Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.     

A comparative analysis of cartilage engineered from different perinatal mesenchymal progenitor cells

We sought to compare engineered cartilaginous constructs derived from different perinatal mesenchymal progenitor cell (MPC) sources. Ovine MPCs isolated from amniotic fluid (AF, n = 8), neonatal bone marrow (BM, n = 6), and preterm umbilical cord blood (CB, n = 12) were expanded and comparably seeded onto synthetic scaffolds. Constructs were maintained in chondrogenic media containing transforming growth factor-beta. After 12-15 weeks, specimens were compared with native fetal hyaline and elastic cartilage by gross inspection, histology, immunohistochemistry, and quantitative extracellular matrix (ECM) assays. MPCs from AF proliferated significantly faster ex vivo when compared to MPCs from the other sources. Chondrogenic differentiation was evident in all groups, as shown by toluidine blue staining and expression of aggrecan, cartilage proteoglycan link protein, and collagen type II. Quantitatively, all engineered specimens had significantly lower levels of glycosaminoglycans than native hyaline cartilage. Elastin levels in AF-based constructs (156.0 +/- 120.4 microg/mg) were comparable to that of native elastic cartilage (235.8 +/- 54.2 microg/mg), both of which were significantly higher than in BM- and CB-based specimens. We conclude that the ECM profile of cartilage engineered from perinatal MPCs is highly dependent on cell source. ECM peculiarities should be considered when designing the optimal cartilaginous bioprosthesis for use in perinatal surgical reconstruction.     

Original approach for cartilage tissue engineering with mesenchymal stem cells

Cartilage tissue engineering gives the ability to product adaptable neocartilage to lesion with autologous cells. Our work aimed to develop a stratified scaffold with a simple and progressive spraying build-up to mimic articular cartilage environment. An Alginate/Hyaluronic Acid (Alg/HA) hydrogel seeded with human Mesenchymal Stem Cells (hMSC) was construct by spray. First, cells repartition and actin organization were study with confocal microscopy. Then, we analyzed cells viability and finally, metabolic activity. Our results indicated a homogenous cells repartition in the hydrogel and a pericellular actin repartition. After 3 days of culture, we observed about 52% of viable cells in the scaffold. Then, from day 7 until the end of culture (D28), the proportion of living cells and their metabolic activity increased, what indicates that culture conditions are not harmful for the cells. We report here that sprayed method allowed to product a scaffold with hMSCs that confer a favorable environment for neocartilage construction: 3D conformation and ability of cells to increase their metabolic activity, therefore with few impact on hMSCs.     

Chondrogenesis from human placenta-derived mesenchymal stem cells in three-dimensional scaffolds for cartilage tissue engineering

Human placenta-derived mesenchymal stem cells (hPMSCs) represent a promising source of stem cells. The application of hPMSCs in cartilage tissue engineering, however, was less reported. In this study, hPMSCs were grown in a three-dimensional (3D) environment for cartilage tissue formation in vitro. To select proper scaffolds for 3D culture of mesenchymal stem cells (MSCs), rat adipose-derived MSCs were initially employed to optimize the composition and condition of the 3D environment. The suitability of a poly(D,L-lactide-co-glycolide) (PLGA) precision scaffold previously developed for seeding and culture of primary chondrocytes was tested for MSCs. It was established that MSCs had to be embedded in alginate gel before seeded in the PLGA precision scaffold for cartilage-like tissue formation. The inclusion of nano-sized calcium-deficient hydroxyapatite (nCDHA) and/or a recombinant protein containing arginine-glycine-aspartate (RGD) into the alginate gel enhanced the chondrogenesis for both rat adipose-derived MSCs and hPMSCs. The amount of extracellular matrix such as glycosaminoglycan and type II collagen accumulated during a period of 21 days was found to be the greatest for hPMSCs embedded in the alginate/nCDHA/RGD gel and injected and cultivated in the precision scaffold. Also, histological analyses revealed the lacunae formation and extracellular matrix production from the seeded hPMSCs. Comparing human bone marrow-derived MSCs (hBMSCs) and hPMSCs grown in the previous composite scaffolds, the secretion of glycosaminoglycan was twice as higher for hPMSCs as that for hBMSCs. It was concluded that the alginate/nCDHA/RGD mixed gel in the aforementioned system could provide a 3D environment for the chondrogenesis of hPMSCs, and the PLGA precision scaffold could provide the dimensional stability of the whole construct. This study also suggested that hPMSCs, when grown in a suitable scaffold, may be a good source of stem cells for building up the tissue-engineered cartilage.     

骨髓间充质干细胞用于软骨组织工程的研究进展

细胞移植技术治疗软骨损伤已成为一项新兴的组织工程学研究热点.骨髓间充质干细胞由于其具有扩增快、便于分离提纯、可以体外诱导分化成为软骨细胞的特性,有可能成为组织工程化软骨的新型种子细胞.随着骨髓间充质干细胞应用于软骨组织工程研究的深入,结合近年的研究文献和成果,就骨髓间充质干细胞的诱导微环境和诱导方式的研究进展进行综述,探讨骨髓间充质干细胞作为种子细胞在构建组织工程软骨中的优越性.     

骨髓基质干细胞在软骨组织工程中的应用

骨关节炎等损坏关节软骨的疾病严重影响人们的正常生活。骨髓基质干细胞具有较强的自我更新能力和多向分化潜能,在特定的诱导条件下分化为软骨细胞,为组织工程软骨修复软骨缺损带来希望。就骨髓基质干细胞分离、诱导分化为软骨细胞的方法、载体材料及目前存在的问题加以综述。     

骨髓基质干细胞在软骨组织工程中的应用

骨关节炎等损坏关节软骨的疾病严重影响人们的正常生活。骨髓基质干细胞具有较强的自我更新能力和多向分化潜能，在特定的诱导条件下分化为软骨细胞，为组织工程软骨修复软骨缺损带来希望。就骨髓基质干细胞分离、诱导分化为软骨细胞的方法、载体材料及目前存在的问题加以综述。     

间充质干细胞应用于软骨组织工程的研究进展

[摘要]本文主要回顾了不同来源的间充质干细胞作为种子细胞的软骨组织工程学研究进展，并讨论自体软骨细胞移植技术和诱导间充质干细胞成软骨分化的软骨组织再生技术各自的优缺点，并展望其临床应用前景。     

脐血间充质干细胞在软骨组织工程中的应用

背景：关节软骨修复再生能力较差,软骨缺损的修复与功能重建是关节外科的一大难题,也是近年研究热点之一,而软骨组织工程技术的发展为其提供了新的思路和方法。 目的：总结和分析脐血间充质干细胞的生物学特性及其在软骨组织工程中的研究与应用。 方法：通过计算机检索中国期刊全文数据库及PubMed数据库2000-01/2010-12的有关文献资料,分别以“脐血间充质干细胞,组织工程,软骨缺损”为中文检索词,“umbilical cord blood mesenchymal stem cells,tissue engineering,cartilage repair”为英文检索词,纳入脐血间充质干细胞和软骨组织工程的相关文献,排除重复性研究,共选取33篇文献做进一步分析。 结果与结论：脐血间充质干细胞以其固有的取材方便、免疫原性较弱、分化能力强以及被病毒细菌污染率低等特有的优势,成为软骨组织工程理想的种子细胞,将在未来软骨组织工程的研究及应用中发挥重要的作用。     

Fetal liver as a source of autologous progenitor cells for perinatal tissue engineering

Mesenchymal progenitor cells, isolated from adult bone marrow, have been shown to have utility for autologous tissue engineering. The possibility of isolating from the fetal hematopoietic system a cell population with similar potential, which could be used for autologous reconstruction of prenatally diagnosed congenital anomalies, has not been explored to date. Liver stromal cells isolated from a portion of the right lateral hepatic lobe of midgestation fetal lambs were expanded in vitro. Passage 1 cells displayed a uniform fibroblast-like morphology but could be induced to differentiate into skeletal muscle, adipocytes, chondrocytes, and endothelial cells by selective medium supplementation. By manipulating the extracellular matrix in vitro, spontaneously contracting cardiac myocyte-like cells could be generated as well. Multilineage differentiation was confirmed by morphology, protein expression, and upregulation of lineage-specific mRNA. The potential for engineering myocardial tissue was then investigated by transplanting early-passage progenitor cells, organized on a three-dimensional matrix, into the ventricle of an immunocompromised rat utilizing a previously described model of left ventricular tissue engineering. Survival, incorporation into the host myocardium, and cardiomyocytic differentiation of the transplanted cells were confirmed. We have demonstrated that mesenchymal progenitor cells with multilineage potential can be isolated from the fetal liver and have potential utility for autologous tissue engineering.     

Assessment of the suitability of chitosan/polybutylene succinate scaffolds seeded with mouse mesenchymal progenitor cells for a cartilage tissue engineering approach

In this work, scaffolds derived from a new biomaterial originated from the combination of a natural material and a synthetic material were tested for assessing their suitability for cartilage tissue engineering applications. In order to obtain a better outcome result in terms of scaffolds' overall properties, different blends of natural and synthetic materials were created. Chitosan and polybutylene succinate (C-PBS) 50/50 (wt%) were melt blended using a twin-screw extruder and processed into 5 x 5 x 5 mm scaffolds by compression moulding with salt leaching. Micro-computed tomography analysis calculated an average of 66.29% porosity and 92.78% interconnectivity degree for the presented scaffolds. The salt particles used ranged in size between 63 and 125 mum, retrieving an average pore size of 251.28 mum. Regarding the mechanical properties, the compressive modulus was of 1.73 +/- 0.4 MPa (E(sec) 1%). Cytotoxicity evaluation revealed that the leachables released by the developed porous structures were not harmful to the cells and hence were noncytotoxic. Direct contact assays were carried out using a mouse bone marrow-derived mesenchymal progenitor cell line (BMC9). Cells were seeded at a density of 5 x 10(5) cells/scaffold and allowed to grow for periods up to 3 weeks under chondrogenic differentiating conditions. Scanning electron microscopy analysis revealed that the cells were able to proliferate and colonize the scaffold structure, and MTS test demonstrated cell viability during the time of the experiment. Finally, Western blot performed for collagen type II, a natural cartilage extracellular matrix component, showed that this protein was being expressed by the end of 3 weeks, which seems to indicate that the BMC9 cells were being differentiated toward the chondrogenic pathway. These results indicate the adequacy of these newly developed C-PBS scaffolds for supporting cell growth and differentiation toward the chondrogenic pathway, suggesting that they should be considered for further studies in the cartilage tissue engineering field.     

A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells

The utilization of adult stem cells in tissue engineering is a promising solution to the problem of tissue or organ shortage. Adult bone marrow derived mesenchymal stem cells (MSCs) are undifferentiated, multipotential cells which are capable of giving rise to chondrocytes when maintained in a three-dimensional culture and treated with members of the transforming growth factor-beta (TGF-beta) family of growth factors. In this study, we fabricated a nanofibrous scaffold (NFS) made of a synthetic biodegradable polymer, poly(-caprolactone) (PCL), and examined its ability to support in vitro chondrogenesis of MSCs. The electrospun PCL porous scaffold was constructed of uniform, randomly oriented nanofibers with a diameter of 700 nm, and structural integrity of this scaffold was maintained over a 21-day culture period. MSCs cultured in NFSs in the presence of TGF-beta1 differentiated to a chondrocytic phenotype, as evidenced by chondrocyte-specific gene expression and synthesis of cartilage-associated extracellular matrix (ECM) proteins. The level of chondrogenesis observed in MSCs seeded within NFSs was comparable to that observed for MSCs maintained as cell aggregates or pellets, a widely used culture protocol for studying chondrogenesis of MSCs in vitro. Due to the physical nature and improved mechanical properties of NFSs, particularly in comparison to cell pellets, the findings reported here suggest that the PCL NFS is a practical carrier for MSC transplantation, and represents a candidate scaffold for cell-based tissue engineering approaches to cartilage repair.     

羊膜上皮细胞和羊膜间充质干细胞构建组织工程皮肤

背景：由于人胎盘来源的间充质干细胞具有多方面的优点,近年来已成为干细胞研究的热点。 目的：分析鉴定羊膜间充质干细胞和羊膜上皮细胞的生物学特性,探讨其作为皮肤种子细胞在三维气液培养构建组织工程皮肤中的应用情况。 方法：用胰酶胶原酶多步消化法获取羊膜间充质干细胞和羊膜上皮细胞,通过流式细胞术、反转录-聚合酶链反应和免疫荧光染色技术,鉴定两种细胞的表面分子标记、干细胞特性、与皮肤角质形成细胞的相似性,并利用两种细胞为种子细胞以鼠Ⅰ型胶原为基质进行三维气液培养。 结果与结论：①流式细胞术检测体外培养羊膜间充质干细胞和羊膜上皮细胞均高表达CD90、CD73、CD105,不表达造血干细胞标志CD34以及MHC-Ⅱ类分子HLA-DR。②反转录-聚合酶链反应检测到羊膜间充质干细胞表达干细胞特性基因CMCY和NANOG,羊膜上皮细胞表达干细胞特性基因CMCY和 KLF4,两种细胞均有干细胞特性。③反转录-聚合酶链反应检测羊膜间充质干细胞表达皮肤角质形成细胞特性基因K19、β1-integrin、K8,羊膜上皮细胞表达K19、β1-integrin、K5、K8,免疫荧光染色见羊膜上皮细胞表达与角质形成细胞增殖相关的的特性蛋白K14,说明羊膜上皮细胞与皮肤角质形成细胞更具相似性, 在特定条件下更易于分化为皮肤角质形成细胞。④利用两种细胞成功构建组织工程皮肤,苏木精-伊红染色切片显示其具有一定的皮肤结构,且羊膜上皮细胞发生了初步分化。以上结果说明羊膜间充质干细胞与羊膜上皮细胞通过三维培养构建人皮肤组织是可行的。     

Characterization of multipotential mesenchymal progenitor cells derived from human trabecular bone

The in vitro culture of human trabecular bone-derived cells has served as a useful system for the investigation of the biology of osteoblasts. The recent discovery in our laboratory of the multilineage mesenchymal differentiation potential of cells derived from collagenase-treated human trabecular bone fragments has prompted further interest in view of the potential application of mesenchymal progenitor cells (MPCs) in the repair and regeneration of tissue damaged by disease or trauma. Similar to human MPCs derived from bone marrow, a clearer understanding of the variability associated with obtaining these bone-derived cells is required in order to optimize the design and execution of applicable studies. In this study, we have identified the presence of a CD73(+), STRO-1(+), CD105(+), CD34(-), CD45(-), CD144(-) cell population resident within collagenase-treated, culture-processed bone fragments, which upon migration established a homogeneous population of MPCs. Additionally, we have introduced a system of culturing these MPCs that best supports and maintains their optimal differentiation potential during long-term culture expansion. When cultured as described, the trabecular bone-derived cells display stem cell-like capabilities, characterized by a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic, adipocytic, and chondrocytic lineages, even when maintained in long-term in vitro culture.     

人羊膜间充质细胞定向分化的研究进展

人羊膜间充质细胞源自胎盘羊膜组织,具有间充质干细胞特性和多向分化潜能。在不同生长因子的调节下,人羊膜间充质细胞可分化成内、中、外三个胚层来源的细胞,如神经细胞、成骨细胞、软骨细胞、脂肪细胞、肝样细胞等。并且人羊膜间充质细胞具有比骨髓间充质干细胞更强的扩增能力和免疫原性低等优势,在组织工程及细胞替代治疗等再生医学领域具有广阔的应用前景。本文就人羊膜间充质细胞分离培养、生物学特性及多向分化潜能方面作一综述。     

Engineered mesenchymal stem cells for cartilage repair

Healthy cartilage is a highly robust tissue, and is resilient against the stringent mechanical and biological constraints imposed upon it. Cartilage defects are common features of joint diseases, but current treatments can rarely restore the full function of native cartilage. Recent studies have provided new perspectives for cartilage engineering using mesenchymal stem cells (MSCs). However, the sequential events occurring during chondrogenesis must be fully understood before we are able to reproduce faithfully the complex molecular events that lead to MSC differentiation and long-term maintenance of cartilage characteristics. Here, we focus on the potential of MSCs to repair cartilage with an emphasis on the factors that are known to be required in inducing chondrogenesis.     

Mesenchymal stem cells for cartilage engineering

Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, cell-based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. Moreover, the lack of efficient modalities of treatment for large chondral defects has prompted research on tissue engineering combining chondrogenic cells, scaffold materials and environmental factors.This paper focuses on the main parameters in tissue engineering and on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. Here we discuss the prospects of using autologous chondrocytes or MSCs in regenerative medicine and summarize the advantages and disadvantages of these cells in articular cartilage engineering.     

In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells

Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-beta3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.     

Modulation of immunological properties of allogeneic mesenchymal stem cells by collagen scaffolds in cartilage tissue engineering

Influence of the structures of some collagen scaffolds on immunological properties of the seeded allogeneic mesenchymal stem cells (MSCs) was studied in this article. Hydrogels, sponge, and membrane were prepared from type-I collagen. These scaffolds were seeded with neonatal rabbit MSCs and cultured for different periods. Changes of the immunological properties associated with different scaffolds were analyzed and compared. It was found that the expression of major histocompatibility complex (MHC) class I and II molecules on MSCs increased gradually in all scaffolds, but the least increment was recorded in hydrogels. Mixed lymphocyte reactions (MLR) showed that the MSC-hydrogel constructs invoked considerably low allogeneic lymphocytes proliferation. Even in presence of interferon-γ (IFN-γ), the hydrogels with higher concentration gave comparatively lower increment of MHC-II expression and allogeneic lymphocytes proliferation. These results suggest that different scaffold structures may provide different microenvironments and extents of isolation from the host immune system for the seed cells, thereby affecting their immunological properties. Therefore, scaffold structures may modulate the immunological properties of tissue-engineered cartilage with allogeneic cells. Hydrogels, especially which were prepared from higher collagen concentrations, were found to be a promising scaffold structure, from the perspective of avoiding severe immune rejection.     

Human mesenchymal progenitor cell-based tissue engineering of a single-unit osteochondral construct

A desirable strategy for articular cartilage repair is to surgically replace the damaged area with an in vitro-engineered osteochondral plug. We report here the development of a novel osteochondral construct using human trabecular bone-derived mesenchymal progenitor cells and a biodegradable poly-D,L-lactic acid scaffold. The cartilage layer was fabricated by press-coating a chondrifying high-density cell pellet onto the scaffold, which was then loaded with cells previously initiated to undergo osteogenesis. The composite was then cultured in a cocktail medium formulated to maintain both chondrogenesis and osteogenesis. Macroscopically, the construct consisted of a cartilage-like layer adherent to, and overlying, a dense bone-like component. RT-PCR, immunohistochemistry, and histology revealed hyaline-like cartilage and bone with an interface resembling the native osteochondral junction. All parameters, including mechanical properties, improved with increased culture time. The single-cell source nature of the construct, which minimizes handling while maximizing biocompatibility, suggests applicability for articular cartilage repair.