Sustained release of bioactive protein from a lyophilized tissue‐engineered construct promotes the osteogenic potential of mesenchymal stem cells

Tissue‐engineered constructs (TECs) seeded with mesenchymal stem cells (MSCs) represent a therapy for large bone defects. However, massive cell death in TECs in the early postimplantation period prompted us to investigate the osteoinductive mechanism of TECs. Previous studies demonstrated that stem cell extracts retained equivalent levels of bioactive proteins and exhibited an osteoinductive nature similar to that of intact cells. These data led us to hypothesize that despite the massive cell death in TECs, devitalized MSC‐derived proteins remain on the scaffolds and are released to improve cell function. Here, TECs were prepared using demineralized bone matrix seeded with human umbilical cord Wharton's jelly‐derived MSCs (hWJMSCs), and the cells seeded in TECs were devitalized by lyophilizing the TECs. Scanning electron microscopy, BCA protein assays, quantitative cytokine array analysis and immunofluorescent staining indicated that approximately 3 mg/cm³ of total protein and 49 types of cytokines derived from hWJMSCs were preserved in the lyophilized TECs (LTECs). The sustainable release of total protein and cytokines from LTECs lasted for more than 2 weeks. The released protein improved the osteogenic behavior of and gene expression in MSCs. Furthermore, the lyophilized hWJMSC‐derived proteins had immunoregulatory properties similar to those of live MSCs in mixed lymphocyte reactions. Collectively, we present a novel perspective on the osteoinductive mechanism of TECs and introduce LTECs as new systems for delivering multiple cytokines to enhance MSC behavior. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:386–394, 2016.     

Xenogeneic transplantation of human WJ‐MSCs rescues mice from acute radiation syndrome via Nrf‐2‐dependent regeneration of damaged tissues

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ‐MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue‐regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ‐MSCs protected mice from a lethal dose of IR even when transplanted up to 24 hours after irradiation, and a combination of WJ‐MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ‐MSCs. This combinatorial approach mitigated IR‐induced damage to the hematopoietic and gastrointestinal system. WJ‐MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony‐stimulating factor (G‐CSF) and IL‐6 in mice. Knockdown of G‐CSF and IL‐6 in WJ‐MSCs before injection to lethally irradiated mice or transplantation of WJ‐MSCs to lethally irradiated Nrf‐2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ‐MSCs could be a potential cell‐based therapy for individuals accidentally exposed to radiation.     

Comparison of the efficacy of three concentrations of retinoic acid for transdifferentiation induction in sheep marrow‐derived mesenchymal stem cells into male germ cells

Recent studies have shown the unique role of retinoic acid (RA) in the induction of transdifferentiation in mesenchymal stem cells (MSCs) into germ cells (GCs). This study is the first study that compares the efficacy of three different concentrations of RA for the production of male GCs in vitro. Male sheep marrow‐derived MSCs (MMSCs) were treated with the following concentrations of RA: 1 μm (RA1), 5 μm (RA2) and 10 μm (RA3) for a period of 21 days. The production of male GCs was evaluated by the assessment of expressions of GC‐specific markers (by RT‐PCR, qRT‐PCR and immunocytochemistry), morphological characteristics and changes in alkaline phosphatase (ALP) activity. All three concentrations created male GC features. RA treatment upregulated the expressions of VASA and beta1 INTEGRIN and downregulated PIWIL2 and OCT4. DAZL was not expressed by RA treatment. Interestingly, immunocytochemistry detected PGP 9.5 expression in all treatment groups, with the highest expression noted in the RA3 group (P < 0.05). GC‐like cells along with increased ALP activity were observed in all treated cultures, too. Finally, results showed that 10 μm RA has the most efficiency for transdifferentiation induction in MMSCs and production of male GCs in vitro.     

Comparison of human umbilical cord blood‐derived mesenchymal stem cells with healthy fibroblasts on wound‐healing activity of diabetic fibroblasts

Various types of skin substitutes composed of fibroblasts and/or keratinocytes have been used for the treatment of diabetic ulcers. However, the effects have generally not been very dramatic. Recently, human umbilical cord blood‐derived mesenchymal stromal cells (hUCB‐MSCs) have been commercialised for cartilage repair as a first cell therapy product using allogeneic stem cells. In a previous pilot study, we reported that hUCB‐MSCs have a superior wound‐healing capability compared with fibroblasts. The present study was designed to compare the treatment effect of hUCB‐MSCs with that of fibroblasts on the diabetic wound healing in vitro. Diabetic fibroblasts were cocultured with healthy fibroblasts or hUCB‐MSCs. Five groups were evaluated: group I, diabetic fibroblasts without coculture; groups II and III, diabetic fibroblasts cocultured with healthy fibroblasts or hUCB‐MSCs; and groups IV and V, no cell cocultured with healthy fibroblasts or hUCB‐MSCs. After a 3‐day incubation, cell proliferation, collagen synthesis levels and glycosaminoglycan levels, which are the major contributing factors in wound healing, were measured. As a result, a hUCB‐MSC‐treated group showed higher cell proliferation, collagen synthesis and glycosaminoglycan level than a fibroblast‐treated group. In particular, there were significant statistical differences in collagen synthesis and glycosaminoglycan levels (P = 0·029 and P = 0·019, respectively). In conclusion, these results demonstrate that hUCB‐MSCs may have a superior effect to fibroblasts in stimulating diabetic wound healing.     

Mesenchymal stem cells and insulin‐like growth factor‐I gene‐enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons

Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin‐like growth factor‐I (IGF‐I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs, as well as IGF‐I gene‐enhanced MSCs (AdIGF‐MSCs) on tendon healing in vivo. Collagenase‐induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Tendons were treated with 10 × 10⁶ MSCs or 10 × 10⁶ AdIGF‐MSCs. Control limbs were injected with 1 mL of phosphate‐buffered saline (PBS). Ultrasound examinations were performed at t = 0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure and evaluated for biochemical composition and histologic characteristics. Expression of collagen types I and III, IGF‐I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase‐3 (MMP‐3), matrix metalloproteinase‐13 (MMP‐13), and aggrecanase‐1 (ADAMTS‐4) were similar in MSC and control tendons. Both MSC and AdIGF‐MSC injection resulted in significantly improved tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF‐MSCs for the treatment of tendinitis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1392–1398, 2009     

Differentiation potential of adipose tissue-derived mesenchymal stem cells into germ cells with and without growth factors

This study aimed to culture the adipose tissue-derived mesenchymal stem cells (AT-MSCs) with and without leukaemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), epidermal growth factor (EGF) and retinoic acid (RA), and investigate their impact on the differentiation of these cells into germ cells. MSCs were separated from adipose tissue of mice, and the nature of these cells is confirmed by flow cytometry. The cells were cultured in different conditions, including MSCs grown in the presence of the growth factors, MSCs without the growth factors, MSCs cultured with combined growth factors and RA, and MSCs cultured with RA. After 2 weeks, the gene expression of c-Kit, Gcnf, Mvh and Scp3 and the protein expression of c-Kit and Gcnf were assessed by real-time PCR and Western blot, respectively. Scp3 was overexpressed in the groups supplemented with RA (p < .01). The expression of c-Kit and Mvh in the growth factor-supplemented groups was increased (p < .01). Western blot analysis confirmed the real-time PCR results. The use of the growth factors for the long-term culture of stem cells can be beneficial. However, to promote germ cell differentiation, the growth factors might be used by other meiosis inducer factors, such as RA.     

Mesenchymal stem cells attenuate liver fibrosis by suppressing Th17 cells – an experimental study

This study investigates molecular and cellular mechanisms involved in mesenchymal stem cell (MSC)‐mediated modulation of IL‐17 signaling during liver fibrosis. Mice received CCl₄ (1 μl/g intraperitoneally) twice/week for 1 month. MSCs (1 × 10⁶), or MSC‐conditioned medium (MSC‐CM), were intravenously injected 24 h after CCl₄ and on every 7th day. Liver fibrosis was determined by macroscopic examination, histological analysis, Sirius red staining, and RT‐PCR. Serum levels of cytokines, indoleamine 2,3‐dioxygenase (IDO), and kynurenine were determined by ELISA. Flow cytometry was performed to identify liver‐infiltrated cells. In vitro, CD4⁺ T cells were stimulated and cultured with MSCs. 1‐methyltryptophan was used for inhibition of IDO. MSCs significantly attenuated CCl₄‐induced liver fibrosis by decreasing serum levels of inflammatory IL‐17, increasing immunosuppressive IL‐10, IDO, and kynurenine, reducing number of IL‐17 producing Th17 cells, and increasing percentage of CD4⁺IL‐10⁺ T cells. Injection of MSC‐CM resulted with attenuated fibrosis accompanied with the reduced number of Th17 cells in the liver and decreased serum levels of IL‐17. MSC‐CM promoted expansion of CD4⁺FoxP3⁺IL‐10⁺ T regulatory cells and suppressed proliferation of Th17 cells. This phenomenon was completely abrogated in the presence of IDO inhibitor. MSCs, in IDO‐dependent manner, suppress liver Th17 cells which lead to the attenuation of liver fibrosis.     

Low‐dose BMP‐2 and MSC dual delivery onto coral scaffold for critical‐size bone defect regeneration in sheep

Tissue‐engineered constructs (TECs) combining resorbable calcium‐based scaffolds and mesenchymal stem cells (MSCs) have the capability to regenerate large bone defects. Inconsistent results have, however, been observed, with a lack of osteoinductivity as a possible cause of failure. This study aimed to evaluate the impact of the addition of low‐dose bone morphogenetic protein‐2 (BMP‐2) to MSC‐coral‐TECs on the healing of clinically relevant segmental bone defects in sheep. Coral granules were either seeded with autologous MSCs (bone marrow‐derived) or loaded with BMP‐2. A 25‐mm‐long metatarsal bone defect was created and stabilized with a plate in 18 sheep. Defects were filled with one of the following TECs: (i) BMP (n = 5); (ii) MSC (n = 7); or (iii) MSC‐BMP (n = 6). Radiographic follow‐up was performed until animal sacrifice at 4 months. Bone formation and scaffold resorption were assessed by micro‐CT and histological analysis. Bone union with nearly complete scaffold resorption was observed in 1/5, 2/7, and 3/6 animals, when BMP‐, MSC‐, and MSC‐BMP‐TECs were implanted, respectively. The amount of newly formed bone was not statistically different between groups: 1074 mm³ [970–2478 mm³], 1155 mm³ [970–2595 mm³], and 2343 mm³ [931–3276 mm³] for BMP‐, MSC‐, and MSC‐BMP‐TECs, respectively. Increased scaffold resorption rate using BMP‐TECs was the only potential side effect observed. In conclusion, although the dual delivery of MSCs and BMP‐2 onto a coral scaffold further increased bone formation and bone union when compared to single treatment, results were non‐significant. Only 50% of the defects healed, demonstrating the need for further refinement of this strategy before clinical use. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2637–2645, 2017.

     

Dexamethasone inhibition of confluence‐induced apoptosis in human mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have been extensively characterized with respect to their in vitro expansion and differentiation potential, especially with respect to osteogenesis. Dexamethasone (Dex) is a well‐known inducer of osteogenic differentiation of MSCs, but little is known about the effect of Dex treatment on apoptosis in MSCs. In this study, apoptosis in MSCs was examined with respect to cell density and Dex supplementation, using DAPI staining and DNA fragmentation ELISA Assay. In MSC cultures initiated at 1.0, 3.0, and 9.0 × 10³ cells per cm², it was found that higher MSC density correlated with increased apoptosis and that this apoptotic effect was diminished in cultures containing 100 nM Dex. MSCs and fibroblasts were co‐cultured, along with empty insert controls, and assayed for apoptosis by ELISA and DAPI counts to determine if soluble factors accounted for the cell density‐related apoptosis. No difference was seen between MSCs cultured with inserts containing either MSCs, fibroblasts, or empty control. To determine cell contact effects, BrdU‐labeled MSCs were cultured alone or with unlabeled chondrocytes at 2× and 8× the number of MSCs, with and without Dex, and apoptosis levels quantified. The results showed increased apoptosis at greater cell densities, and that the amount of apoptosis was greatly diminished in cultures containing Dex. These results show that apoptosis in MSCs is cell density‐related, requires direct cell contact, and that Dex treatment reduces or eliminates this density‐related apoptosis. These results may impact how MSCs should be cultured for clinical applications. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:216–221, 2009     

脐带Wharton胶来源MSCs生物学特性及其优越性的研究进展

目的总结脐带Wharton胶来源MSCs(Wharton’s jelly-MSCs,WJ-MSCs)生物学特性及其优越性的研究进展。方法查阅近年来关于WJ-MSCs的生物学特性及其与脐带血MSCs(umbilical cord blood MSCs,UBMSCs)和BMSCs相比较的文献,进行综述分析。结果从脐带Wharton胶中可分离获得大量具有自我复制、自我更新、高度增殖和多向分化潜能的MSCs。与UBMSCs和BMSCs相比较,WJ-MSCs在分离时间、分离成功率、倍增时间、传代数量和扩增潜能等方面均具有优越性。结论 WJ-MSCs具有取材简便、来源丰富、相对纯净、无伦理问题等优点,是细胞移植治疗、基因治疗和组织工程器官构建的理想种子细胞,为组织再生修复与原位重建提供了新思路。     

Mesenchymal stem cell‐conditioned medium accelerates wound healing with fewer scars

Mesenchymal stem cells (MSCs) derived from umbilical cord s (UC‐MSCs) have been shown to enhance cutaneous wound healing by means of the paracrine activity. Fibroblasts are the primary cells involved in wound repair. The paracrine effects of UC‐MSCs on dermal fibroblasts have not been fully explored in vitro or in vivo. Dermal fibroblasts were treated with conditioned media from UC‐MSCs (UC‐MSC‐CM). In this model, UC‐MSC‐CM increased the proliferation and migration of dermal fibroblasts. Moreover, adult dermal fibroblasts transitioned into a phenotype with a low myofibroblast formation capacity, a decreased ratio of transforming growth factor‐β1,3 (TGF‐β1/3) and an increased ratio of matrix metalloproteinase/tissue inhibitor of metalloproteinases (MMP/TIMP). Additionally, UC‐MSC‐CM‐treated wounds showed accelerated healing with fewer scars compared with control groups. These observations suggest that UC‐MSC‐CM may be a feasible strategy to promote cutaneous repair and a potential means to realise scarless healing.     

Differential Effector Response of Amnion‐ and Adipose‐Derived Mesenchymal Stem Cells to Inflammation; Implications for Intradiscal Therapy

Intervertebral disc degeneration (IVDD) is a progressive condition marked by tissue destruction and inflammation. The therapeutic effector functions of mesenchymal stem cells (MSCs) makes them an attractive therapy for patients with IVDD. While several sources of MSCs exist, the optimal choice for use in the inflamed IVD remains a significant question. Adipose (AD)‐ and amnion (AM)‐derived MSCs have several advantages compared with other sources, however, no study has directly compared the impact of IVDD inflammation on their effector functions. Human MSCs were cultured in media with or without supplementation of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α at concentrations reportedly produced by IVDD cells. MSC proliferation and production of pro‐ and anti‐inflammatory cytokines were quantified following 24 and 48 h of culture. Additionally, the osteogenic and chondrogenic potential of AD‐ and AM‐MSCs was characterized via histology and biochemical analysis following 28 days of culture. In inflammatory culture, AM‐MSCs produced significantly more anti‐inflammatory IL‐10 (14.47 ± 2.39 pg/ml; p = 0.004) and larger chondrogenic pellets (5.67 ± 0.26 mm²; p = 0.04) with greater percent area staining positively for glycosaminoglycan (82.03 ± 3.26%; p < 0.001) compared with AD‐MSCs (0.00 ± 0.00 pg/ml; 2.76 ± 0.18 mm²; 34.75 ± 2.49%; respectively). Conversely, AD‐MSCs proliferated more resulting in higher cell numbers (221,000 ± 8,021 cells; p = 0.048) and produced higher concentrations of pro‐inflammatory cytokines prostaglandin E₂ (1,118.30 ± 115.56 pg/ml; p = 0.030) and IL‐1β (185.40 ± 7.63 pg/ml; p = 0.010) compared with AM‐MSCs (109,667 ± 5,696 cells; 1,291.40 ± 78.47 pg/ml; 144.10 ± 4.57 pg/ml; respectively). AD‐MSCs produced more mineralized extracellular matrix (3.34 ± 0.05 relative absorbance units [RAU]; p < 0.001) compared with AM‐MSCs (1.08 ± 0.06 RAU). Under identical inflammatory conditions, a different effector response was observed with AM‐MSCs producing more anti‐inflammatories and demonstrating enhanced chondrogenesis compared with AD‐MSCs, which produced more pro‐inflammatory cytokines and demonstrated enhanced osteogenesis. These findings may begin to help inform researchers which MSC source may be optimal for IVD regeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2445–2456, 2019     

Collagen‐containing scaffolds enhance attachment and proliferation of non‐cultured bone marrow multipotential stromal cells

Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen‐containing bovine bone scaffold (Orthoss^® Collagen) with a non‐collagen‐containing bovine bone scaffold, Orthoss^®. Another collagen‐containing synthetic scaffold, Vitoss^® was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit‐fibroblast assay and flow‐cytometry. The number of BM MSCs initially attached to Orthoss^® Collagen and Vitoss^® was similar but greater than Orthoss^® (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss^® Collagen and Vitoss^® after 2‐week culture was also higher compared to Orthoss^® (p = 0.010 and p = 0.023, respectively). Interestingly, collagen‐containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture‐expanded MSCs on Orthoss^® collagen and Vitoss^® was greater compared to Orthoss^® (p = 0.047 and p = 0.004, respectively). Collectively, collagen‐containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:597–606, 2016.     

脐血间充质干细胞移植对大鼠局灶性脑缺血的影响

目的　从人脐血中分离纯化间充质干细胞(MSC) ,观察其移植对大鼠大脑中动脉栓塞后神经功能恢复的影响及细胞的存活、迁移向神经细胞分化的情况。方法　雄性SD大鼠45只,用线栓法建立大鼠大脑中动脉栓塞(MCAO)模型,大鼠随机分为3组:MSC移植组、单核细胞组和生理盐水组。移植后1、7、14、2 1、2 8d采用改良神经功能损害评分(mNSS)观察大鼠神经功能恢复情况,应用免疫组织化学和免疫荧光双标记技术检测5溴 2脱氧尿核苷(BrdU )标记的MSC细胞的存活、迁移及其胶质纤维酸性蛋白(GFAP)和神经元特异性核蛋白(NeuN)的表达。结果　人脐血MSC细胞移植可显著提高大鼠局灶性脑缺血后神经功能的恢复(P <0 .0 5 )。移植的MSC细胞可在大鼠脑组织中存活,并向缺血区域迁移,11.67%MSC细胞表达GFAP ,3 .72 %MSC细胞表达NeuN。结论　人脐血中含有MSC细胞并可促进局灶性脑缺血大鼠的神经功能恢复,移植细胞可在大鼠脑缺血区域中存活、迁移并向星形胶质细胞或神经元分化     

Induction of fracture repair by mesenchymal cells derived from human embryonic stem cells or bone marrow

Development of novel therapeutic approaches to repair fracture non‐unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)‐derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non‐union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM‐MSCs) or a no cell control group (n = 10–12 per group). Preliminary studies demonstrated that both for hESC‐derived MSCs and hBM‐MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque, and stiffness were significantly greater in the hBM‐MSC as compared to the control group that received no cells; values for these parameters in the hESC‐derived MSC group were intermediate between the hBM‐MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X‐ray in the hESC‐derived MSC group. Our results thus indicate that while hESC‐derived MSCs may have potential to induce fracture healing in non‐unions, hBM‐MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1804–1811, 2011     

Bone marrow‐derived mesenchymal stem cell attenuates skin fibrosis development in mice

Recent studies showed that mesenchymal stem cell (MSC) transplantation significantly alleviated tissue fibrosis; however, little is known about the efficacy on attenuating cutaneous scar formation. In this study, we established a dermal fibrosis model induced by bleomycin and evaluated the benefit of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) on skin fibrosis development. Tracing assay of green fluorescent protein (GFP⁺)BM‐MSCs showed that the cells disappeared gradually within 24 hours upon administration, which hinted the action of BM‐MSCs in vivo was exerted in the initial phase of repair in this model. Therefore, we repeatedly transplanted syngeneic BM‐MSCs in the process of skin fibrosis formation. After 3 weeks, it was found that BM‐MSC‐treated lesional skin demonstrated a unanimous basket‐weave organisation of collagen arrangement similar to normal skin, with few inflammatory cells. In addition, lesional skin with BM‐MSC treatment exhibited a significant down‐regulation of transforming growth factor‐β1 (TGF‐β1), type I collagen and heat‐shock protein 47 (HSP47), with higher expression of matrix metalloproteinases (MMPs)‐2, ‐9 and ‐13. Further experiments showed that α‐smooth muscle actin (α‐SMA) positive cells, the most reliable marker of myofibroblasts, apparently decreased after BM‐MSC transplantation, which revealed that BM‐MSCs could attenuate myofibroblast proliferation and differentiation as well as matrix production. Taken together, these findings suggested that BM‐MSCs can inhibit the formation process of bleomycin‐induced skin fibrosis, alleviate inflammation and favour the remodelling of extracellular matrix.     

细胞因子表达谱在人脐带Wharton胶间质干细胞诱导分化成软骨细胞的变化

[目的]观察从人脐带Wharton' s胶中分离的间质干细胞(WMSC)诱导分化成软骨细胞时表达细胞因子及生长因子的异同,从而为WMSC诱导的软骨细胞用于组织工程的构建以及其在临床的应用打下基础.[方法]从人正常足月分娩的脐带中分离间质干细胞,在体外以条件培基诱导分化成软骨细胞,免疫组化法鉴定其特性,应用细胞因子蛋白芯片方法检测WMSC及其诱导分化的软骨细胞所表达细胞因子及生长因子,并取成年人正常关节软骨为对照.[结果]78种细胞因子和生长因子在WMSC及其诱导分化的软骨细胞均有表达,两者表达谱相同,而表达量则多少有不同.诱导分化后表达量增多的因子为:MIP-3α,ENA-78,VEGF,PDGF-BB,SCF,FGF-7,GCP-2及SDF-1.减少的因子为MIP-1βHGF,NT-4,IL-10及MIP-δ.其共同表达的具免疫抑制作用的因子对降低异体移植时的免疫排斥可能有一定作用,其共同表达的金属蛋白酶抑制因子对其移植于体内时可具有保护作用.而共同表达的肿瘤生长抑制因子则可减少对干细胞移植后瘤变的担心.[结论]从人脐带wharton' s胶中分离的间质干细胞(WMSC)诱导分化成软骨细胞时表达细胞因子及生长因子谱相同,是一种理想的可替代自身软骨细胞的新型种子细胞,用于构建工程软骨组织.     

Optimising proliferation and migration of mesenchymal stem cells using platelet products: A rational approach to bone regeneration

This study investigates how mesenchymal stem cell's (MSCs) proliferation and migration abilities are influenced by various platelet products (PP). Donor‐matched, clinical‐, and control laboratory‐standard PPs were generated and assessed based on their platelet and leukocyte concentrations. Bone marrow derived MSCs were exposed to these PP to quantify their effect on in vitro MSC proliferation and migration. An adapted colony forming unit fibroblast (CFU‐F) assay was carried out on bone marrow aspirate using clinical‐standard PP‐loaded electrospun poly(?‐caprolactone) (PCL) membrane to mimic future clinical applications to contain bone defects. Clinical‐standard PP had lower platelet (2.5 fold, p < 0.0001) and higher leukocyte (14.1 fold, p < 0.0001) concentrations compared to laboratory‐standard PP. It induced suboptimal MSC proliferation compared to laboratory‐standard PP and fetal calf serum (FCS). All PP induced significantly more MSC migration than FCS up to 24 h. The removal of leukocytes from PP had no effect on MSC proliferation or migration. The PP‐loaded membranes successfully supported MSC colony formation. This study indicates that platelet concentrations in PP impact MSC proliferation more than the presence of leukocytes, whilst MSC migration in response to PP is not influenced by platelet or leukocyte numbers. Clinical‐standard PP could be applied alongside manufactured membranes in the future treatment of bone reconstruction. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1329–1338, 2019.     

Stromal cell‐derived factor‐1 and monocyte chemotactic protein‐3 improve recruitment of osteogenic cells into sites of musculoskeletal repair

Homing of osteogenic cells through the systemic circulation represents an alternative to traditional orthopedic tissue engineering approaches that focus on local cell populations. We hypothesize that expression of the chemokine, stromal cell‐derived factor‐1 (SDF‐1) or monocyte chemotactic protein‐3 (MCP‐3) may enhance homing of osteogenic cells into sites of fracture repair, as both have demonstrated promise in recruitment of marrow stromal cells (MSCs). This hypothesis was tested by transplantation of culture expanded MSCs expressing these factors adjacent to a fracture site on a collagen scaffold. One green fluorescent protein positive (GFP⁺) and one wild‐type mouse were surgically conjoined as parabiots at 7–8 weeks of age. Fibular osteotomy was performed 4 weeks after parabiosis on the hind limb of the wild‐type mouse. Mice were randomly allocated to receive one of the following five treatments: control (no scaffold), empty scaffold (no cells), or scaffold containing MSCs, scaffold containing MSCs expressing SDF‐1, or scaffold containing MSCs expressing MCP‐3. Fracture callus was harvested 2 weeks after injury, and analyzed with confocal microscopy and cell‐counting software. When compared to fracture callus treated with nontransfected MSCs, the fracture callus of mice treated with both SDF‐1 and MCP‐3 secreting MSCs demonstrated a significant increase in the number of both GFP⁺ cells (p = 0.0003, p = 0.02) and GFP⁺/AP⁺ cells (p = 0.0005, p = 0.01). These data suggest that homing of osteogenic cells from systemic circulation participate in fracture repair and that homing pathways might be modulated to enhance the contribution of circulating progenitors at the site of skeletal injury. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1064–1069, 2011     

Fibrillin‐1 Regulates Skeletal Stem Cell Differentiation by Modulating TGFβ Activity Within the Marrow Niche

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age‐related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin‐1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin‐1 (Fbn1^Prx1–/– mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1^Prx1–/– mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1^Prx1–/– mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1‐silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin‐1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1^Prx1–/– limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin‐1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches. © 2015 American Society for Bone and Mineral Research.