Cartilage Repair Strategies Research Articles

Ex vivo model unravelling cell distribution effect in hydrogels for cartilage repair

SummaryThe implantation of chondrocyte-laden hydrogels is a promising cartilage repair strategy. Chondrocytes can be spatially positioned in hydrogels and thus in defects, while current clinical cell therapies introduce chondrocytes in the defect depth. The main aim of this study was to evaluate the effect of spatial chondrocyte distribution on the reparative process. To reduce animal experiments, an ex vivo osteochondral plug model was used and evaluated. The role of the delivered and endogenous cells in the repair process was investigated.Full thickness cartilage defects were created in equine osteochondral plugs. Defects were filled with (A) chondrocytes at the bottom of the defect, covered with a cell-free hydrogel, (B) chondrocytes homogeneously encapsulated in a hydrogel, and (C, D) combinations of A and B with different cell densities. Plugs were cultured for up to 57 days, after which the cartilage and repair tissues were characterized and compared to baseline samples. Additionally, at day 21, the origin of cells in the repair tissue was evaluated.Best outcomes were obtained with conditions C and D, which resulted in well-integrated cartilage-like tissue that completely filled the defect, regardless of the initial cell density. A critical role of the spatial chondrocyte distribution in the repair process was observed. Moreover, the osteochondral plugs stimulated cartilage formation in the hydrogels when cultured in the defects. The resulting repair tissue originated from the delivered cells.These findings confirm the potential of the osteochondral plug model for the optimization of the composition of cartilage implants and for studying repair mechanisms.

Honokiol improved chondrogenesis and suppressed inflammation in human umbilical cord derived mesenchymal stem cells via blocking nuclear factor-\u03baB pathway

BackgroundCartilage degradation is the significant pathological process in osteoarthritis (OA). Inflammatory cytokines, such as interleukin-1β (IL-1β), activate various downstream mediators contributing to OA pathology. Recently, stem cell-based cartilage repair emerges as a potential therapeutic strategy that being widely studied, whereas, the outcome is still far from clinical application. In this study, we focused on an anti-inflammatory agent, honokiol, which is isolated from an herb, investigated the potential effects on human umbilical cord derived mesenchymal stem cells (hUC-MSCs) in IL-1β stimulation.MethodsSecond passage hUC-MSCs were cultured for multi-differentiation. Flow cytometry, qRT-PCR, von Kossa stain, alcian blue stain and oil red O stain were used for characterization and multi-differentiation determination. Honokiol (5, 10, 25, 50 μM) and IL-1β (10 ng/ml) were applied in hUC-MSCs during chondrogenesis. Analysis was performed by MTT, cell apoptosis evaluation, ELISA assay, qRT-PCR and western blot.ResultshUC-MSC was positive for CD73, CD90 and CD105, but lack of CD34 and CD45. Remarkable osteogenesis, chondrogenesis and adipogenesis were detected in hUC-MSCs. IL-1β enhanced cell apoptosis and necrosis and activated the expression of caspase-3, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6) and matrix metalloproteinase (MMP)-1, −9, 13 in hUC-MSCs. Moreover, the expression of SRY-related high-mobility group box 9 (SOX-9), aggrecan and col2α1 was suppressed. Honokiol relieved these negative impacts induced by IL-1β and suppressed Nuclear factor-κB (NF-κB) pathway by downregulating expression of p-IKKα/β, p-IκBα and p-p65 in dose-dependent and time-dependent manner.ConclusionsHonokiol improved cell survival and chondrogenesis of hUC-MSCs and inhibited IL-1β-induced inflammatory response, which suggested that combination of anti-inflammation and stem cell can be a novel strategy for better cartilage repair.

Autologous nasal chondrocytes delivered by injectable hydrogel for in vivo articular cartilage regeneration

Cell based tissue engineering serves as a promising strategy for articular cartilage repair, which remains a challenge both for researchers and clinicians. The aim of this research was to assess the potential of autologous nasal chondrocytes (NCs) combined with alginate hydrogel as injectable constructs for rabbit articular cartilage repair. Autologous nasal chondrocytes were isolated from rabbit nasal septum, expanded either on monolayer or in 3D alginate hydrogel. In vitro, DNA quantification revealed that NCs can proliferate stable in 3D alginate matrix, but slower than that cultured in monolayer. Further, a higher synthesis rate of glycosaminoglycans (GAGs) was detected by GAG measurement in 3D alginate culture. Gene expression analysis at different time point (day 1, 7, 14) showed that 3D culture of NCs in alginate up-regulated chondrogenic markers (Col2A1, ACAN SOX9), meanwhile down-regulated dedifferentiation related gene (Col1A1). In vivo, autologous nasal chondrocytes combined with alginate hydrogel were used for repairing rabbit knee osteochondral defect (Alg + NC group). Histological staining indicated that Alg + NC group obtained superior and more hyaline-like repaired tissue both at 3 and 6 months after surgery. Mechanical analysis showed that the repaired tissue in the Alg + NC group possessed similar mechanical properties to the native cartilage. In conclusion, nasal chondrocytes appeared to be a very promising seed cell source for cartilage tissue engineering, and alginate hydrogel can serve as suitable delivery system.

Effect of NGF From Venom of Chinese Cobra (Naja Atra)on Chondrocytes Proliferation and Metabolism In Vitro.

Autologous chondrocyte implantation (ACI) is promising strategy for cartilage repair. However, chondrocyte phenotype is easily lost when expanded in vitro which defined as "dedifferentiation." To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. In the present study, we investigated the chondro-protective effect of NGF from Chinese cobra venom on human chondrocytes by determination of its specific effect on cell viability, proliferation, morphology, GAG production, and cartilage specific gene expression. The results suggested that NGF showed no cytotoxicity to chondrocytes below the concentration of 16 µg/mL. DNA and glycosaminoglycan (GAG) content were, respectively, improved in NGF groups comparing to the control (P < 0.05). NGF up-regulate the gene expression of ACAN, SOX9, and COL2A1 while down-regulate the expression level of COL1A1 (P < 0.05). Moreover, the results of viability assay, hematoxylin-eosin, safranin O, and immunohistochemical staining also suggested better performances in NGF groups. NGF of 6 µg/mL shown lower cytotoxicity on chondrocytes, more glycosaminoglycans (GAGs) synthesis and up-regulated chondrogenic gene expression. This study may provide a basis for the development of a novel agent for the treatment of articular cartilage defects. J. Cell. Biochem. 118: 4308-4316, 2017. © 2017 Wiley Periodicals, Inc.

The LncRNA ZBED3-AS1 induces chondrogenesis of human synovial fluid mesenchymal stem cells

Human synovial fluid-derived mesenchymal stem cells (SFMSCs) have great potential for cartilage induction and are promising for cell-based strategies for articular cartilage repair. Many long non-coding RNAs (lncRNAs) regulate chondrogenesis of MSCs. We hypothesized that the divergent lncRNA ZBED3-AS1, which binds locally to chromatin, could promote the expression of zbed3, a novel Axin-interacting protein that activates Wnt/β-catenin signaling, involved in chondrogenesis. However, the function of ZBED3-AS1 in SFMSCs is unclear. In this study, the expression, biological function, and roles of ZBED3-AS1 in SFMSC chondrogenesis were examined by multilineage differentiation, flow cytometry, and gain-of-function studies. We found that ZBED3-AS1 promotes chondrogenesis. Furthermore, ZBED3-AS1 could directly increase zbed3 expression. Finally, the wnt-inhibitor DKK1 could reverse the stimulatory effect of ZBED3-AS1 on chondrogenesis. These findings demonstrate the role of a new lncRNA, ZBED3-AS1, in SFMSC chondrogenesis and may improve osteoarthritis treatment.

The Absence of Detectable ADAMTS-4 (Aggrecanase-1) Activity in Synovial Fluid Is a Predictive Indicator of Autologous Chondrocyte Implantation Success

Background: Autologous chondrocyte implantation (ACI) is used worldwide in the treatment of cartilage defects in the knee. Several demographic and injury-specific risk factors have been identified that can affect the success of ACI treatment. However, the discovery of predictive biomarkers in this field has thus far been overlooked. Purpose: To identify potential biomarkers in synovial fluid and plasma that can be used in the preoperative setting to help optimize patient selection for cell-based cartilage repair strategies. Study Design: Controlled laboratory study. Methods: Fifty-four ACI-treated patients were included. Cartilage oligomeric matrix protein (COMP), hyaluronan, soluble CD14 levels, and aggrecanase-1 (ADAMTS-4) activity in synovial fluid and COMP and hyaluronan in plasma were measured. Baseline and postoperative functional outcomes were determined using the patient-reported Lysholm score. To find predictors of postoperative function, linear and logistic regression analyses were performed. The dependent variables were the baseline and postoperative Lysholm score; the independent variables were patient age and body mass index, defect location, defect area, having a bone-on-bone defect, type of defect patch (periosteum or collagen), requirement of an extra procedure, and baseline biomarker levels. Results: The mean baseline Lysholm score was 47.4 ± 17.0, which improved to 64.6 ± 21.7 postoperatively. The activity of ADAMTS-4 in synovial fluid was identified as an independent predictor of the postoperative Lysholm score. Indeed, simply the presence or absence of ADAMTS-4 activity in synovial fluid appeared to be the most important predictive factor. As determined by contingency analysis, when ADAMTS-4 activity was detectable, the odds of being a responder were 3 times smaller than when ADAMTS-4 activity was not detectable. Other predictive factors were the baseline Lysholm score, age at ACI, and defect patch type used. Conclusion: The absence of ADAMTS-4 activity in the synovial fluid of joints with cartilage defects may be used in conjunction with known demographic risk factors in the development of an ACI treatment algorithm to help inform the preclinical decision.

3D Printing of Cytocompatible Water-Based Light-Cured Polyurethane with Hyaluronic Acid for Cartilage Tissue Engineering Applications.

Diseases in articular cartilages have affected millions of people globally. Although the biochemical and cellular composition of articular cartilages is relatively simple, there is a limitation in the self-repair ability of the cartilage. Therefore, developing strategies for cartilage repair is very important. Here, we report on a new liquid resin preparation process of water-based polyurethane based photosensitive materials with hyaluronic acid with application of the materials for 3D printed customized cartilage scaffolds. The scaffold has high cytocompatibility and is one that closely mimics the mechanical properties of articular cartilages. It is suitable for culturing human Wharton’s jelly mesenchymal stem cells (hWJMSCs) and the cells in this case showed an excellent chondrogenic differentiation capacity. We consider that the 3D printing hybrid scaffolds may have potential in customized tissue engineering and also facilitate the development of cartilage tissue engineering.

Advances and Prospects in Stem Cells for Cartilage Regeneration.

The histological features of cartilage call attention to the fact that cartilage has a little capacity to repair itself owing to the lack of a blood supply, nerves, or lymphangion. Stem cells have emerged as a promising option in the field of cartilage tissue engineering and regenerative medicine and could lead to cartilage repair. Much research has examined cartilage regeneration utilizing stem cells. However, both the potential and the limitations of this procedure remain controversial. This review presents a summary of emerging trends with regard to using stem cells in cartilage tissue engineering and regenerative medicine. In particular, it focuses on the characterization of cartilage stem cells, the chondrogenic differentiation of stem cells, and the various strategies and approaches involving stem cells that have been used in cartilage repair and clinical studies. Based on the research into chondrocyte and stem cell technologies, this review discusses the damage and repair of cartilage and the clinical application of stem cells, with a view to increasing our systematic understanding of the application of stem cells in cartilage regeneration; additionally, several advanced strategies for cartilage repair are discussed.

Electrical stimulation drives chondrogenesis of mesenchymal stem cells in the absence of exogenous growth factors.

Electrical stimulation (ES) is known to guide the development and regeneration of many tissues. However, although preclinical and clinical studies have demonstrated superior effects of ES on cartilage repair, the effects of ES on chondrogenesis remain elusive. Since mesenchyme stem cells (MSCs) have high therapeutic potential for cartilage regeneration, we investigated the actions of ES during chondrogenesis of MSCs. Herein, we demonstrate for the first time that ES enhances expression levels of chondrogenic markers, such as type II collagen, aggrecan, and Sox9, and decreases type I collagen levels, thereby inducing differentiation of MSCs into hyaline chondrogenic cells without the addition of exogenous growth factors. ES also induced MSC condensation and subsequent chondrogenesis by driving Ca2+/ATP oscillations, which are known to be essential for prechondrogenic condensation. In subsequent experiments, the effects of ES on ATP oscillations and chondrogenesis were dependent on extracellular ATP signaling via P2X4 receptors, and ES induced significant increases in TGF-β1 and BMP2 expression. However, the inhibition of TGF-β signaling blocked ES-driven condensation, whereas the inhibition of BMP signaling did not, indicating that TGF-β signaling but not BMP signaling mediates ES-driven condensation. These findings may contribute to the development of electrotherapeutic strategies for cartilage repair using MSCs.

Ex vivo culture platform for assessment of cartilage repair treatment strategies.

There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone, which better represents the in vivo situation and enables supply of factors specific to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue. Next, we evaluated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture over 84 days. Finally, we determined the optimal defect size that does not show spontaneous self-healing in this culture system. It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allow for long-term culture of osteochondral explants while maintaining cartilage viability, matrix tissue content, structure and mechanical properties for at least 56 days. Furthermore, we could create critical size cartilage defects of different sizes in the model. The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials, for their regenerative potential, for evaluation of drug and cell therapies, or to study mechanisms of cartilage regeneration. It will undoubtedly reduce the number of animals needed for in vivo testing.

Cartilage repair strategies in the knee: A survey of Turkish surgeons

ObjectivesThe purpose of this study was to analyze the trends in cartilage repair strategies among Turkish orthopedic surgeons for isolated focal (osteo)chondral lesions of the knee joint. Materials and methodsA web-based survey of 21 questions consisting of surgical indications, techniques and time to return to sports was developed to investigate the preferences of members of the TOTBID and the TUSYAD. ResultsA total of 147 surgeons answered the questionnaire.70% of the respondents were TUSYAD members. 82% of respondents had at least five years experience in arthroscopy. Half of the surgeons indicated that patient age of 50 was the upper limit for cartilage repair. Irrespective of activity level, microfracture (60–67%) was the most frequently used technique for lesions smaller 2.5 cm2. In lesions larger than 4 cm2, MACI was the most commonly advocated procedure (67%). In patients with high activity levels, mosaicplasty was the first choice (69%) for lesions between 2.5 and 4 cm2 in size, followed by MACI (27%). ConclusionPatient age, activity level, BMI and lesion size were important determinants for the choice of treatment of isolated chondral lesions in the knee. These results reflect the choices of experienced knee surgeons in the country. Although not widely performed in Turkey and has limited reimbursement by the health care system, the first choice for defects over 4 cm2 was second generation ACI. Third party payers & health reimbursement authorities should take into account that large defects require methods which are relatively expensive and need high technology. Cross-sectional survey, Level II.

PCL‐PEG‐PCL film promotes cartilage regeneration in vivo

Management of chondral defects has long been a challenge due to poor self-healing capacity of articular cartilage. Many approaches, ranging from symptomatic treatment to structural cartilage regeneration, have obtained very limited satisfactory results. Cartilage tissue engineering, which involves optimized combination of novel scaffolds, cell sources and growth factors, has emerged as a promising strategy for cartilage regeneration and repair. In this study, the aim was to investigate the role of poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL, PCEC) PCEC scaffold in cartilage repair. First, PCEC film was fabricated, and its characteristics were tested using SEM and AFM. Cell (rASC - rat adipose-derived stem cells, and mASCs - green fluorescent mouse adipose-derived stem cells) morphologies on PCEC film were observed using SEM and fluorescence microscopy, after cell seeding. Tests of cell viability on PCEC film were conducted using the CCK-8 assay. Furthermore, full cartilage defects in rats were created, and PCEC films were implanted, to evaluate their healing effects, over 8weeks. It was found that PCEC film, as a biomaterial implant, possessed good in vitro properties for cell adhesion, migration and proliferation. Importantly, in the in vivo experiment, PCEC film exhibited desirable healing outcomes. These results demonstrated that PCEC film was a good scaffold for cartilage tissue engineering for improving cell proliferation and adhesion and could lead to excellent repair of cartilage defects.

Learn, simplify and implement: developmental re-engineering strategies for cartilage repai.

The limited self-healing capacity of cartilage in adult individuals, and its tendency to deteriorate once structurally damaged, makes the search for therapeutic strategies following cartilage-related traumas relevant and urgent. To date, autologous cell-based therapies represent the most advanced treatments, but their clinical success is still hampered by the long-term tendency to form fibrous as opposed to hyaline cartilage tissue. Would the efficiency and robustness of therapies be enhanced if cartilage regeneration approaches were based on the attempt to recapitulate processes occurring during cartilage development ("developmental engineering")? And from this perspective, shouldn't cartilage repair strategies be inspired by development, but adapted to be effective in a context (an injured joint in an adult individual) that is different from the embryo ("developmental re-engineering")? Here, starting from mesenchymal stem/stromal cells (MSCs) as an adult cell source possibly resembling features of the embryonic mesenchyme, we propose a developmental re-engineering roadmap based on the following three steps: (i) learn from embryonic cartilage development which are the key pathways involved in MSC differentiation towards stable cartilage, (ii) simplify the complex developmental events by approximation to essential molecular pathways, possibly by using in vitro high-throughput models and, finally, (iii) implement the outcomes at the site of the injury by establishing an appropriate interface between the delivered signals and the recipient environment (e.g., by controlling inflammation and angiogenesis). The proposed re-design of developmental machinery by establishing artificial developmental events may offer a chance for regeneration to those tissues, like cartilage, with limited capacity to recover from injuries.

Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage.

Recent regenerative medicine and tissue engineering strategies for bone and cartilage repair have led to fascinating progress of translation from basic research to clinical applications. In this context, the use of gene therapy is increasingly being considered as an important therapeutic modality and regenerative technique. Indeed, in the last 20 years, nucleic acids (plasmid DNA, interferent RNA) have emerged as credible alternative or complement to proteins, which exhibited major issues including short half-life, loss of bioactivity in pathologic environment leading to high dose requirement and therefore high production costs. The relevance of gene therapy strategies in combination with a scaffold, following a so-called "Gene-Activated Matrix (GAM)" approach, is to achieve a direct, local and sustained delivery of nucleic acids from a scaffold to ensure efficient and durable cell transfection. Among interesting cells sources, Mesenchymal Stem Cells (MSC) are promising for a rational use in gene/cell therapy with more than 1700 clinical trials approved during the last decade. The aim of the present review article is to provide a comprehensive overview of recent and ongoing work in non-viral genetic engineering of MSC combined with scaffolds. More specifically, we will show how this inductive strategy can be applied to orient stem cells fate for bone and cartilage repair.

Cartilage claims a permanent home

Bone It is unclear whether certain tissues in our bodies are permanent or refreshed over time. Nuclear bomb testing more than 50 years ago released the carbon-14 isotope into the atmosphere, which allows researchers to determine the turnover of human tissues in people over 50 years old. Heinemeier et al. used this “14C bomb pulse” method to determine the regenerative potential of cartilage. They examined human knee joints in both healthy individuals and those with osteoarthritis, taking cartilage samples from both high- and moderate-load areas. The collagen matrix of cartilage was essentially permanent, even in disease. Thus, tissue engineering and regenerative medicine need to take this structural stability of collagen into account when designing cartilage repair strategies. Sci. Transl. Med. 8 , 346ra90 (2016).

A Synthetic Thermosensitive Hydrogel for Cartilage Bioprinting and Its Biofunctionalization with Polysaccharides.

Hydrogels based on triblock copolymers of polyethylene glycol and partially methacrylated poly[N-(2-hydroxypropyl) methacrylamide mono/dilactate] make up an attractive class of biomaterials because of their biodegradability, cytocompatibility, and tunable thermoresponsive and mechanical properties. If these properties are fine-tuned, the hydrogels can be three-dimensionally bioprinted, to generate, for instance, constructs for cartilage repair. This study investigated whether hydrogels based on the polymer mentioned above with a 10% degree of methacrylation (M10P10) support cartilage formation by chondrocytes and whether the incorporation of methacrylated chondroitin sulfate (CSMA) or methacrylated hyaluronic acid (HAMA) can improve the mechanical properties, long-term stability, and printability. Chondrocyte-laden M10P10 hydrogels were cultured for 42 days to evaluate chondrogenesis. M10P10 hydrogels with or without polysaccharides were evaluated for their mechanical properties (before and after UV photo-cross-linking), degradation kinetics, and printability. Extensive cartilage matrix production occurred in M10P10 hydrogels, highlighting their potential for cartilage repair strategies. The incorporation of polysaccharides increased the storage modulus of polymer mixtures and decreased the degradation kinetics in cross-linked hydrogels. Addition of HAMA to M10P10 hydrogels improved printability and resulted in three-dimensional constructs with excellent cell viability. Hence, this novel combination of M10P10 with HAMA forms an interesting class of hydrogels for cartilage bioprinting.

A Biomimetic Poly(vinyl alcohol)-Carrageenan Composite Scaffold with Oriented Microarchitecture.

In general, the design of a scaffold should imitate certain advantageous properties of native extracellular matrix (ECM) to operate as a temporary ECM for cells. From this perspective, a biomimetic scaffold was prepared using poly(vinyl alcohol) and carrageenan in which axially oriented pore structure can be formed through a facile unidirectional freeze-thaw method. We examined the feasibility of this oriented scaffold, which has better physicochemical properties than a non-oriented scaffold fabricated by the conventional method. The microenvironment of this oriented scaffold could imitate biochemical and physical cues of natural cartilage ECM for guiding spatial organization and proliferation of cells in vitro, indicating its potential in cartilage repair strategy. Furthermore, the biocompatibility of the scaffold in vivo was demonstrated in a subcutaneous rat model, which revealed uniform infiltration and survival of newly formed tissue into the oriented scaffold after 4 weeks with only a minimal inflammatory response being observed over the course of the experiments. These results together indicated that the present biomimetic scaffold with oriented microarchitecture could be a promising candidate for cartilage tissue engineering.

Silencing of Antichondrogenic MicroRNA-221 in Human Mesenchymal Stem Cells Promotes Cartilage Repair In Vivo.

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-β. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs toward chondrogenic differentiation in the absence of TGF-β. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. Stem Cells 2016;34:1801-1811.

Bioenergetic reprogramming of articular chondrocytes by exposure to exogenous and endogenous reactive oxygen species and its role in the anabolic response to low oxygen.

Monolayer culture is integral to many cell‐based cartilage repair strategies, but chondrocytes lose regenerative potential with increasing duration in vitro. This coincides with elevated reactive oxygen species (ROS) levels and a bioenergetic transformation characterized by increasing mitochondrial function. This study investigates ROS as stimuli for bioenergetic reprogramming and the effect of antioxidants on the propensity of chondrocytes to regenerate a cartilaginous matrix. Articular chondrocytes were cultured in monolayer under a 2% O2 atmosphere. Oxidative stress was increased using 50 μm H2O2 or a 20% O2 culture atmosphere, or decreased using the antioxidant N‐acetyl‐cysteine (NAC). Mitochondrial function was characterized using 200 nm Mitotracker green and an oxygen biosensor. After two population doublings ± NAC, chondrocytes were encapsulated in alginate beads (1 × 107 cells/ml) for an additional 10 days before DMB assay of glycosaminoglycan content. The beads were cultured under both 20% O2 and the more physiological 5% O2 condition. Chondrocytes expanded in 20% O2 exhibited elevated mitochondrial mass and functional capacity, which was partially mimicked by the exogenous ROS, H2O2. Oligomycin treatment revealed that the increased oxygen consumption was coupled to oxidative phosphorylation. NAC limited these markers of bioenergetic reprogramming during culture‐expansion with no significant effect on subsequent GAG production under 20% O2. However, NAC treatment in monolayer abolished the hypoxic induction of GAG in alginate beads. This supports the hypothesis of a causal relationship between exposure to ROS and acquired mitochondrial function in chondrocytes. Additionally, mitochondrial function may be required for the hypoxic induction of GAG synthesis by chondrocytes. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

Enzymatic control of chitosan gelation for cell delivery vehicle

Event Abstract Back to Event Enzymatic control of chitosan gelation for cell delivery vehicle Carla Tim1, Cintia C. Martignago2, Lia M. Neves3, Nivaldo A. Parizotto2 and Ana Claudia Rennó1 1 Federal University of São Paulo, Bioscience, Brazil 2 Federal University of São Carlos, Physiotherapy, Brazil 3 Federal University of São Carlos, Physiologic, Brazil Osteoarthritis (OA) is a common chronic disease among older people, which leads to a loss of articular cartilage. Due to the very limited cartilage regenerative capacity and consequently the limited efficacy of the standard treatments, the investigation of strategic innovative approaches to prevent the development of the clinical condition of OA is of great interest. Thus, development in strategies for cartilage repair have increasingly focused on cell-assisted repair, proposing to use autologous chondrocytes or other cell types to regenerate articular cartilage in situ. One promising cell delivery vehicle is chitosan hydrogels, mainly due to its several advantages including easy handling, high cell seeding efficiency and good formability to irregular defects in a minimally invasive manner. Chitosan hydrogels can be formed homogeneously by neutralization of chitosan solutions using ammonia produced in situ from enzymatic hydrolysis of urea. Therefore, this study aimed to investigate the possibility to control chitosan gelation using enzymatic hydrolysis of urea for the delivery of chodrocytes. Chitosan (Sigma–Aldrich) was dissolved at a concentration of 2.5 w/v% in 0.100M HCl. Urease (Sigma–Aldrich) was dissolved in phosphate buffered saline and Urea (Sigma–Aldrich) was dissolved in MilliQ water. To prepare 1.5 w/v% chitosan hydrogels, 1mL of chitosan was mixed with 0.67mL urease. Subsequently, 10 ul of the urea solution was added. The vials were inverted every 30 sec and the gelation time was recorded as the one at which the solution did not flow in 5 different conditions, i.e., urease concentration of 30 U/mL and urea concentrations of 7.5, 8.5, 9.5,10.5 and11.5 M. The speed at which the pH changed under different gel compositions was investigated and recorded with the use of a pHmeter electrode placed in a freshly prepared mixture of starting solutions immediately after addition of the urea. The results revealed that the gelation time of the chitosan hydrogels can be precisely controlled by varying the urea concentrations as shown in table 1. The pH during gelation of the chitosan hydrogels is displayed, which confirmed that the pH increased from initial values of about 5.7–6.8 to final values of 6.9–7.3 within 5–28 min after addition of urea. Final pH values were controlled by the concentration of urea as main source for alkaline ammonia groups, but did not depend on the concentration of urease, which allows for independent control over final pH and gelation kinetics by varying the concentrations of urea and urease, respectively. Thus, it is possible to set the desired gelation time and final pH of the gel for various applications. The results demonstrated that physically crosslinked chitosan hydrogels for biomedical purpose can be easily fabricated via enzymatic control, and such control over the gelation of chitosan hydrogels could be an option for the delivery of chondrocytes via an injectable system. We would like to acknowledge the contributions of Brazilian funding agency Fapesp (FAPESP project#2014/13702-6) for the financial support of this research.