Role of nanotopography in the development of tissue engineered 3D organs and tissues using mesenchymal stem cells

Recent regenerative medicine and tissue engineering strategies(using cells, scaffolds, medical devices and gene therapy) have led to fascinating progress of translation of basic research towards clinical applications. In the past decade, great deal of research has focused on developing various three dimensional(3D) organs, such as bone, skin, liver, kidney and ear,using such strategies in order to replace or regenerate damaged organs for the purpose of maintaining or restoring organs' functions that may have been lost due to aging, accident or disease. The surface properties of a material or a device are key aspects in determining the success of the implant in biomedicine, as the majority of biological reactions in human body occur on surfaces or interfaces. Furthermore, it has been established in the literature that cell adhesion and proliferation are, to a great extent, influenced by the micro- and nanosurface characteristics of biomaterials and devices. In addition, it has been shown that the functions of stem cells, mesenchymal stem cells in particular, could be regulated through physical interaction with specific nanotopographical cues. Therefore, guided stem cell proliferation, differentiation and function are of great importance in the regeneration of 3D tissues and organs using tissue engineering strategies. This review will provide an update on the impact of nanotopography on mesenchymal stem cells for the purpose of developing laboratory-based 3D organs and tissues, as well as the most recent research and case studies on this topic.

Role of adipose tissue grafting and adipose-derived stem cells in peripheral nerve surgery

The application of autologous fat grafting in reconstructive surgery is commonly used to improve functional form.This review aims to provide an overview of the scientific evidence on the biology of adipose tissue,the role of adipose-derived stem cells,and the indications of adipose tissue grafting in peripheral nerve surgery.Adipose tissue is easily accessible through the lower abdomen and inner thighs.Non-vascularized adipose tissue grafting does not support oxidative and ischemic stress,resulting in variable survival of adipocytes within the first 24 hours.Enrichment of adipose tissue with a stromal vascular fraction is purported to increase the number of adipose-derived stem cells and is postulated to augment the long-term stability of adipose tissue grafts.Basic science nerve research suggests an increase in nerve regeneration and nerve revascularization,and a decrease in nerve fibrosis after the addition of adipose-derived stem cells or adipose tissue.In clinical studies,the use of autologous lipofilling is mostly applied to secondary carpal tunnel release revisions with promising results.Since the use of adipose-derived stem cells in peripheral nerve reconstruction is relatively new,more studies are needed to explore safety and long-term effects on peripheral nerve regeneration.The Food and Drug Administration stipulates that adipose-derived stem cell transplantation should be minimally manipulated,enzyme-free,and used in the same surgical procedure,e.g.adipose tissue grafts that contain native adipose-derived stem cells or stromal vascular fraction.Future research may be shifted towards the use of tissue-engineered adipose tissue to create a supportive microenvironment for autologous graft survival.Shelf-ready alternatives could be enhanced with adipose-derived stem cells or growth factors and eliminate the need for adipose tissue harvest.

Endometrial mesenchymal stem cells as a cell based therapy for pelvic organ prolapse

Pelvic organ prolapse(POP) occurs when the pelvic organs(bladder, bowel or uterus) herniate into the vagina, causing incontinence, voiding, bowel and sexual dysfunction, negatively impacting upon a woman's quality of life. POP affects 25% of all women and results from childbirth injury. For 19% of all women, surgical reconstructive surgery is required for treatment, often augmented with surgical mesh. The surgical treatment fails in up to 30% of cases or results in adverse effects, such as pain and mesh erosion into the bladder, bowel or vagina. Due to these complications the Food and Drug Administration cautioned against the use of vaginal mesh and several major brands have been recently been withdrawn from market. In this review we will discuss new cell-based approaches being developed for the treatment of POP. Several cell types have been investigated in animal models, including a new source of mesenchymal stem/stromal cells(MSC) derived from human endometrium. The unique characteristics of endometrial MSC, methods for their isolation and purification and steps towards their development for good manufacturing practice production will be described. Animal models that could be used to examine the potential for this approach will also be discussed as will a rodent model showing promise in developing an endometrial MSC-based therapy for POP. The development of a preclinical large animal model for assessing tissue engineering constructs for treating POP will also be mentioned.

Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells

We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.

Xenogeneic stem cell transplantation:Research progress and clinical prospects

Organ transplantation is the ultimate treatment for end-stage diseases such as heart and liver failure.However,the severe shortage of donor organs has limited the organ transplantation progress.Xenogeneic stem cell transplantation provides a new strategy to solve this problem.Researchers have shown that xenogeneic stem cell transplantation has significant therapeutic effects and broad application prospects in treating liver failure,myocardial infarction,advanced type 1 diabetes mellitus,myelosuppression,and other end-stage diseases by replacing the dysfunctional cells directly or improving the endogenous regenerative milieu.In this review,the sources,problems and solutions,and potential clinical applications of xenogeneic stem cell transplantation will be discussed.

Perspectives on endogenous and exogenous tissue engineering following injury to tissues of the knee

The knee is a multi-component organ system comprised of several tissues which function coordinately to provide mobility. Injury to any one component compromises the integrity of the system and leads to adaptation of the other components. Over time, such events often lead to dysfunction and degeneration of the knee. Therefore, there has been considerable research emphasis to repair injured components in the knee including cartilage, menisci, and ligaments. Approaches to improving healing and repair/regeneration of knee tissues have included surgery, anti-sense gene therapy, injection of growth factors and inflammatory cytokine antagonists, transplantation of in vitro expanded chondrocytes, enhancement of endogenous cells via microfracture, injection of mesenchymal stem cells, and implantation of in vitro tissue engineered constructs. Some of these approaches have lead to temporary improvement in knee functioning, while others offer the potential to restore function and tissue integrity for longer periods of time. This article will review the status of many of these approaches, and provide a perspective on their limitations and potential to contribute to restoration of knee function across the lifespan.

颌骨来源骨髓间充质干细胞成骨分化的特点、优势与应用

背景:颌面部骨组织缺损修复是目前研究的热点与难点,而种子细胞的选择是关键。颌骨来源骨髓间充质干细胞是存在于颌骨内的成体间充质干细胞,在颌面部组织再生方面的应用更具优势。目的:综述颌骨来源骨髓间充质干细胞的生物学特性和成骨分化优势以及药物、体内环境、微小RNA对其成骨分化影响的相关研究进展。方法:利用计算机在PubMed和中国知网进行文献检索。中文检索词为“口腔,骨组织工程,干细胞”,英文检索词为“oral,bone tissue engineering,stem cells”。共检索下载文章405篇,根据纳入与排除标准对文章进行筛选,最终纳入70篇文献进行综述。结果与结论:颌骨来源骨髓间充质干细胞是口腔骨组织工程的优良种子细胞,与长骨骨髓间充质干细胞相比,颌骨来源骨髓间充质干细胞具有更强的增殖能力和成骨分化能力。药物、体内环境、微小RNA均可以调控颌骨来源骨髓间充质干细胞的成骨分化,但目前对颌骨来源骨髓间充质干细胞的研究尚处于初始阶段,因此需要更多论证力较强的研究来证实其在颌面部骨组织再生领域的应用更具优势。

血管化类器官的构建策略

背景:有效促进类器官内部血管发生是目前类器官培养中的焦点问题,作为一种新近发展的生物培养技术,血管化的类器官在研究活体组织的发育、疾病形成的机制、组织替代疗法以及药物筛选等方面,都有很大的研究和应用价值。目的:对近年来类器官血管化的方法或策略进行归纳总结,分析类器官血管化形成机制以及构建策略,以期为更加深入地研究类器官的发生机制和为临床转化提供可靠的思路。方法:检索PubMed及中国知网数据库收录的相关文献。英文检索词为“organoids,Vascularization,Vascular,Vascular development,vessel”,中文检索词为“血管发生,血管生成,类器官,干细胞,血管化,预血管化”,最终纳入77篇文献进行归纳总结。结果与结论:①类器官血管化形成机制涉及3个关键因素,即种子细胞、细胞因子与细胞外基质。种子细胞为血管化类器官提供了关键的细胞来源,细胞因子为类器官内部的血管发生起了重要的信号引导作用,细胞外基质为血管细胞提供了外在的生长环境,促进类器官血管化的发生。②血管化类器官的构建策略包括细胞自我重组、微血管碎片渗入、宿主体内移植以及微流控芯片等。体外诱导多能干细胞向血管内皮祖细胞分化能顺利与邻近组织整合并具有血管生成的潜力,故可利用多能干细胞的自我重组功能构建血管化类器官。微血管碎片保留了其细胞复杂性、天然结构和表型可塑性,更利于模拟天然微血管从而促进类器官的血管化。宿主体内移植是目前类器官实现完整血液灌流的最佳方法,而微流控芯片则为实现类器官体外血液供应提供了解决方案。③类器官的多种构建策略如多类干细胞共分化、信号分子的精准调控、微血管渗入和活体宿主移植等,一定程度上在类器官中引入血管成分,使得类器官在功能和成熟度上更接近相应组织。然而缺乏血流灌注仍然是一个难题,迄今为止,仅宿主体内移植才能在类器官中实现有效的血流灌注,因此类器官在血管化方面仍面临许多挑战。

生物活性静电纺丝纳米纤维支架在乳房脂肪组织工程中的研究与应用进展

乳房重建手术是乳腺癌综合治疗中常用的方法。随着外科技术和材料学的进步,术后仿真乳房重建已成为可实现的目标。为解决当前手术存在的问题,乳腺整形外科医师积极研究组织工程技术。乳房脂肪组织工程是一种新兴技术,基于自体脂肪移植、生物学和组织工程领域的经验,为尚未满足的乳房重建临床需求提供了潜在的解决方案。脂肪组织工程可以制备具有仿生结构的软组织,为乳房重建提供了新的可能性。通过将稳定增殖、低免疫原性的种子细胞植入支架,脂肪组织工程可以构建具有生物活性的支架,实现脂肪组织的再生和修复。其中,采用静电纺丝技术制备的纳米纤维支架具有优异的细胞外基质结构和力学性能,不仅提供了脂肪组织生长的空间,还通过调节细胞表型与启动细胞外基质之间的相互作用,为细胞分化和增殖提供诱导平台。因此,静电纺丝纳米纤维支架被认为是乳房脂肪组织工程有前景的应用材料之一。本文综述了乳房脂肪组织工程中生物活性静电纺丝纳米纤维支架的研究进展,旨在更好地了解乳房重建手术的发展趋势,并为未来的临床应用提供指导和借鉴。

尿道组织工程中常用的种子细胞

组织工程学是当前治疗尿道损伤的研究热点。基于种子细胞的组织工程修复促进了多种治疗方法的发展,组织工程修复时,将选择的合适的种子细胞与支架材料结合并移植到损伤部位,通过种子细胞的增殖和分化修复组织缺损。近年来,尿道组织工程的发展较明显,如来自阴茎组织、膀胱和口腔的自体细胞,以及来自骨髓、脂肪组织和尿液等的干细胞均在尿道修复和重建过程中发挥了巨大作用。未来,针对尿道组织工程中常用种子细胞的深入研究将进一步促进尿道修复和重建的发展。

组织工程技术在阴道重建中的应用及进展

阴道缺损对患者造成的身心压力巨大,传统的阴道重建方法能在一定程度上恢复生理功能,但往往伴随着长治疗周期和多种并发症。随着组织工程技术的进步,其在阴道重建领域的应用展现出了解决女性生殖系统缺损的优势和潜能。本文分析了近年来组织工程在阴道重建领域的研究进展,探究种子细胞、生物材料、信号因子和动物模型的应用前景,以期为临床阴道修复提供启发和参考。

角膜缘微环境细胞的分离培养研究进展

角膜缘微环境细胞(LNCs)是位于角膜缘、与角膜缘上皮干细胞(LESCs)紧密相连的一类间充质干细胞,能同时表达间充质标志物和多种胚胎干细胞标志物,对调节LESCs的静息、自我更新以及分化状态起着重要作用。近年研究表明,LNCs可通过胶原酶消化法、上皮块消化法、中性蛋白酶-胶原酶消化法以及组织块培养法进行体外分离培养,通过3D Matrigel共培养、Transwell共培养可研究LNCs与LESCs之间的相互作用关系。LNCs与LESCs可通过SDF-1/CXCR4、Notch、BMP、Wnt、Sonic Hedgehog、KIT/AKT等多种信号通路以及神经生长因子、角质形成细胞因子、胰岛素样生长因子等多种细胞因子相互作用。LNCs现已成为角膜上皮组织工程、眼表重建以及角膜再生等研究中的一大热点。本文就LNCs的研究背景、体外分离培养方法、与LESCs的相互作用机制及其应用前景等进行综述。

Generation of functional organs from stem cells

We are now well entering the exciting era of stem cells.Potential stem cell therapy holds great promise for the treatment of many diseases such as stroke,traumatic brain injury,Alzheimer’s disease,Parkinson’s disease,amyotrophic lateral-sclerosis,myocardial infarction,muscular dystrophy,diabetes,and etc..It is generally believed that transplantation of specific stem cells into the injured tissue to replace the lost cells is an effective way to repair the tissue.In fact,organ transplantation has been successfully practiced in clinics for liver or kidney failure.However,the severe shortage of donor organs has been a major obstacle for the expansion of organ transplantation programs.Toward that direction,generation of transplantable organs using stem cells is a desirable approach for organ replacement and would be of great interest for both basic and clinical scientists.Here we review recent progress in the field of organ generation using various methods including single adult tissue stem cells,a blastocyst complementation system,tissue decellularization/recellularization and a combination of stem cells and tissue engineering.

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575 mu m in thickness during the monitoring period. A 45 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.

心脏类器官技术要点及特征分析

心脏类器官技术通过在体外3D培养获得具备心脏部分生理及结构特征的细胞结构,是在科研领域对传统的体外2D培养及动物实验技术的重要补充。其在药物筛选、疾病模型构建等多领域存在巨大潜力。但因心脏发育的复杂性,心脏类器官技术的发展相较于其他器官更缓慢。目前已有的心脏类器官技术主要依赖于组织工程技术和干细胞自组织性。本文将就不同心脏类器官技术的特点进行论述,总结已有的技术进展及待完善的方向。

浅谈间充质干细胞结合新型生物补片在盆腔器官脱垂治疗中的优势

盆腔器官脱垂(POP)作为困扰全世界中老年女性的常见疾病,其病因及最佳治疗方案尚不明确,但使用替代方法加强自身组织强度是重建盆底支撑网络的研究热点。间充质干细胞(MSCs)结合组织工程技术被视为POP治疗的新途径。各种类型的MSCs因来源、储备、获取及特有功能不同,在POP的治疗中具有不同的价值。MSCs利用其强大的免疫调节及旁分泌能力促使网片成功植入及募集整合,网片材料为MSCs提供合适的生存环境及机械支持,二者优势互补,促使组织工程技术研发的生物补片具有良好的生物相容性、机械稳定性、弹性及柔韧性等特点,一跃成为当前理想的新型治疗材料。本文通过阐述POP的病因和治疗现状,比较不同类型MSCs的特点,综述了利用干细胞特性结合生物材料治疗POP的新型模式。

Domesticated cynomolgus monkey embryonic stem cells allow the generation of neonatal interspecies chimeric pigs

Blastocyst complementation by pluripotent stem cell(PSC)injection is believed to be the most promising method to generate xenogeneic organs.However,ethical issues prevent the study of human chimeras in the late embryonic stage of development.Primate embryonic stem cells(ESCs),which have similar pluripotency to human ESCs,are a good model for studying interspecies chimerism and organ generation.However,whether primate ESCs can be used in xenogenous grafts remains unclear.In this study,we evaluated the chimeric ability of cynomolgus monkey(Macaca fascicularis)ESCs(cmESCs)in pigs,which are excellent hosts because of their many similarities to humans.We report an optimized culture medium that enhanced the anti-apoptotic ability of cmESCs and improved the development of chimeric embryos,in which domesticated cmESCs(D-ESCs)injected into pig blastocysts differentiated into cells of all three germ layers.In addition,we obtained two neonatal interspecies chimeras,in which we observed tissue-specific D-ESC differentiation.Taken together,the results demonstrate the capability of D-ESCs to integrate and differentiate into functional cells in a porcine model,with a chimeric ratio of 0.001-0.0001 in different neonate tissues.We believe this work will facilitate future developments in xenogeneic organogenesis,bringing us one step closer to producing tissue-specific functional cells and organs in a large animal model through interspecies blastocyst complementation.

Scaffold strategies combined withmesenchymal stem cells in vaginalconstruction: a review

Tissue engineering has provided new treatment alternatives for tissue reconstruction. Advances in the tissue engineeringfield have resulted in mechanical support and biological substitutes to restore, maintain or improve tissue/organs structuresand functions. The application of tissue engineering technology in the vaginal reconstruction treatment can not onlyprovide mechanical requirements, but also offer tissue repairing as an alternative to traditional approaches. In this review, wediscuss recent advances in cell-based therapy in combination with scaffolds strategies that can potentially be adopted forgynaecological transplantation.

不同部位脂肪源性干细胞的生物学特性比较

背景: 大鼠不同部位来源的脂肪源性干细胞在体外培养时的特性是否存在差异目前尚无定论。目的: 比较同一只大鼠不同部位来源的脂肪源性干细胞在体外培养时的生长特性和成脂诱导分化能力的差异。方法: 无菌操作下取F344大鼠腹股沟及腹腔大网膜脂肪组织各5mL,Ⅰ型胶原酶酶解法分离出脂肪源性干细胞,细胞计数后进行体外培养,观察其形态特征和生长状态,MTT法测定不同部位细胞的倍增时间。取不同部位来源的第2代脂肪源性干细胞进行成脂诱导,诱导14d进行油红O染色,观察不同部位来源的脂肪源性干细胞的成脂分化能力。结果与结论: 在同一只大鼠内脏大网膜脂肪获得的脂肪源性干细胞数目为(281±10)×107L-1,明显多于腹股沟皮下脂肪的(85±5)×107L-1(P<0.01)。从内脏大网膜脂肪与腹股沟皮下脂肪获得的脂肪源性干细胞分别于第5,6天进入指数增长期;第9,10天到达平台期;倍增时间为50h和60h左右。传代后的细胞生长分化活跃,呈成纤维细胞样,成脂诱导后,大网膜组织来源的脂肪源性干细胞的成脂诱导分化率明显高于腹股沟组织来源的脂肪源性干细胞[(38.90±2.86)%,(35.30±3.29)%,P<0.01]。可见同一只大鼠不同部位脂肪组织分离得到的脂肪源性干细胞数目不同,体外成脂诱导分化能力亦存在差异。