Passaged human chondrocytes accumulate extracellular matrix when induced by bovine chondrocytes

A source of sufficient number of cells is a major limiting factor for cartilage tissue engineering. To circumvent this problem, we developed a co‐culture method to induce redifferentiation in bovine articular chondrocytes, which had undergone dedifferentiation following serial passage in monolayer culture. In this study we determine whether human osteoarthritic (OA) and non‐diseased passaged dedifferentiated chondrocytes will respond similarly. Human passaged chondrocytes were co‐cultured for 4 weeks with primary bovine chondrocytes and their redifferentiation status was determined. Afterwards the cells were cultured either independently or in co‐culture with cryopreserved passaged cells for functional analysis. The co‐culture of passaged cells with primary chondrocytes resulted in reversion of their phenotype towards articular chondrocytes, as shown by increased gene expression of type II collagen and COMP, decreased type I collagen expression and extracellular matrix formation in vitro. Furthermore, this redifferentiation was stable, as those cells not only formed hyaline‐like cartilage tissue when grown on their own but also they could induce redifferentiation of passaged chondrocytes in co‐culture. These data suggest that it may be possible to use autologous chondrocytes obtained from osteoarthritic cartilage to form tissue suitable to use for cartilage repair. Copyright © 2009 John Wiley & Sons, Ltd.     

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors

The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell‐based therapies and cell‐loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two‐step process reported here first generates hiPSC aggregates of 324 ± 71 μm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA‐1‐60, TRA‐1‐81, SSEA‐4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16‐fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10‐fold increase was achieved. Taken together, 16 × 10⁶ hiPSCs were expanded into 2 × 10⁹ hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell‐based therapies and brings their clinical potential closer to fruition.     

Expansion and long-term maintenance of induced pluripotent stem cells in stirred suspension bioreactors

Induced pluripotent stem cells (iPSCs) can provide an important source of cells for the next-generation of cell therapies in regenerative medicine, in part due to their similarity to embryonic stem cells (ESCs). Patient-specific iPSCs represent an opportunity for autologous cell therapies that are not restricted by immunological, ethical and technical obstacles. One of the technical hurdles that must be overcome before iPSCs can be clinically implemented is the scalable, reproducible production of iPSCs and their differentiated progeny. All of the iPSC lines established thus far have been generated and expanded with static tissue culture protocols, which are time-consuming and suffer from batch-to-batch variability. Alternatively, stirred suspension bioreactors propose several benefits and their homogeneous culture environment facilitates the large-scale expansion required for clinical studies at less cost. We have previously developed protocols for expanding murine and human ESCs as undifferentiated aggregates in stirred suspension bioreactors. The resulting cells were karyotypically normal, expressed pluripotency markers and could be differentiated into all three germ lineages, both in vitro and in vivo. In this study, we demonstrate that stirred suspension bioreactors yield 58-fold expansion of undifferentiated pluripotent iPSCs over 4 days. In vitro differentiation into cartilage, bone and cardiomyocytes lineages, in addition to in vivo teratoma formation, further confirmed the existence of fully functional and undifferentiated pluripotent iPSC aggregates following long-term passaging. Stirred suspension bioreactor culture represents an efficient process for the large-scale expansion and maintenance of iPSCs, which is an important first step in their clinical application.     

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.     

Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1

The idea of rescuing the body self‐repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re‐entry in the cell cycle of confluent, growth‐arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up‐regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short‐ and long‐term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an “in vitro” tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia‐inducible factor (HIF‐1) alpha increase, its nuclear relocation and the binding to HIF‐1 responsive elements. These events were possibly related to the cell proliferation because the HIF‐1 inhibitor acriflavine inhibited HIF‐1 binding to HIF‐1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage.     

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.     

A papain‐induced disc degeneration model for the assessment of thermo‐reversible hydrogel–cells therapeutic approach

Nucleus pulposus (NP) regeneration by the application of injectable cell‐embedded hydrogels is an appealing approach for tissue engineering. We investigated a thermo‐reversible hydrogel (TR‐HG), based on a modified polysaccharide with a thermo‐reversible polyamide [poly(N‐isopropylacrylamide), pNIPAM], which is made to behave as a liquid at room temperature and hardens at > 32 °C. In order to test the hydrogel, a papain‐induced bovine caudal disc degeneration model (PDDM), creating a cavity in the NP, was employed. Human mesenchymal stem cells (hMSCs) or autologous bovine NP cells (bNPCs) were seeded in TR‐HG; hMSCs were additionally preconditioned with rhGDF‐5 for 7 days. Then, TR‐HG was reversed to a fluid and the cell suspension injected into the PDDM and kept under static loading for 7 days. Experimental design was: (D1) fresh disc control + PBS injection; (D2) PDDM + PBS injection; (D3) PDDM + TR‐HG (material control); (D4) PDDM + TR‐HG + bNPCs; (D5) PDDM + TR‐HG + hMSCs. Magnetic resonance imaging performed before and after loading, on days 9 and 16, allowed imaging of the hydrogel‐filled PDDM and assessment of disc height and volume changes. In gel‐injected discs the NP region showed a major drop in volume and disc height during culture under static load. The RT–PCR results of injected hMSCs showed significant upregulation of ACAN, COL2A1, VCAN and SOX9 during culture in the disc cavity, whereas the gene expression profile of NP cells remained unchanged. The cell viability of injected cells (NPCs or hMSCs) was maintained at over 86% in 3D culture and dropped to ~72% after organ culture. Our results underline the need for load‐bearing hydrogels that are also cyto‐compatible. Copyright © 2013 John Wiley & Sons, Ltd.     

Endothelial invasive response in a co‐culture model with physically‐induced osteodifferentiation

Manipulation of stem cells using physicochemical stimuli has emerged as an important tool in regenerative medicine. While 2D substrates with tunable elasticity have been studied for control of stem cell differentiation, we recently developed a stratified co‐culture model of angiogenesis of human mesenchymal stem cells (hMSCs) that differentiate on a tunable polydimethylsiloxane (PDMS) substrate, thereby creating a physiologic context for elasticity‐induced differentiation. Endothelial cells (EC) were cultured on top of the hMSC construct on a collagen gel to monitor network formation. Media composition influenced EC invasion due to the conditioning media, the reduction of serum and supplemental growth factors, and the addition of recombinant growth factors. Conditioned media, recombinant growth factors and direct co‐culture were compared for endothelial cell invasive response using quantitative image analysis. As anticipated, use of recombinant vascular endothelial growth factor (VEGF) induced the deepest EC invasions while direct co‐culture caused shallow invasions compared to other conditions. However, endothelial cells displayed lumen‐like morphology, suggesting that cell‐cell interaction in the co‐culture model could mimic sprouting behaviour. In summary, an engineered suitable biochemical and physical environment facilitated endothelial cells to form 3D vessel structures onto hMSCs. These structures were plated on a stiff surface known to induce osteodifferentiation of stem cells. This low cost co‐culture system, with its minimal chemical supplementation and physically controllable matrix, could potentially model in vivo potential in engineered and pre‐vascularized bone grafts. Copyright © 2012 John Wiley & Sons, Ltd.     

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

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

In vitro and in vivo co‐culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL–PEG–PCL hydrogels enhances cartilage formation

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co‐culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL–PEG–PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co‐cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co‐culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co‐culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co‐culture system in rabbit models. The co‐culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co‐culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Cartilage regeneration by selected chondrogenic clonal mesenchymal stem cells in the collagenase‐induced monkey osteoarthritis model

Osteoarthritis (OA) is the most common form of arthritis, in which cartilage is irreversibly degraded, causing severe pain and disability. Current therapeutic strategies cannot repair damaged cartilage. We evaluated the repair potential of selected chondrogenic clonal MSCs (sC‐MSCs) by delivering them into the injured cartilage site in a collagenase‐induced OA model in Cynomolgus monkeys. In vitro characterization showed that the isolated monkey sC‐MSCs and polyclonal MSCs (P‐MSCs) expressed mesenchymal stem cell markers and could differentiate into chondrocytes. The articular cartilage lesions in animals were treated with normal saline (NS), autologous P‐MSCs and sC‐MSCs, respectively, by direct delivery. The clinical parameters, radiographic images, histological and immunohistochemical examinations at weeks 8, 16 and 24 post‐treatment demonstrated that the abrasions of articular cartilage were significantly improved and repaired by MSC‐based treatment, particularly in the sC‐MSC‐treated group, which displayed consistently higher histological scores than those of other groups. In summary, treatment with sC‐MSCs can effectively improve the healing of cartilage lesions in the Cynomolgus monkey collagenase‐induced OA model. Due to the genetic proximity of monkey and human, the therapeutic strategy presented in this study will have broad applications in clinical practice. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Fate of bone marrow mesenchymal stem cells following the allogeneic transplantation of cartilaginous aggregates into osteochondral defects of rabbits

The purpose of this study was to track mesenchymal stem cells (MSCs) labelled with internalizing quantum dots (i‐QDs) in the reparative tissues, following the allogeneic transplantation of three‐dimensional (3D) cartilaginous aggregates into the osteochondral defects of rabbits. QDs were conjugated with a unique internalizing antibody against a heat shock protein‐70 (hsp70) family stress chaperone, mortalin, which is upregulated and expressed on the surface of dividing cells. The i‐QDs were added to the culture medium for 24 h. Scaffold‐free cartilaginous aggregates formed from i‐QD‐labelled MSCs (i‐MSCs), using a 3D culture system with chondrogenic supplements for 1 week, were transplanted into osteochondral defects of rabbits. At 4, 8 and 26 weeks after the transplantation, the reparative tissues were evaluated macroscopically, histologically and fluoroscopically. At as early as 4 weeks, the defects were covered with a white tissue resembling articular cartilage. In histological appearance, the reparative tissues resembled hyaline cartilage on safranin‐O staining throughout the 26 weeks. In the deeper portion, subchondral bone and bone marrow were well remodelled. On fluoroscopic evaluation, QDs were tracked mainly in bone marrow stromata, with some signals detected in cartilage and the subchondral bone layer. We showed that the labelling of rabbit MSCs with anti‐mortalin antibody‐conjugated i‐QDs is a tolerable procedure and provides a stable fluorescence signal during the cartilage repair process for up to 26 weeks after transplantation. The results suggest that i‐MSCs did not inhibit, and indeed contributed to, the regeneration of osteochondral defects. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of chondrocyte sheets prepared using a co‐culture method with temperature‐responsive culture inserts

Conventional culture methods using temperature‐responsive culture dishes require 4–5 weeks to prepare layered chondrocyte sheets that can be used in articular cartilage repair and regeneration. This study investigated whether the use of synovial tissue obtained from the same joint as the chondrocyte nutritive supply source could more quickly facilitate the preparation of chondrocyte sheets. After culturing derived synoviocytes and chondrocytes together (i.e. combined culture or co‐culture) on temperature‐responsive inserts, chondrocyte growth was assessed and a molecular analysis of the chondrocyte sheets was performed. Transplantable tissue could be obtained more quickly using this method (average 10.5 days). Real‐time polymerase chain reaction and immunostaining of the three‐layer chondrocyte sheets confirmed the significant expression of genes critical to cartilage maintenance, including type II collagen (COL2), aggrecan‐1 and tissue metallopeptidase inhibitor 1. However, the expression of COL1, matrix metalloproteinase 3 (MMP3), MMP13 and A‐disintegrin and metalloproteinase with thrombospondin motifs 5 was suppressed. The adhesive factor fibronectin‐1 (FN1) was observed in all sheet layers, whereas in sheets generated using conventional preparation methods positive FN1 immunostaining was observed only on the surface of the sheets. The results indicate that synoviocyte co‐cultures provide an optimal environment for the preparation of chondrocyte sheets for tissue transplantation and are particularly beneficial for shortening the required culture period. Copyright © 2013 John Wiley & Sons, Ltd.     

Clinical‐scale expansion of adipose‐derived stromal cells starting from stromal vascular fraction in a single‐use bioreactor: proof of concept for autologous applications

Adipose‐derived stromal cells (ASCs) are adult multipotent cells increasingly used for cell therapy due to their differentiation potential, their paracrine effect and their convenience. ASCs are currently selected from stromal vascular fractions (SVFs) of adipose tissue and expanded in 2D flasks following good manufacturing practices. This process is limited in surface area, labour‐intensive and expensive, especially for autologous applications requiring selection and expansion steps for every patient. Closed and automated bioreactors offer an alternative for scalable and cost‐effective production of ASCs. This study investigated a single‐use stirred‐tank bioreactor that can expand ASCs from SVFs on microcarriers. A preliminary microcarrier screening in static and spinner flask conditions was performed to evaluate the best candidate for adhesion, amplification and harvest. The selected microcarrier was used for process development in the bioreactor. The first experiments showed poor selectivity and growth of the ASCs from the SVF (n  =  2). The process was then adjusted by two means: (1) decreasing the platelet lysate in the medium for enhancing cell adherence; and (2) adding a shear protectant (Pluronic F68). Following these modifications, we demonstrated that the number of population doublings of ASCs from SVFs was not significantly different between the bioreactor and the 2D controls (n  =  3). In addition, the ASC characterization after culture showed that cells maintained their clonogenic potential, phenotype, differentiation potential and immunosuppressive capacities. This study provides the proof of concept that isolation and amplification of functional ASCs from SVFs can be performed in a stirred‐tank bioreactor combined with microcarriers. Copyright © 2016 John Wiley & Sons, Ltd.     

Human chondrocyte migration behaviour to guide the development of engineered cartilage

Tissue‐engineering techniques have been successful in developing cartilage‐like tissues in vitro using cells from animal sources. The successful translation of these strategies to the clinic will likely require cell expansion to achieve sufficient cell numbers. Using a two‐dimensional (2D) cell migration assay to first identify the passage at which chondrocytes exhibited their greatest chondrogenic potential, the objective of this study was to determine a more optimal culture medium for developing three‐dimensional (3D) cartilage‐like tissues using human cells. We evaluated combinations of commonly used growth factors that have been shown to promote chondrogenic growth and development. Human articular chondrocytes (AC) from osteoarthritic (OA) joints were cultured in 3D environments, either in pellets or encapsulated in agarose. The effect of growth factor supplementation was dependent on the environment, such that matrix deposition differed between the two culture systems. ACs in pellet culture were more responsive to bone morphogenetic protein (BMP2) alone or combinations containing BMP2 (i.e. BMP2 with PDGF or FGF). However, engineered cartilage development within agarose was better for constructs cultured with TGFβ3. These results with agarose and pellet culture studies set the stage for the development of conditions appropriate for culturing 3D functional engineered cartilage for eventual use in human therapies. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro chondrogenesis with lysozyme susceptible bacterial cellulose as a scaffold

A current focus of tissue engineering is the use of adult human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes for cartilage repair. Several natural and synthetic polymers (including cellulose) have been explored as a biomaterial scaffold for cartilage tissue engineering. While bacterial cellulose (BC) has been used in tissue engineering, its lack of degradability in vivo and high crystallinity restricts widespread applications in the field. Recently we reported the formation of a novel bacterial cellulose that is lysozyme‐susceptible and ‐degradable in vivo from metabolically engineered Gluconacetobacter xylinus. Here we report the use of this modified bacterial cellulose (MBC) for cartilage tissue engineering using hMSCs. MBC's glucosaminoglycan‐like chemistry, combined with in vivo degradability, suggested opportunities to exploit this novel polymer in cartilage tissue engineering. We have observed that, like BC, MBC scaffolds support cell attachment and proliferation. Chondrogenesis of hMSCs in the MBC scaffolds was demonstrated by real‐time RT–PCR analysis for cartilage‐specific extracellular matrix (ECM) markers (collagen type II, aggrecan and SOX9) as well as histological and immunohistochemical evaluations of cartilage‐specific ECM markers. Further, the attachment, proliferation, and differentiation of hMSCs in MBC showed unique characteristics. For example, after 4 weeks of cultivation, the spatial cell arrangement and collagen type‐II and ACAN distribution resembled those in native articular cartilage tissue, suggesting promise for these novel in vivo degradable scaffolds for chondrogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

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.