A Novel Approach via Surface Modification of Degradable Polymers With Adhesive DOPA-IGF-1 for Neural Tissue Engineering

The highly damaging state of spinal cord injuries has provided much inspiration for the design of surface modification of the implants that can promote nerve regeneration and functional reconstruction. DOPA-IGF-1, a new recombinant protein designed in our previous study, exhibited strong binding affinity to titanium and significantly enhanced the growth of NIH3T3 cells on the surface of titanium with the same biological activity as IGF-1. In this article, surface modification of poly(lactide-co-glycolide) (PLGA) films with recombinant DOPA-IGF-1 was performed to promote the paracrine activity of human umbilical cord mesenchymal stem cells (hUCMSCs) by secreting neurotrophic factors. DOPA-IGF-1 exhibited the strongest binding ability to PLGA films than commercial IGF-1 and nonhydroxylated YKYKY-IGF-1. In vitro cultures of hUCMSCs on the modified PLGA films showed that DOPA-IGF-1@PLGA substrates significantly improved the proliferation, adhesion, and neurotrophic factors secretion of hUCMSCs, especially for nerve growth factor, as confirmed by qRT-PCR and western blot analysis. Subsequently, the acquired neurotrophic factors secreted by the hUCMSCs cultured on the DOPA-IGF-1@PLGA films obviously enhanced neurite outgrowth of PC12 cells. Taken together, PLGA substrates with DOPA-IGF-1 immobilization is a promising platform for neural tissue engineering via neurotrophic factors secretion from MSCs and should be further tested in vivo.     

In vitro differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs), derived from Wharton's jelly, into choline acetyltransferase (ChAT)-positive cells

We isolated and expanded fibroblast-like cells from the Wharton's jelly of human umbilical cord successfully. Immunocytochemistry showed that they were positive for several markers of mesenchymal stem cells (CD73, CD90, and CD105) and integrin markers (CD29 and CD44), but negative for a hematopoietic cell maker (CD45) and an endothelial cell marker (CD31). Their differentiation into osteocytes and adipocytes under specific conditions indicated that they had multi-lineage differentiation potential. Therefore these results proved that the cells we obtained from Wharton's jelly were human umbilical cord mensenchymal stem cells (hUCMSCs). Using immunocytochemistry and Western blotting analysis, we found that after treatment with neuronal induction medium [NIM; consisting of brain-derived neurotrophic factor (BDNF) and low-serum media] for 14 days, hUCMSCs expressed a neuronal specific marker, microtubule associated protein 2 (MAP2), and extended neurite-like processes. After treatment with NIM, supplemented with hippocampal cholinergic neurostimulating peptide (HCNP) or rat denervated hippocampal extract [rDHE; derived from rat fimbria fornix (FF) transected hippocampus], hUCMSCs expressed choline acetytransferase (ChAT) and this action could be enhanced when cells were cultured with NIM, supplemented with HCNP and rDHE in combination. ELISA showed that these ChAT-positive cells could secrete acetylcholine (ACh). These findings indicate that hUCMSCs possess the potential of differentiation into functional ChAT-positive cells in vitro and provide a new candidate of cells for the cell transplantation to treat Alzheimer's disease (AD).     

miR-92b-3p促进人脐带间充质干细胞的成骨分化

目的探究miR-92b-3p在人脐带间充质干细胞(h UCMSCs)成骨分化过程中的作用及其作用机制。方法通过转染h UCMSCs过表达或抑制miR-92b-3p,qRT-PCR检测各组成骨相关基因OCN、RUNX2、OSTERIX mRNA表达以明确miR-92b-3p对h UCMSCs成骨分化的体外调控作用,用茜素红染色比较各处理组的骨化能力。通过生物信息学分析miR-92b-3p可能的靶基因,并通过双荧光素酶基因报告系统以及Western blot验证。结果 miR-92b-3p在h UCMSCs成骨过程中表达量较对照组明显升高(P0.05);过表达miR-92b-3p能促进h UCMSCs体外成骨分化及体内的异位成骨能力(P0.05);而抑制miR-92b-3p则能降低h UCMSCs体外成骨分化(P0.05)。过表达miR-92b-3p能显著降低DKK1的表达量(P0.05),抑制则能明显提高DKK1的表达(P0.05)。结论 miR-92b-3p可通过抑制DKK1表达,促进h UCMSCs成骨分化。     

不同分离方法及培养条件对人脐带间充质干细胞生物活性的影响

目的:观察不同分离方法及培养条件对人脐带间充质干细胞(Human umbilical cord-mesenchymal stem cells,hUCMSCs)生物活性的影响。方法:分别用组织块贴壁法、酶消化法获取hUCMSCs,进行原代培养,记录各组首次传代时间,相差显微镜观察细胞的形态,流式细胞仪鉴定其CD29、CD44、HLA-ABC、CD34、CD45、HLA-DR抗原表达。分别用DMEM-LG、DMEM-HG和DMEM-F123种培养基培养第3代、第7代细胞,MTT法比较不同时间点3种培养基培养细胞的生长情况。结果:两种分离方法均能得到较均一的梭形或者多角形贴壁细胞,胞核大胞浆丰富,CD29、CD44、HLA-ABC抗原表达阳性,CD34、CD45、HLA-DR抗原表达阴性。用DMEM-F12培养的细胞活力更好,细胞生长更迅速。结论:两种分离方法均能得到hUCMSCs,DMEM-F12培养基更能够促进细胞的增殖,较适合于培养hUCMSCs。     

Exosome loaded genipin crosslinked hydrogel facilitates full thickness cutaneous wound healing in rat animal model

Full thickness cutaneous wound therapy and regeneration remains a critical challenge in clinical therapeutics. Recent reports have suggested that mesenchymal stem cells exosomes therapy is a promising technology with great potential to efficiently promote tissue regeneration. Multifunctional hydrogel composed of both synthetic materials and natural materials is an effective carrier for exosomes loading. Herein, we constructed a biodegradable, dual-sensitive hydrogel encapsulated human umbilical cord-mesenchymal stem cells (hUCMSCs) derived exosomes to facilitate wound healing and skin regeneration process. The materials characterization, exosomes identification, and in vivo full-thickness cutaneous wound healing effect of the hydrogels were performed and evaluated. The in vivo results demonstrated the exosomes loaded hydrogel had significantly improved wound closure, re-epithelialization rates, collagen deposition in the wound sites. More skin appendages were observed in exosomes loaded hydrogel treated wound, indicating the potential to achieve complete skin regeneration. This study provides a new access for complete cutaneous wound regeneration via a genipin crosslinked dual-sensitive hydrogel loading hUCMSCs derived exosomes.     

磷酸钙骨水泥复合物/脐带间充质干细胞凝胶构建组织工程骨

目的构建新型可注射强化型磷酸钙骨水泥复合物/脐带间充质干细胞凝胶组织工程骨,探讨其力学性能,细胞活性和成骨作用。方法选用第四代hUCMSCs,1.2%海藻酸钠水凝胶构建hUCMSCs水凝胶微球。高温煅烧钙/磷比约为1.9的磷酸氢钙和碳酸钙混合物,按摩尔质量比以1：1混合制备CPC粉末。15%Chitosan,8mm长度可吸收纤维用于提高CPC复合物力学强度。实验组分为四组：（1）单纯hUCMSCs微球;（2）CPC＋hUCMSCs微球;（3）CPC＋chitosan＋hUCMSCs微球;（4）CPC＋chitosan＋可吸收纤维＋hUCMSCs微球,分别检测力学性能,细胞活性和成骨作用。结果新型组织工程骨力学性能显著增强,抗弯曲强度提高到（11.7±2.1）MPa,弹性模量提高到（2.0±0.4）GPa,断裂功提高到（1.65±0.66）kj/m2（P〈0.05）。hUCMSCs的ALP活性第7天时明显增高,第14天时达峰,第21天时有所减弱,各组间比较无明显统计学差异（P〉0.1）。第7天时,所有实验组中的hUCMSCs均见少量矿物合成。第14天和第21天时,矿物合成数量明显增多。hUCMSCs中ALP基因表达培养第1天最低,第4天达峰,第8天时稍减弱。OC基因表达第8天达峰。结论构建完成新型可注射强化型磷酸钙骨水泥/脐带间充质干细胞凝胶构建组织工程骨。水凝胶微球中hUCMSCs在CPC中具有良好的成骨作用。CPC-chitosan-可吸收纤维组织工程骨力学性能满足松质骨力学要求,支持水凝胶微球中hUCMSCs的细胞活性和成骨作用。为组织工程骨研究和临床应用提供新思路和新方法。     

共培养环境中大鼠胚胎中脑细胞诱导人脐带间充质干细胞分化

目的:检测人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)在与大鼠胚胎中脑细胞共培养环境中酪氨酸羟化酶(tyrosine hydroxylase,TH)和多巴胺转运蛋白(dopamine transporter,DAT)表达的变化。方法:(1)植块法分离培养hUCMSCs,以细胞免疫化学法鉴定其表面特异性标记物的表达;(2)无菌条件下分离E15大鼠胚胎中脑组织,制成单细胞悬液及中脑组织提取液;(3)用大鼠胚胎中脑组织提取液做培养液,将5-溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)标记的hUCMSCs与大鼠胚胎中脑细胞共培养14 d,细胞免疫化学法检测hUCMSCs中TH和DAT的表达。结果:HUCMSCs高表达CD29、CD44、CD73、CD90和CD105,极低表达CD31和CD45。在与大鼠胚胎中脑细胞共培养14 d后,hUCMSCs中TH阳性表达率为(18.06±2.29)%,DAT阳性表达率为(11.14±2.10)%。结论:用植块法可成功分离并体外培养hUCMSCs,其免疫表型符合间充质干细胞(mesenchymal stem cells,MSCs)的特征。在以大鼠胚胎中脑组织提取液作为培养液,并与大鼠胚胎中脑细胞共培养的诱导条件下,hUCMSCs可部分向多巴胺能神经元分化。     

脐带间充质干细胞复合β-磷酸三钙生物陶瓷体内外成骨能力的初步研究

目的:观察人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)和支架材料β-磷酸三钙(β-TCP)生物陶瓷复合的体内外成骨情况。方法:在体外构建hUCMSCs和β-TCP的复合物,将细胞以3×105/mL的浓度接种到支架材料上进行复合体构建,并进行电镜观察、特异性免疫荧光染色、MTT、ALP检测。将复合物植入裸鼠体内,进行成骨能力研究。实验分为3组,即植入单纯β-TCP支架组、植入体外成骨诱导2周的hUCMSCs和β-TCP复合物组、单纯植入体外成骨诱导2周的hUCMSCs组。植入后2个月取出标本,进行大体观察、X线片观察和组织学观察。采用SPSS16.0软件包对实验数据进行重复测量随机区组设计的方差分析。结果:hUCMSCs植入支架4h后,即可见细胞在支架上附着,1周后可见细胞在支架中大量增殖,β-TCP具有一定的骨诱导性。复合物植入裸鼠内2个月,hUCMSCs和β-TCP复合组X线密度最高。HE染色显示,2个月后,hUCMSCs和β-TCP复合组见不规则新骨和血管形成,单纯β-TCP组未见明显新骨和血管形成。Masson染色显示,2个月后,hUCMSCs和β-TCP复合组见大量胶原形成,单纯β-TCP组未见明显胶原形成。VG染色显示,2个月后,hUCMSCs和β-TCP复合组孔隙中有大量类骨质及少量骨陷窝形成,单纯β-TCP组未见类骨质形成。结论:hUCMSCs和β-TCP具有良好的生物相容性,两者构建的复合物在植入裸鼠体内2个月时可见新骨及血管化形成。     

人脐带间充质干细胞对耐亚胺培南铜绿假单胞菌耐药性形成的影响

目的探讨人脐带间充质干细胞（hUCMSCs）体外对耐亚胺培南铜绿假单胞菌（IRPA）耐药性形成及对OprD2基因的影响。 方法本实验设立3个组,实验组为hUCMSCs组,对照组为细胞对照组（即人肺成纤维细胞组,NHLF组）和空白对照组。次抑菌浓度肉汤诱导PA耐药传导过程中,hUCMSCs组和NHLF组分别加入其与PA共培育所得的上清液,空白对照组加入细胞培养液,观察3组诱导耐药所需代数以及抑菌圈的大小。诱导耐药前后分别采用K-B法及real-time PCR法测定PA对常见抗菌药物的敏感性及OprD2基因的表达量。 结果经次抑菌浓度的亚胺培南诱导后,hUCMSCs组PA耐药性的出现较NHLF组及空白对照组延迟。NHLF组和空白对照组PA于诱导的第17代出现亚胺培南耐药,而hUCMSCs组PA于第19代出现耐药性。real-time PCR结果显示,诱导耐药后PA中OprD2表达量较诱导前出现减少或消失。其中hUCMSCs组PA OprD2的表达量减少至诱导耐药前的10.96%,而NHLF及空白对照组OprD2无表达,即诱导后出现OprD2基因缺失。 结论人脐带间充质干细胞具有延迟PA耐药性形成的作用,其机制可能是通过分泌抗菌肽LL-37和人β防御素-2从而抑制OprD2表达的减少,而外膜蛋白OprD2表达量减少或缺失是引起PA对亚胺培南耐药的原因。