Chondrogenesis by bone marrow‐derived mesenchymal stem cells grown in chondrocyte‐conditioned medium for auricular reconstruction

Bone marrow‐derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell‐based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte‐conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2 weeks with or without transforming growth factor‐beta 3 (TGF‐β3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12 weeks and evaluated with real‐time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real‐time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12 weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF‐β3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF‐β3. Osteogenesis was only observed in the SM group with TGF‐β3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF‐β3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

Investigating cellulose derived glycosaminoglycan mimetic scaffolds for cartilage tissue engineering applications

Articular cartilage has a limited capacity to heal and, currently, no treatment exists that can restore normal hyaline cartilage. Creating tissue engineering scaffolds that more closely mimic the native extracellular matrix may be an attractive approach. Glycosaminoglycans, which are present in native cartilage tissue, provide signalling and structural cues to cells. This study evaluated the use of a glycosaminoglycan mimetic, derived from cellulose, as a potential scaffold for cartilage repair applications. Fully sulfated sodium cellulose sulfate (NaCS) was initially evaluated in soluble form as an additive to cell culture media. Human mesenchymal stem cell (MSC) chondrogenesis in pellet culture was enhanced with 0.01% NaCS added to induction media as demonstrated by significantly higher gene expression for type II collagen and aggrecan. NaCS was combined with gelatine to form fibrous scaffolds using the electrospinning technique. Scaffolds were characterized for fibre morphology, overall hydrolytic stability, protein/growth factor interaction and for supporting MSC chondrogenesis in vitro. Scaffolds immersed in phosphate buffered saline for up to 56 days had no changes in swelling and no dissolution of NaCS as compared to day 0. Increasing concentrations of the model protein lysozyme and transforming growth factor‐β3 were detected on scaffolds with increasing concentrations of NaCS (p < 0.05). MSC chondrogenesis was enhanced on the scaffold with the lowest NaCS concentration as seen with the highest collagen type II production, collagen type II immunostaining, and expression of cartilage‐specific genes. These studies demonstrate the feasibility of cellulose sulfate as a scaffolding material for cartilage tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.     

Human corneal fibroblast migration and extracellular matrix synthesis during stromal repair: Role played by platelet‐derived growth factor‐BB,basic fibroblast growth factor,and transforming growth factor‐β1

The development of treatments that modulate corneal wound healing to avoid fibrosis during tissue repair is important for the restoration of corneal transparency after an injury. To date, few studies have studied the influence of growth factors (GFs) on human corneal fibroblast (HCF) expression of extracellular matrix (ECM) proteins such as collagen types I and III, proteoglycans such as perlecan, or proteins implicated in cellular migration such as α5β1‐integrin and syndecan‐4. Using in vitro HCFs, a mechanical wound model was developed to study the influence of the GFs basic fibroblast GF (bFGF), platelet‐derived GF (PDGF‐BB) and transforming GF‐β1 (TGFβ1) on ECM protein production and cellular migration. Our results show that mechanical wounding provokes the autocrine release of bFGF and TGFβ1 at different time points during the wound closure. The HCF response to PDGF‐BB was a rapid closure due to fast cellular migration associated with a high focal adhesion replacement and a high expression of collagen and proteoglycans, producing nonfibrotic healing. bFGF stimulated nonfibrotic ECM production and limited the migration process. Finally, TGFβ1 induced expression of the fibrotic markers collagen type III and α5β1 integrin, and it inhibited cellular migration due to the formation of focal adhesions with a low turnover rate. The novel in vitro HCF mechanical wound model can be used to understand the role played by GFs in human corneal repair. The model can also be used to test the effects of different treatments aimed at improving the healing process. Copyright © 2016 John Wiley & Sons, Ltd.     

A mixed co‐culture of mesenchymal stem cells and transgenic chondrocytes in alginate hydrogel for cartilage tissue engineering

To regenerate articular cartilage tissue from degeneration and trauma, synovial mesenchymal stem cells (SMSCs) were used in this study as therapeutic progenitor cells to induce therapeutic chondrogenesis. To accomplish this, chondrocytes pre‐transduced with adenoviral vectors carrying the transforming growth factor (TGF) β3 gene were selected as transgenic companion cells and co‐cultured side‐by‐side with SMSCs in a 3D environment to provide chondrogenic growth factors in situ. We adopted a mixed co‐culture strategy for this purpose. Transgenic delivery of TGF‐β3 in chondrocytes was performed via recombinant adenoviral vectors. The mixed co‐culture of SMSCs and transgenic chondrocytes was produced in alginate gel constructs. Gene expression in both SMSCs and chondrocytes were characterized. Biochemical assays in vitro and in vivo showed that release of TGF‐ß3 from transgenic chondrocytes not only induced SMSC differentiation into chondrocytic cells but also preserved the chondrocytic phenotype of chondrocytes from suspected dedifferentiation. As a result, this mixed co‐culture strategy in conjunction with TGF‐ß3 gene delivery could be a promising approach in cartilage tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Electromagnetic fields counteract IL‐1β activity during chondrogenesis of bovine mesenchymal stem cells

Osteoarthritis (OA) is a common joint disease associated with articular cartilage degeneration. To improve the therapeutic options of OA, tissue engineering based on the use of mesenchymal stem cells (MSCs) has emerged. However, the presence of inflammatory cytokines, such as interleukin‐1β (IL‐1β), during chondrogenesis reduces the efficacy of cartilage engineering repair procedures by preventing chondrogenic differentiation. Previous studies have shown that electromagnetic fields (EMFs) stimulate anabolic processes in OA cartilage and limit IL‐1β catabolic effects. We investigated the role of EMFs during chondrogenic differentiation of MSCs, isolated from bovine synovial fluid, in the absence and presence of IL‐1β. Pellets of MSCs were differentiated for 3 and 5 weeks with transforming growth factor‐β3 (TGFβ3), in the absence and presence of IL‐1β and exposed or unexposed to EMFs. Biochemical, quantitative real‐time RT–PCR and histological results showed that EMFs alone or in the presence of TGFβ3 play a limited role in promoting chondrogenic differentiation. Notably, in the presence of IL‐1β and TGFβ3 a recovery of proteoglycan (PG) synthesis, PG content and aggrecan and type II collagen mRNA expression in the EMF‐exposed compared to unexposed pellets was observed. Also, histological and immunohistochemical results showed an increase in staining for alcian blue, type II collagen and aggrecan in EMF‐exposed pellets. In conclusion, this study shows a significant role of EMFs in counteracting the IL‐1β‐induced inhibition of chondrogenesis, suggesting EMFs as a therapeutic strategy for improving the clinical outcome of cartilage engineering repair procedures, based on the use of MSCs. Copyright © 2012 John Wiley & Sons, Ltd.     

FGF,TGFβ and Wnt crosstalk: embryonic to in vitro cartilage development from mesenchymal stem cells

Articular cartilage is easily damaged, yet difficult to repair. Cartilage tissue engineering seems a promising therapeutic solution to restore articular cartilage structure and function, with mesenchymal stem cells (MSCs) receiving increasing attention for their promise to promote cartilage repair. It is known from embryology that members of the fibroblast growth factor (FGF), transforming growth factor‐β (TGFβ) and wingless‐type (Wnt) protein families are involved in controlling different differentiation stages during chondrogenesis. Individually, these pathways have been extensively studied but so far attempts to recapitulate embryonic development in in vitro MSC chondrogenesis have failed to produce stable and functioning articular cartilage; instead, transient hypertrophic cartilage is obtained. We believe a better understanding of the simultaneous integration of these factors will improve how we relate embryonic chondrogenesis to in vitro MSC chondrogenesis. This narrative review attempts to define current knowledge on the crosstalk between the FGF, TGFβ and Wnt signalling pathways during different stages of mesenchymal chondrogenesis. Connecting embryogenesis and in vitro differentiation of human MSCs might provide insights into how to improve and progress cartilage tissue engineering for the future. Copyright © 2013 John Wiley & Sons, Ltd.     

Fibroblast growth factor improves the motility of human mesenchymal stem cells expanded in a human plasma‐derived xeno‐free medium through αVβ3 integrin

Human mesenchymal stem cells (MSC) are being explored for cell therapies targeting varied human diseases. For that, cells are being expanded in vitro, many times with fetal bovine serum (FBS) as the main source of growth factors. However, animal‐derived components should not be used, to avoid immune rejection from the patient that receives the MSC. To solve this issue, different xeno‐free media are being developed, and an industrial‐grade human plasma fraction (SCC) is a promising candidate to substitute FBS. Indeed, we have previously shown that MSC expanded in SCC‐medium maintain their phenotype and genetic stability. However, a reduction on MSC motility was observed when comparing with MSC motility on FBS‐medium. Thus, in this present study, we have tested different factors to improve the motility of MSC in SCC‐medium. Time lapse assays and experiments with transwells revealed that supplementation of the xeno‐free medium with FGF or PDGF, but not TNF‐α or SDF‐1, increased MSC motility. Interestingly, FGF and PDGF supplementation also led to alterations on MSC morphology to a shape similar to the one observed when using FBS. The mechanism behind the effect of FGF on MSC motility involved the increased expression of αVβ3 integrin. Furthermore, assays with small molecule inhibitors revealed that the signalling molecule p38 MAPK is important for MSC motility and that MEK/ERK and PI3K/AKT also have a role on FGF‐supplemented expanded MSC. Thus, it was found that FGF supplementation can improve the motility of xeno‐free‐expanded MSC and that the cells motility is regulated by αVβ3 integrin.     

Resveratrol counteracts IL‐1β‐mediated impairment of extracellular matrix deposition in 3D articular chondrocyte constructs

When aiming at cell‐based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL‐1β need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti‐inflammatory properties. However, long‐term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long‐term model cultures for cell‐based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL‐1β (1–10 ng/ml) or RSV (50 μM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL‐1β (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL‐1β‐treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL‐1β treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL‐1β‐mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long‐term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques.     

Application of marrow mesenchymal stem cell‐derived extracellular matrix in peripheral nerve tissue engineering

To advance molecular and cellular therapy into the clinic for peripheral nerve injury, modification of neural scaffolds with the extracellular matrix (ECM) of peripheral nerves has been established as a promising alternative to direct inclusion of support cells and/or growth factors within a neural scaffold, while cell‐derived ECM proves to be superior to tissue‐derived ECM in the modification of neural scaffolds. Based on the fact that bone marrow mesenchymal stem cells (BMSCs), just like Schwann cells, are adopted as support cells within a neural scaffold, in this study we used BMSCs as parent cells to generate ECM for application in peripheral nerve tissue engineering. A chitosan nerve guidance conduit (NGC) and silk fibroin filamentous fillers were respectively prepared for co‐culture with purified BMSCs, followed by decellularization to stimulate ECM deposition. The ECM‐modified NGC and lumen fillers were then assembled into a chitosan–silk fibroin‐based, BMSC‐derived, ECM‐modified neural scaffold, which was implanted into rats to bridge a 10 mm‐long sciatic nerve gap. Histological and functional assessments after implantation showed that regenerative outcomes achieved by our engineered neural scaffold were better than those achieved by a plain chitosan–silk fibroin scaffold, and suggested the benefits of BMSC‐derived ECM for peripheral nerve repair. Copyright © 2016 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.     

Effect of the small compound TD‐198946 on glycosaminoglycan synthesis and transforming growth factor β3‐associated chondrogenesis of human synovium‐derived stem cells in vitro

As an alternative to chondrocytes‐based cartilage repair, stem cell‐based therapies have been investigated. Specifically, human synovium‐derived stem cells (hSSCs) are a promising cell source based on their highly capacities for chondrogenesis, but some methodological improvements are still required towards optimal cartilage regeneration. Recently, a small compound, TD‐198946, was reported to promote chondrogenesis of several stem cells, but the effect on hSSCs is still unknown. This study aimed to examine the effects of TD‐198946 on chondrocyte differentiation and cartilaginous tissue formation with hSSCs. A range of concentrations of TD‐198946 were examined in chondrogenic cultures of hSSC‐derived cell pellets. The effect of TD‐198946 on glycosaminoglycan (GAG) production, chondrocyte marker expression, and cartilaginous tissue formation was assessed. At concentrations >1 nM, TD‐198946 dose‐dependently enhanced GAG production, particularly hyaluronan, whereas chondrocyte differentiation was not impacted. When combined with transforming growth factor β3 (TGFβ3), TD‐198946 promoted chondrocyte differentiation and production of cartilaginous matrices at doses <1 nM as judged by SOX9, S100, and type 2 collagen upregulation. Conversely, doses >1 nM TD‐198946 attenuated TGFβ3‐associated chondrocyte differentiation, but aggrecan was efficiently produced at 1 to 10 nM TD‐198946 as judged by safranin O staining. Thus, TD‐198946 exhibited different dose ranges for either GAG synthesis or chondrocyte differentiation. Regarding use of TD‐198946 for in vitro engineering of cartilage, cartilaginous particles rich in type 2 collagen and GAG were predominately created with TGFβ3 + 0.25 nM TD‐198946. These studies have demonstrated that TD‐198946 synergistically enhances chondrogenesis of hSSCs in a unique dose range, and such findings may provide a novel strategy for stem cell‐based cartilage therapy.     

Transforming growth factor‐beta1 inhibits tissue engineering cartilage absorption via inducing the generation of regulatory T cells

The objective of the present study was to explore the mechanisms of transforming growth factor (TGF)‐β1 inhibiting the absorption of tissue engineering cartilage. We transfected TGF‐β1 gene into bone marrow mesenchymal stem cells (BMMSCs) and co‐cultured with interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α and CD4⁺CD25^? T lymphocytes. We then characterized the morphological changes, apoptosis and characterization of chondrogenic‐committed cells from TGF‐β1⁺BMMSCs and explored their mechanisms. Results showed that BMMSCs apoptosis and tissue engineering cartilage absorption in the group with added IFN‐γ and TNF‐α were greater than in the control group. In contrast, there was little BMMSC apoptosis and absorption by tissue engineering cartilage in the group with added CD4⁺CD25^? T lymphocytes; Foxp3⁺T cells and CD25⁺CD39⁺ T cells were found. In contrast, no type II collagen or Foxp3⁺T cells or CD25⁺CD39⁺ T cells was found in the TGF‐β1^–BMMSC group. The data suggest that IFN‐γ and TNF‐α induced BMMSCs apoptosis and absorption of tissue engineering cartilage, but the newborn regulatory T (Treg) cells inhibited the function of IFN‐γ and TNF‐α and protected BMMSCs and tissue engineering cartilage. TGF‐β1not only played a cartilage inductive role, but also inhibited the absorption of tissue engineering cartilage. The pathway proposed in our study may simulate the actual reaction procedure after implantation of BMMSCs and tissue engineering cartilage in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Matrix‐directed differentiation of human adipose‐derived mesenchymal stem cells to dermal‐like fibroblasts that produce extracellular matrix

Commercially available skin substitutes lack essential non‐immune cells for adequate tissue regeneration of non‐healing wounds. A tissue‐engineered, patient‐specific, dermal substitute could be an attractive option for regenerating chronic wounds, for which adipose‐derived mesenchymal stem cells (ADMSCs) could become an autologous source. However, ADMSCs are multipotent in nature and may differentiate into adipocytes, osteocytes and chondrocytes in vitro, and may develop into undesirable tissues upon transplantation. Therefore, ADMSCs committed to the fibroblast lineage could be a better option for in vitro or in vivo skin tissue engineering. The objective of this study was to standardize in vitro culture conditions for ADMSCs differentiation into dermal‐like fibroblasts which can synthesize extracellular matrix (ECM) proteins. Biomimetic matrix composite, deposited on tissue culture polystyrene (TCPS), and differentiation medium (DM), supplemented with fibroblast‐conditioned medium and growth factors, were used as a fibroblast‐specific niche (FSN) for cell culture. For controls, ADMSCs were cultured on bare TCPS with either DM or basal medium (BM). Culture of ADMSCs on FSN upregulated the expression of differentiation markers such as fibroblast‐specific protein‐1 (FSP‐1) and a panel of ECM molecules specific to the dermis, such as fibrillin‐1, collagen I, collagen IV and elastin. Immunostaining showed the deposition of dermal‐specific ECM, which was significantly higher in FSN compared to control. Fibroblasts derived from ADMSCs can synthesize elastin, which is an added advantage for successful skin tissue engineering as compared to fibroblasts from skin biopsy. To obtain rapid differentiation of ADMSCs to dermal‐like fibroblasts for regenerative medicine, a matrix‐directed differentiation strategy may be employed. Copyright © 2014 John Wiley & Sons, Ltd.     

Effective expansion of human adipose‐derived stromal cells and bone marrow‐derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles‐coated substratum with human platelet‐rich plasma

Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet‐rich plasma (PRP). In this study, we investigated the capability of F/P NP‐coated plates to act as a substratum for the proliferation of human adipose‐derived stromal cells (ASCs) and bone marrow‐derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP‐coated plates and grew optimally, with a doubling time of 30 and 32 h in low‐concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor‐2 (FGF‐2) on the F/P NP‐coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF‐2 on F/P NP‐coated plates pretreated with PRP and FGF‐2 in a concentration‐dependent manner. Thus, F/P NP‐coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low‐concentration PRP medium supplemented with FGF‐2. No xenogeneic serum is required. Copyright © 2012 John Wiley & Sons, Ltd.