Cord-blood mesenchymal stem cells and tissue engineering

Cord-blood-derived stem cells have proven clinically useful for numerous disease states, as have mesenchymal stem cells (MCSs) derived from bone marrow and adipose tissue. The recent identification of MSCs in cord-blood heralds cord-blood as an untapped resource for nonhematopoietic stem cell-based therapeutic strategies for the replacement of injured or disease connective tissue. This review discusses the potential for tissue engineering applications of MSCs, highlighting the development of vascularized tissue engineering constructs using microvascular free flaps as a novel tissue engineering strategy.     

Cartilage tissue engineering: From hydrogel to mesenchymal stem cells

Articular cartilage does not repair itself spontaneously. To promote its repair, the transfer of stem cells from adipose tissue (ATSC) using an injectable self-setting cellulosic-hydrogel (Si-HPMC) appears promising. In this context, the objective of this work was to investigate the influence of in vitro chondrogenic differentiation of ATSC on the in vivo cartilage formation when combined with Si-HPMC. In a first set of experiments, we characterized ATSC for their ability to proliferate, self renew and express typical mesenchymal stem cell surface markers. Then, the potential of ATSC to differentiate towards the chondrogenic lineage and the optimal culture conditions to drive this differentiation were evaluated. Real-time RT-PCR and histological analysis for sulphated glycosaminoglycans and type II collagen revealed that 3-dimensional culture and hypoxic condition favored ATSC chondrogenesis regarding mRNA expression level and the corresponding proteins production. In order to assess the phenotypic stability of chondrogenically-differentiated ATSC, real-time RT-PCR for specific terminal chondrogenic markers and alkaline phosphatase activity assay were performed. In addition to promote chondrogenesis, our culture conditions seem to prevent the terminal differentiation of ATSC. Histological examination of ATSC/Si-HPMC implants suggested that the in vitro chondrogenic pre-commitment of ATSC in monolayer is sufficient to obtain cartilaginous tissue in vivo.     

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.     

间充质干细胞在组织工程气管中的研究进展

组织工程学通过细胞或组织重建,修复缺损组织并保留其生物功能,成为气管替代治疗的新途径.种子细胞、生长因子及气管支架材料是组织工程气管的三大要素,学者们一直在寻求理想的种子细胞.间充质干细胞(MSCs)是一种具有高度自我更新能力和多向分化潜能的干细胞,广泛分布于骨髓、脐带、脂肪组织、心肌组织、大脑、肌肉和皮肤等,可向骨、软骨、脂肪、神经等多种细胞定向分化.MSCs具有增殖能力强、分化范围广、免疫调节等功能特点,能够修复损伤的组织,有望成为应用于组织工程气管的理想种子细胞.旨在对MSCs在组织工程气管中的应用作一综述.     

Adipose tissue engineering using mesenchymal stem cells attached to injectable PLGA spheres

The reconstruction of soft tissue defects remains a challenge in plastic and reconstructive surgery, and a real clinical need exists for an adequate solution. This study was undertaken in order to differentiate mesenchymal stem cells (MSCs) into adipocytes, and to then assess the possibility of constructing adipose tissue via the attachment of MSCs to injectable PLGA spheres. We also designed injectable PLGA spheres for scar-free transplantation. In this study, MSCs and adipo-MSCs (MSCs cultured in adipogenic medium for 7 days) were attached to PLGA spheres and cultured for 7 days, followed by injection into nude mice for 2 weeks. As a result, the difference between lipid accumulation in adipo-MSCs at 1 and 7 days was much higher in vitro than in the MSCs. Two weeks after injection, a massive amount of new tissue was formed in the APLGA group, whereas only a small amount was formed in the MPLGA group. We verified that the newly formed tissue originated from the injected MSCs via GFP testing, and confirmed that the created tissue was actual adipose tissue by oil red O staining and Western blot (PPAR(gamma) and C/EBP(alpha) were expressed only in APLGA groups). Therefore, this study presents an efficient model of adipose tissue engineering using MSCs and injectable PLGA spheres.     

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.     

Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells

Silk fibroin scaffolds were studied as a new biomaterial option for tissue-engineered cartilage-like tissue. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on silk, collagen, and crosslinked collagen scaffolds and cultured for 21 days in serum-free chondrogenic medium. Cells proliferated more rapidly on the silk fibroin scaffolds than on the collagen matrices. The total content of glycosaminoglycan deposition was three times higher on silk as compared to collagen scaffolds. Glycosaminoglycan deposition coincided with overexpression of collagen type II and aggrecan genes. Cartilage-like tissue was homogeneously distributed throughout the entire silk scaffolds, while on the collagen and crosslinked collagen systems tissue formation was restricted to the outer rim, leaving a doughnut appearance. Round or angular-shaped cells resided in deep lacunae in the silk systems and stained positively for collagen type II. The aggregate modulus of the tissue-engineered cartilage constructs was more than 2-fold higher than that of the unseeded silk scaffold controls. These results suggest that silk fibroin scaffolds are suitable biomaterial substrates for autologous cartilage tissue engineering in serum-free medium and enable mechanical improvements along with compositional features suitable for durable implants to generate or regenerate cartilage.     

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.     

Tissue engineering of small intestinal tissue using collagen sponge scaffolds seeded with smooth muscle cells

In a previously reported attempt to regenerate small intestine with autologous tissues, collagen scaffolds were used without cell seeding or with autologous mesenchymal stem cell seeding. However the regenerated intestine lacked a smooth muscle layer. To accomplish regeneration of a smooth muscle layer, this present study used collagen scaffolds seeded with the smooth muscle cells (SMC) in a canine model. Autologous SMC were isolated from stomach wall and cultured. Two types of scaffolds were fabricated: in SMC (+), cultured SMCs were mixed with collagen solution and poured into a collagen sponge; and in SMC (-), SMCs were omitted. Both scaffolds were implanted into defects of isolated ileum as a patch graft. Animals were euthanized at 4, 8, and 12 weeks; for the last time point, the ileal loop had been reanastomosed at 8 weeks. At 12 weeks, the SMC (-) group showed a luminal surface covered by a regenerated epithelial cell layer with very short villi; however only a thin smooth muscle layer was observed, representing the muscularis mucosae. In the SMC (+) group, the luminal surface was covered completely by a relatively well-developed epithelial layer with numerous villi. Implanted SMCs were seen in the lamina propria and formed a smooth muscle layer. Thus, we concluded that collagen sponge scaffolds seeded with autologous SMCs have a potential for small intestine regeneration.     

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。     

Collagen-calcium phosphate cement scaffolds seeded with umbilical cord stem cells for bone tissue engineering

Human umbilical cord mesenchymal stem cells (hUCMSCs) avoid the invasive procedure required to harvest bone marrow MSCs. The addition of collagen fibers into self-setting calcium phosphate cement (CPC) may increase the scaffold strength, and enhance cell attachment and differentiation. The objectives of this study were to develop a novel class of collagen-CPC composite scaffolds, and to investigate hUCMSC attachment, proliferation, and osteogenic differentiation on collagen-CPC scaffolds for the first time. Collagen fibers in CPC improved the load-bearing capability. Flow cytometry showed that the hUCMSCs expressed cell surface markers characteristic of MSCs, and were negative for hematopoietic and endothelial cell markers. hUCMSCs proliferated rapidly in all CPC composite scaffolds, with cell number increasing by sevenfold in 8 days. Cellular function was enhanced with collagen fibers in CPC scaffolds. Cell density increased from (645±60) cells/mm(2) on CPC with 0% collagen, to (1056±65) cells/mm(2) on CPC with 8% collagen (p<0.05). The actin stress fibers inside the hUCMSCs were stained, and the fluorescence intensity was doubled when the collagen in CPC was increased by 0% to 8%. RT-PCR showed that hUCMSCs on CPC with collagen had higher osteogenic expression than those on CPC without collagen. Alizarin Red S staining revealed a great increase in mineralization by hUCMSCs on CPC with collagen than that without collagen. In conclusion, hUCMSCs showed excellent proliferation, differentiation, and synthesis of bone minerals in collagen-CPC composite scaffolds for the first time. The novel hUCMSC-seeded collagen-CPC construct with superior cell function and load-bearing capability is promising to enhance bone regeneration in a wide range of orthopedic and craniofacial applications.     

脐带间充质干细胞在骨组织工程中的应用进展

背景：在骨组织工程领域,对新型种子细胞脐带间充质干细胞的研究逐渐增多。 目的：针对脐带间充质干细胞的分离培养、生物学特征及其在骨组织中所用载体进行综述。 方法：由第一作者应用计算机检索1999-01/2011-03 PubMed数据库及中国期刊全文数据库中有关脐带间充质干细胞分离培养、生物学特征、载体方面的文章。英文检索词为“umbilical cord stem cell,tissue engineering”,中文检索词为“脐带间充质干细胞,骨组织工程”。排除重复性研究及无关研究,共保留46篇文章进行综述。 结果与结论：脐带间充质干细胞具备胚胎和成体干细胞的多重特点,并且符合国际细胞疗法协会制定的间充质干细胞标准,但是脐带间充质干细胞的分离培养、诱导分化方法等尚不完善,应进一步深入分析。在脐带干细胞的载体方面,单一的材料支架目前研究已很多,对于复合材料载体与可注射性载体的研究可能是今后的热点。     

Homogeneous seeding of mesenchymal stem cells into nonwoven fabric for tissue engineering

Mesenchymal stem cells (MSCs) were isolated from the bone marrow of rats and seeded into a nonwoven fabric of polyethylene terephtalate (PET) by agitation and static methods. MSC attachment was investigated in terms of the number of cells attached to the fabric, their distribution inside the fabric, and cell damage. The number of MSCs attached was greater for the agitation seeding method than for the static seeding method. The higher the rotating speed in the agitation seeding method, the greater the number of cells attached. When the cell suspension was seeded into the fabric in culture medium volumes of 50 and 200 microL per well of the culture plate or per culture tube, the best cell attachment was observed for the tube culture group at the larger volume. These cells attached more homogeneously throughout the fabric in greater numbers than was the case for the other culture groups. It is possible that agitation of the cell suspension allows cells to infiltrate uniformly inside the fabric, resulting in a homogeneous distribution of the cells in the fabric. A biochemical study revealed that neither the agitation nor static seeding method damaged cells, irrespective of the medium volume and the type of culture vessel. We conclude that the agitation seeding method is a promising method by which to formulate a homogeneous construct of fabric and MSCs.     

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。     

脂肪组织工程的新视角——脂肪间充质干细胞

文章对脂肪组织的来源，细胞的分离、纯化、培养及分子调控，细胞外基质的作用，细胞外微环境的影响进行综述。种子细胞的最佳来源是胚胎干细胞。研究表明过氧化物酶增殖体复合物在成脂分化中起主要调控作用，CCAT增强结合蛋白与ADD-1可启动和维持向脂肪方向分化作用，PPAR与配体结合激活脂肪发育过程中关键基因的转录，而AhR基因起负向调节作用。体外微环境培养中，起作用的是氧分压、pH值和黏附因子。     

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。     

Magnetic resonance imaging of the brain stem

Magnetic resonance imaging of 30 normal volonteers' posterior fossae was performed on a CGR Magniscan machine with a magnetic field strength of 0.5 Tesla. We chose the inversion recovery signal with a Tr of 2,000 ms, a TE of 21 ms and an inverse time of 500 ms. This sequence gives a better definition of the grey matter, the white matter and the CSF. To study the brain stem we suggest 8 horizontal and 3 sagittal sections.