组在移植4周后免疫组化显示VEGF表达较对照组明显升高（15.02±1.87 VS 5.45±0.90,P<0.05）, SDF－1较对照组明显升高（20.02±3.87 VS 8.24±1.17, P<0.01）、TGF-β1较对照组降低（13.24±2.07 VS 26.33±4.17,P<0.01）;心脏功能亦较对照组显著改善（P<0.05）。结论 骨髓间充质干细胞移植到梗死心肌后可以干预VEGF、SDF－1、TGF-β1的分泌促进梗死后心脏功能的恢复。     

骨髓间充质干细胞联合活性水凝胶治疗2型糖尿病大鼠心肌梗死研究

目的利用水凝胶作为骨髓间充质干细胞(BM-MSCs)移植的载体,治疗2型糖尿病大鼠急性心肌梗死。方法 SD大鼠20只,3周龄,体质量50 g左右,用于制造2型糖尿病模型。将造模成功的SD大鼠随机分为水凝胶联合细胞移植组、细胞移植组、生理盐水组、假手术组,每组5只。将水凝胶联合细胞移植组、细胞移植组、生理盐水组2型糖尿病大鼠冠状动脉左前降支结扎,制造急性心肌梗死模型,同时做不结扎的假手术组。前3组分别移植100μL Matrigel和1×106 BM-MSCs的混合物、100μL含有1×106 BM-MSCs的0.9%氯化钠溶液、100μL 0.9%氯化钠溶液。术后28 d,检测大鼠血糖、血脂及血胰岛素水平;对术后28 d的大鼠心脏样本进行组织学染色评价心室重构情况;v WF(von Willebrand factor)免疫荧光染色观察大鼠心脏梗死区血管新生情况。结果 SD大鼠的BM-MSCs呈典型的长梭形且贴壁生长;经过8周高脂高糖饲料喂养后,血样生物化学指标显示,SD大鼠血液中总胆固醇、低密度脂蛋白、胰岛素等显著升高;后注射链脲霉素,大鼠空腹血糖在7.0 mmol/L以上,说明成功建立糖尿病大鼠模型;BM-MSCs移植糖尿病大鼠体内后,与不治疗组相比,BM-MSCs移植能显著改善心肌梗死后大鼠左心室重构;免疫荧光染色结果显示,BM-MSCs能促进梗死区内的血管新生。联合Matrigel移植能进一步提升BM-MSCs的治疗效果。结论联合Matrigel移植能显著提升BM-MSCs对糖尿病大鼠心肌梗死的治疗效果。     

神经干细胞NgR基因沉默立体定向移植治疗大鼠脑损伤

背景:神经干细胞具有自我增殖能力和多向分化潜能,一定条件下可以分化成神经系统的各种细胞,因此在神经损伤修复方面有着良好的应用前景。而RNA干扰避免了永久基因沉默的弊病,最有希望与神经干细胞移植相结合治疗颅脑损伤。目的:检测是否可以通过沉默NgR基因的方法提高神经干细胞立体定向移植对重型颅脑损伤大鼠的治疗效果。方法:60只雄性Wistar大鼠制成重型液压颅脑损伤模型后随机区组法分为3组,每组20只。实验组:造模24h后向损伤的大鼠脑组织内注射NgR基因沉默的神经干细胞悬液6μL;对照组:同法注射等量的神经干细胞悬液;空白组:同法注射等量的不含干细胞的培养液。伤后24h,3d,1,2周行动物神经学缺损评分。2周后处死行免疫组织化学和苏木精-伊红染色。结果与结论:转染小分子干扰RNA后,与对照组相比,实验组NgR基因蛋白表达量明显降低,移植后1周和2周,接受神经干细胞移植的大鼠神经学缺损评分明显低于对照组(P0.05);且其脑组织切片中的神经元数量较对照组明显增多(P0.01)。伤后2周苏木精-伊红染色空白组可见损伤处脑组织断裂,为瘢痕连接,有明显空洞形成;对照组在移植部位出现典型的神经细胞样形态学改变;实验组出现典型的神经细胞样形态学改变且空洞消失。免疫组织化学染色观察空白组BrdU标记的阳性细胞为(37.92±16.02)个/高倍视野,对照组为(89.68±15.34)个/高倍视野,实验组为(102.67±13.52)个/高倍视野,各组间两两比较,差异均有显著性意义(P0.01)。提示神经干细胞NgR基因沉默后立体定向移植治疗大鼠脑损伤可明显改善重型颅脑损伤后大鼠的神经学功能。     

负载软骨细胞的透明质酸可注射材料修复软骨缺损北大核心

背景:在软骨组织工程材料中,以透明质酸为代表的多聚糖材料因具备良好的材料细胞作用界面,有利于软骨细胞的生长,成为近年来的研究热点。目的:探究负载软骨细胞的透明质酸可注射材料修复大鼠软骨缺损的可行性。方法:取90只SD大鼠,制备软骨缺损模型,造模后第2天随机分3组干预,实验组关节腔内注射负载软骨细胞的透明质酸水凝胶,对照组关节腔内注射透明质酸水凝胶,空白对照组不进行干预。注射后1,3,6周取修复部位软骨组织,进行苏木精-伊红染色、Masson染色、扫描电镜观察及血红素氧合酶表达、胶原水平检测。结果与结论:①苏木精-伊红染色:注射后6周,空白对照组以肉芽组织填充修复区;对照修复部位被黄白色修复组织填满,与正常软骨边界明显,表面不平整,淋巴细胞较3周时减少;实验组修复组织呈半透明状,与正常软骨边界模糊,淋巴细胞较3周时明显减少;②Masson染色:注射后6周,实验组修复区胶原纤维合成状况最佳,其次为对照组,均强于空白对照组;③扫描电镜观察:注射后6周,空白对照组修复区胶原纤维排列不规则,部分断裂;对照组修复区胶原纤维排列较整齐,仍可见部分断裂;实验组实验部位胶原纤维排列整齐,与正常软骨边缘分界不明;④血红素氧合酶表达与胶原水平:注射后6周,实验组血红素氧合酶表达高于对照组、空白对照组(P<0.05);实验组注射后1,3,6周的胶原水平高于对照组、空白对照组(P<0.05);⑤结果表明:负载软骨细胞的透明质酸可注射材料可促进大鼠软骨缺损的修复。     

神经干细胞移植治疗脊髓损伤

背景：研究已证实神经干细胞能促进脊髓损伤大鼠神经功能的恢复,但对移植细胞在体内的增殖、分化、迁移的研究有限。 目的：观察神经干细胞移植对脊髓损伤大鼠后肢运动功能修复的影响。 方法：SD大鼠制成T10脊髓全横断损伤模型,于造模成功后1周采用局部微量注射法。随机数字表法分为3组：损伤对照组仅打开椎管暴露脊髓;移植对照组：注射10 μL DMEM/F12培养液;细胞移植组：造模后移植浓度为1.0×109 L-1的神经干细胞悬液10 μL。移植后通过不同时间点BBB行为评分、病理组织学、免疫荧光技术评价大鼠脊髓功能修复情况及移植细胞在体内的存活、迁移、分化。 结果与结论：在体外成功建立SD大鼠海马源性神经干细胞培养体系;移植对照组、细胞移植组大鼠随着时间延长BBB评分均不同程度提高,从移植后2周起细胞移植组大鼠评分明显高于移植对照组(P < 0.05);神经干细胞移植后能够在体内继续存活、迁移并且分化为NF-200、GFAP表达阳性的神经元及星形胶质细胞。提示神经干细胞移植治疗脊髓损伤是一种有效的方法。     

异体骨髓间充质干细胞治疗大鼠糖尿病足溃疡及血管内皮生长因子的表达

背景:目前在临床多采用自体骨髓干细胞及自体外周血干细胞治疗糖尿病足溃疡,少见利用骨髓间充质干细胞治疗糖尿病足溃疡的基础研究。目的:观察局部移植异体骨髓间充质干细胞治疗大鼠糖尿病足溃疡的效果及血管内皮生长因子的全身及局部表达情况。方法:取雄性Wistar大鼠90只,随机数字表法分为3组:对照组(正常足部溃疡)、干细胞治疗组、糖尿病对照组。干细胞治疗组、糖尿病对照组建立2型糖尿病足溃疡模型,造模后分别注射同种异体骨髓间充质干细胞、干细胞培养基DMEM。造模后第1,4,8天观察各组大鼠溃疡面积、细胞核染色示踪及病理学检查,ELISA法测外周血血管内皮生长因子浓度、Western-blotting法测局部血管内皮生长因子浓度。结果与结论:与糖尿病对照组比较,干细胞治疗组溃疡愈合速度迅速,但仍较对照组愈合缓慢,外周血中血管内皮生长因子表达增高,也未达到正常水平。异体干骨髓间充质干细胞治疗前期(1～4d)能明显提高局部血管内皮生长因子浓度,但在后期(8d)局部浓度提高不足。干细胞治疗组愈合缓慢,表皮覆盖不完全,但较糖尿病对照组愈合明显提前,表皮覆盖较明显;细胞核染色示移植后干细胞均聚集于溃疡区周围。提示异体移植骨髓间充质干细胞可促进大鼠糖尿病足溃疡的愈合,其促进愈合的可能机制为其上调了血管内皮生长因子在全身尤其局部的表达。     

骨髓间充质干细胞移植治疗失神经肌萎缩

背景:外科显微镜手术和一些辅助治疗方法均无法通过修复损伤的神经细胞来有效延缓或治疗失神经肌萎缩。研究发现骨髓间充质干细胞具有定向分化潜能,并且在一定环境因素下能对损伤的组织进行修复,由此推测其可以对失神经萎缩肌肉起到一定的修复作用。目的:探讨移植骨髓间充质干细胞是否能够减轻和延缓失神经肌肉组织萎缩。方法:分离培养SD大鼠骨髓间充质干细胞,取第3代骨髓间充质干细胞经BrdU标记后用于移植治疗。将30只SD大鼠分为3组,每组10只,对每只大鼠左后肢进行手术。假手术组只暴露坐骨神经主干,不钳夹神经,移植治疗组、模型对照组钳夹坐骨神经主干后,向其支配的腓肠肌注射骨髓间充质干细胞悬液和不含胎牛血清的DMEM培养液。骨髓间充质干细胞移植后1,2周,采用BBB评分评价各组大鼠左后肢运动功能;骨髓间充质干细胞移植后14 d,取腓肠肌组织进行苏木精-伊红染色和BrdU免疫组化染色。结果与结论:第3代骨髓间充质干细胞BrdU标记为阳性;标记的骨髓间充质干细胞能在移植治疗组失神经损伤的肌肉组织中存活并起修复作用;相对于模型对照组,移植治疗组失神经肌纤维由相互融合重新恢复规整。结果表明移植骨髓间充质干细胞能够减轻和延缓失神经肌肉组织萎缩。     

Craniofacial defect regeneration using engineered bone marrow mesenchymal stromal cells

Large craniofacial bony defects remain a significant clinical challenge. Bone marrow mesenchymal stromal cells (BM-MSCs) constitute a multipotent population. Previously, we developed a novel approach for BM-MSC expansion on 3D CultiSpher-S gelatin microcarrier beads in spin culture with preservation of their multipotentiality, reduction of apoptosis, and enhancement of bone formation in vivo. Here, we hypothesized that such cultured BM-MSCs without exogenous growth factors would respond to the orthopedic microenvironment, thus promoting craniofacial defect regeneration. BM-MSCs isolated from green fluorescent protein (GFP) transgenic rats were ex vivo expanded and transplanted into critical-sized (5-mm diameter) rat calvaria defects. Gelatin beads or defect alone served as controls. By 28 and 42 days, rats were sacrificed for microcomputed tomography (microCT), histologic, and immunohistochemistry examination. MicroCT results demonstrated that BM-MSCs were a statistically significant factor contributing to new bone volume regeneration. Histologic assessment showed that the BM-MSCs group produced more and higher quality new bone compared with beads or defect-alone groups in both osteoinductive and osteoconductive manners. Specifically, immunohistochemical staining identified GFP(+) cells residing in new bone lacunae in conjunction with non-GFP(+) cells. Therefore, ex vivo expanded BM-MSCs at least in part regenerated critical-sized calvaria defects by osteogenic differentiation in vivo.     

Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration

Bone marrow mesenchymal stem cells can trans-differentiate into neuronal phenotypes. We examined the differentiation of marrow stromal cells (MSCs) in culture and during nerve regeneration. MSCs from adult rats were exposed to glial growth factor (GGF) to stimulate glial differentiation. Subsequently differentiated MSCs were retrovirally labelled with green fluorescent protein and transplanted into 1 cm nerve conduits in the rat sciatic nerve. Fifteen days post-operatively the conduits were examined for axonal and Schwann cell regeneration and MSC integration. In vitro, MSCs exposed to GGF expressed S100 and glial fibrillary acidic protein. Following transplantation, MSCs maintained S100 expression and enhanced nerve regeneration, with significant Schwann cell regeneration compared to control (2.7 +/- 0.21 vs. 2.05 +/- .21 mm; P < 0.05). MSCs not exposed to GGF prior to transplantation expressed S100 in vivo indicating glial differentiation in response to local cytokines and growth factors.     

Transplantation of bone marrow-derived mesenchymal stem cells into the developing mouse eye

Mesenchymal stem cells (MSCs) have been studied widely for their potential to differentiate into various lineage cells including neural cells in vitro and in vivo. To investigate the influence of the developing host environment on the integration and morphological and molecular differentiation of MSCs, human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the developing mouse retina. Enhanced green fluorescent protein (GFP)-expressing BM-MSCs were transplanted by intraocular injections into mice, ranging in ages from 1 day postnatal (PN) to 10 days PN. The survival dates ranged from 7 days post-transplantation (DPT) to 28DPT, at which time an immunohistochemical analysis was performed on the eyes. The transplanted BM-MSCs survived and showed morphological differentiation into neural cells and some processes within the host retina. Some transplanted cells expressed microtubule associated protein 2 (MAP2ab, marker for mature neural cells) or glial fibrillary acid protein (GFAP, marker for glial cells) at 5PN 7DPT. In addition, some transplanted cells integrated into the developing retina. The morphological and molecular differentiation and integration within the 5PN 7DPT eye was greater than those of other-aged host eye. The present findings suggest that the age of the host environment can strongly influence the differentiation and integration of BM-MSCs.     

Transplantation of bone marrow mesenchymal stem cells on collagen scaffolds for the functional regeneration of injured rat uterus

Serious injuries of endometrium in women of reproductive age are often followed by uterine scar formation and a lack of functional endometrium predisposing to infertility or miscarriage. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown great promise in clinical applications. In the present study, BM-MSCs loaded onto degradable collagen membranes were constructed. Collagen membranes provided 3-dimmensional architecture for the attachment, growth and migration of rat BM-MSCs and did not impair the expression of the stemness genes. We then investigated the effect of collagen/BM-MSCs constructs in the healing of severe uterine injury in rats (partial full thickness uterine excision). At four weeks after the transplantation of collagen/BM-MSCs constructs, BM-MSCs were mainly located to the basal membrane of regenerative endometrium. The wounded tissue adjacent to collagen/BM-MSCs constructs expressed higher level of bFGF, IGF-1, TGFβ1 and VEGF than the corresponding tissue in rats receiving collagen construct alone or in spontaneous regeneration group. Moreover, the collagen/BM-MSCs system increased proliferative abilities of uterine endometrial and muscular cells, facilitated microvasculature regeneration, and restored the ability of endometrium to receive the embryo and support its development to a viable stage. Our findings indicate that BM-MSCs may support uterine tissue regeneration.     

骨髓间充质干细胞参与A549肺腺癌的组织修复

背景：肿瘤被认为是一种特殊的不愈合创伤,骨髓间充质干细胞通过向肿瘤组织归巢和向间质成分分化,参与肿瘤间质重构,从而改变肿瘤微环境,影响肿瘤的生长和转移。 目的：在A549肺癌荷瘤小鼠模型上证实骨髓间充质干细胞参与其损伤修复,探讨骨髓间充质干细胞参与肿瘤组织修复的机制。 方法：体外分离、培养人骨髓间充质干细胞,使用流式细胞术鉴定,制造A549肺癌荷瘤小鼠模型。实验组采用瘤周注射人骨髓间充质干细胞,对照组注射等量PBS,对比观察动物生活情况,肿瘤生长大小。4周后取材,苏木精-伊红染色对比观察肿瘤组织,Masson染色对比胶原纤维含量,RT-PCR检测两组α平滑肌收缩蛋白的表达,免疫组织化学检测两组成纤维细胞特异蛋白、成纤维细胞活化蛋白的表达情况,反映两组肿瘤组织的间质纤维的程度。免疫组织化学方法对比两组中血管内皮生长因子、肝细胞生长因子、白细胞介素6、肌糖蛋白C的表达高低。 结果与结论：骨髓间充质干细胞促进荷瘤小鼠肿瘤的生长,实验组肿瘤组织生长速度明显快于对照组(P < 0.05)。RT-PCR检测α平滑肌收缩蛋白的表达：与对照组比较,实验组α平滑肌收缩蛋白 mRNA的表达水平显著升高。免疫组织化学方法检测实验组肿瘤组织中TAFs标志物：成纤维细胞特异蛋白、成纤维细胞活化蛋白的表达,IHC检测血管内皮生长因子、肝细胞生长因子、白细胞介素6、肌糖蛋白C的表达明显高于对照组,差异有显著性意义(P < 0.05)。说明骨髓间充质干细胞在肿瘤微环境中向成纤维细胞方向分化,参与肿瘤间质的形成和构建,分泌血管内皮生长因子、肝细胞生长因子、白细胞介素6、肌糖蛋白C等促进肿瘤的生长修复。     

Localized expression of human BMP-7 by BM-MSCs enhances renal repair in an in vivo model of ischemia-reperfusion injury

Ischemia and subsequent reperfusion (I/R) damage kidney tubular cells and consequently impair renal function. Rabbit bone marrow mesenchymal stem cells (BM-MSCs) expressing human bone morphogenic protein-7 (hBMP-7) regenerated tubular cells and improved renal function in a kidney I/R model. Rabbits were injected immediately after I/R with one of the following: (i) hBMP-7-transduced BM-MSCs (BM-MSCshBMP-7); (ii) enhanced green fluorescent protein-transduced BM-MSCs (BM-MSCsEGFP); or (iii) PBS. The activity of superoxide dismutase (SOD) was higher, and the amount of malondialdehyde (MDA) was lower in the BM-MSCshBMP-7 group than in the BM-MSCsEGFP group. Both the BM-MSCshBMP-7 group and the BM-MSCsEGFP group had higher SOD activity and lower amounts of MDA than the PBS group. Bcl-2- and Bcl-2-associated X protein levels, and other variables, indicated the regeneration of the kidney in both experimental groups. However, the BM-MSCs (hBMP-7) group showed higher activity than the BM-MSCsEGFP group, indicating that the combined strategy of BM-MSC transplantation with hBMP-7 gene therapy could be a useful approach for the treatment of renal IRI.     

Controlling the fibroblastic differentiation of mesenchymal stem cells via the combination of fibrous scaffolds and connective tissue growth factor

Controlled differentiation of multi-potent mesenchymal stem cells (MSCs) into vocal fold-specific, fibroblast-like cells in vitro is an attractive strategy for vocal fold repair and regeneration. The goal of the current study was to define experimental parameters that can be used to control the initial fibroblastic differentiation of MSCs in vitro. To this end, connective tissue growth factor (CTGF) and micro-structured, fibrous scaffolds based on poly(glycerol sebacate) (PGS) and poly(?-caprolactone) (PCL) were used to create a three-dimensional, connective tissue-like microenvironment. MSCs readily attached to and elongated along the microfibers, adopting a spindle-shaped morphology during the initial 3 days of preculture in an MSC maintenance medium. The cell-laden scaffolds were subsequently cultivated in a conditioned medium containing CTGF and ascorbic acids for up to 21 days. Cell morphology, proliferation, and differentiation were analyzed collectively by quantitative PCR analyses, and biochemical and immunocytochemical assays. F-actin staining showed that MSCs maintained their fibroblastic morphology during the 3 weeks of culture. The addition of CTGF to the constructs resulted in an enhanced cell proliferation, elevated expression of fibroblast-specific protein-1, and decreased expression of mesenchymal surface epitopes without markedly triggering chondrogenesis, osteogenesis, adipogenesis, or apoptosis. At the mRNA level, CTGF supplement resulted in a decreased expression of collagen I and tissue inhibitor of metalloproteinase 1, but an increased expression of decorin and hyaluronic acid synthesase 3. At the protein level, collagen I, collagen III, sulfated glycosaminoglycan, and elastin productivity was higher in the conditioned PGS-PCL culture than in the normal culture. These findings collectively demonstrate that the fibrous mesh, when combined with defined biochemical cues, is capable of fostering MSC fibroblastic differentiation in vitro.     

Angiogenic cell therapy for hepatic fibrosis

Progression of liver fibrosis has been linked with injuries associated with hypoxia and neovascularization. Neovascularization consists of angiogenesis and vasculogenesis, representing formation of blood vessels by differentiation of endothelial progenitor cells (EPCs). We investigated antifibrogenic and regenerative effects of EPC transplantation in chronic liver injury. Rat EPCs were isolated from bone marrow cells and examined in vitro for lineage markers. Recipient rats were injected intraperitoneally with dimethylnitrosamine (DMN) three times weekly for 4 weeks, plus EPC transplantation once weekly for 4 weeks. Transplanted rats showed suppression of liver fibrogenesis. Expression of growth factors promoting liver regeneration such as hepatocyte growth factor (HGF), transforming growth factor (TGF)-α, epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) was increased in transplanted rats, together with hepatocyte proliferation. Normal liver function parameters such as transaminase, total bilirubin, total protein, and albumin were maintained in transplanted rats. EPC transplantation is effective not only for preventing liver fibrosis but also for promoting regeneration in chronically damaged livers. Also, recently it has been reported that green fluorescent protein-positive bone marrow cells contribute to the liver tissue repair of fibrosis model rats. EPC transplantation might become an alternative if further preclinical investigation finds it to be effective in severely cirrhotic livers.