Matrilin‐3 codelivery with adipose‐derived mesenchymal stem cells promotes articular cartilage regeneration in a rat osteochondral defect model

Matrilin‐3 is an essential extracellular matrix component present only in cartilaginous tissues. Matrilin‐3 exerts chondroprotective effects by regulating an anti‐inflammatory function and extracellular matrix components. We hypothesized that the codelivery of matrilin‐3 with infrapatellar adipose‐tissue‐derived mesenchymal stem cells (Ad‐MSCs) may enhance articular cartilage regeneration. Matrilin‐3 treatment of Ad‐MSCs in serum‐free media induced collagen II and aggrecan expression, and matrilin‐3 in chondrogenic media also enhanced in vitro chondrogenic differentiation. Next, the in vivo effect of matrilin‐3 codelivery with Ad‐MSCs on cartilage regeneration was assessed in an osteochondral defect model in Sprague Dawley rats: Ad‐MSCs and hyaluronic acid were implanted at the defect site with or without matrilin‐3 (140, 280, and 700 ng). Safranin O staining revealed that matrilin‐3 (140 and 280 ng) treatment significantly improved cartilage regeneration and glycosaminoglycan accumulation. In the animals treated with 140‐ng matrilin‐3, in particular, the defect site exhibited complete integration with surrounding tissue and a smooth glistening surface. The International Cartilage Repair Society macroscopic and O'Driscoll microscopic scores for regenerated cartilage were furthermore shown to be considerably higher for this group (matrilin‐3; 140 ng) compared with the other groups. Furthermore, the defects treated with 140‐ng matrilin‐3 revealed significant hyaline‐like cartilage regeneration in the osteochondral defect model; in contrast, the defects treated with 700‐ng matrilin‐3 exhibited drastically reduced cartilage regeneration with mixed hyaline–fibrocartilage morphology. Codelivery of matrilin‐3 with Ad‐MSCs significantly influenced articular cartilage regeneration, supporting the potential use of this tissue‐specific protein for a cartilage‐targeted stem cell therapy.     

Modulation of chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium

Studies on tissue‐engineering approaches for the regeneration of traumatized cartilage focus increasingly on multipotent human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes. The present study applied porous scaffolds made of collagen from the jellyfish Rhopilema esculentum for the in vitro chondrogenic differentiation of hMSCs. Culture conditions in those scaffolds differ from conditions in high‐density pellet cultures, making a re‐examination of these data necessary. We systematically investigated the influence of seeding density, basic culture media [Dulbecco's modified Eagle's medium (DMEM), α‐minimum essential medium (α‐MEM)] with varying glucose content and supplementation with fetal calf serum (FCS) or bovine serum albumin (BSA) on the chondrogenic differentiation of hMSCs. Gene expression analyses of selected markers for chondrogenic differentiation and hypertrophic development were conducted. Furthermore, the production of cartilage extracellular matrix (ECM) was analysed by quantification of sulphated glycosaminoglycan and collagen type II contents. The strongest upregulation of chondrogenic markers, along with the highest ECM deposition was observed in scaffolds seeded with 2.4 × 10⁶ cells/cm³ after cultivation in high‐glucose DMEM and 0.125% BSA. Lower seeding densities compared to high‐density pellet cultures were sufficient to induce in vitro chondrogenic differentiation of hMSCs in collagen scaffolds, which reduces the amount of cells required for the seeding of scaffolds and thus the monolayer expansion period. Furthermore, examination of the impact of FCS and α‐MEM on chondrogenic MSC differentiation is an important prerequisite for the development of an osteochondral medium for simultaneous osteogenic and chondrogenic differentiation in biphasic scaffolds for osteochondral tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Hyperosmolarity and hypoxia induce chondrogenesis of adipose-derived stem cells in a collagen type 2 hydrogel

Apart from soluble growth factors, various other biophysicochemical cues are known to promote chondrogenesis. Under physiological conditions, cartilage in the joint comprises a hyperosmotic and hypoxic environment. Therefore, in this study, we examined the inductive effects of hyperosmotic and/or hypoxic conditions on adipose stem cells (ASCs) and compared them with conventional TGFβ1‐induction. After encapsulation in collagen type II hydrogels and specific induction, ASCs were assessed for viability, proliferation, morphology and chondrogenic differentiation potential. Viability was similar under all conditions, with low proliferative activity. After 4 days, hypoxia and/or hyperosmolarity did not affect round cell morphology, while cells were mainly stretched in the TGFβ1‐induced group. At 21 days, the TGFß1‐treated group had aggregated into a cell nodule. Hyperosmolarity mimicked this aggregation to a lesser extent, whereas cells under hypoxia stretched out after 21 days, with a combined effect in the hypoxic/hyperosmotic group. Both individual and combined hyperosmotic and/or hypoxic conditions significantly upregulated SOX5, SOX9, COMP and Link‐p gene expression compared with the non‐induced group, and to similar levels as the TGFβ1‐induced group. GAG synthesis in both hydrogel and medium was increased under hypoxic conditions, whereas hyperosmolarity decreased GAG formation in the hydrogels, but increased GAG formation in the medium. We conclude that in a joint mimicking the three‐dimensional (3D) micro‐environment, a combination of hyperosmolarity and hypoxia is able to induce chondrogenesis to the same extent as TGFβ1. This might lead to an interesting alternative when considering short‐term triggering in a one‐step surgical procedure for the treatment of cartilaginous defects. Copyright © 2011 John Wiley & Sons, Ltd.     

Micropatterned three‐dimensional hydrogel system to study human endothelial–mesenchymal stem cell interactions

The creation of vascularized engineered tissues of clinically relevant size is a major challenge of tissue engineering. While it is known that endothelial and mural vascular cells are integral to the formation of stable blood vessels, the specific cell types and optimal conditions for engineered vascular networks are poorly understood. To this end, we investigated the vasculogenic potential of human mesenchymal stem cell (MSC) populations derived from three different sources: (a) bone marrow aspirates; (b) perivascular cells from the umbilical cord vein; and (c) perivascular cells from the umbilical cord artery. Cell populations were isolated and identified as MSCs according to their phenotypes and differentiation potential. Human umbilical vein endothelial cells (HUVECs) were used as a standard for endothelial cells. A novel co‐culture system was developed to study cell–cell interactions in a spatially controlled three‐dimensional (3D) fibrin hydrogel model. Using microfluidic patterning, it was possible to localize hydrogel‐encapsulated HUVECs and MSCs within separate channels spaced at 500, 1000 or 2000 µm. All three MSC populations had similar expression profiles of mesenchymal cell markers and similar capacity for osteogenic and adipogenic differentiation. However, bone marrow‐derived MSCs (but not umbilical vein or artery derived MSCs) showed strong distance‐dependent migration toward HUVECs and supported the formation of stable vascular networks resembling capillary‐like vasculature. The presented approach provides a simple and robust model to study the cell–cell communication of relevance to engineering vascularized tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

骨髓间充质干细胞复合温敏水凝胶支架的制备

背景：壳聚糖及其衍生物制备的支架对细胞迁移和神经轴突再生有重要作用。壳聚糖及其衍生物的组织相容性好,易使干细胞在其表面附着生长,在神经组织工程具有较为广阔的应用前景。
　　目的：制备适宜骨髓间充质干细胞生长的壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性水凝胶细胞支架,观察骨髓间充质干细胞在细胞支架中的生长情况。
　　方法：将壳聚糖进行季铵盐化改性处理,通过傅里叶变换红外光谱分析谱检测确定其生成。实验以壳聚糖与壳聚糖季铵盐配比为8∶1成功制备出较为稳定的壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性温敏水凝胶细胞支架,观察成胶情况,并进行生物安全性检测。
　　结果与结论：实验在傅里叶变换红外光图谱上发现了季铵基基团的特征峰。细胞毒性实验显示,水凝胶浸提液干预的大鼠骨髓间充质干细胞无毒性。急性全身毒性实验显示,浸提液对大鼠体质量增加无明显影响,支架生物安全性较好。扫描电镜观察显示,骨髓间充质干细胞在细胞支架中能正常的生长和增殖。结果证实,实验成功制备了壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性水凝胶细胞支架,适合骨髓间充质干细胞生长和增殖。     

Differential bone‐forming capacity of osteogenic cells from either embryonic stem cells or bone marrow‐derived mesenchymal stem cells

For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue‐engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in immune‐deficient mice, the osteogenic potential of embryonic stem cells has been mainly assessed in vitro. Therefore, we performed a series of studies to compare the in vitro and in vivo osteogenic capacities of human and mouse embryonic stem cells to those of hMSCs. Embryonic and mesenchymal stem cells showed all characteristic signs of osteogenic differentiation in vitro when cultured in osteogenic medium, including the deposition of a mineralized matrix and expression of genes involved in osteogenic differentiation. As such, based on the in vitro results, osteogenic ES cells could not be discriminated from osteogenic hMSCs. Nevertheless, although osteogenic hMSCs formed bone upon implantation, osteogenic cells derived from both human and mouse embryonic stem cells did not form functional bone, indicated by absence of osteocytes, bone marrow and lamellar bone. Although embryonic stem cells show all signs of osteogenic differentiation in vitro, it appears that, in contrast to mesenchymal stem cells, they do not possess the ability to form bone in vivo when a similar culture method and osteogenic differentiation protocol was applied. Copyright © 2010 John Wiley & Sons, Ltd.     

Multiphoton microscopy analysis of extracellular collagen I network formation by mesenchymal stem cells

Collagen I is the major fibrous extracellular component of bone responsible for its ultimate tensile strength. In tissue engineering, one of the most important challenges for tissue formation is to get cells interconnected via a strong and functional extracellular matrix (ECM), mimicking as closely as possible the natural ECM geometry. Still missing in tissue engineering are: (a) a versatile, high‐resolution and non‐invasive approach to evaluate and quantify different aspects of ECM development within engineered biomimetic scaffolds online; and (b) deeper insights into the mechanism whereby cellular matrix production is enhanced in 3D cell–scaffold composites, putatively via enhanced focal adhesion linkage, over the 2D setting. In this study, we developed sensitive morphometric detection methods for collagen I‐producing and bone‐forming mesenchymal stem cells (MSCs), based on multiphoton second harmonic generation (SHG) microscopy, and used those techniques to compare collagen I production capabilities in 2D‐ and 3D‐arranged cells. We found that stimulating cells with 1% serum in the presence of ascorbic acid is superior to other medium conditions tested, including classical osteogenic medium. In contrast to conventional 2D culture, having MSCs packed closely in a 3D environment presumably stimulates cells to produce strong and complex collagen I networks with defined network structures (visible in SHG images) and improves collagen production. Copyright © 2015 John Wiley & Sons, Ltd.     

Mathematical modelling of glycosaminoglycan production by stem cell aggregates incorporated with growth factor‐releasing polymer microspheres

Systems composed of high density cells incorporated with growth factor‐releasing polymer microspheres have recently been shown to promote chondrogenic differentiation and cartilage formation. Within these systems, the effects of spatial and temporal patterning of growth factor release on hyaline cartilage‐specific extracellular matrix production have been examined. However, at present, it is unclear which microsphere densities and growth factor delivery profiles are optimal for inducing human mesenchymal stem cell differentiation and glycosaminoglycan production. A mathematical model to describe glycosaminoglycan production as a function of initial microsphere loading and microsphere degradation rate over a period of 3 weeks is presented. Based on predictions generated by this model, it may be feasible to design a bioactive microsphere system with specific spatiotemporal growth factor presentation characteristics to promote glycosaminoglycan production at controllable rates. Copyright © 2014 John Wiley & Sons, Ltd.     

Chondrogenic potential of human mesenchymal stem cells and expression of Slug transcription factor

The scientific literature rarely reports experimental failures or inconsistent outcomes in the induction of cell differentiation; however, researchers commonly experience poor or unsuccessful responses to differentiating agents when culturing stem cells. One way of investigating the underlying reasons for such responses is to look at the basal expression levels of specific genes in multipotent stem cells before the induction of differentiation. In addition to shedding light on the complex properties of stem cells and the molecular modulation of differentiation pathways, this strategy can also lead to the development of important time‐ and money‐saving tools that aid the efficient selection of cellular specimens – in this case, stem cells that are more prone to differentiate towards specific lineages and are therefore more suitable for cell‐based therapeutic protocols in regenerative medicine. To address this latter aspect, this study focused on understanding the reasons why some human mesenchymal stem cell (hMSC) samples are less efficient at differentiating towards chondrogenesis. This study shows that analysis of the basal expression levels of Slug, a negative regulator of chondrogenesis in hMSC, provides a rapid and simple tool for distinguishing stem cell samples with the potential to form a cartilage‐like matrix, and that are therefore suitable for cartilage tissue engineering. It is shown that high basal levels of Slug prevent the chondrogenic differentiation of hMSCs, even in the presence of transforming growth factor‐β and elevated levels of Sox9. Copyright © 2013 John Wiley & Sons, Ltd.     

Monitoring immune response after allogeneic transplantation of mesenchymal stem cells for osteochondral repair

The aim of this study was to investigate the safety of using allogeneic magnetically labelled mesenchymal stem cells (m‐MSCs) to ameliorate osteochondral repair, with immune surveillance using a mixed lymphocyte reaction (MLR) assay. Twenty knees of Japanese white rabbits were randomly divided into two groups: the control (autologous) group, where 2 × 10⁵ autologous m‐MSCs were transplanted into the defect site; the experimental (allogeneic) group, where 2 × 10⁵ m‐MSCs from Dutch rabbits were transplanted into the defect of Japanese white rabbits. The rabbits were then euthanized after 12 weeks. The repaired tissue was stained with toluidine blue stain in order to produce histological scoring on the Fortier scale. Splenocytes were used to evaluate anti‐donor alloreactivity by a MLR assay using the carboxyfluorescein diacetate succimidyl ester (CFSE) labelling technique (CFSE‐MLR). In both groups, complete repair of the subchondral bone covered by a layer of chondrogenic tissue was confirmed. Also, there was no histologically significant difference on the Fortier scale. Using the CFSE‐MLR assay, CD4+ T‐cells showed alloreactivity in each combination, while the stimulation index of CD4+ T‐cells was not statistically different between the control and experimental groups. Allogeneic m‐MSCs are a safe alternative source to autologous m‐MSCs, upholding repair of an osteochondral defect for clinical application using universal donor MSCs through a one‐stage surgical procedure. Copyright © 2017 John Wiley & Sons, Ltd.     

In vitro interaction between mouse breast cancer cells and mouse mesenchymal stem cells during adipocyte differentiation

Surgical treatment following breast cancer, i.e. lumpectomy and mastectomy, may not efficiently remove all cancerous cells. As such, when mesenchymal stem cells (MSCs) are incorporated into the breast reconstruction process, it is likely that those MSCs will encounter remnant cancerous cells after transplantation into the defect site. The potential interaction between breast cancer cells and MSCs remains unclear. We hypothesized that paracrine interactions might occur between cells and various proteinases, growth factors and other cytokine molecules in the local microenvironment. Conditioned media (CM) from two mouse mammary cancer cell lines (4T1 and 4T07) and one mouse mammary epithelial cell line (NMuMG) were studied in the experimental model. Post‐confluent mouse MSCs (D1 cells) were differentiated with an adipogenic hormonal cocktail. Conditioned media from the three cell types did not have an inhibitory effect on D1 cell viability; however, triglyceride (TG) and Oil red O (ORO) analysis results showed that 4T1‐CM significantly inhibited D1 adipocyte differentiation and reduced lipid vesicle accumulation in the differentiating D1 cells. Preliminary analysis of the conditioned media revealed that a higher presence of matrix metalloprotease‐9 (MMP‐9) and urokinase plasminogen activator (uPA) was present in the 4T1‐CM as compared to the levels found in 4T07‐CM and NMuMG‐CM, which were below the detection limit. Additionally, the conditioned medium of differentiated D1 cells on day 12 had a negative effect on 4T1 and 4T07 cell viability but no effect on NMuMG cell viability. The results suggest that mouse breast cancer cells modulate mouse MSC adipogenic differentiation, the level of modulation specific to the metastatic level. Copyright © 2009 John Wiley & Sons, Ltd.     

Enhanced osteogenesis of human mesenchymal stem cells by self‐assembled peptide hydrogel functionalized with glutamic acid templated peptides

Self‐assembling peptide (SAP) hydrogel has been shown to be an excellent biological material for three‐dimensional cell culture and stimulatie cell migration and differentiation into the scaffold, as well as for repairing bone tissue defects. Herein, we designed one of the SAP scaffolds KLD (KLDLKLDLKLDL) through direct coupling to short bioactive motif O1 (EEGGC) and O2 (EEEEE) of which bioactivity on osteogenic differentiation was previously demonstrated and self‐assembled in different concentrations (0.5%, 1%, and 2%). Our aim was to enhance osteogenesis and biomineralization of injectable SAP hydrogels with controlled mechanical properties so that the peptide hydrogel also becomes capable of being injected to bone defects. The molecular integration of the nanofibrous peptide scaffolds was observed using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The rheological properties and degradation profile of SAP hydrogels were evaluated to ensure stability of SAPs. Compared with pure KLD scaffold, we found that these designed bioactive peptide scaffolds significantly promoted hMSCs proliferation depicted by biochemical analysis of alkaline phosphatase (ALP) activity, total calcium deposition. Moreover, key osteogenic markers of ALP activity, collagen type I (COL‐1), osteopontin (OP), and osteocalcin (OCN) expression levels determined by real‐time polymerase chain reaction (PCR) and immunofluorescence analysis were also significantly increased with the addition of glutamic acid residues to KLD. We demonstrated that the designed SAP scaffolds promoted the proliferation and osteogenic differentiation of hMSCs. Our results suggest that these designed bioactive peptide scaffolds may be useful for promoting bone tissue regeneration.     

脂肪干细胞与骨髓间充质干细胞成软骨能力的比较

背景：脂肪干细胞与骨髓间充质干细胞是软骨组织工程中应用较多细胞,二者在生物学特性上有诸多相似之处。目的：比较脂肪来源和骨髓来源的2种间充质干细胞的成软骨分化能力。方法：选取3月龄新西兰大白兔,取腹部脂肪,分离提取脂肪干细胞。取兔双侧股骨,采用贴壁筛选法分离提取骨髓间充质干细胞。绘制第3代脂肪干细胞和骨髓间充质干细胞的生长曲线,比较2种细胞的倍增时间。对第3代脂肪干细胞和骨髓间充质干细胞进行成软骨诱导,分别对诱导14d的2种细胞行甲苯胺蓝染色和Ⅱ型胶原免疫组化染色。结果与结论：骨髓间充质干细胞原代细胞呈聚集样生长,而脂肪干细胞原代细胞呈单个、散在生长。脂肪干细胞增殖速度要快于骨髓间充质干细胞,倍增时间短于骨髓间充质干细胞h。2种细胞成软骨诱导14d后,均表达糖胺聚糖和Ⅱ型胶原,且骨髓间充质干细胞成软骨诱导后表达Ⅱ型胶原水平高于脂肪干细胞。说明脂肪干细胞与骨髓间充质干细胞体外增殖皆迅速且稳定,但是脂肪干细胞的生长增殖速度更快。单层培养时,特定条件下,脂肪干细胞与骨髓间充质干细胞均能向软骨细胞转化,但是骨髓间充质干细胞比脂肪干细胞具有更高的潜能。     

骨髓间充质干细胞体外诱导分化为神经样细胞的机制

目的探索骨髓间充质干细胞 (MSCs)在体外诱导分化为神经样细胞的机制。方法分离培养大鼠MSCs ,用二甲基亚砜 (DSMO)和丁羟茴醚 (BHA)诱导分化 ,检测诱导分化前、预诱导 2 4h、诱导分化后 6h、2 4h和 48h神经细胞和神经干细胞的特异性标记蛋白的表达。结果诱导分化后 ,大部分MSCs变成双极、多极和锥形 ,并相互交织成网络结构 ,巢蛋白 (Nestin)在诱导分化前不表达 ,在诱导分化后 6h达到最高 ,2 4h和 48h逐渐降低。神经元特异性核蛋白 (NeuN )在诱导分化前不表达 ,在诱导分化后 6h出现表达 ,2 4h和 48h表达增强。结论MSCs经体外诱导先分化为神经干细胞 ,然后分化为神经元样细胞。     

富血小板血浆及脐带间充质干细胞修复软骨损伤

背景：研究者直接将富血小板血浆作为支架材料与骨髓基质干细胞、软骨细胞等复合后体外培养发现软骨细胞在富血小板血浆三维支架呈现增殖生长,骨髓基质干细胞在增殖的同时有向软骨细胞分化的倾向。目的：观察富血小板血浆和人脐带间充质干细胞对受损软骨修复的影响。方法：正常健康新西兰大白兔40只,制备兔软骨损伤模型。2次离心法制备富血小板血浆,制备3代人脐带间充质干细胞。随机将动物分为4组,造模后生理盐水组关节腔一次性注入生理盐水0.5 mL;富血小板血浆组注入12.5%富血小板血浆0.5 mL;人脐带间充质干细胞组注入1×107人脐带间充质干细胞0.5 mL;富血小板血浆(12.5%)联合人脐带间充质干细胞(1×107)组注入两种物质0.5 mL。造模后第12周,大体观察软骨损伤修复情况;苏木精-伊红染色光镜下观察损伤部位细胞修复情况;根据O’Driscol组织学评分标准对造模后第12周切片进行组织学评分。结果与结论：软骨损伤后大体观察、苏木精-伊红染色组织学观察及组织学评分均显示富血小板血浆组、人脐带间充质干细胞组、富血小板血浆联合人脐带间充质干细胞组对软骨损伤的修复效果优于生理盐水组,差异有显著性意义(P<0.05),富血小板血浆组、富血小板血浆联合人脐带间充质干细胞组的修复效果优于人脐带间充质干细胞组,差异有显著性意义(P<0.05)。结果说明富血小板血浆、人脐带间充质干细胞均能促进软骨损伤的修复,而且富血小板血浆、富血小板血浆联合人脐带间充质干细胞较单独应用人脐带间充质干细胞效果好。     

人间充质干细胞成骨诱导培养后的细胞成分研究

目的了解构建组织工程化骨的人间充质干细胞(MSCs),在体外成骨诱导环境中培养2代后,成骨细胞、软骨细胞、脂肪细胞的比例。方法体外扩增培养人MSCs,然后在含地塞米松、β-甘油磷酸钠、维生素C等的培养液中诱导培养2代,进行成骨细胞、软骨细胞、脂肪细胞的鉴定。结果人MSCs体外成骨诱导培养2代后,碱性磷酸酶阳性细胞数65%,Ⅰ型胶原免疫组化阳性细胞数55%、骨钙素阳性细胞数52%,Ⅱ型胶原免疫组化染色为阴性,油红O法染色阳性细胞数2.5%。结论人MSCs体外成骨诱导培养2代后,可有约50%的成骨细胞,少量脂肪细胞形成,无成熟的软骨细胞出现。该结论可为利用间充质干细胞构建组织工程化骨提供理论参考。