Human adipose tissue‐derived tenomodulin positive subpopulation of stem cells: A promising source of tendon progenitor cells

Cell‐based therapies are of particular interest for tendon and ligament regeneration given the low regenerative potential of these tissues. Adipose tissue is an abundant source of stem cells, which may be employed for the healing of tendon lesions. However, human adult multipotent adipose‐derived stem cells (hASCs) isolated from the stromal vascular fraction of adipose tissue originate highly heterogeneous cell populations that hinder their use in specific tissue‐oriented applications. In this study, distinct subpopulations of hASCs were immunomagnetic separated and their tenogenic differentiation capacity evaluated in the presence of several growth factors (GFs), namely endothelial GF, basic‐fibroblast GF, transforming GF‐β1 and platelet‐derived GF‐BB, which are well‐known regulators of tendon development, growth and healing. Among the screened hASCs subpopulations, tenomodulin‐positive cells were shown to be more promising for tenogenic applications and therefore this subpopulation was further studied, assessing tendon‐related markers (scleraxis, tenomodulin, tenascin C and decorin) both at gene and protein level. Additionally, the ability for depositing collagen type I and III forming extracellular matrix structures were weekly assessed up to 28 days. The results obtained indicated that tenomodulin‐positive cells exhibit phenotypical features of tendon progenitor cells and can be biochemically induced towards tenogenic lineage, demonstrating that this subset of hASCs can provide a reliable source of progenitor cells for therapies targeting tendon regeneration.     

Differentiation of adipose stem cells seeded towards annulus fibrosus cells on a designed poly(trimethylene carbonate) scaffold prepared by stereolithography

Cell‐based therapies could potentially restore the biomechanical function and enhance the self‐repair capacity of annulus fibrosus (AF) tissue. However, choosing a suitable cell source and scaffold design are still key challenges. In this study, we assessed the in vitro ability of human adipose stem cells (hASCs), an easily available cell source to produce AF‐like matrix in novel AF‐mimetic designed scaffolds based on poly(trimethylene carbonate) and built by stereolithography. To facilitate efficient differentiation of hASCs towards AF tissue, we tested different culture medium compositions and cell seeding techniques. This is the first study to report that medium supplementation with transforming growth factor (TGF)‐β3 is essential to support AF differentiation of hASCs while TGF‐β1 has negligible effect after 21 days of culture. Fibrin gel seeding resulted in superior cell distribution, proliferation and AF‐like matrix production of hASCs compared to direct and micromass seeding under TGF‐β3 stimulation. Not only the production of sulphated glycosaminoglycans (sGAG) and collagen was significantly upregulated, but the formed collagen was also oriented and aligned into bundles within the designed pore channels. The differentiated hASCs seeded with fibrin gel were also found to have a comparable sGAG:collagen ratio and gene expression profile as native AF cells demonstrating the high potential of this strategy in AF repair. Copyright © 2016 John Wiley & Sons, Ltd.     

TGF‐β1 enhances contractility in engineered skeletal muscle

Scaffoldless engineered 3D skeletal muscle tissue created from satellite cells offers the potential to replace muscle tissue that is lost due to severe trauma or disease. Transforming growth factor‐beta 1 (TGF‐β1) plays a vital role in mediating migration and differentiation of satellite cells during the early stages of muscle development. Additionally, TGF‐β1 promotes collagen type I synthesis in the extracellular matrix (ECM) of skeletal muscle, which provides a passive elastic substrate to support myofibres and facilitate the transmission of force. To determine the role of TGF‐β1 in skeletal muscle construct formation and contractile function in vitro, we created tissue‐engineered 3D skeletal muscle constructs with varying levels of recombinant TGF‐β1 added to the cell culture medium. Prior to the addition of TGF‐β1, the primary cell population was composed of 75% Pax7‐positive cells. The peak force for twitch, tetanus and spontaneous force were significantly increased in the presence of 2.0 ng/ml TGF‐β1 when compared to 0, 0.5 and 1.0 ng/ml TGF‐β1. Visualization of the cellular structure with H&E and with immunofluorescence staining for sarcomeric myosin heavy chains and collagen type I showed denser regions of better organized myofibres in the presence of 2.0 ng/ml TGF‐β1 versus 0, 0.5 and 1.0 ng/ml. The addition of 2.0 ng/ml TGF‐β1 to the culture medium of engineered 3D skeletal muscle constructs enhanced contractility and extracellular matrix organization. Copyright © 2012 John Wiley & Sons, Ltd.     

Preferential tendon stem cell response to growth factor supplementation

Tendon injuries are increasingly prevalent around the world, accounting for more than 100 000 new clinical cases/year in the USA alone. Cell‐based therapies have been proposed as a therapeutic strategy, with recent data advocating the use of tendon stem cells (TSCs) as a potential cell source with clinical relevance for tendon regeneration. However, their in vitro expansion is problematic, as they lose their multipotency and change their protein expression profile in culture. Herein, we ventured to assess the influence of insulin‐like growth factor 1 (IGF‐1), growth and differentiation factor‐5 (GDF‐5) and transforming growth factor‐β1 (TGFβ1) supplementation in TSC culture. IGF‐1 preserved multipotency for up to 28 days. Upregulation of decorin and scleraxis expression was observed as compared to freshly isolated cells. GDF‐5 treated cells exhibited reduced differentiation along adipogenic and chondrogenic pathways after 28 days, and decorin, scleraxis and collagen type I expression was increased. After 28 days, TGFβ1 supplementation led to increased scleraxis, osteonectin and collagen type II expression. The varied responses to each growth factor may reflect their role in tendon repair, suggesting that: GDF‐5 promotes the transition of tendon stem cells towards tenocytes; TGFβ1 induces differentiation along several pathways, including a phenotype indicative of fibrocartilage or calcified tendon, common problems in tendon healing; and IGF‐1 promotes proliferation and maintenance of TSC phenotypes, thereby creating a population sufficient to have a beneficial effect. Copyright © 2014 John Wiley & Sons, Ltd.     

转化生长因子β和调节性T细胞在急性脑出血患者外周血中的变化及相关性研究

目的探讨CD4~+CD25~+CD127~(low/-)调节性T细胞和转化生长因子β在急性脑出血患者外周血中的表达及其相关性。方法采用流式细胞术检测33例试验组急性脑出血患者和30例健康对照者的外周血CD4~+CD25~+CD127~(low/-)调节性T细胞百分率,同时采用酶联免疫吸附试验检测63例研究对象的血清转化生长因子β(TGF-β)表达水平,将所得数据进行统计分析。结果试验组CD4+CD25+T细胞百分率、CD4~+CD25~+CD127~(low/-)调节性T细胞百分率均显著低于对照组,差异有统计学意义(P0.05)。试验组TGF-β表达水平与对照组相比显著升高,差异有统计学意义(P0.05)。分析急性脑出血患者的调节性T细胞数量、TGF-β表达水平与临床指标,发现其与体温、白细胞相关性不大,与空腹血糖和血压具有明显的相关性。结论急性脑出血患者CD4~+CD25~+CD127~(low/-)调节性T细胞数量异常减少可能与其免疫功能紊乱相关。     

Growth differentiation factor‐5 regulation of extracellular matrix gene expression in murine tendon fibroblasts

The synthesis and organization of extracellular matrix (ECM) of tendon, in resting and states of repair, are governed by fibroblasts. Growth differentiation factor‐5 (GDF‐5) may enhance the cellular response to tendon injury, thus improving the structural outcome of the regenerative tissue. This study was an attempt to identify potential mechanisms controlling the response of fibroblasts to injury and GDF‐5, in the pursuit of improved tissue regeneration. There were two sets of experiments. Isolated mice Achilles tendon fibroblasts were treated with different concentrations of rGDF‐5 (0–100 ng/ml) for 0–12 days in cell culture. The temporal effect of rGDF‐5 on ECM gene expression was analysed for type I collagen and aggrecan expression. Microarray and gene expression analysis were performed on cells treated with 100 ng/ml for 4 days. Forty‐five mice underwent bilateral mid‐substance Achilles tendon tenotomy and suture repair. Repair sites were injected with 10 µg rGDF‐5 or saline. Tendons were assessed histologically at 2, 4 and 6 weeks. Expression of ECM genes procollagen IX, aggrecan, matrix metalloproteinase 9 and fibromodulin were upregulated. Proinflammatory reaction genes were downregulated. rGDF‐5 led to an increase in total DNA, glycosaminoglycan (GAG) and hydroxyproline (OHP). The OHP:DNA ratio of fibroblast cultures was increased over all time points, with increased GAG:DNA at day 12. rGDF‐5 treatment showed improved collagen organization over controls. The results delineate the mode of action of rGDF‐5 at the cellular and gene level. rGDF‐5 could play a role in tendon repair and be used for future therapies that promote tendon healing. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of AT1- and β-adrenergic receptor antagonists on TGF-β1-induced fibrosis in transgenic mice

Background Transforming growth factor‐β1 (TGF‐β1) is involved in interstitial remodelling promoting collagen synthesis and suppressing collagen degradation by inhibition of collagenases. TGF‐β1 mediates angiotensin II‐dependent effects and modulates β1‐adrenergic signalling. To study the effect of neuroendocrine antagonism on TGF‐β‐induced hypertrophic and fibrotic phenotype, we treated TGF‐β1 (Cys223,225Ser) transgenic mice (TGF‐β1‐TG) with either the β1‐receptor blocker metoprolol (MET), the angiotensin II type I (AT1)‐receptor antagonist telmisartan (TEL) or an antibody blocking TGF‐β1 signalling (TGFβ1‐sR‐Ab). Material and Methods Transforming growth factor‐β1‐TG mice (8 weeks) overexpressing TGF‐β1 were treated with either TEL (10 mg kg^?1), MET (350 mg kg^?1) or a soluble TGF‐β1 receptor antibody (1 mg kg^?1) for 6 weeks. Morphological analyses of interstitium and cardiomyocytes were related to expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) by immunoblotting and zymography. Results In TGF‐β1‐TG mice, myocardial interstitial total collagen content was fourfold elevated compared to that of controls (P < 0·05) and was lowered under the treatment with TEL (P < 0·05). Protein expression of TIMP‐1 and ‐4 was increased in TGF‐β1‐TG but inhibited by TEL (TIMP‐1 and TIMP‐4) and MET (TIMP‐1), while collagenase activity was decreased in TGF‐β1‐TG and normalized by treatment with TEL (MMP‐1 and MMP‐13) and MET (MMP‐1) (P < 0·05). Morphometric measurements of cardiomyocyte diameter and area demonstrated similar antihypertrophic effects for all treatment groups. Conclusion The AT1‐antagonist TEL reduced myocardial hypertrophy and interstitial fibrosis in TGF‐β1‐TG mice by normalizing MMP/TIMP ratio. β1‐Adrenergic inhibition by MET as well as TGF‐β1 antagonism induced antihypertrophic rather than antifibrotic effects. Inhibition of both renin‐angiotensin system and β1‐adrenergic system may exert different but synergistic effects to reduce myocardial remodelling.     

The mechanobiology of tendon fibroblasts under static and uniaxial cyclic load in a 3D tissue engineered model mimicking native extracellular matrix

Tendon mechanobiology plays a vital role in tendon repair and regeneration; however, this mechanism is currently poorly understood. We tested the role of different mechanical loads on extracellular matrix (ECM) remodelling gene expression and the morphology of tendon fibroblasts in collagen hydrogels, designed to mimic native tissue. Hydrogels were subjected to precise static or uniaxial loading patterns of known magnitudes and sampled to analyse gene expression of known mechano‐responsive ECM‐associated genes (Collagen I, Collagen III, Tenomodulin, and TGF‐β). Tendon fibroblast cytomechanics was studied under load by using a tension culture force monitor, with immunofluorescence and immunohistological staining used to examine cell morphology. Tendon fibroblasts subjected to cyclic load showed that endogenous matrix tension was maintained, with significant concomitant upregulation of ECM remodelling genes, Collagen I, Collagen III, Tenomodulin, and TGF‐β when compared with static load and control samples. These data indicate that tendon fibroblasts acutely adapt to the mechanical forces placed upon them, transmitting forces across the ECM without losing mechanical dynamism. This model demonstrates cell‐material (ECM) interaction and remodelling in preclinical a platform, which can be used as a screening tool to understand tendon regeneration.     

Multiphasic collagen fibre–PLA composites seeded with human mesenchymal stem cells for osteochondral defect repair: an in vitro study

A promising approach for the repair of osteochondral defects is the use of a scaffold with a well‐defined cartilage–bone interface. In this study, we used a multiphasic composite scaffold with an upper collagen I fibre layer for articular cartilage repair, separated by a hydrophobic interface from a lower polylactic acid (PLA) part for bone repair. Focusing initially on the engineering of cartilage, the upper layer was seeded with human mesenchymal stem cells (hMSCs) suspended in a collagen I hydrogel for homogeneous cell distribution. The constructs were cultured in a defined chondrogenic differentiation medium supplemented with 10 ng/ml transforming growth factor‐β1 (TGFβ1) or in DMEM with 10% fetal bovine serum as a control. After 3 weeks a slight contraction of the collagen I fibre layer was seen in the TGFβ1‐treated group. Furthermore, a homogeneous cell distribution and chondrogenic differentiation was achieved in the upper third of the collagen I fibre layer. In the TGFβ1‐treated group cells showed a chondrocyte‐like appearance and were surrounded by a proteoglycan and collagen type II‐rich extracellular matrix. Also, a high deposition of glycosaminoglycans could be measured in this group and RT–PCR analyses confirmed the induction of chondrogenesis, with the expression of cartilage‐specific marker genes, such as aggrecan and collagen types II and X. This multiphasic composite scaffold with the cartilage layer on top might be a promising construct for the repair of osteochondral defects. Copyright © 2009 John Wiley & Sons, Ltd.     

Tenogenic differentiation of stem cells for tendon repair—what is the current evidence?

Tendon/ligament injuries are very common in sports and other rigorous activities. Tendons regenerate and repair slowly and inefficiently in vivo after injury. The limited ability of tendon to self-repair and the general inefficiencies of current treatment regimes have hastened the motivation to develop tissue-engineering strategies for tissue repair. Of particular interest in recent years has been the use of adult mesenchymal stem cells (MSCs) to regenerate functional tendons and ligaments. Different sources of MSCs have been studied for their effects on tendon repair. However, ectopic bone and tumour formation has been reported in some special circumstances after transplantation of MSCs. The induction of MSCs to differentiate into tendon-forming cells in vitro prior to transplantation is a possible approach to avoid ectopic bone and tumour formation while promoting tendon repair. While there are reports about the factors that might promote tenogenic differentiation, the study of tenogenic differentiation is hampered by the lack of definitive biomarkers for tendons. This review aims to summarize the cell sources currently used for tendon repair as well as their advantages and limitations. Factors affecting tenogenic differentiation were summarized. Molecular markers currently used for assessing tenogenic differentiation or neotendon formation are summarized and their advantages and limitations are commented upon. Finally, further directions for promoting and assessing tenogenic differentiation of stem cells for tendon repair are discussed.     

Joint mimicking mechanical load activates TGFβ1 in fibrin‐poly(ester‐urethane) scaffolds seeded with mesenchymal stem cells

Transforming growth factor‐β1 (TGF‐β1) is widely used in an active recombinant form to stimulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). Recently, it has been shown that the application of multiaxial load, that mimics the loading within diarthrodial joints, to MSCs seeded in to fibrin‐poly(ester‐urethane) scaffolds leads to the endogenous production and secretion of TGF‐β1 by the mechanically stimulated cells, which in turn drives the chondrogenic differentiation of the cells within the scaffold. The work presented in this short communication provides further evidence that the application of joint mimicking multiaxial load induces the secretion of TGF‐β1 by mechanically stimulated MSCs. The results of this work also show that joint‐like multiaxial mechanical load activates latent TGF‐β1 in response to loading in the presence or absence of cells; this activation was not seen in non‐loaded control scaffolds. Despite the application of mechanical load to scaffolds with different distributions/numbers of cells no significant differences were seen in the percentage of active TGF‐β1 quantified in the culture medium of scaffolds from different groups. The similar level of activation in scaffolds containing different numbers of cells, cells at different stages of differentiation or with different distributions of cells suggests that this activation results from the mechanical forces applied to the culture system rather than differences in cellular behaviour. These results are relevant when considering rehabilitation protocols after cell therapy or microfracture, for articular cartilage repair, where increased TGF‐β1 activation in response to joint mobilization may improve the quality of developing cartilaginous repair material. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd     

风湿性心脏病二尖瓣置换手术前后患者Treg及IL-6、IL-10、TGF-β1的变化分析

目的探讨风湿性心脏病二尖瓣置换手术前后患者的血清浓度以及白细胞介素(IL)-6、IL-10、转化生长因子-β1(TGF-β1)的变化分析。方法选取2013年4月至2014年6月在该院接受治疗的45例风湿性心脏病患者为观察组以及本院体检中心的45例健康体检者为对照组作为本次的研究对象。观察两组受试者外周血Treg细胞的变化情况、血清浓度IL-6、IL-10、TGF-β1以及二尖瓣置换组织的mRNA表达水平。结果术后,观察组风湿性心脏病患者的外周血中CD4+CD25+调节性T细胞(Treg)/CD4+T细胞以及CD4+CD25+细胞/淋巴细胞的所占比例明显低于对照组(P0.05);观察组患者外周血清浓度IL-6、IL-10、TGF-β1水平均低于对照组(P0.05);观察组患者的二尖瓣MMP-2、MMP-9和TIMP-2(表达水平均高于对照组(P0.05)。结论风湿性心脏病二尖瓣置换术后患者的外周血中CD4+CD25+Treg/CD4+T细胞水平明显的下降,血清浓度IL-6、IL-10、TGF-β1也相应地出现下降的现象。     

The profibrotic cytokine transforming growth factor‐β1 increases endothelial progenitor cell angiogenic properties

Summary. Background: Transforming growth factor‐β1 (TGF‐β1) is a profibrotic cytokine that plays a major role in vascular biology, and is known to regulate the phenotype and activity of various vascular cell populations. Because most fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), are associated with vascular remodeling, and as endothelial progenitor cells (EPCs) may be involved in this process, we investigated the impact of TGF‐β1 modulation of EPC angiogenic properties. Methods: TGF‐β1 plasma levels were determined in 64 patients with IPF and compared with those in controls. The effect of TGF‐β1 on angiogenesis was studied in vivo in a Matrigel plug model and in vitro on endothelial colony‐forming cells (ECFCs). We studied the effects of inhibiting the expression of the three main receptors of TGF‐β1 in ECFCs by using short interfering RNA. Results: Total TGF‐β1 plasma levels were significantly increased in patients with IPF as compared with controls (P < 0.0001). TGF‐β1 had proangiogenic effects in vivo by increasing hemoglobin content and blood vessel formation in Matrigel plugs implanted in C57/Bl6 mice, and in vitro by enhancing ECFC viability and migration. The effects were abolished by silencing the three main TGF‐β1 receptors. Conclusions: TGF‐β1 is proangiogenic in vivo and induces ECFC angiogenic properties in vitro, suggesting that TGF‐β1 may play a role during vascular remodeling in fibrotic disease states via EPCs.     

Interactive endothelial phenotype maintenance and osteogenic differentiation of adipose tissue stromal vascular fraction SSEA‐4+‐derived cells

Bone formation relies on complex processes that require well‐orchestrated interactions between several cell types, such as bone‐forming cells (osteoblasts, OBs) and endothelial cells (ECs). Their co‐culture has been proved relevant to mimicking specific features of the bone niche. Here we propose the co‐culture of microvascular‐like ECs and pre‐OBs, both derived from the SSEA‐4⁺ cell subpopulation from the stromal vascular fraction of human adipose tissue (SSEA‐4⁺ hASCs), to define the conditions in which cells synergistically communicate to support the full differentiation of pre‐OBs and maintenance of the EC phenotype. Co‐cultures of different ratios of the two cell types were established and maintained for up to 21 days in standard endothelial maintenance (ENDO) and osteogenic differentiation (OST) media, as well as in a mixture of these (MIX). The osteogenic maturation of pre‐OBs (ALP activity, OPN and OCN expression, calcium deposition), the evolution of EC numbers (CD31⁺ cells) and maintenance of the endothelial phenotype (CD31 and vWF expression, LDL uptake) were assessed throughout the culture time as a function of cell ratio and culture media. The results obtained demonstrate that EC number has a significant effect on the osteogenic differentiation of pre‐OBs, depending on the medium used. While in ENDO medium the osteogenic differentiation was not observed, in the OST and MIX media it was attained at similar levels, except for the co‐culture with a higher number of ECs in MIX medium. These findings demonstrate that the use of SSEA‐4⁺ hASCs as a single‐cell source is promising to attain 3D bone‐like models with the potential to promote vascularized bone tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

The fate of bovine bone marrow stromal cells in hydrogels: a comparison to nucleus pulposus cells and articular chondrocytes

Bone marrow‐derived stromal cells (BMSCs) are good candidates for cell‐based tissue regeneration. For such purposes, cell survival within three‐dimensional (3D) scaffolds is often desirable. We hypothesize that undifferentiated BMSCs will have difficulties thriving within these gels, in contrast to articular chondrocytes (ACs) and nucleus pulposus cells (NPCs), but that early chondrogenic differentiation of the former will increase their survival. BMSCs, ACs and NPCs cast in 1.2% alginate or 2% agarose were cultured for 21 days in serum‐containing media. BMSCs were also cultured in medium with 10 ng/ml TGF‐β1. By day 21, NPCs and ACs proliferated, maintained upregulation of aggrecan and collagen type II, produced glycosaminoglycans and stained positively for collagen type II in both scaffolds. In contrast, the number of living BMSCs and the DNA content of their constructs decreased in both scaffolds. Addition of TGF‐β₁ resulted in cell survival and behaviour more similar (gene expression, glycosaminoglycan production and collagen type II synthesis) to ACs and NPCs. This study demonstrated that, unlike ACs and NPCs, undifferentiated BMSCs have more difficulty thriving within hydrogels, but that this can be improved by chondrogenic induction. Hence, immediate conditioning of BMSCs could be a worthwhile strategy. Copyright © 2009 John Wiley & Sons, Ltd.     

TGF-β1 serum concentration as a complementary diagnostic biomarker of lung cancer: establishment of a cut-point value

Lung cancer is a malignant disease with increasing mortality rates. Cytokines play a role in normal cell growth regulation and differentiation and are also implicated in malignant disease. Among these cytokines, Transforming Growth Factor β type 1 (TGF‐β1) acts as a tumor promoter in malignant cells. Several clinical studies have found high levels of TGF‐β1 in various cancer types. The aim of this study was to establish a TGF‐β1 cut‐off point as a complementary diagnostic tool in lung cancer detection. Therefore, 72 clinically well‐characterized individuals were studied, 41 lung cancer patients and 31 healthy subjects. Serum TGF‐β1 concentration was measured by an enzyme‐linked immunosorbent assay (ELISA). We compared statistically the serum TGF‐β1 concentration between both groups with analysis of variance, linear regression and receiver operating curve analysis. We observed that lung cancer patients produced higher TGF‐β1 levels than healthy individuals (37,225±9,436 vs. 28,416±9,324 pg/ml, P<0.001). The cut‐point diagnostic value was 30,500 pg/ml with 80.5% sensitivity, 64.5% specificity and odds ratio: 7.5, 95% CI: 2.6–21.8. Conclusions: We found significantly higher TGF‐β1 levels in lung cancer patients than in healthy individuals. We propose the measurement of serum TGF‐β1 levels as a complementary diagnostic test in lung cancer detection. J. Clin. Lab. Anal. 25:238–243, 2011. © 2011 Wiley‐Liss, Inc.