Porcine Articular Chondrocytes Research Articles

The Phenotype of Mesenchymal Stromal Cell and Articular Chondrocyte Cocultures on Highly Porous Bilayer Poly-L-Lactic Acid Scaffolds Produced by Thermally Induced Phase Separation and Supplemented with Hydroxyapatite.

Bilayer scaffolds could provide a suitable topology for osteochondral defect repair mimicking cartilage and subchondral bone architecture. Hence, they could facilitate the chondro- and osteogenic lineage commitment of multipotent mesenchymal stromal cells (MSCs) with hydroxyapatite, the major inorganic component of bone, stimulating osteogenesis. Highly porous poly-L-lactic acid (PLLA) scaffolds with two layers of different pore sizes (100 and 250 µm) and hydroxyapatite (HA) supplementation were established by thermally induced phase separation (TIPS) to study growth and osteogenesis of human (h) MSCs. The topology of the scaffold prepared via TIPS was characterized using scanning electron microscopy (SEM), a microCT scan, pycnometry and gravimetric analysis. HMSCs and porcine articular chondrocytes (pACs) were seeded on the PLLA scaffolds without/with 5% HA for 1 and 7 days, and the cell attachment, survival, morphology, proliferation and gene expression of cartilage- and bone-related markers as well as sulfated glycosaminoglycan (sGAG) synthesis were monitored. All scaffold variants were cytocompatible, and hMSCs survived for the whole culture period. Cross-sections revealed living cells that also colonized inner scaffold areas, producing an extracellular matrix (ECM) containing sGAGs. The gene expression of cartilage and bone markers could be detected. HA represents a cytocompatible supplement in PLLA composite scaffolds intended for osteochondral defects.

A COMPARATIVE STUDY OF THREE DIFFERENT BIOACTIVE GLASS SCAFFOLDS TAILORED FOR CARTILAGE TISSUE ENGINEERING

The regenerative capacity of hyaline cartilage is greatly limited. To prevent the onset of osteoarthritis, cartilage defects have to be properly treated. Cartilage, tissue engineered by mean of bioactive glass (BG) scaffolds presents a promising approach. Until now, conventional BGs have been used mostly for bone regeneration, as they are able to form a hydroxyapatite (HA) layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to compare two BGs based on a novel BG composition tailored specifically for cartilage (CAR12N) and patented by us with conventional BG (BG1393) with a similar topology. The highly porous scaffolds consisting of 100% BG (CAR12N, CAR12N with low Ca2+/Mg2+ and BG1393) were characterized and dynamically seeded with primary porcine articular chondrocytes (pACs) or primary human mesenchymal stem cells (hMSCs) for up to 21 days. Subsequently, cell viability, DNA and glycosaminoglycan contents, cartilage-specific gene and protein expression were evaluated. The manufacturing process led to a comparable high (over 80%) porosity in all scaffold variants. Ion release and pH profiles confirmed bioactivity for them. After both, 7 and 21 days, more than 60% of the total surfaces of all three glass scaffold variants was densely colonized by cells with a vitality rate of more than 80%. The GAG content was significantly higher in BG1393 colonized with pACs. In general, the GAG content was higher in pAC colonized scaffolds in comparison to those seeded with hMSCs. The gene expression of cartilage-specific collagen type II, aggrecan, SOX9 and FOXO1 could be detected in all scaffold variants, irrespectively whether seeded with pACs or hMSCs. Cartilage-specific ECM components could also be detected at the protein level. In conclusion, all three BGs allow the maintenance of the chondrogenic phenotype or chondrogenic differentiation of hMSCs and thus, they present a high potential for cartilage regeneration.

Synovial fluid profile dictates nanoparticle uptake into cartilage - implications of the protein corona for novel arthritis treatments

Drug delivery strategies for joint diseases need to overcome the negatively charged cartilage matrix. Previous studies have extensively investigated particle approaches to increase uptake efficiency by harnessing the anionic charge of the cartilage but have neglected to address potential interactions with the protein-rich biological environment of the joint space. We aimed to evaluate the effects of hard protein coronas derived from osteoarthritis (OA) and rheumatoid arthritis (RA) patient synovial fluids as well as the commonly used fetal calf serum (FCS) on nanoparticle (NP) uptake into tissues and cells. We developed a NP panel with varying PEGylation and incubated them with synovial fluid from either OA, RA patients or FCS. We evaluated the effects of the formed NP-biocorona complex uptake into the porcine articular cartilage explants, chondrocytes and monocyte cell lines and primary patient FLS cells. Proteins composing hard biocoronas were identified using a quantitative proteomics approach. Formed biocoronas majorly impacted NP uptake into cartilage tissue and dictated their uptake in chondrocytes and monocytes. The most suitable NP for potential OA applications was identified. A variety of proteins that were found on all NPs, irrespective of surface modifications. NP-, and protein-specific differences were also observed between the groups, and candidate proteins were identified that could account for the observed differences. This study demonstrates the impact of protein coronas from OA and RA patient synovial fluids on NP uptake into cartilage, emphasizing the importance of biological microenvironment considerations for successful translation of drug delivery vehicles into clinics.

Biodegradable Poly(D-L-lactide-co-glycolide) (PLGA)-Infiltrated Bioactive Glass (CAR12N) Scaffolds Maintain Mesenchymal Stem Cell Chondrogenesis for Cartilage Tissue Engineering.

Regeneration of articular cartilage remains challenging. The aim of this study was to increase the stability of pure bioactive glass (BG) scaffolds by means of solvent phase polymer infiltration and to maintain cell adherence on the glass struts. Therefore, BG scaffolds either pure or enhanced with three different amounts of poly(D-L-lactide-co-glycolide) (PLGA) were characterized in detail. Scaffolds were seeded with primary porcine articular chondrocytes (pACs) and human mesenchymal stem cells (hMSCs) in a dynamic long-term culture (35 days). Light microscopy evaluations showed that PLGA was detectable in every region of the scaffold. Porosity was greater than 70%. The biomechanical stability was increased by polymer infiltration. PLGA infiltration did not result in a decrease in viability of both cell types, but increased DNA and sulfated glycosaminoglycan (sGAG) contents of hMSCs-colonized scaffolds. Successful chondrogenesis of hMSC-colonized scaffolds was demonstrated by immunocytochemical staining of collagen type II, cartilage proteoglycans and the transcription factor SOX9. PLGA-infiltrated scaffolds showed a higher relative expression of cartilage related genes not only of pAC-, but also of hMSC-colonized scaffolds in comparison to the pure BG. Based on the novel data, our recommendation is BG scaffolds with single infiltrated PLGA for cartilage tissue engineering.

Highly porous novel chondro-instructive bioactive glass scaffolds tailored for cartilage tissue engineering

Cartilage injuries remain challenging since the regenerative capacity of cartilage is extremely low. The aim was to design a novel type of bioactive glass (BG) scaffold with suitable topology that allows the formation of cartilage-specific extracellular matrix (ECM) after colonization with chondrogenic cells for cartilage repair. Highly porous scaffolds with interconnecting pores consisting of 100 % BG were manufactured using a melting, milling, sintering and leaching technique. Scaffolds were colonized with porcine articular chondrocytes (pAC) and undifferentiated human mesenchymal stromal cells (hMSC) for up to 35 days.Scaffolds displayed high cytocompatibility with no major pH shift. Scanning electron microscopy revealed the intimate pAC-scaffold interaction with typical cell morphology. After 14 days MSCs formed cell clusters but still expressed cartilage markers. Both cell types showed aggrecan, SOX9 gene and protein expression, cartilage proteoglycan and sulfated glycosaminoglycan synthesis for the whole culture time. Despite type II collagen gene expression could not anymore be detected at day 35, protein synthesis was visualized for both cell types during the whole culturing period, increasing in pAC and declining after day 14 in hMSC cultures.The novel BG scaffold was stable, cytocompatible and cartilage-specific protein synthesis indicated maintenance of pAC's differentiated phenotype and chondro-instructive effects on hMSCs.

Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23-32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114-E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263-273 (2017)]. We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+ at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA.

Are Chondrocytes Overlying Cam Morphology Suitable for Therapeutic Use?

Objectives:Cam-type femoroacetabular impingement (FAI) results in extra bone and cartilage on the anterolateral femoral head-neck junction, causing abnormal contact between the femur and the acetabulum. We investigated whether chondrocytes isolated from cam lesions in FAI patients are metabolically similar to normal chondrocytes and therefore a potential option for therapeutic use via autologous implantation.Methods:Cam cartilage was obtained intraoperatively from 7 human patients with cam lesions (Alpha angle > 60 degrees) (4 arthroscopy, 2 arthroplasty, and 1 periacetabular osteotomy). Healthy femoral and acetabular articular cartilage was obtained from 8 Yucatan minipig hips for comparison. Chondrocytes were extracted from the cartilage then cultured at a high density for up to 1 week. Mitochondrial stress testing procedures (with 2 mM oligomycin, 2.5 mM carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), 2 mM rotenone, 5 mM antimycin A) for determination of the chondrocytes basal and maximal respiration were performed.Results:No significant differences in basal (p = 0.73) or maximum (p = 0.40) respiration were found when comparing chondrocytes from cam lesions to normal porcine articular chondrocytes. There was a significant difference in maximum respiration when comparing chondrocytes from cam lesions in patients who underwent a total hip arthroplasty to chondrocytes from other cam lesions (p = 0.007).Conclusion:Basal and maximum respiration in chondrocytes from cartilage overlying cam lesions suggests a similar level of respiration to healthy articular chondrocytes. Given the importance of mitochondria to cartilage homeostasis, this study supports that chondrocytes overlying non-arthritic femoral cam lesions may be a novel source of healthy chondrocytes.

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.

Rat Model of Intra-articular Chondrocyte Xenotransplantation.

Current treatment options are not providing an adequate solution for cartilage defects. Articular cartilage lesions in particular are not able to repair spontaneously and progressively degenerate with an arthrosic pattern. Aiming to solve this pressing medical need, xenotransplantation of porcine chondrocytes could be developed as a new therapeutic approach. Xenotransplantation is gaining much attention, thanks to the advances in animal genetic engineering and progress in the characterization of the rejection mechanisms that prevent long-term graft survival. In this regard, our team has identified various targets for intervention that should be tested in a meaningful animal model to prove their relevance in rejection of xenogeneic cartilage. To this end, we have recently established a discordant xenotransplantation model by injecting three million porcine articular chondrocytes (PAC) into the femorotibial joint of Lewis rats. This chapter describes this new model, which can be used to assess the immunoregulatory effect of a variety of strategies designed to inhibit rejection of xenogeneic PAC both at the humoral and cellular levels.

Chondrogenic phenotype in responses to poly(ɛ-caprolactone) scaffolds catalyzed by bioenzymes: effects of surface topography and chemistry

Biodegradable poly(ε-caprolactone) (PCL) has been increasingly investigated as a promising scaffolding material for articular cartilage tissue repair. However, its use can be limited due to its surface hydrophobicity and topography. In this study, 3D porous PCL scaffolds fabricated by a fused deposition modeling (FDM) machine were enzymatically hydrolyzed using two different biocatalysts, namely Novozyme®435 and Amano lipase PS, at varied treatment conditions in a pH 8.0 phosphate buffer solution. The improved surface topography and chemistry of the PCL scaffolds were anticipated to ultimately boost the growth of porcine articular chondrocytes and promote the chondrogenic phenotype during cell culture. Alterations in surface roughness, wettability, and chemistry of the PCL scaffolds after enzymatic treatment were thoroughly investigated using several techniques, e.g., SEM, AFM, contact angle and surface energy measurement, and XPS. With increasing enzyme content, incubation time, and incubation temperature, the surfaces of the PCL scaffolds became rougher and more hydrophilic. In addition, Novozyme®435 was found to have a higher enzyme activity than Amano lipase PS when both were used in the same enzymatic treatment condition. Interestingly, the enzymatic degradation process rarely induced the deterioration of compressive strength of the bulk porous PCL material and slightly reduced the molecular weight of the material at the filament surface. After 28 days of culture, both porous PCL scaffolds catalyzed by Novozyme®435 and Amano lipase PS could facilitate the chondrocytes to not only proliferate properly, but also function more effectively, compared with the non-modified porous PCL scaffold. Furthermore, the enzymatic treatments with 50 mg of Novozyme®435 at 25 °C from 10 min to 60 min were evidently proven to provide the optimally enhanced surface roughness and hydrophilicity most significantly favorable for induction of chondrogenic phenotype, indicated by the greatest expression level of cartilage-specific gene and the largest production of total glycosaminoglycans.

Secreted factors from synovial fibroblasts immediately regulate gene expression in articular chondrocytes

Although articular cartilage degeneration in osteoarthritis represents a major public health problem, there is still no molecular approach to prevent this pathology by blocking specific molecules. We have previously applied genome-wide expression analyses with porcine samples to identify specific markers of either growth plate or articular cartilage. Since the molecular differences were also found in cultured chondrocytes derived from both sites, we utilized primary porcine articular chondrocytes (PPACs) for the present study and analyzed, if and how they respond to synoviocyte-derived molecules. PPACs were treated by conditioned medium from porcine synovial fibroblasts (SF-CM) for 2, 6 and 24 h. Gene expression was subsequently monitored by qRT-PCR and microarray analysis. We found that short-term administration of SF-CM to PPACs significantly reduced expression of chondrocyte markers, while it induced expression of SDC4, encoding syndecan-4, a positive regulator of articular cartilage breakdown. Consistently, expression of MMP3, a putative downstream effector of syndecan-4 was strongly induced by SF-CM in PPACs. We identified an MMP3-inducing fraction in the range of 40 kDa after gel filtration, and we confirmed our findings in three-dimensional PPAC cultures, where SF-CM also reduced the glycosaminoglycan content. Taken together, our data suggest that synovial fibroblasts secrete one or more molecule(s) that activate specific signaling events in articular chondrocytes. Identifying a responsible ligand receptor pair(s) might pave the way to develop molecular therapies to reduce the severity of osteoarthritis.

Biodistribution and Immunogenicity of Allogeneic Mesenchymal Stem Cells in a Rat Model of Intraarticular Chondrocyte Xenotransplantation.

Xenogeneic chondrocytes and allogeneic mesenchymal stem cells (MSC) are considered a potential source of cells for articular cartilage repair. We here assessed the immune response triggered by xenogeneic chondrocytes when injected intraarticularly, as well as the immunoregulatory effect of allogeneic bone marrow-derived MSC after systemic administration. To this end, a discordant xenotransplantation model was established by injecting three million porcine articular chondrocytes (PAC) into the femorotibial joint of Lewis rats and monitoring the immune response. First, the fate of MSC injected using various routes was monitored in an in vivo imaging system. The biodistribution revealed a dependency on the injection route with MSC injected intravenously (i.v.) succumbing early after 24 h and MSC injected intraperitoneally (i.p.) lasting locally for at least 5 days. Importantly, no migration of MSC to the joint was detected in rats previously injected with PAC. MSC were then administered either i.v. 1 week before PAC injection or i.p. 3 weeks after to assess their immunomodulatory function on humoral and adaptive immune parameters. Anti-PAC IgM and IgG responses were detected in all PAC-injected rats with a peak at week 2 postinjection and reactivity remaining above baseline levels by week 18. IgG2a and IgG2b were the predominant and long-lasting IgG subtypes. By contrast, no anti-MSC antibody response was detected in the cohort injected with MSC only, but infusion of MSC before PAC injection temporarily augmented the anti-PAC antibody response. Consistent with a cellular immune response to PAC in PAC-injected rats, cytokine/chemokine profiling in serum by antibody array revealed a distinct pattern relative to controls characterized by elevation of multiple markers at week 2, as well as increases in proliferation in draining lymph nodes. Notably, systemic administration of allogeneic MSC under the described conditions did not diminish the immune response. IL-2 measurements in cocultures of rat peripheral blood lymphocytes with PAC indicated that PAC injection induced some T-cell hyporesponsiveness that was not enhanced in the cohorts additionally receiving MSC. Thus, PAC injected intraarticularly in Lewis rats induced a cellular and humoral immune response that was not counteracted by the systemic administration of allogeneic MSC under the described conditions.

Towards biofabrication of a viable modular auricular cartilage implant using a polymer fiber-reinforced hydrogel

Event Abstract Back to Event Towards biofabrication of a viable modular auricular cartilage implant using a polymer fiber-reinforced hydrogel Iris Otto1, 2, Pedro Ferreira Da Costa1, 3, Corstiaan C. Breugem2, Moshe Kon2 and Jos Malda1, 4 1 University Medical Center Utrecht, Orthopaedics, Netherlands 2 University Medical Center Utrecht, Plastic, Reconstructive & Hand Surgery, Netherlands 3 University Medical Center Utrecht, Utrecht Biofabrication Facility, Netherlands 4 Faculty of Veterinary Sciences, Utrecht University, Equine Sciences, Netherlands Introduction: The past two decades have seen increased interest in the use of tissue engineering approaches towards the treatment of auricular deformations. These approaches utilize a combination of cells, bioactive cues, and supporting (bio)materials. Although neocartilage tissue production can be attained, engineering the human auricle keeps facing a number of challenges. These challenges include preserving cellular viability and the complex auricular shape after implantation under the tight skin envelope. Two factors play a role here: soft hydrogels in support of chondrocytes, and nutrient limitation in large non-vascularized constructs. The inferior mechanical properties of the resulting constructs lead to deformation and degradation, demonstrating the need for an adequate nutrient supply, as well as mechanical reinforcement. Biofabrication approaches have the potential to satisfy these requirements. Especially 3D-bioprinting has the ability to combine and deposit multiple materials into intricate architectures. In this study, we propose a novel approach to allow sufficient nutrient diffusion in large constructs by means of modular constructs. Furthermore, we created hybrid constructs for increased mechanical strength by combining gelatin methacrylate (gelMA) bioink with biodegradable polycaprolactone (PCL) fiber-reinforcement. Materials and Methods: Porcine articular and auricular chondrocytes were embedded in 10% gelMA hydrogels (6mm diameter) and cultured in vitro for 2 weeks before subcutaneous implantation in nude mice. Regenerative performance of these implants was evaluated after 6 and 12 weeks in vivo by means of histological and biochemical analysis. In addition, 3D-printed reinforcing PCL-fiber scaffolds were included within the cell-laden hydrogel constructs. This way, hybrid bio-constructs are generated in the shape of the human auricle, for which histological and biochemical evaluation is performed after in vitro maturation. Results and Discussion: In line with previous findings, articular chondrocytes adequately produced extracellular matrix [1],[2]. Nevertheless, auricular chondrocytes embedded within gelMA showed even more prominent neocartilage production as was visualized by proteoglycan and collagen stainings, and a significantly higher GAG/DNA ratio. Strong yet flexible modular PCL scaffolds could reproducibly be fabricated in the complex 3D shape of the auricle with the bioprinter. Hybrid constructs with considerable mechanical properties and with convincing structure and shape were successfully created. Conclusions: This study demonstrated that auricular chondrocytes in gelMA outperform articular chondrocytes. In addition, biofabrication of complex 3D modular auricular shapes was feasible, resulting in flexible yet strong scaffold for the reinforcement of cell-laden hydrogels. This combinatory approach holds promise for tissue engineering of auricular cartilage implants.

Effects of resveratrol on collagen type II protein in the superficial and middle zone chondrocytes of porcine articular cartilage

Ethnopharmacological relevanceResveratrol (RSV) was first isolated in 1940 from the roots of white hellebore (Veratrum grandiflorum (Maxim. ex Miq) O. Loes) and in 1963 from the roots of Japanese knotweed (Polygonum cuspidatum Siebold & Zucc.). These species have been used traditionally to treat arthritis, gout or inflammation. RSV (3,5,4-trihydroxystilbene) is a polyphenolic phytoalexin compound found in various plants, such as grape vines, berries, peanuts, seeds and roots; the highest concentration is in the skin of red grapes. This component of red wine has potent anti-inflammatory properties and may reduce the side effects of non-steroidal anti-inflammatory drugs that are currently used for pain amelioration in osteoarthritis (OA). In early degeneration of articular cartilage, which may lead to OA there is a loss of the tensile properties, indicative of damage to the fibrillar network. Damage to this fibrillar meshwork, made up of primarily collagen type II (90–95%), may be a critical event in the pathology of many arthritides, due in part to the very slow rate of collagen turnover within the cartilage. Collagen type II is the pre-dominant protein of the cartilage middle zone matrix mainly responsible for tensile strength of articular cartilage. The aim of the study was to investigate the effects of RSV on the expression of collagen type II from the superficial and middle zone chondrocytes of porcine articular cartilage. Materials and methodsPorcine articular chondrocytes were isolated from the superficial and middle zone of articular cartilage, cultured as monolayers in serum-free chemically defined medium for four days. Effects of RSV on porcine articular chondrocytes were studied by assessing expression of collagen type II mRNA by RT-PCR and protein levels of collagen type II by ELISA; as well as localisation of collagen type II on cartilage tissue sections using immunohistochemistry. ResultsRSV significantly stimulated the expression of collagen type II at the mRNA and protein levels in the superficial and middle zone. Immunohistochemistry revealed that collagen type II was present along the whole cartilage tissue sections. The staining was strong in the superficial zone, mild in the middle zone and less around hypertrophic chondrocytes in the deep zone. Histological analysis confirmed that cartilage slices were obtained from specific articular cartilage zones. ConclusionThis study revealed the importance of RSV in the regulation of collagen type II protein in different zones of articular cartilage.

Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition, but Causes a Transient Reduction of Articular Cartilage Thickness.

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.

Genetic engineering strategies to prevent the effects of antibody and complement on xenogeneic chondrocytes.

Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.

Biopolymer-based functional inks for the preparation of artificial cartilage via bioprinting technology

We present bioinks based on biopolymers from the native extracellular matrix, namely gelatin and chondroitin sulfate, which are suitable for bioprinting technology applying living mammalian cells. The bioinks can be crosslinked due to chemical modification of the biopolymers with methacrylic functions. Such insoluble hydrogels with tunable physico-chemical propertie are able to closely mimic the native extracellular matrix of biological tissues (ECM). Highly methacrylated gelatin (GM10) resulted in solutions with low viscosities (e.g. 15 wt%: 6.7 ± 1.0 mPa s at 37 °C) within the inkjet-printable range and crosslinked hydrogels with high mechanical strength. Less methacrylated gelatin (GM2) was proper for preparation of soft hydrogels and highly viscous bioinks for pneumatic dispensing technology (e.g. 15 wt%: 39.3 ± 3.6 mPa s at 37 °C). By additional acetylation of GM2 (GM2A8) the solution viscosity could be significantly lowered (e.g. 15 wt%: η = 5.2 ± 0.5 mPa s, 37 °C) and, thus, inkjet-printable bioinks with low crosslinking capacity were also gained. Consequently, a versatile bioink system for bioprinting technology was created. by adjustable twofold modification of gelatin with photochemically reactive and inert groups. Introduction of an additional methacrylated component of the ECM, methacrylated chondroitin sulfate (CSM), further tuned the chemical and physical properties of bioink and hydrogels. GM(A)-CSM hybrid hydrogels possessed for example significantly higher swellability than pure GM(A) hydrogels of the same mass fraction. CSM also influenced bioink viscosity: For unmodified gelatin and gelatin with low degree of modification CSM induced a decrease in bioink viscosity (e.g. 15 wt% GM2: 39.3 ± 3.6 mPa s versus 14 wt% GM2 + 1 wt% CSM: 32.9 ± 6.5 mPa s). In contrast to this, addition of CSM to bioinks made of gelatins with high degree of modification, GM2A8 or GM10, induced an increase in solution viscosity (e.g. 15 wt% GM2A8: 5.2 ± 0.5 mPa s versus 14 wt% GM2A8 + 1 wt% CSM: 6.5 ± 1.0 mPa s). The developed bioinks were proven to be suitable for bioprinting with viable mammalian cells, in this case porcine articular chondrocytes, and for fabrication of tissue models with intrinsic structure. Thus, biomaterials presented in this study can be used for biofabrication of artificial three-dimensional tissues with biomimetic organization, such as articular cartilage with its hierarchical structure.

Sprifermin (rhfgf18) has an anabolic effect on human osteoarthritic chondrocytes involving fgfr3 and erk1/2 but not p38α and JNKS

Purpose: Restoring the articular surface of degraded or largely injured cartilage necessitates cartilage re-cellularization and the stimulation of cartilage matrix molecule production. Sprifermin (currently in a phase II clinical trial for knee osteoarthritis) is a growth factor that is involved in chondrogenesis and exerts an anabolic effect on cartilage. Using a 3D cell culture system, we previously demonstrated the ability of Sprifermin to stimulate cartilage matrix molecule expression and to promote the chondrocyte phenotype in bovine and porcine articular chondrocytes. In the present work, we aimed to confirm this effect in osteoarthritic chondrocytes of human origin (hOA). In parallel, the pathways involved in Sprifermin signaling were investigated in porcine chondrocytes. Methods: Primary osteoarthritic chondrocytes were isolated from the cartilage of patients who underwent total knee replacement. Cells were cultivated for a few days in monolayer first and then for one week in scaffold-free 3D culture before starting the treatment. The latter consisted in the incubation with Sprifermin [100 ng/mL] permanently or one day/week for a total period of four weeks. Results were compared to a control culture without Sprifermin. Biochemical assays, quantitative PCR (qPCR) and histology were used to characterize the 3D constructs. FGFR expression by qPCR and activation of various kinases (with the human phospho-MAPK array kit from R&D Systems) upon Sprifermin stimulation were assessed on freshly isolated hOA chondrocytes. The effects of a panel of inhibitors (MAPK inhibitors and one pan-FGFR inhibitor) were tested in porcine cells. For this, freshly isolated chondrocytes from a pig hip were cultivated for 7 days in monolayer with 100 ng/mL of Sprifermin in presence of three concentrations of the inhibitors (0.1, 0.3 and 1 μM or 1, 3 and 10 μM depending on their potency). Sprifermin has been previously shown to strongly decrease the collagen I expression (monitored by qPCR) and to promote a round cell morphology (evaluated by actin staining) of porcine chondrocytes in monolayer. These parameters were used to evaluate the impact of the various inhibitors. Results: A 3D scaffold-free culture was used to test the effect of Sprifermin on hOA chondrocytes. In this setting Sprifermin [1day/week] has been found to have a positive effect on the cell content and to greatly increase the size and matrix content of the 3D constructs. In accordance with the literature, FGFR1 and 3 exhibited the highest expression among FGFRs in hOA chondrocytes. In addition, Sprifermin effects were suppressed by FGFR inhibition. In porcine chondrocytes cultivated in monolayer, Sprifermin strongly inhibited collagen I expression and promoted a round cell shape. Both effects were completely inhibited in presence of the pan-FGFR inhibitor PD173074 in a dose-dependent manner. Because FGF18 does not bind FGFR1, in chondrocytes Sprifermin signaling involves mainly FGFR3. Upon incubation with Sprifermin [100 ng/mL], ERK1/2 was activated as well as p38γ, but not p38α or the JNKs. The ERK inhibitor PD325901 completely inhibited the effect of Sprifermin on cell morphology at 100 nM. The JNK inhibitor (SR3306) and p38 inhibitors (BIRB-796 and SB203580) did not have any effect. The effect of these inhibitors on collagen I expression is still under investigation. Taken together these results indicate that ERK1/2 is involved in Sprifermin signaling whereas p38α and JNKs are not. Conclusions: As observed in previous studies with bovine and porcine chondrocytes, Sprifermin was found to exert anabolic effects in hOA chondrocytes. These effects seem to be mediated by FGFR3 and ERK1/2 activation. Others demonstrated that ERK1/2 is also involved in FGF2-FGFR1 signaling but in this case led to a catabolic response. This difference can be possibly explained by the fact that JNK and p38 are also activated by FGF2-FGFR1 signaling but not by FGF18-FGFR3.

Cartilage Tissue Engineering with Silk Fibroin Scaffolds Fabricated by Indirect Additive Manufacturing Technology.

Advanced tissue engineering (TE) technology based on additive manufacturing (AM) can fabricate scaffolds with a three-dimensional (3D) environment suitable for cartilage regeneration. Specifically, AM technology may allow the incorporation of complex architectural features. The present study involves the fabrication of 3D TE scaffolds by an indirect AM approach using silk fibroin (SF). From scanning electron microscopic observations, the presence of micro-pores and interconnected channels within the scaffold could be verified, resulting in a TE scaffold with both micro- and macro-structural features. The intrinsic properties, such as the chemical structure and thermal characteristics of SF, were preserved after the indirect AM manufacturing process. In vitro cell culture within the SF scaffold using porcine articular chondrocytes showed a steady increase in cell numbers up to Day 14. The specific production (per cell basis) of the cartilage-specific extracellular matrix component (collagen Type II) was enhanced with culture time up to 12 weeks, indicating the re-differentiation of chondrocytes within the scaffold. Subcutaneous implantation of the scaffold-chondrocyte constructs in nude mice also confirmed the formation of ectopic cartilage by histological examination and immunostaining.

Anti‐inflammatory effects of hydrophilic and lipophilic statins with hyaluronic acid against LPS‐induced inflammation in porcine articular chondrocytes

The objective of the study is to understand the therapeutic effects of lipophilic (simvastatin) and hydrophilic statins (pravastatin) combined with/without hyaluronic acid for osteoarthritis by an in vitro LPS-induced inflammatory model of articular chondrocytes. HA in combination with different doses of simvastatin or pravastatin were used. Beside cytotoxicity, the influence of statins on NO production, pro-inflammatory cytokine, inflammatory mediators, and NF-κB p50 protein were analyzed. Finally, TUNEL assay was performed to detect DNA strand breakage. Two statins were less able to lower NF-κB activity when they were administrated along without HA. The gene expression demonstrates that simvastatin and pravastatin had the ability to decrease pro-inflammatory and inflammatory mediator levels. High dose simvastatin with or without HA down regulated inflammatory cytokines, but resulted in higher cytotoxicity. TUNEL assay confirms the regulatory effect of statins with or without HA over the apoptosis of chondrocytes, especially in hydrophilic statins. The significant down-regulation of inflammatory mediators suggests that intra-articular injection of HA in combination with statins might feasibly slow the progress of osteoarthritis. Administration of simvastatin or pravastatin with hyaluronic acid may produce beneficial effects for OA treatment, but with better results when hydrophilic statin was used.