The use of mesenchymal stem cells in tissue engineering

Mesenchymal stem cells (MSCs) are of great interest to both clinicians and researchers for their great potential to enhance tissue engineering. Their ease of isolation, manipulability, and potential for differentiation are specifically what have made them so attractive. These multipotent cells have been found to differentiate into cartilage, bone, fat, muscle, tendon, skin, hematopoietic-supporting stroma and neural tissue. Their diverse in vivo distribution includes bone marrow, adipose, periosteum, synovial membrane, skeletal muscle, dermis, pericytes, blood, trabecular bone, human umbilical cord, lung, dental pulp, and periodontal ligament. Despite their frequent use in research, no standardized criteria exist for the identification of mesenchymal stem cells; The International Society for Cellular Therapy has sought to change this with a set of guidelines elucidating the major surface markers found on these cells. While many studies have shown MSCs to be just as effective as unipotent cells for certain types of tissue regeneration, limitations do exist due to their immunosuppressive properties. This paper serves as a review pertaining to these issues, as well as others related to the use of MSCs in tissue engineering.     

The use of mesenchymal stem cells in tissue engineering: A global assessment

Mesenchymal stem cells (MSCs) are of great interest to both clinicians and researchers for their great potential to enhance tissue engineering. Their ease of isolation, manipulability and potential for differentiation are specifically what have made them so attractive. These multipotent cells have been found to differentiate into cartilage, bone, fat, muscle, tendon, skin, hematopoietic-supporting stroma and neural tissue. Their diverse in vivo distribution includes bone marrow, adipose, periosteum, synovial membrane, skeletal muscle, dermis, pericytes, blood, trabecular bone, human umbilical cord, lung, dental pulp and periodontal ligament. Despite their frequent use in research, no standardized criteria exist for the identification of mesenchymal stem cells; The International Society for Cellular Therapy has sought to change this with a set of guidelines elucidating the major surface markers found on these cells. While many studies have shown MSCs to be just as effective as unipotent cells for certain types of tissue regeneration, limitations do exist due to their immunosuppressive properties. This paper serves as a review pertaining to these issues, as well as others related to the use of MSCs in tissue engineering.Key words: mesenchymal stem cells, tissue engineering, regenerative medicine     

Gene therapy and tissue engineering for sports medicine

Sports injuries usually involve tissues that display a limited capacity for healing. The treatment of sports injuries has improved over the past 10 to 20 years through sophisticated rehabilitation programs, novel operative techniques, and advances in the field of biomechanical research. Despite this considerable progress, no optimal solution has been found for treatment of various sports-related injuries, including muscle injuries, ligament and tendon ruptures, central meniscal tears, cartilage lesions, and delayed bone fracture healing. New biological approaches focus on the treatment of these injuries with growth factors to stimulate and hasten the healing process. Gene therapy using the transfer of defined genes encoding therapeutic proteins represents a promising way to efficiently deliver suitable growth factors into the injured tissue. Tissue engineering, which may eventually be combined with gene therapy, may potentially result in the creation of tissues or scaffolds for regeneration of tissue defects following trauma. In this article we will discuss why gene therapy and tissue engineering are becoming increasingly important in modern orthopaedic sports medicine practice. We then will review recent research achievements in the area of gene therapy and tissue engineering for sports-related injuries, and highlight the potential clinical applications of this technology in the treatment of patients with musculoskeletal problems following sports-related injuries.     

组织工程产品的种类及应用——组织工程连载之六

组织工程产品包括人造皮肤、血管、软骨、骨、角膜、心脏瓣膜、气管、肌腱、韧带、神经、肌肉、骨髓、生殖道、尿道、肠、乳房、肝脏、肾脏、胰脏、心脏、膀胱、手等,但绝大部分处于实验室研究探索阶段,正在进行临床实验或批准应用还不多。已经获得批准的主要是皮肤产品、软骨及骨产品、心血管产品、神经系统产品、人工器官等,其临床应用较多。今后将会有越来越多的组织工程产品面世,其临床应用也会越来越广泛。     

Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine

The use of tissue-engineering techniques such as stem-cell therapy to renew injured tissues is a promising strategy in regenerative medicine. As a cell-delivery vehicle, fibrin glues (FG) facilitate cell attachment, growth and differentiation and, ultimately, tissue formation and organization by its three-dimensional structure. Numerous studies have provided evidence that stromal cells derived from bone marrow (bone marrow stromal cells; BMSC) and adipose tissue (adipose-derived stromal cells; ADSC) contain a population of adult multipotent mesenchymal stromal cells (MSC) and endothelial progenitor cells that can differentiate into several lineages. By combining MSC with FG, the implantation could take advantage of the mutual benefits. Researchers and physicians have pinned their hopes on stem cells for developing novel approaches in regenerative medicine. This review focuses on the therapeutic potential of MSC with FG in bone defect reconstruction, cartilage and tendon injury repair, ligament, heart and nerve regeneration, and, furthermore, wound healing.     

Pluripotent mesenchymal stem cells reside within avian connective tissue matrices

Summary Recent studies have noted the presence of putative stem cells derived from the connective tissues associated with skeletal muscle, heart, and dermis. Long-term continuous cultures of these cells from each tissue demonstrated five distinct phenotypes of mesodermal origin, i.e. muscle, fat, cartilage, bone, and connective tissue. Clonal analysis was performed to determine whether these morphologies were the result of a mixed population of lineage-committed stem cells or the differentiation of pluripotent stem cells or both. Putative stem cells from four tissues (skeletal muscle, dermis, atria, and ventricle) were isolated and cloned. Combined, 1158 clones were generated from the initial cloning and two subsequent subclonings. Plating efficiency approximated 5.8%. Approximately 70% of the 1158 clones displayed a pure stellate morphology, while the remaining clones contained a mixture of stellate, chondrogenic- or osteogenic-like morphologies or both. When cultured in the presence of dexamethasone, cells from all clones differentiated in a time- and concentration-dependent manner into muscle, fat, cartilage, and bone. These results suggest that pluripotent mesenchymal stem cells are present within the connective tissues of skeletal muscle, dermis, and heart and may prove useful for studies concerning the regulation of stem cell differentiation, wound healing, and tissue restoration, replacement and repair.     

去细胞方法及其在组织工程中的应用

去细胞基质在组织工程及再生医学的大量应用为解决组织器官的修复和重建等难题带来了希望。去细胞方法大致可以分为三类:化学处理法、物理处理法及酶学处理法,且已经应用于组织工程及再生医学的各个方面。本文总结并分类目前常用的去细胞方法及其在组织工程各方面的应用,对目前国内外常用的去细胞方法及其在组织工程及再生医学中的应用进行回顾总结与分析。     

Decellularized adipose matrix provides an inductive microenvironment for stem cells in tissue regeneration

Stem cells play a key role in tissue regeneration due to their self-renewal and multidirectional differentiation, which are continuously regulated by signals from the extracellular matrix (ECM) microenvironment. Therefore, the unique biological and physical characteristics of the ECM are important determinants of stem cell behavior. Although the acellular ECM of specific tissues and organs (such as the skin, heart, cartilage, and lung) can mimic the natural microenvironment required for stem cell differentiation, the lack of donor sources restricts their development. With the rapid development of adipose tissue engineering, decellularized adipose matrix (DAM) has attracted much attention due to its wide range of sources and good regeneration capacity. Protocols for DAM preparation involve various physical, chemical, and biological methods. Different combinations of these methods may have different impacts on the structure and composition of DAM, which in turn interfere with the growth and differentiation of stem cells. This is a narrative review about DAM. We summarize the methods for decellularizing and sterilizing adipose tissue, and the impact of these methods on the biological and physical properties of DAM. In addition, we also analyze the application of different forms of DAM with or without stem cells in tissue regeneration (such as adipose tissue), repair (such as wounds, cartilage, bone, and nerves), in vitro bionic systems, clinical trials, and other disease research.     

去细胞方法及其在组织工程中的应用

去细胞基质在组织工程及再生医学的大量应用为解决组织器官的修复和重建等难题带来了希望。去细胞方法大致可以分为三类：化学处理法、物理处理法及酶学处理法,且已经应用于组织工程及再生医学的各个方面。本文总结并分类目前常用的去细胞方法及其在组织工程各方面的应用,对目前国内外常用的去细胞方法及其在组织工程及再生医学中的应用进行回顾总结与分析。     

Mesenchymal stem cells: clinical applications and biological characterization

Mesenchymal stem cells (MSCs) have been isolated from bone marrow, periosteum, trabecular bone, adipose tissue, synovium, skeletal muscle and deciduous teeth. These cells have the capacity to differentiate into cells of connective tissue lineages, including bone, fat, cartilage and muscle. A great deal has been learned in recent years about the isolation and characterization of MSCs, and control of their differentiation. These cells have generated a great deal of interest because of their potential use in regenerative medicine and tissue engineering and there are some dramatic examples, derived from both pre-clinical and clinical studies, that illustrate their therapeutic value. This review summarizes recent findings regarding the potential clinical use of MSCs in cardiovascular, neural and orthopaedic applications. As new methods are developed, there are several aspects to the implanted cell-host interaction that need to be addressed before we can fully understand the underlying mechanisms. These include the host immune response to implanted cells, the homing mechanisms that guide delivered cells to a site of injury and the differentiation in vivo of implanted cells under the influence of local signals.     

Biomimetic approach to tissue engineering

The overall goal of tissue engineering is to create functional tissue grafts that can regenerate or replace our defective or worn out tissues and organs. Examples of grafts that are now in pre-clinical studies or clinical use include engineered skin, cartilage, bone, blood vessels, skeletal muscle, bladder, trachea, and myocardium. Engineered tissues are also finding applications as platforms for pharmacological and physiological studies in vitro. To fully mobilize the cell's biological potential, a new generation of tissue engineering systems is now being developed to more closely recapitulate the native developmental milieu, and mimic the physiologic mechanisms of transport and signaling. We discuss the interactions between regenerative biology and engineering, in the context of (i) creation of functional tissue grafts for regenerative medicine (where biological input is critical), and (ii) studies of stem cells, development and disease (where engineered tissues can serve as advanced 3D models).     

Cytocentrifugation: a convenient and efficient method for seeding tendon-derived cells into monolayer cultures or 3-D tissue engineering scaffolds

Tendon and ligament injuries are very common, requiring some 200,000 reconstructions per year in the USA. Autografting can be used to repair these but donor tissue is limited and harvesting leads to morbidity at the graft sites. Tissue engineering has been used to grow simple tissues such as skin, cartilage and bone and due to their low vascularity and simple structure, tendons should be ideal candidates for such an approach. Scaffolds are essential for tissue engineering as they provide structure and signals that regulate growth. However, they present a physical barrier to cell seeding with the majority of the cells congregating at the scaffold surface. To address this we used centrifugation to enhance penetration of tendon-derived cells to the centres of 3-D scaffolds. The process had no apparent deleterious effects on the cells and both plating efficiency and cell distribution improved. After attachment the cells continued to proliferate and deposit a collagenous matrix. Scaffold penetration was investigated using layers of Azowipes allowing the separation and examination of individual leaves. At relatively low g-forces, cells penetrated a stack of 6 Azowipes leaving cells attached to each leaf. These data suggest that cytocentrifugation improves the penetration and homogeneity of tendon derived cells in 3-D and monolayer cultures.     

外胚间充质干细胞构建组织工程骨骼肌的应用研究

目的:探讨利用大鼠颌突外胚间充质干细胞构建组织工程骨骼肌的可行性,并观察对骨骼肌缺损的修复重建的促进效应。方法:取妊娠E 11.5胎鼠颌突外胚间充质干细胞,纯化后在含5ml/L体积浓度二甲基亚砜的DMEM/F12培养基中诱导分化为骨骼肌样细胞,将细胞种植于BAM膜上培养形成组织工程骨骼肌。将其移植入大鼠骨骼肌缺损模型,手术后14 d观察骨骼肌恢复情况,同期进行组织学及免疫组化染色鉴定。结果:经诱导后外胚间充质干细胞可向骨骼肌样细胞转化,构建的组织工程骨骼肌可加速缺损的修复重建,组织学染色显示外胚间充质干细胞具有正常骨骼肌的组织形态,可表达成肌相关蛋白MyOD。结论:诱导后的外胚间充质干细胞可作为种子细胞构建组织工程骨骼肌,本实验为临床肌肉的缺损修复奠定了理论基础。     

Formation of cartilage by non-chondrogenic cell types

Freshly excised embryonic rat skeletal muscle has been shown to form hyaline cartilage when organ cultured upon demineralized rat bone (bone matrix). Since skeletal muscle is composed of fibrous connective tissue (C.T.) as well as muscle cells, the cartilage could arise from either of these sources. The object of this study was to determine whether cartilage arose from fibrous connective tissue or muscle cells, or both, and whether the ability to form cartilage is limited to tissues derived from somatic mesoderm. Control experiments demonstrated that 19-day embryonic rat skeletal muscle formed cartilage when organ cultured on bone matrix after dissociation and cultivation in vitro, and that 11-day embryonic chick muscle also formed cartilage, although less reproducibly (3 out of 10 cases). Fibroblasts and skeletal muscle were cloned from similar suspensions of dissociated muscle in order to test these purified cell types. Dermis, vascular tissue, and tendons were mechanically removed prior to dissociation in order to eliminate fibroblasts from contaminant sources. Cloned fibroblasts, derived from rat skeletal muscle, formed cartilage in three out of three cases. It was not possible to clone sufficient rat skeletal muscle to place an aggregate onto bone matrix. An aggregate of several hundred chick skeletal muscle clones formed cartilage on bone matrix. The freshly excised C.T. capsules of embryonic chick thyroid and lung were tested for the ability to form cartilage as nonskeletal C.T. derivatives. The epithelial rudiments of thyroid and lung were also tested as endodermal derivatives. Chick cornea was similarly tested as an ectodermal derivative. Of these tissues, only the C.T. capsules formed cartilage. The results demonstrate that various C.T. cell types may alter their phenotype well after that stage at which their differentiation is thought to be stabilized, and that the ability to differentiate as cartilage may be common to all C.T. cells. The option of differentiating along a certain variety of pathways may depend more upon local conditions than on a predetermined pattern.     

Confocal laser scanning microscopy in orthopaedic research

Confocal laser scanning microscopy (CLSM) is a type of high-resolution fluorescence microscopy that overcomes the limitations of conventional widefield microscopy and facilitates the generation of high-resolution 3D images from relatively thick sections of tissue. As a comparatively non-destructive imaging technique, CLSM facilitates the in situ characterization of tissue microstructure. Images generated by CLSM have been utilized for the study of articular cartilage, bone, muscle, tendon, ligament and menisci by the foremost research groups in the field of orthopaedics including those teams headed by Bush, Errington, Guilak, Hall, Hunziker, Knight, Mow, Poole, Ratcliffe and White. Recent evolutions in techniques and technologies have facilitated a relatively widespread adoption of this imaging modality, with increased "user friendliness" and flexibility. Applications of CLSM also exist in the rapidly advancing field of orthopaedic implants and in the investigation of joint lubrication.     

Fat tissue: an underappreciated source of stem cells for biotechnology

Adipose tissue can be harvested in large amounts with minimal morbidity. It contains numerous cells types, including adipocytes, preadipocytes, vascular endothelial cells and vascular smooth muscle cells; it also contains cells that have the ability to differentiate into several lineages, such as fat, bone, cartilage, skeletal, smooth, and cardiac muscle, endothelium, hematopoietic cells, hepatocytes and neuronal cells. Cloning studies have shown that some adipose-derived stem cells (ADSCs) have multilineage differentiation potential. ADSCs are also capable of expressing multiple growth factors, including vascular endothelial growth factor and hepatocyte growth factor. Early, uncontrolled, non-randomized clinical research, applying fresh adipose-derived cells into a cranial defect or undifferentiated ADSCs into fistulas in Crohn's disease, has shown healing and an absence of side effects. The combination of these properties, and the large quantity of cells that can be obtained from fat, suggests that this tissue will be a useful tool in biotechnology.     

Scaffolds in tissue engineering of blood vessels

Tissue engineering of small diameter (<5?mm) blood vessels is a promising approach for developing viable alternatives to autologous vascular grafts. It involves in vitro seeding of cells onto a scaffold on which the cells attach, proliferate, and differentiate while secreting the components of extracellular matrix that are required for creating the tissue. The scaffold should provide the initial requisite mechanical strength to withstand in vivo hemodynamic forces until vascular smooth muscle cells and fibroblasts reinforce the extracellular matrix of the vessel wall. Hence, the choice of scaffold is crucial for providing guidance cues to the cells to behave in the required manner to produce tissues and organs of the desired shape and size. Several types of scaffolds have been used for the reconstruction of blood vessels. They can be broadly classified as biological scaffolds, decellularized matrices, and polymeric biodegradable scaffolds. This review focuses on the different types of scaffolds that have been designed, developed, and tested for tissue engineering of blood vessels, including use of stem cells in vascular tissue engineering.     

Informing tendon tissue engineering with embryonic development

Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering.     

Morphofunctional characterization of decellularized vena cava as tissue engineering scaffolds

Clinical experience for peripheral arterial disease treatment shows poor results when synthetic grafts are used to approach infrapopliteal arterial segments. However, tissue engineering may be an option to yield surrogate biocompatible neovessels. Thus, biological decellularized scaffolds could provide natural tissue architecture to use in tissue engineering, when the absence of ideal autologous veins reduces surgical options. The goal of this study was to evaluate different chemical induced decellularization protocols of the inferior vena cava of rabbits. They were decellularized with Triton X100 (TX100), sodium dodecyl sulfate (SDS) or sodium deoxycholate (DS). Afterwards, we assessed the remaining extracellular matrix (ECM) integrity, residual toxicity and the biomechanical resistance of the scaffolds. Our results showed that TX100 was not effective to remove the cells, while protocols using SDS 1% for 2 h and DS 2% for 1 h, efficiently removed the cells and were better characterized. These scaffolds preserved the original organization of ECM. In addition, the residual toxicity assessment did not reveal statistically significant changes while decellularized scaffolds retained the equivalent biomechanical properties when compared with the control. Our results concluded that protocols using SDS and DS were effective at obtaining decellularized scaffolds, which may be useful for blood vessel tissue engineering.