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.     

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.     

In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells,employing a two‐chambered co‐culture well design

The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk–RADA peptide scaffold in a specially designed two‐chambered co‐culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two‐chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co‐culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage‐like and subchondral bone‐like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single‐step approach that highlights the feasibility of rabbit BMSCs as a single‐cell source for multilayered osteochondral construct generation in vitro. Copyright © 2013 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.     

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.     

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.     

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.     

Effects of transforming growth factor-β subtypes on in vitro cartilage production and mineralization of human bone marrow stromal-derived mesenchymal stem cells

Human bone marrow stromal-derived mesenchymal stem cells (hBMSCs) will differentiate into chondrocytes in response to defined chondrogenic medium containing transforming growth factor-β (TGFβ). Results in the literature suggest that the three mammalian subtypes of TGFβ (TGFβ1, TGFβ2 and TGFβ3) provoke certain subtype-specific activities. Therefore, the aim of our study was to investigate whether the TGFβ subtypes affect chondrogenic differentiation of in vitro cultured hBMSCs differently. HBMSC pellets were cultured for 5 weeks in chondrogenic media containing either 2.5, 10 or 25 ng/ml of TGFβ1, TGFβ2 or TGFβ3. All TGFβ subtypes showed a comparable dose-response curve, with significantly less cartilage when 2.5 ng/ml was used and no differences between 10 and 25 ng/ml. Four donors with variable chondrogenic capacity were used to evaluate the effect of 10 ng/ml of either TGFβ subtype on cartilage formation. No significant TGFβ subtype-dependent differences were observed in the total amount of collagen or glycosaminoglycans. Cells from a donor with low chondrogenic capacity performed equally badly with all TGFβ subtypes, while a good donor overall performed well. After addition of β-glycerophosphate during the last 2 weeks of culture, the expression of hypertrophy markers was analysed and mineralization was demonstrated by alkaline phosphatase activity and alizarin red staining. No significant TGFβ subtype-dependent differences were observed in expression collagen type X or VEGF secretion. Nevertheless, pellets cultured with TGFβ1 had significantly less mineralization than pellets cultured with TGFβ3. In conclusion, this study suggests that TGFβ subtypes do affect terminal differentiation of in vitro cultured hBMSCs differently.     

Stem cells display a donor dependent response to escalating levels of growth factor release from extracellular matrix‐derived scaffolds

Numerous growth factor delivery systems have been developed for tissue engineering. However, little is known about how the dose of a specific protein will influence tissue regeneration, or how different patients will respond to altered levels of growth factor presentation. The objective of the present study was to assess stem cell chondrogenesis within extracellular‐matrix (ECM)‐derived scaffolds loaded with escalating levels of transforming growth factor (TGF)‐β3. It was also sought to determine if stem cells display a donor‐dependent response to different doses of TGF‐β3, from low (5 ng) to high (200 ng), released from such scaffolds. It was found that ECM‐derived scaffolds possess the capacity to bind and release increasing amounts of TGF‐β3, with between 60% and 75% of this growth factor released into the media over the first 12 days of culture. After seeding these scaffolds with human infrapatellar fat pad‐derived stem cells (FPSCs), it was found that cartilage‐specific ECM accumulation was greatest for the higher levels of growth factor loading. Importantly, soak‐loading cartilage ECM‐derived scaffolds with high levels of TGF‐β3 always resulted in at least comparable levels of chondrogenesis to controls where this growth factor was continuously added to the culture media. Similar results were observed for FPSCs from all donors, although the absolute level of secreted matrix did vary from donor to donor. Therefore, while no single growth factor release profile will be optimal for all patients, the results of this study suggest that the combination of a highly porous cartilage ECM‐derived scaffold coupled with appropriate levels of TGF‐β3 can consistently drive chondrogenesis of adult stem cells. Copyright © 2016 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.     

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     

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.     

Activated platelet‐rich plasma improves cartilage regeneration using adipose stem cells encapsulated in a 3D alginate scaffold

In the current study, the effect of superimposing platelet‐rich plasma (PRP) on different culture mediums in a three‐dimensional alginate scaffold encapsulated with adipose‐derived mesenchymal stem cells for cartilage tissue repair is reported. The three‐dimensional alginate scaffolds with co‐administration of PRP and/or chondrogenic supplements had a significant effect on the differentiation of adipose mesenchymal stem cells into mature cartilage, as assessed by an evaluation of the expression of cartilage‐related markers of Sox9, collagen II, aggrecan and collagen, and glycosaminoglycan assays. For in vivo studies, following induction of osteochondral lesion in a rabbit model, a high degree of tissue regeneration in the alginate plus cell group (treated with PRP plus chondrogenic medium) compared with other groups of cell‐free alginate and untreated groups (control) were observed. After 8 weeks, in the alginate plus cell group, functional chondrocytes were observed, which produced immature matrix, and by 16 weeks, the matrix and hyaline‐like cartilage became completely homogeneous and integrated with the natural surrounding cartilage in the defect site. Similar effect was also observed in the subchondral bone. The cell‐free scaffolds formed fibrocartilage tissue, and the untreated group did not form a continuous cartilage over the defect by 16 weeks.     

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.     

Micro‐aggregates do not influence bone marrow stromal cell chondrogenesis

Although bone marrow stromal cells (BMSCs) appear promising for cartilage repair, current clinical results are suboptimal and the success of BMSC‐based therapies relies on a number of methodological improvements, among which is better understanding and control of their differentiation pathways. We investigated here the role of the cellular environment (paracrine vs juxtacrine signalling) in the chondrogenic differentiation of BMSCs. Bovine BMSCs were encapsulated in alginate beads, as dispersed cells or as small micro‐aggregates, to create different paracrine and juxtacrine signalling conditions. BMSCs were then cultured for 21 days with TGFβ₃ added for 0, 7 or 21 days. Chondrogenic differentiation was assessed at the gene (type II and X collagens, aggrecan, TGFβ, sp7) and matrix (biochemical assays and histology) levels. The results showed that micro‐aggregates had no beneficial effects over dispersed cells: matrix production was similar, whereas chondrogenic marker gene expression was lower for the micro‐aggregates, under all TGFβ conditions tested. This weakened chondrogenic differentiation might be explained by a different cytoskeleton organization at day 0 in the micro‐aggregates. Copyright © 2014 John Wiley & Sons, Ltd.     

PGA‐associated heterotopic chondrocyte cocultures: implications of nasoseptal and auricular chondrocytes in articular cartilage repair

The availability of autologous articular chondrocytes remains a limiting issue in matrix assisted autologous chondrocyte transplantation. Non‐articular heterotopic chondrocytes could be an alternative autologous cell source. The aims of this study were to establish heterotopic chondrocyte cocultures to analyze cell‐cell compatibilities and to characterize the chondrogenic potential of nasoseptal chondrocytes compared to articular chondrocytes. Primary porcine and human nasoseptal and articular chondrocytes were investigated for extracellular cartilage matrix (ECM) expression in a monolayer culture. 3D polyglycolic acid‐ (PGA) associated porcine heterotopic mono‐ and cocultures were assessed for cell vitality, types II, I, and total collagen‐, and proteoglycan content. The type II collagen, lubricin, and Sox9 gene expressions were significantly higher in articular compared with nasoseptal monolayer chondrocytes, while type IX collagen expression was lower in articular chondrocytes. Only β1‐integrin gene expression was significantly inferior in humans but not in porcine nasoseptal compared with articular chondrocytes, indicating species‐dependent differences. Heterotopic chondrocytes in PGA cultures revealed high vitality with proteoglycan‐rich hyaline‐like ECM production. Similar amounts of type II collagen deposition and type II/I collagen ratios were found in heterotopic chondrocytes cultured on PGA compared to articular chondrocytes. Quantitative analyses revealed a time‐dependent increase in total collagen and proteoglycan content, whereby the differences between heterotopic and articular chondrocyte cultures were not significant. Nasoseptal and auricular chondrocytes monocultured in PGA or cocultured with articular chondrocytes revealed a comparable high chondrogenic potential in a tissue engineering setting, which created the opportunity to test them in vivo for articular cartilage repair. Copyright © 2011 John Wiley & Sons, Ltd.     

Biomimetic scaffolds and dynamic compression enhance the properties of chondrocyte‐ and MSC‐based tissue‐engineered cartilage

Adult chondrocytes are surrounded by a protein‐ and glycosaminoglycan‐rich extracellular matrix and are subjected to dynamic mechanical compression during daily activities. The extracellular matrix and mechanical stimuli play an important role in chondrocyte biosynthesis and homeostasis. In this study, we aimed to develop scaffold and compressive loading conditions that mimic the native cartilage micro‐environment and enable enhanced chondrogenesis for tissue engineering applications. Towards this aim, we fabricated porous scaffolds based on silk fibroin (SF) and SF with gelatin/chondroitin sulfate/hyaluronate (SF‐GCH), seeded the scaffolds with either human bone marrow mesenchymal stromal cells (BM‐MSCs) or chondrocytes, and evaluated their performance with and without dynamic compression. Human chondrocytes derived from osteoarthritic joints and BM‐MSCs were seeded in scaffolds, precultured for 1 week, and subjected to compression with 10% dynamic strain at 1 Hz, 1 hr/day for 2 weeks. When dynamic compression was applied, chondrocytes significantly increased expression of aggrecan (ACAN) and collagen X (COL10A1) up to fivefold higher than free‐swelling controls. In addition, dynamic compression dramatically improved the chondrogenesis and chondrocyte biosynthesis cultured in both SF and SF‐GCH scaffolds evidenced by glycosaminoglycan (GAG) content, GAG/DNA ratio, and immunostaining of collagen type II and aggrecan. However, both chondrocytes and BM‐MSCs cultured in SF‐GCH scaffolds under dynamic compression showed higher GAG content and compressive modulus than those in SF scaffolds. In conclusion, the micro‐environment provided by SF‐GCH scaffolds and dynamic compression enhances chondrocyte biosynthesis and matrix accumulation, indicating their potential for cartilage tissue engineering applications.     

In vitro generation of a multilayered osteochondral construct with an osteochondral interface using rabbit bone marrow stromal cells and a silk peptide‐based scaffold

Tissue engineering of a biological osteochondral multilayered construct with a cartilage‐interface subchondral bone layer is a key challenge. This study presented a rabbit bone marrow stromal cell (BMSC)/silk fibroin scaffold‐based co‐culture approach to generate tissue‐engineered osteochondral grafts with an interface. BMSC‐seeded scaffolds were first cultured separately in osteogenic and chondrogenic stimulation media. The two differentiated pieces were then combined using an RADA self‐assembling peptide and subsequently co‐cultured. Gene expression, histological and biochemical analyses were used to evaluate the multilayered structure of the osteochondral graft. A complete osteochondral construct with a cartilage‐subchondral bone interface was regenerated and BMSCs were used as the only cell source for the osteochondral construct and interface regeneration. Furthermore, in the intermediate region of co‐cultured samples, hypertrophic chondrogenic gene markers type X collagen and MMP‐13 were found on both chondrogenic and osteogenic section edges after co‐culture. However, significant differences gene expression profile were found in distinct zones of the construct during co‐culture and the section in the intermediate region had significantly higher hypertrophic chondrocyte gene expression. Biochemical analyses and histology results further supported this observation. This study showed that specific stimulation from osteogenic and chondrogenic BMSCs affected each other in this co‐culture system and induced the formation of an osteochondral interface. Moreover, this system provided a possible approach for generating multilayered osteochondral constructs. Copyright © 2013 John Wiley & Sons, Ltd.     

Translating the application of transforming growth factor‐β1, chondroitinase‐ABC,and lysyl oxidase‐like 2 for mechanically robust tissue‐engineered human neocartilage

Strategies to overcome the limited availability of human articular chondrocytes and their tendency to dedifferentiate during expansion are required to advance their clinical use and to engineer functional cartilage on par with native articular cartilage. This work sought to determine whether a biochemical factor (transforming growth factor‐β1 [T]), a biophysical agent (chondroitinase‐ABC [C]), and a collagen crosslinking enzyme (lysyl oxidase‐like 2 [L]) are efficacious in forming three‐dimensional human neocartilage from expanded human articular chondrocytes. Among the treatment regimens, the combination of the three stimuli (TCL treatment) led to the most robust glycosaminoglycan content, total collagen content, and type II collagen production. In particular, TCL treatment synergistically increased tensile stiffness and strength of human neocartilage by 3.5‐fold and 3‐fold, respectively, over controls. Applied to two additional donors, the beneficial effects of TCL treatment appear to be donor independent; tensile stiffness and strength were increased by up to 8.5‐fold and 3‐fold, respectively, over controls. The maturation of human neocartilage in response to TCL treatment was examined following 5 and 8 weeks of culture, demonstrating maintenance or further enhancement of functional properties. The present study identifies a novel strategy for engineering human articular cartilage using serially passaged chondrocytes.     

Scaffold‐free 3D cellulose acetate membrane‐based cultures form large cartilaginous constructs

Scaffold‐free three‐dimensional (3D) cultures provide clinical potential in cartilage regeneration. The purpose of this study was to characterize a scaffold‐free 3D membrane‐based culture system, in which human articular chondrocytes were cultivated on a cellulose acetate membrane filter, and compare it to pellet and monolayer cultures. Chondrocytes were expanded in monolayer culture for up to 5 passages, transferred to membrane‐based or pellet cultures and harvested after 7 or 21 days. The chondrogenic potential was assessed by histology (toluidine blue, safranin‐O), immunohistochemistry for collagen type II and quantitative analysis of collagen type II α₁ (COL2A1). Membrane‐based cultures (P1) formed flexible disc‐like constructs (diameter 4000 µm, thickness 150 µm) with a large smooth surface after 7 days. Positive safranin‐O and collagen type II staining was found in membrane‐based and pellet cultures at P1–3. Expression of COL2A1 after 7 days was increased in both culture systems compared to monolayer culture up to P3, whereas cells from monolayer > P3 did not redifferentiate. The best results for COL2A1 expression were obtained from membrane‐based cultures at P1. After 21 days the membrane‐based cultures did not express COL2A1. We concluded that membrane‐based and pellet cultures showed the ability to promote redifferentiation of chondrocytes expanded in monolayer culture. The number of cell passages had an impact on the chondrogenic potential of cells. Membrane‐based cultures provided the highest COL2A1 expression and a large, smooth and cartilage‐like surface. As these are appropriate features for clinical applications, we assume that membrane‐based cultures might be of use in cartilage regeneration if they displayed similar results in vivo. Copyright © 2010 John Wiley & Sons, Ltd.