Osteoarthritic human chondrocytes proliferate in 3D co‐culture with mesenchymal stem cells in suspension bioreactors

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage‐generating cells. Previous studies in static culture have shown that hACs co‐cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co‐culture of hMSCs and OA hACs under serum‐free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125‐ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75‐fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co‐culture as a 2D monolayer in static culture flasks, bioreactor co‐culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum‐free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell‐based cartilage repair.     

Co-culturing mesenchymal stem cells from bone marrow and periosteum enhances osteogenesis and neovascularization of tissue-engineered bone

Mesenchymal stem cells (MSCs) isolated from bone marrow and periosteum are often used as cellular sources for bone tissue engineering. This study showed that co‐cultured human bone marrow stem cells (hBMSCs) and periosteal‐derived stem cells (hPCs) resulted in a synergistic effect on osteogenic differentiation both in vitro and in vivo. Compared to hBMSCs and hPCs, co‐culturing MSCs showed abundant mineralization, robust calcium deposition, steadily increasing ALP activity, and upgraded mRNA expression of osteogenic specific genes (COL1A1, BMP‐2, osteopontin, osteocalcin) in vitro. Eight weeks after implantation of cellular β‐TCP scaffolds in immunodeficient mice, similar synergistic effects were confirmed during in vivo evaluation of total new bone formation, mature bone formation, and neovascularization. Based on these findings, the use of co‐cultured hBMSCs and hPCs can be recommended as a promising new approach for bone tissue engineering applications. Copyright © 2011 John Wiley & Sons, Ltd.     

Multilineage co‐culture of adipose‐derived stem cells for tissue engineering

Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue‐engineering strategies with adipose‐derived stem cells (ASCs), the focus of this study was on developing indirect co‐culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co‐culture with each of the mature cell populations was shown to successfully induce or enhance lineage‐specific differentiation of the ASCs. In general, a more homogeneous but lower‐level differentiation response was observed in co‐culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non‐canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF‐1 and DKK‐1 on multilineage differentiation in co‐culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co‐culture with the mature cell populations. Copyright © 2012 John Wiley & Sons, Ltd.     

Advances in tissue engineering through stem cell‐based co‐culture

Stem cells are the future in tissue engineering and regeneration. In a co‐culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co‐culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co‐culture systems are discussed. Direct and indirect co‐culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell–cell contact, cell–extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co‐culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co‐culture use. Copyright © 2014 John Wiley & Sons, Ltd.     

基因修饰骨髓间充质干细胞修复关节软骨损伤

背景：随着生物技术的发展,通过转基因技术修饰细胞,从而获得长期稳定表达的生物活性因子以治疗关节软骨损伤逐渐引起重视。目的：就基因修饰的骨髓间充质干细胞在修复关节软骨损伤中的应用作一综述。方法：由第一作者检索1990至2011年PubMed数据库（http：／／www．ncbi．nlm．nih.gov／PubMed）有关基因修饰骨髓间充质干细胞修复关节软骨损伤的文献,英文检索词为“cartilage,genetherapy,mesenchymalstemcells,tissueengineering,bioactivefactor,vector”。共纳入15篇文献归纳总结。结果与结论：骨髓间充质干细胞已被广泛应用于修复关节软骨损伤。通过转基因技术将特定外源基因导入骨髓间充质干细胞,联合细胞治疗和基因治疗可达到更好的治疗效果,在关节软骨损伤的治疗中有广阔的应用前景。     

多样本骨髓间充质干细胞分离培养方法的量化比较

目的比较不同方法获取骨髓间充质干细胞的效率,为其在组织工程的应用确立最佳培养方案。方法以3个月龄新西兰大白兔为实验对象,通过对全血培养法、溶血纯化法、密度梯度离心法进行比较性研究,对克隆形成率、首次传代时间、扩增成功率等指标进行比较。结果对克隆形成率和扩增成功率而言,溶血纯化方法最低传代时间为20.5d,全血培养方法有部分提高传代时间为(13.9±2.9)d;而密度梯度离心方法的效率最高,首次传代时间为(7.5±0.7)d。结论对于成年动物穿刺获取骨髓间充质干细胞而言,密度离心>全血培养>溶血纯化方法,明确了一种实用有效的骨髓间充质干细胞分离培养方法。     

A hydrolytically‐tunable photocrosslinked PLA‐PEG‐PLA/PCL‐PEG‐PCL dual‐component hydrogel that enhances matrix deposition of encapsulated chondrocytes

In this study, a series of photocrosslinked hydrogels were designed composed of both poly(lactide)‐poly(ethylene glycol)‐poly(lactide) (PEL) and poly(ε‐caprolactone)‐poly(ethylene glycol)‐poly(ε‐caprolactone) (PEC) macromers. The PEL/PEC hydrogels at ratios of 100:0, 75:25, 50;50, 25:75 and 0:100 were studied for their degradation characteristics and their ability to support chondrogenesis of encapsulated chondrocytes. Difference in hydrolytic susceptibility between copolymers led to different degradation patterns where higher PEC content correlated with slower degradation. Increased chondrogenic gene expression was observed in chondrocyte‐laden hydrogels within a 4‐week culture period. Biochemical and histological evaluations revealed significant accumulation of extracellular matrix proteins such as glycosaminoglycans and collagen in the 50/50 hydrogel owing to appropriate tuning of hydrogel degradation. These results demonstrate that the dual‐component photocrosslinked hydrogel system is suitable for use as scaffold to support chondrogenesis and, moreover, the tunability of these systems opens up possibilities for use in different cell culturing applications. Copyright © 2014 John Wiley & Sons, Ltd.     

Composite clinoptilolite/PCL‐PEG‐PCL scaffolds for bone regeneration: In vitro and in vivo evaluation

In this study, clinoptilolite (CLN) was employed as a reinforcement in a polymer‐based composite scaffold in bone tissue engineering and evaluated in vivo for the first time. Highly porous, mechanically stable, and osteogenic CLN/PCL‐PEG‐PCL (CLN/PCEC) scaffolds were fabricated with modified particulate leaching/compression molding technique with varying CLN contents. We hypothesized that CLN reinforcement in a composite scaffold will improve bone regeneration and promote repair. Therefore, the scaffolds were analyzed for compressive strength, biodegradation, biocompatibility, and induction of osteogenic differentiation in vitro. CLN inclusion in PC‐10 (10% w/w) and PC‐20 (20% w/w) scaffolds revealed 54.7% and 53.4% porosity, higher dry (0.62 and 0.76 MPa), and wet (0.37 and 0.45 MPa) compressive strength, greater cellular adhesion, alkaline phosphatase activity (2.20 and 2.82 mg/g_DNA/min), and intracellular calcium concentration (122.44 and 243.24 g Ca/mg_DNA). The scaffolds were evaluated in a unicortical bone defect at anterior aspect of proximal tibia of adult rabbits 4 and 8 weeks postimplantation. Similar to in vitro results, CLN‐containing scaffolds led to efficient regeneration of bone in a dose‐dependent manner. PC‐20 demonstrated highest quality of bone union, cortex development, and bone‐scaffold interaction at the defect site. Therefore, higher CLN content in PC‐20 permitted robust remodeling whereas pure PCEC (PC‐0) scaffolds displayed fibrous tissue formation. Consequently, CLN was proven to be a potent reinforcement in terms of promoting mechanical, physical, and biological properties of polymer‐based scaffolds in a more economical, easy‐to‐handle, and reproducible approach.     

Matrix formation is enhanced in co‐cultures of human meniscus cells with bone marrow stromal cells

The ultimate aim of this study was to assess the feasibility of using human bone marrow stromal cells (BMSCs) to supplement meniscus cells for meniscus tissue engineering and regeneration. Human menisci were harvested from three patients undergoing total knee replacements. Meniscus cells were released from the menisci after collagenase treatment. BMSCs were harvested from the iliac crest of three patients and were expanded in culture until passage 2. Primary meniscus cells and BMSCs were co‐cultured in vitro in three‐dimensional (3D) pellet culture at three different cell–cell ratios for 3 weeks under normal (21% O₂) or low (3% O₂) oxygen tension in the presence of serum‐free chondrogenic medium. Pure BMSCs and pure meniscus cell pellets served as control groups. The tissue generated was assessed biochemically, histochemically and by quantitative RT–PCR. Co‐cultures of primary meniscus cells and BMSCs resulted in tissue with increased (1.3–1.7‐fold) deposition of proteoglycan (GAG) extracellular matrix (ECM) relative to tissues derived from BMSCs or meniscus cells alone under 21% O₂. GAG matrix formation was also enhanced (1.3–1.6‐fold) under 3% O₂ culture conditions. Alcian blue staining of generated tissue confirmed increased deposition of GAG‐rich matrix. mRNA expression of type I collagen (COL1A2), type II collagen (COL2A1) and aggrecan were upregulated in co‐cultured pellets. However, SOX9 and HIF‐1α mRNA expression were not significantly modulated by co‐culture. Co‐culture of primary meniscus cells with BMSCs resulted in increased ECM formation. Co‐delivery of meniscus cells and BMSCs can, in principle, be used in tissue engineering and regenerative medicine strategies to repair meniscus defects. Copyright © 2012 John Wiley & Sons, Ltd.     

全骨髓贴壁法培养兔骨髓间充质干细胞体外定向成骨诱导分化及鉴定

背景：流式细胞仪分离法和免疫磁珠分离法对细胞活性影响较大,密度梯度离心法虽然能够获得纯度高的单核细胞,但由于多次离心可造成细胞的大量流失且对细胞活性有一定的影响使其应用值得商榷。目的：采用全骨髓贴壁法分离兔骨髓间充质干细胞进行成骨诱导分化及鉴定。方法：采用全骨髓贴壁法体外分离培养兔骨髓间充质干细胞,倒置显微镜下观察细胞形态学特征。在成骨诱导剂作用下,通过碱性磷酸酶染色试剂盒行碱性磷酸酶染色,I型胶原免疫细胞化学染色,VonKossa法及茜素红进行矿化结节染色以及电镜下检测兔骨髓间充质干细胞成骨诱导后的形态结构。结果与结论：经诱导后细胞出现与成骨细胞相似的形态学特征,碱性磷酸酶染色阳性,I型胶原免疫细胞化学染色,Von-Kossa法及茜素红矿化结节染色阳性。表明经成骨诱导剂诱导后全骨髓贴壁法体外分离纯化培养的兔骨髓间充质干细胞能向成骨细胞方向分化增殖。     

Engineering cartilaginous grafts using chondrocyte‐laden hydrogels supported by a superficial layer of stem cells

During postnatal joint development, progenitor cells that reside in the superficial region of articular cartilage first drive the rapid growth of the tissue and later help direct the formation of mature hyaline cartilage. These developmental processes may provide directions for the optimal structuring of co‐cultured chondrocytes (CCs) and multipotent stromal/stem cells (MSCs) required for engineering cartilaginous tissues. The objective of this study was to engineer cartilage grafts by recapitulating aspects of joint development where a population of superficial progenitor cells drives the development of the tissue. To this end, MSCs were either self‐assembled on top of CC‐laden agarose gels (structured co‐culture) or were mixed with CCs before being embedded in an agarose hydrogel (mixed co‐culture). Porcine infrapatellar fat pad‐derived stem cells (FPSCs) and bone marrow‐derived MSCs (BMSCs) were used as sources of progenitor cells. The DNA, sGAG and collagen content of a mixed co‐culture of FPSCs and CCs was found to be lower than the combined content of two control hydrogels seeded with CCs and FPSCs only. In contrast, a mixed co‐culture of BMSCs and CCs led to increased proliferation and sGAG and collagen accumulation. Of note was the finding that a structured co‐culture, at the appropriate cell density, led to greater sGAG accumulation than a mixed co‐culture for both MSC sources. In conclusion, assembling MSCs onto CC‐laden hydrogels dramatically enhances the development of the engineered tissue, with the superficial layer of progenitor cells driving CC proliferation and cartilage ECM production, mimicking certain aspects of developing cartilage. Copyright © 2015 John Wiley & Sons, Ltd.     

兔骨髓间充质干细胞的分离培养及成骨诱导

背景：骨髓间充质干细胞具有多向分化潜能,且可大量体外扩增培养,是重要的组织工程种子细胞。但尚无统一的体外培养及定向诱导方法。目的：探讨体外定向诱导兔骨髓间充质干细胞分化为成骨细胞的可行性。方法：应用密度梯度离心法从兔四肢骨中分离纯化间充质干细胞,应用密度为1．073g／mL的Percoll分离液,3000r／minx30min离心,区别于相关报道的Ficoll分离液,2000—2500r／min×（20—30）min离心以及全骨髓培养法体外扩增至第3代,分别在普通培养基（对照组）和成骨诱导培养基（实验组）中培养。结果与结论：成功获得大量高纯度骨髓间充质干细胞。经成骨诱导后,实验组骨钙素含量明显高于对照组（P〈0．05）。实验组碱性磷酸酶和钙结节染色阳性,对照组均阴性。结果表明使用密度梯度离心法可成功建立兔骨髓间充质干细胞的分离培养体系,骨髓间充质干细胞可定向诱导为成骨细胞。     

Cartilage graft engineering by co‐culturing primary human articular chondrocytes with human bone marrow stromal cells

Co‐culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small‐scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold‐based constructs generated by combining primary human ACs with human bone marrow MSCs (BM‐MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM‐MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co‐culture of ACs (25%) with BM‐MSCs (75%) in scaffolds resulted in up to 1.4‐fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8‐fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM‐MSCs with a relatively low fraction of non‐expanded ACs in porous scaffolds. The proposed co‐culture strategy is directly relevant towards a single‐stage surgical procedure for cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd.     

兔骨髓基质细胞的体外分离培养及诱导成骨的实验研究

目的 探讨兔骨髓基质细胞（BMSCs）的体外分离培养及定向诱导成骨的方法。方法 选择10周龄新西兰兔，抽取其股骨大转子部骨髓，体外分离纯化得到BMSCs，再利用条件培养液将其向成骨方向定向诱导培养。采用碱性磷酸酶（ALP）和冯库萨（Von Kossa）染色方法，鉴定其成骨性能。结果 BMSCs在培养皿中贴壁生长、增殖，经ALP和Yon Kossa染色证实其有成骨潜能。结论 兔BMSCs的体外培养增殖能力强，能诱导分化为成骨细胞，可以作为骨组织工程的种子细胞。     

猪骨髓间质干细胞的分离培养及分化潜能的鉴定

目的：建立猪骨髓间质干细胞(MSCs)的体外分离培养和鉴定的方法，探讨体外培养的间充质干细胞的一些生物学特点，为利用猪的实验研究提供实验基础。方法：猪的髂嵴穿刺吸取骨髓，经密度梯度离心得到骨髓单个核细胞，接种后形成单层贴壁的成纤维样的细胞。检测细胞周期，多向诱导分化鉴定分离的细胞。结果：体外培养的原代MSCs12～14d达到融合，传代后仍具有分化成骨的能力，细胞周期显示有80％的细胞处于GO／G1期。结论：体外培养猪的MSCs具有分化成骨的潜能，生长稳定，传代后仍保持未分化状态．猪骨髓间充质干细胞分离培养体系的建立为基础研究和组织工程提供了一个有价值的动物模型。     

成人骨髓间充质干细胞体外定向诱导分化为神经元样细胞的研究

目的研究人骨髓间充质干细胞（mesenchymal stem cells，MSCs）向神经细胞分化的可能性。方法利用Per-coil梯度分离及贴壁筛选法分离培养和扩增MSCs；利用bFGF、化学诱导剂DMSO和BHA联合诱导MSCs向神经元转化，观察分化过程中细胞形态的变化，利用免疫细胞化学和RT-PCR方法检测神经元特异性标志物的表达情况。结果经Perecoll梯度分离及贴壁筛选法获得了纯度较高的人MSCs，诱导分化后的细胞呈现双极、多极和锥形的典型神经元细胞的形态，并且分别从mRNA和蛋白水平上证明诱导分化后的细胞表达神经元标记物NSE和NF，不表达神经胶质细胞标记物GFAP。结论人MSCs可以在体外诱导分化为神经元样细胞，这种潜能使其有可能成为神经系统疾病细胞移植治疗的种子细胞。     

Review of vascularised bone tissue‐engineering strategies with a focus on co‐culture systems

Poor angiogenesis within tissue‐engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue‐engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue‐engineered constructs and are briefly discussed in this review. Recent investigations relating to co‐culture systems of endothelial and osteoblast‐like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co‐culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co‐culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long‐lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Copyright © 2012 John Wiley & Sons, Ltd.     

Low‐oxygen conditions promote synergistic increases in chondrogenesis during co‐culture of human osteoarthritic stem cells and chondrocytes

There has been increased interest in co‐cultures of stem cells and chondrocytes for cartilage tissue engineering as there are the limitations associated with using either cell type alone. Drawbacks associated with the use of chondrocytes include the limited numbers of cells available for isolation from damaged or diseased joints, their dedifferentiation during in vitro expansion, and a diminished capacity to synthesise cartilage‐specific extracellular matrix components with age and disease. This has motivated the use of adult stem cells with either freshly isolated or culture‐expanded chondrocytes for cartilage repair applications; however, the ideal combination of cells and environmental conditions for promoting robust chondrogenesis remains unclear. In this study, we compared the effect of combining a small number of freshly isolated or culture‐expanded human chondrocytes with infrapatellar fat pad–derived stem cells (FPSCs) from osteoarthritic donors on chondrogenesis in altered oxygen (5% or 20%) and growth factor supplementation (TGF‐β3 only or TGF‐β3 and BMP‐7) conditions. Both co‐cultures, but particularly those including freshly isolated chondrocytes, were found to promote cell proliferation and enhanced matrix accumulation compared to the use of FPSCs alone, resulting in the development of a tissue that was compositionally more similar to that of the native articular cartilage. Local oxygen levels were found to impact chondrogenesis in co‐cultures, with more robust increases in proteoglycan and collagen deposition observed at 5% O₂. Additionally, collagen type I synthesis was suppressed in co‐cultures maintained at low‐oxygen conditions. This study demonstrates that a co‐culture of freshly isolated human chondrocytes and FPSCs promotes robust chondrogenesis and thus is a promising cell combination for cartilage tissue engineering.