壳聚糖-胶原多孔支架的制备研究

本研究的目的就是利用细胞亲和力较好的胶原与壳聚糖复合,制备具有较好细胞亲和力的组织工程复合支架。在研究中采用冷冻诱导相分离的方法制备了壳聚糖(Cs)和胶原溶胀液(Co)质量比为1：2、1：3、1：4、1：5的4种成分的复合多孔支架,并对支架的孔结构、吸水性、孔隙率和溶胀性等特性进行了评价。结果显示,制备的复合多孔支架孔隙率均在90％以上;不同成分的支架孔径大小不同,胶原含量越高,孔径越大;支架内部孔隙连通：残留的醋酸在超声波作用下用碱液中和并干燥后,支架浸入水中基本无溶胀,保持了原有的形貌。     

旋转壁式生物反应器中微囊化骨髓间充质干细胞支持造血干/祖细胞的体外扩增

为了构建一种新型的造血干细胞和基质细胞动态共培养体系,脐带血单个核细胞和包埋有兔骨髓间充质干细胞的海藻酸钠-壳聚糖-海藻酸钠(alginate-chitosan-alginate, ACA)微胶囊在旋转壁式生物反应器(rotating wall vessel, RWV)中进行了7d动态悬浮共培养.培养液不含血清,补充多种生长因子(SCF 50ng·mL-1,FL 50ng·mL-1,TPO 50ng·mL-1及IL-325ng·mL-1).每24h进行总有核细胞计数,并测量培养液的pH和渗透压,在0h、72h和168h进行流式CD34 细胞分析以及集落形成能力检验.结果表明在RWV动态共培养过程中,培养液的pH保持在7.2~7.4,渗透压保持在280~310mmOsmol·kg-1,均适合造血干/祖细胞的体外扩增.经过7d的无血清动态共培养,总有核细胞、CD34 细胞以及混合集落(colony-forming units in culture, CFU-Cs)分别扩增了107.05±6.08,26.52±1.5和19.2±3.18倍.这种新型的动态微囊化共培养体系支持了造血干/祖细胞的大规模体外扩增,基质细胞抑制了造血干/祖细胞的分化,微胶囊对造血细胞和基质细胞进行了免疫隔离,RWV则为细胞和微胶囊提供了优良的低剪切力的悬浮培养环境.     

新型胶原支架材料的制备

根据支架材料的要求及猪皮的特点,采用多种化学、生物与物理综合方法处理猪皮得到了一种新型胶原支架材料,并探讨了酶处理、复合、交联和Co60辐照对其性能的影响,对其制备工艺进行了优化.该材料具有良好的微观渗透性、适当的降解速度和抗张强度,有望成为一种较理想的组织工程支架材料.     

体外构建壳聚糖/β-甘油磷酸钠/明胶仿生梯度支架

针对软骨特定结构缺损后不能自我修复的特点,通过调控单层原料质量比,构建具有复杂分层结构的壳聚糖/β-甘油磷酸钠/明胶(chitosan/β-sodium glycerophosphates/gelatin,Cs/GP/Gel)仿生复合梯度支架。通过对比物理性能优选适宜比例的支架材料,并将骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)接种到梯度支架上考察其生物相容性。结果表明,Cs/GP/Gel复合梯度支架具有良好的吸水性能[(584.24±3.79)%～(677.47±1.70)%]、孔隙率[(86.34±5.10)%～(95.20±2.86)%]和降解性能[(86.09±2.46)%～(92.48±3.86)%]。扫描电镜(SEM)结果表明,支架材料在纵向维度呈现出明显的生理分层结构和孔径梯度渐进性,可有效模拟真实软骨的天然生理分层结构。比例为9:1:5的Cs/GP/Gel复合梯度支架适于作为骨软骨的支撑材料。对BMSCs-支架复合物Live/Dead染色后发现,细胞在梯度支架材料上存活、分布及伸展良好。该仿生梯度支架为开发新型生物医用材料提供了重要依据。     

聚乙烯醇/蒙脱石支架用作生物催化剂的研究

PVA/MMT复合材料制备组织工程支架包衣片,用作新型的生物催化剂,同时以面包酵母作为模型细胞,对新型催化剂的各种性能进行了考察研究。结果表明,碟状三维多孔支架孔隙率达90%以上,细胞载量可达45 mg/cm3gel,具有很好的连通性。采用PVA/OMMT纳米复合材料作为包衣材料,多次使用后无细胞泄露现象。亲水与疏水分子在新型生物催化剂中都表现出很好的扩散性,同时包衣还可以降低底物和产物对细胞毒害作用。新型的生物催化剂具有很好的储存稳定性,储存30 d后残余活力为75.4%。     

脐血造血干/祖细胞与脂肪干细胞共培养的作用距离研究

设计了一种细胞间距可调的transwell共培养方法,以研究脐带血来源的造血干/祖细胞(HS/PCs)和人脂肪干细胞(human-adipose derived stem cells,h-ADSCs)体外共培养时细胞间作用距离对造血干细胞扩增能力和脂肪干细胞在共培养后干细胞特性的影响.采用不同规格的砂纸打磨孔板的上壁面...     

气升式环流中空纤维膜生物反应器内骨髓间充质干细胞的培养与扩增

骨髓间充质干细胞(MSCs)具有多向分化的潜能,是重要的组织工程"种子细胞"和理想的基因治疗靶细胞,但在骨髓中的含量非常少,需体外扩增才能满足临床需求.今将第四代MSCs与Ⅰ型胶原溶液混合后接种于中空纤维膜(HFMs)内,置37℃、5% CO2、饱和湿度的培养箱内1 h后形成凝胶,然后将此HFMs接种至气升式环流生物反应器(ALB)内,进行MSCs的三维动态培养与扩增.实验过程中每24 h取样测吸光度、计细胞数、绘制细胞生长曲线,测定细胞代谢参数,并对扩增的细胞进行流式细胞仪分析及多向诱导分化检测.结果表明:在气升式环流中空纤维膜生物反应器(ALHFMB)内,O2含量基本保持恒定;细胞代谢旺盛,MSCs扩增倍数明显增加,7 d后扩增近16倍;扩增的细胞仍保持MSCs表型并具有较强的成骨、成软骨及成脂肪的多向分化能力.     

同时扩增与收获脐带血来源造血干细胞及间充质干细胞的研究

目的为利用生物反应器,实现脐带血(Umbilical cord blood,UCB)来源的造血干细胞(Hematopoietic stem cells,HSCs)与间充质干细胞(Mesenchymal stem cells,MSCs)的同时扩增与收获。方法为在不添加血清、只添加细胞因子组合(SCF15ng·mL-1,FL5ng·mL-1,TPO6ng·mL-1,IL-315ng·mL-1,G-CSF1ng·mL-1,GM-CSF5ng·mL-1)及海藻酸钙壳聚糖胶珠包被基质细胞支持的条件下,采用玻璃包被的聚苯乙烯(Glass coated styrene copolymer,GCSC)微载体与生物反应器相结合的策略,考察了UCB-HSCs与UCB-MSCs在转瓶及旋转壁式生物反应器(Rotating wall vessel bioreactor,RWVB)内的共培养。结果RWVB中的扩增效果最佳,12天内有核细胞(Nuclear cells,NCs)扩增了3.7±0.3倍;集落形成细胞(Colony-forming units in culture,CFU-Cs)扩增了5.1±1.2倍;CD34+CD45+CD105-(HSCs)细胞扩增了5.2±0.4倍;CD34-CD45-CD105+(MSCs)细胞扩增了13.9±1.2倍。培养结束后,通过自由沉降的方法分离UCB-HSCs和粘附在GCSC微载体表面的UCB-MSCs。同时,细胞多向诱导分化及免疫表型分析结果显示,粘附在GCSC微载体表面上的细胞能够向骨、软骨及脂肪细胞分化;并能够表达间质细胞相关表面标志CD13,CD44,CD73和CD105,而不表达造血细胞的相关表面标志CD34,CD45及HLA-DR,与骨髓MSCs相一致。结论为添加细胞因子、基质细胞及微载体支持的条件下,在生物反应器内能够实现UCB-HSCs和UCB-MSCs的同时扩增与收获。     

生物可降解聚合物多孔支架的制备研究进展

组织工程的关键技术之一在于运用生物可降解聚合物制备出具有特定结构、内部连通性好并具有良好力学性能的三维多孔支架,本文对近几年来制备支架的方法以及研究热点做了综述,并对组织工程用生物可降解聚合物多孔支架的发展方向做了展望。     

静电纺丝法制备丝素蛋白/季铵盐壳聚糖复合纤维支架

将丝素蛋白(SF)水溶液和季铵盐壳聚糖(HACC)水溶液以100∶0、99∶1、98∶2和97∶3的溶质比共混作为纺丝液,测试了其质量分数为30%时的表面张力和电导率,并通过应力控制流变仪对其静态剪切和动态剪切作用下的流变性能进行了分析。采用静电纺丝技术制备出静电纺SF/HACC复合纤维支架,通过扫描电子显微镜对其表面形貌进行表征。结果表明,随着HACC含量的增加,纺丝液的黏度和电导率逐步提升;HACC能促进纺丝液的凝胶化;得到的静电纺纤维支架有着较均一的形貌,纤维较扁平,在组织工程修复领域具有良好的应用前景。     

Zone-Dependent Architecture and Biochemical Composition of Decellularized Porcine Nasal Cartilage Modulate the Activity of Adipose Tissue-Derived Stem Cells in Cartilage Regeneration

Previous anatomical studies have shown different functional zones in human nasal septal cartilage (NC). These zones differ in respect to histological architecture and biochemical composition. The aim of this study was to investigate the influence of these zones on the fate of stem cells from a regenerative perspective. Therefore, decellularized porcine septal cartilage was prepared and subjected to histological assessment to demonstrate its equivalence to human cartilage. Decellularized porcine NC (DPNC) exposed distinct surfaces depending on two different histological zones: the outer surface (OS), which is equivalent to the superficial zone, and the inner surface (IS), which is equivalent to the central zone. Human adipose tissue-derived stem cells (ASCs) were isolated from the abdominal fat tissue of five female patients and were seeded on the IS and OS of DPNC, respectively. Cell seeding efficiency (CSE), vitality, proliferation, migration, the production of sulfated glycosaminoglycans (sGAG) and chondrogenic differentiation capacity were evaluated by histological staining (DAPI, Phalloidin, Live-Dead), biochemical assays (alamarBlue^®, PicoGreen^®, DMMB) and the quantification of gene expression (qPCR). Results show that cell vitality and CSE were not influenced by DPNC zones. ASCs, however, showed a significantly higher proliferation and elevated expression of early chondrogenic differentiation, as well as fibrocartilage markers, on the OS. On the contrary, there was a significantly higher upregulation of hypertrophy marker MMP13 (p < 0.0001) and GAG production (p = 0.0105) on the IS, whereas cell invasion into the three-dimensional DPNC was higher in comparison to the OS. We conclude that the zonal-dependent distinct architecture and composition of NC modulates activities of ASCs seeded on DPNC. These findings might be used for engineering of cartilage substitutes needed in facial reconstructive surgery that yield an equivalent histological and functional structure, such as native NC.     

Improvement of an Effective Protocol for Directed Differentiation of Human Adipose Tissue-Derived Adult Mesenchymal Stem Cells to Corneal Endothelial Cells

Corneal disease affects 12.5 million individuals worldwide, with 2 million new cases each year. The standard treatment consists of a corneal transplantation from a human donor; however, the worldwide demand significantly exceeds the available supply. Lamellar endothelial keratoplasty, the replacement of only the endothelial layer of the cornea, can partially solve the problem. Progressive efforts have succeeded in expanding hCECs; however, the ability to expand hCECs is still limited, and new sources of CECs are being sought. Crucial advances have been achieved by the directed differentiation of embryonic or induced pluripotent stem cells, but these cells have disadvantages, such as the use of oncogenes, and are still difficult to establish. We aimed to transfer such knowledge to obtain hCECs from adipose tissue-derived adult mesenchymal stem cells (ADSC) by modifying four previously published procedures. We present several protocols capable of the directed differentiation of human ADSCs to hCECs. In our hands, the protocol by Ali et al. was the best adapted to such differentiation in terms of efficiency, time, and financial cost; however, the protocol by Wagoner et al. was the best for CEC marker expression. Our results broaden the type of cells of autologous extraocular origin that could be employed in the clinical setting for corneal endothelial deficiency.     

Adipose Tissue Uses in Peripheral Nerve Surgery

Currently, many different techniques exist for the surgical repair of peripheral nerves. The degree of injury dictates the repair and, depending on the defect or injury of the peripheral nerve, plastic surgeons can perform nerve repairs, grafts, and transfers. All the previously listed techniques are routinely performed in human patients, but a novel addition to these peripheral nerve surgeries involves concomitant fat grafting to the repair site at the time of surgery. Fat grafting provides adipose-derived stem cells to the injury site. Though fat grafting is performed as an adjunct to some peripheral nerve surgeries, there is no clear evidence as to which procedures have improved outcomes resultant from concomitant fat grafting. This review explores the evidence presented in various animal studies regarding outcomes of fat grafting at the time of various types of peripheral nerve surgery.     

Bioengineering of Dental Pulp Stem Cells in a Microporous PNIPAAm-PLGA Scaffold

In the present article a novel bio absorbable polymeric scaffold using poly(N-isopropyl acrylamide-block-poly(L-lactide-co-glycolide) (PNIPAAm-b-PLGA) copolymers is developed for in vitro culture of human dental pulp stem cells (DPSCs). The processing of porous scaffolds has been carried out by emulsion freeze-drying and salt leaching out methods. DPSCs were cultured on scaffolds for up to 14 days. The morphology of the scaffolds, cell viability and interaction between DPSCs and scaffold was characterized by using SEM. The results of cells implantation indicated that scaffold has good cell biocompatibility. Therefore PNIPAAm-PLGA scaffolds have great potential to be used as cell carrier in tissue engineering.     

Individual Variabilities in Adipose Stem Cell Proliferation,Gene Expression and Responses to Lipopolysaccharide Stimulation

Adipose stem cells (ASCs) are reported to play a role in normal physiology as well as in inflammation and disease. The objective of this work was to elucidate inter-individual differences in growth, gene expression and response to inflammatory stimuli in ASCs from different donors. Human ASC1 (male donor) and ASC2 (female donor) were purchased from Lonza (Walkersville, MD). Cell proliferation was determined by the sulforhodamine B assay. After time-dependent treatment of ASCs with or without bacterial lipopolysaccharide (LPS), marker gene mRNAs for proliferation, steroid hormones, and xenobiotic and immune pathways were determined using RT-PCR, and secreted cytokine levels in media were measured using the Bio-Plex cytokine assay kit. ASCs from both donors expressed androgen receptors but not estrogen receptors. ASC2 had a 2-fold higher proliferation rate and a 6-fold higher level of proliferation marker Ki67 mRNA than ASC1. ASC2 exhibited significantly greater fold induction of TNF-α and CCL2 by LPS compared to ASC1. TNF-α and GM-CSF protein levels were also significantly higher in the LPS-induced ASC2 media, but IL-6 secretion was higher in the LPS-induced ASC1 media. Our findings suggest that inter-individual variability and/or possible sex differences exist in ASCs, which may serve as a key determinant to inflammatory responses of ASCs.     

Polymeric Gelatin Scaffolds Affect Mesenchymal Stem Cell Differentiation and Its Diverse Applications in Tissue Engineering

Studies using polymeric scaffolds for various biomedical applications, such as tissue engineering, implants and medical substitutes, and drug delivery systems, have attempted to identify suitable material for tissue regeneration. This study aimed to investigate the biocompatibility and effectiveness of a gelatin scaffold seeded with human adipose stem cells (hASCs), including physical characteristics, multilineage differentiation in vitro, and osteogenic potential, in a rat model of a calvarial bone defect and to optimize its design. This functionalized scaffold comprised gelatin-hASCs layers to improve their efficacy in various biomedical applications. The gelatin scaffold exhibited excellent biocompatibility in vitro after two weeks of implantation. Furthermore, the gelatin scaffold supported and specifically regulated the proliferation and osteogenic and chondrogenic differentiation of hASCs, respectively. After 12 weeks of implantation, upon treatment with the gelatin-hASCs scaffold, the calvarial bone harboring the critical defect regenerated better and displayed greater osteogenic potential without any damage to the surrounding tissues compared to the untreated bone defect. These findings suggest that the present gelatin scaffold is a good potential carrier for stem cells in various tissue engineering applications.     

Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells and Their Contribution to Angiogenic Processes in Tissue Regeneration

Mesenchymal stem/stromal cells (MSCs) are widely described in the context of their regenerative and immunomodulatory activity. MSCs are isolated from various tissues and organs. The most frequently described sources are bone marrow and adipose tissue. As stem cells, MSCs are able to differentiate into other cell lineages, but they are usually reported with respect to their paracrine potential. In this review, we focus on MSCs derived from adipose tissue (AT-MSCs) and their secretome in regeneration processes. Special attention is given to the contribution of AT-MSCs and their derivatives to angiogenic processes described mainly in the context of angiogenic dysfunction. Finally, we present clinical trials registered to date that concern the application of AT-MSCs and their secretome in various medical conditions.     

Therapeutic Potential of Exosomes Derived from Adipose Tissue-Sourced Mesenchymal Stem Cells in the Treatment of Neural and Retinal Diseases

Therapeutic agents that are able to prevent or attenuate inflammation and ischemia-induced injury of neural and retinal cells could be used for the treatment of neural and retinal diseases. Exosomes derived from adipose tissue-sourced mesenchymal stem cells (AT-MSC-Exos) are extracellular vesicles that contain neurotrophins, immunoregulatory and angio-modulatory factors secreted by their parental cells. AT-MSC-Exos are enriched with bioactive molecules (microRNAs (miRNAs), enzymes, cytokines, chemokines, immunoregulatory, trophic, and growth factors), that alleviate inflammation and promote the survival of injured cells in neural and retinal tissues. Due to the nano-sized dimension and bilayer lipid envelope, AT-MSC-Exos easily bypass blood–brain and blood–retinal barriers and deliver their cargo directly into the target cells. Accordingly, a large number of experimental studies demonstrated the beneficial effects of AT-MSC-Exos in the treatment of neural and retinal diseases. By delivering neurotrophins, AT-MSC-Exos prevent apoptosis of injured neurons and retinal cells and promote neuritogenesis. AT-MSC-Exos alleviate inflammation in the injured brain, spinal cord, and retinas by delivering immunoregulatory factors in immune cells, suppressing their inflammatory properties. AT-MSC-Exos may act as biological mediators that deliver pro-angiogenic miRNAs in endothelial cells, enabling re-vascularization of ischemic neural and retinal tissues. Herewith, we summarized current knowledge about molecular mechanisms which were responsible for the beneficial effects of AT-MSC-Exos in the treatment of neural and retinal diseases, emphasizing their therapeutic potential in neurology and ophthalmology.     

Adipose Stromal/Stem Cell-Derived Extracellular Vesicles: Potential Next-Generation Anti-Obesity Agents

Over the last decade, several compounds have been identified for the treatment of obesity. However, due to the complexity of the disease, many pharmacological interventions have raised concerns about their efficacy and safety. Therefore, it is important to discover new factors involved in the induction/progression of obesity. Adipose stromal/stem cells (ASCs), which are mostly isolated from subcutaneous adipose tissue, are the primary cells contributing to the expansion of fat mass. Like other cells, ASCs release nanoparticles known as extracellular vesicles (EVs), which are being actively studied for their potential applications in a variety of diseases. Here, we focused on the importance of the contribution of ASC-derived EVs in the regulation of metabolic processes. In addition, we outlined the advantages/disadvantages of the use of EVs as potential next-generation anti-obesity agents.     

The Impact of Human Lipoaspirate and Adipose Tissue-Derived Stem Cells Contact Culture on Breast Cancer Cells: Implications in Breast Reconstruction

Background: Autologous fat transfer in the form of lipoaspirates for the reconstruction of the breast after breast cancer surgery is a commonly used procedure in plastic surgery. However, concerns regarding the oncologic risk of nutrient-rich fat tissue are widely debated. Previous studies have primarily focused on studying the interaction between adipose-derived stem cells (ASCs) and breast cancer cells. Methods: In this study, we performed a comprehensive analysis of the paracrine- and contact-based interactions between lipoaspirates, ASCs and breast cancer cell lines. An inverted flask culture method was used to study the contact-based interaction between lipoaspirates and breast cancer cells, while GFP-expressing breast cancer cell lines were generated to study the cell–cell contact interaction with ASCs. Three different human breast cancer cell lines, MCF-7, MDA-MB-231 and BT-474, were studied. We analyzed the impact of these interactions on the proliferation, cell cycle and epithelial-to-mesenchymal (EMT) transition of the breast cancer cells. Results: Our results revealed that both lipoaspirates and ASCs do not increase the proliferation rate of the breast cancer cells either through paracrine- or contact-dependent interactions. We observed that lipoaspirates selectively inhibit the proliferation of MCF-7 cells in contact co-culture, driven by the retinoblastoma (Rb) protein activity mediating cell cycle arrest. Additionally, ASCs inhibited MDA-MB-231 breast cancer cell proliferation in cell–cell contact-dependent interactions. Quantitative real-time PCR revealed no significant increase in the EMT-related genes in breast cancer cells upon co-culture with ASCs. Conclusion: In conclusion, this study provides evidence of the non-oncogenic character of lipoaspirates and supports the safety of clinical fat grafting in breast reconstruction after oncological surgical procedures. In vivo studies in appropriate animal models and long-term post-operative clinical data from patients are essential to reach the final safety recommendations.