Bovine Articular Chondrocytes Research Articles

Cyclic compressive loading activates angiotensin II type 1 receptor in articular chondrocytes and stimulates hypertrophic differentiation through a G-protein-dependent pathway.

Angiotensin II type 1 receptor (AT1R) appears to have a mechanosensing function in a number of cell types. The purpose of this study was to examine whether AT1R expressed in articular chondrocytes is involved in osteoarthritis (OA) progression in vivo and whether cyclic compressive loading activates the AT1R and stimulates hypertrophic differentiation of chondrocytes in vitro. The relationships between the modified Mankin score for cartilage degeneration and the expression of AT1R and type X collagen (Col X) were studied in mouse knees with OA induced using the destabilization‐of‐medial‐meniscus model. Cyclic compressive loads were applied to cultured bovine articular chondrocytes in three‐dimensional agarose scaffolds. Expression of Col X and runt‐related transcription factor 2 (Runx2) was analyzed using RT‐PCR and western blotting. We dissected the downstream pathway for intracellular signal transductions of AT1R including G‐protein‐dependent and G‐protein‐independent pathways. Positive significant correlations between the Mankin score and the rate of AT1R‐immunopositive cells and between the rates of AT1R and Col X expression were noted. The expression of Col X and Runx2 was increased by compressive loading but suppressed by addition of olmesartan, an Ang II receptor blocker, to the agarose scaffolds. Compressive loading upregulated the phosphorylation of c‐Jun N‐terminal kinase (JNK), Src, and STAT1, but olmesartan significantly suppressed only JNK phosphorylation. We conclude that AT1R expressed by articular chondrocytes may be involved in OA progression in vivo. Mechanical stress can activate AT1R and stimulate hypertrophic differentiation of chondrocytes through the G‐protein‐dependent pathway. AT1R has a mechanosensing function in chondrocytes and may be a new therapeutic target in OA.

Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture.

AimsCombining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (hAMSCs) and bone-marrow-derived MSCs (hBMSCs) combined with bovine articular chondrocytes (bACs) was compared.MethodshAMSCs or hBMSCs were combined with bACs in alginate and cultured in vitro or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between bACs and hMSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted.ResultsThe presence of hMSCs–either hAMSCs or hBMSCs—increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in hBMSC/bACs. This effect was similar when hMSCs and bAC were combined in pellet culture, in alginate culture or when conditioned media of hMSCs were used on bAC. Species-specific gene-expression analyses demonstrated that aggrecan was expressed by bACs only, indicating a predominantly trophic role for hMSCs. Collagen-10-gene expression of bACs was not affected by hBMSCs, but slightly enhanced by hAMSCs. After in-vivo implantation, hAMSC/bACs and hBMSC/bACs had similar cartilage matrix production, both appeared stable and did not calcify.ConclusionsThis study demonstrates that replacing 80% of bACs by either hAMSCs or hBMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by hMSCs.

Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding

BackgroundInsulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. MethodsHuman IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. ResultsSecreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. ConclusionThe biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. General significanceThese data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis.

Autologous Chondrocyte Implantation in Osteoarthritic Surroundings: TNFα and Its Inhibition by Adalimumab in a Knee-Specific Bioreactor

Background: Autologous chondrocyte implantation (ACI) fails in up to 20% of cases. Advanced intra-articular degeneration paired with an inflammatory environment may be closely related to implantation failure. Certain cytokines have been identified to play a major role during early osteoarthritis. Purpose: To investigate the effects of tumor necrosis factor α (TNFα) and its potential inhibition by adalimumab on cartilage regeneration in an in vitro model of ACI. Study Design: Controlled laboratory study. Methods: Bovine articular chondrocytes were cultivated and transferred at passage 3 to fibrin-polyurethane scaffolds. Constructs were loaded by compression (10%-20% scaffold height) and shear (±25°) in a fully characterized multiaxial load (L) bioreactor to simulate clinical ACI or were subjected to free swelling (FS) conditions for a duration of 2 weeks. TNFα (20 ng/mL), adalimumab (10 µg/mL), or both were added to the medium. To assess the outcome, DNA, GAG (glycosaminoglycan), and total collagen were quantified, and gene expression of anabolic (collagen 2, aggrecan, cartilage oligomeric protein, proteoglycan 4), catabolic (matrix metalloproteinases [MMP] 3 and 13), dedifferentiation (collagen 1), and hypertrophy (collagen 10) markers and proinflammatory cytokines (TNFα, IL-1β) was analyzed. Histological evaluation was performed with safranin O/fast green, toluidine blue, and immunohistochemistry of collagen 1 and 2. Apoptosis was analyzed by immunolabeling of anti-active caspase 3. For statistical evaluation, nonparametric tests were chosen with a significance level of P < .05. Results: A general downregulation of anabolic and upregulation of catabolic markers was detected in the TNFα groups. Collagen 2 was suppressed by TNFα (FS, P = .029; L, P = .006), while MMP 3 was significantly upregulated (FS, P = .035; L, P = .001). Dynamic loading induced a chondrogenic response, which could not fully antagonize the effect of the cytokine. Adalimumab antagonized all effects of TNFα. The histological and immunohistochemical assessments demonstrated less matrix formation in the cytokine-only groups. TNFα induced apoptosis, and this effect was increased by loading. Conclusion: TNFα does negatively affect chondrogenesis under simulated ACI conditions. Both dynamic load and, more potentially, adalimumab showed the capability of antagonizing the negative effects. Clinical Relevance: Catabolic cytokine suppression (ie, TNFα inhibition) combined with compression and shear load may best meet the conditions for chondrogenesis in an osteoarthritic environment.

Chondrocyte primary cilium is mechanosensitive and responds to low-intensity-ultrasound by altering its length and orientation

The primary cilium has been implicated in mechanotransduction, however, its mechanosensory role in transducing signals under low-intensity ultrasound (LIUS) which has the potential to repair fractures and cartilage, remains uninvestigated. This study examined the impact of continuous-wave US, at the cell resonance frequency of 5.0MHz and pressure amplitude of either 14 or 60kPa, on the incidence, length and orientation of primary cilium in bovine articular chondrocytes. Visualization of primary cilium with acetylated α-tubulin staining demonstrated that the primary cilium was elongated, bent under US and these changes were reversible. Basal expression of phospho-ERK1/2 was lower in deciliated chondrocytes, thus implicating the role for the primary cilium in transducing signals via the MAPK/ERK pathway. This study demonstrates that the chondrocyte primary cilium is mechanosensitive and responds to US by altering its length and orientation.

Fabrication of Highly Crosslinked Gelatin Hydrogel and Its Influence on Chondrocyte Proliferation and Phenotype.

Gelatin methacrylate (GelMA) hydrogels have been widely studied for biomedical applications, such as tissue engineering and drug delivery, because of their good biocompatibility and injectability. However, the quick degradation and low mechanical property of GelMA hydrogels need to be improved for further applications, especially for long-term implantation. In this study, a sequential double modification of gelatin was used to achieve high density of photocrosslinkable double bonds in gelatin derivatives. The amino groups in gelatin were first reacted with methacrylic anhydride. After this, the hydroxyl and carboxyl groups in gelatin were reacted with glycidyl methacrylate to obtain the double modified gelatin macromer. The double modified gelatin macromer was used to prepare gelatin hydrogels with high crosslinking density. The hydrogels exhibited high storage modulus and low degradation. Culture of bovine articular chondrocytes in the gelatin hydrogels showed that chondrocytes had round morphology and maintained a cartilaginous phenotype while cell proliferation was hampered. This method for increasing crosslinking density should be useful for preparation of stable hydrogels for cartilage tissue engineering.

Combined effects of oscillating hydrostatic pressure, perfusion and encapsulation in a novel bioreactor for enhancing extracellular matrix synthesis by bovine chondrocytes.

The influence of combined shear stress and oscillating hydrostatic pressure (OHP), two forms of physical forces experienced by articular cartilage (AC) in vivo, on chondrogenesis, is investigated in a unique bioreactor system. Our system introduces a single reaction chamber design that does not require transfer of constructs after seeding to a second chamber for applying the mechanical forces, and, as such, biochemical and mechanical stimuli can be applied in combination. The biochemical and mechanical properties of bovine articular chondrocytes encapsulated in agarose scaffolds cultured in our bioreactors for 21 days are compared to cells statically cultured in agarose scaffolds in addition to static micromass and pellet cultures. Our findings indicate that glycosaminoglycan and collagen secretions were enhanced by at least 1.6-fold with scaffold encapsulation, 5.9-fold when adding 0.02Pa of shear stress and 7.6-fold with simultaneous addition of 4MPa of OHP when compared to micromass samples. Furthermore, shear stress and OHP have chondroprotective effects as evidenced by lower mRNA expression of β1 integrin and collagen X to non-detectable levels and an absence of collagen I upregulation asobserved in micromass controls. These collective results are further supported by better mechanical properties as indicated by 1.6-19.8-fold increases in elastic moduli measured by atomic force microscopy.

Photocrosslinkable polyaspartamide/polylactide copolymer and its porous scaffolds for chondrocytes

With the aim to produce, by a simple and reproducible technique, porous scaffolds potentially employable for tissue engineering purposes, in this work, we have synthesized a methacrylate (MA) copolymer of α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) and polylactic acid (PLA). PHEA-PLA-MA has been dissolved in organic solvent at different concentrations in the presence of NaCl particles with different granulometry, and through UV irradiation and further salt leaching technique, various porous scaffolds have been prepared. Obtained samples have been characterized by scanning electron microscopy and their porosity has been evaluated as well as their degradation profile in aqueous medium in the absence or in the presence of esterase from porcine liver. PHEA-PLA-MA scaffold that has shown homogeneous porosity and the best degradation profile has been further characterized to study its mechanical properties along with its capacity to incorporate and to control the release of dexamethasone. Finally, the ability to allow a three-dimensional culture of bovine articular chondrocytes have been also investigated.

Cell compaction influences the regenerative potential of passaged bovine articular chondrocytes in an ex vivo cartilage defect model

The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in exvivo systems than in invitro cell cultures. I have therefore established an exvivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p=0.0083) and P2 (p<0.05). Suspension-cultured cells did not promote cartilage repair when compared with implanted monolayer-cultured chondrocytes and pellets: 24.0±0.66% for suspension cells, 46.4±2.9% for monolayer cells, and 127.64±0.90% for pellets (p<0.0001) of the original defect volume (percentage of defect). Additional cultivation with chondrogenesis-promoting growth factors TGF-β1 and BMP-2 revealed an enhancing effect on cartilage repair in all settings. The advantage and innovation of this system over invitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such exvivo explants might serve as a better model system to mimic clinical situations.

SOX9 protein is stabilized by TGF-β and regulates PAPSS2 mRNA expression in chondrocytes

We previously identified 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) as a transcriptional target of transforming growth factor β (TGF-β) in chondrocytes. PAPSS2 is required for proper sulfation of proteoglycans in cartilage. Defective sulfation in the matrix results in alterations in mechanical properties of the cartilage that would be expected to result in degeneration. The objective of this study was to identify factors that regulate PAPSS2 expression and compare to a known TGF-β responsive gene, proteoglycan 4/lubricin (PRG4). In this study, TGF-β-mediated regulation of SOX9 was characterized, and the involvement of SOX9 in regulation of PAPSS2 mRNA was investigated. Primary bovine articular chondrocytes grown in micromass culture and ATDC5 cells were used as the model system. Adenoviruses were used to express SOX9 and SMAD3. siRNA was used to knock-down Sox9 and Smad3. Western blot and real-time quantitative RT-PCR (qPCR) were used to measure changes in protein and mRNA levels in response to treatment. Over-expression of SOX9 was sufficient to up-regulate PAPSS2 mRNA. TGF-β treatment of SOX9-expressing cells resulted in enhanced up-regulation of PAPSS2 mRNA, suggesting that SOX9 cooperates with TGF-β signaling. Furthermore, Sox9 was required for full TGF-β-mediated induction of Papss2. In contrast, PRG4 was regulated by SMAD3 but not SOX9. SOX9 protein levels were increased after treatment with TGF-β, although SOX9 mRNA was not. SOX9 protein was post-translationally stabilized after treatment with TGF-β. TGF-β stabilizes SOX9 protein, and SOX9 is sufficient and necessary for TGF-β-mediated regulation of PAPSS2 mRNA, providing a novel mechanism for TGF-β-mediated gene regulation in chondrocytes.

Covalent and injectable chitosan-chondroitin sulfate hydrogels embedded with chitosan microspheres for drug delivery and tissue engineering

Injectable hydrogels and microspheres derived from natural polysaccharides have been extensively investigated as drug delivery systems and cell scaffolds. In this study, we report a preparation of covalent hydrogels basing polysaccharides via the Schiff' base reaction. Water soluble carboxymethyl chitosan (CMC) and oxidized chondroitin sulfate (OCS) were prepared for cross-linking of hydrogels. The mechanism of cross-linking is attributed to the Schiff' base reaction between amino and aldehyde groups of polysaccharides. Furthermore, bovine serum albumin (BSA) loaded chitosan-based microspheres (CMs) with a diameter of 3.8–61.6μm were fabricated by an emulsion cross-linking method, followed by embedding into CMC-OCS hydrogels to produce a composite CMs/gel scaffold. In the current work, gelation rate, morphology, mechanical properties, swelling ratio, in vitro degradation and BSA release of the CMs/gel scaffolds were examined. The results show that mechanical and bioactive properties of gel scaffolds can be significantly improved by embedding CMs. The solid CMs can serve as a filler to toughen the soft CMC-OCS hydrogels. Compressive modulus of composite gel scaffolds containing 20mg/ml of microspheres was 13KPa, which was higher than the control hydrogel without CMs. Cumulative release of BSA during 2weeks from CMs embedded hydrogel was 30%, which was significantly lower than those of CMs and hydrogels. Moreover, the composite CMs/gel scaffolds exhibited lower swelling ratio and slower degradation rate than the control hydrogel without CMs. The potential of the composite hydrogel as an injectable scaffold was demonstrated by encapsulation of bovine articular chondrocytes in vitro. These results demonstrate the potential of CMs embedded CMC-OCS hydrogels as an injectable drug and cell delivery system in cartilage tissue engineering.

Ammonium-Chloride-Potassium Lysing Buffer Treatment of Fully Differentiated Cells Increases Cell Purity and Resulting Neotissue Functional Properties.

Juvenile and fetal, primary, fully differentiated cells are widely considered to be ideal cell types for tissue engineering applications. However, their use in tissue engineering may be hindered through contamination by undesirable cell types. These include blood-associated cells as well as unwanted resident cell types found both in healthy and pathologic donor tissues. Ammonium-chloride-potassium (ACK) lysing buffer is used to lyse red blood cells (RBCs) during the isolation of stem cell populations, but has not been explored for the purification of fully differentiated cells. This study sought to investigate the effect of ACK buffer treatment of freshly isolated, fully differentiated cells to increase cell purity and enhance the formation of biofunctional engineered neotissues; this was tested in the well-established cartilage tissue engineering model of the self-assembling process using fetal ovine articular chondrocytes (foACs) and juvenile bovine articular chondrocytes (jbACs). ACK buffer treatment of foACs and jbACs decreased the number of contaminating RBCs by over 60% and additionally reduced the number of apoptotic chondrocytes in the cell isolates. Reducing the number of contaminating RBCs removed cellular detractors to the self-assembling process and eliminated an apoptotic stimulus, thus improving neocartilage homogeneity, chondrocyte distribution, and extracellular matrix deposition within the neotissues. For example, in foAC neocartilage, ACK buffer treatment ultimately led to a 170% increase in compressive aggregate modulus, a 130% increase in shear modulus, an 80% increase in tensile modulus, and a 130% increase in ultimate tensile strength of the neocartilage. This work represents the first time that ACK buffer has been used to purify fully differentiated cells and subsequently increase the functional properties of neotissue.

Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of Fetal Calf Serum.

Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood, however, pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology, and clustering of cultured bovine articular chondrocytes within stiff (2% w/v) and soft (0.2% w/v) three-dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; fetal calf serum (FCS) 1-10%;380 mOsm) for up to 7 days. Chondrocytes were fluorescently labeled after 1, 3, and 7 days with 5-chloromethylfluorescein-diacetate (CMFDA) and propidium iodide (PI) or 1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology, and clustering were quantified using Volocity™ software. In stiff gels after 7 d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However, in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P < 0.0001) and enhanced clustering (P < 0.05), compared to stiff gels. FCS exacerbated changes to density (P < 0.01), abnormal morphology (P < 0.001) and clustering (P < 0.01) compared to lower concentrations at the same gel strength. Reduced gel stiffness and/or increased FCS concentrations promoted chondrocyte proliferation and clustering, increased cell volume, and stimulated abnormal morphology, producing similar changes to those occurring in OA. The increased penetration of factors in FCS into soft gels may be important in the development of these abnormal chondrocyte properties. J. Cell. Physiol. 232: 1041-1052, 2017. © 2016 Wiley Periodicals, Inc.

3D Culture of Chondrocytes in Gelatin Hydrogels with Different Stiffness.

Gelatin hydrogels can mimic the microenvironments of natural tissues and encapsulate cells homogeneously, which makes them attractive for cartilage tissue engineering. Both the mechanical and biochemical properties of hydrogels can affect the phenotype of chondrocytes. However, the influence of each property on chondrocyte phenotype is unclear due to the difficulty in separating the roles of these properties. In this study, we aimed to study the influence of hydrogel stiffness on chondrocyte phenotype while excluding the role of biochemical factors, such as adhesion site density in the hydrogels. By altering the degree of methacryloyl functionalization, gelatin hydrogels with different stiffnesses of 3.8, 17.1, and 29.9 kPa Young’s modulus were prepared from the same concentration of gelatin methacryloyl (GelMA) macromers. Bovine articular chondrocytes were encapsulated in the hydrogels and cultured for 14 days. The influence of hydrogel stiffness on the cell behaviors including cell viability, cell morphology, and maintenance of chondrogenic phenotype was evaluated. GelMA hydrogels with high stiffness (29.9 kPa) showed the best results on maintaining chondrogenic phenotype. These results will be useful for the design and preparation of scaffolds for cartilage tissue engineering.

The Chondroprotective Role of Erythromycin in a Murine Joint Destruction Model

Inflammation is a major player in the joint destruction process. Macrolide antibiotics have recently been found to have a novel anti-inflammatory function, but their effects on the joint are unknown. Our objective was to investigate the effect of macrolide antibiotic erythromycin on cartilage gene expression under inflammatory conditions as well as on joint pathology in an in vivo inflammatory joint destruction model. In our in vivo studies, mouse knee joints were injected with monosodium iodoacetate (MIA), a chemical that inhibits glycolysis and causes joint inflammation and matrix loss. Erythromycin was administered by daily intraperitoneal injection. Changes in joint cartilage and synovium were evaluated by histological analysis. In our in vitro studies, primary bovine articular chondrocytes were treated with erythromycin in the presence of pro-inflammatory cytokine IL-1β or lipopolysaccharide (LPS), and cartilage gene expression analysis was performed. Regional differences in cartilage matrix destruction along the medial-lateral axis were observed in joints of MIA-injected mice. Erythromycin treatment inhibited cartilage matrix loss and synovitis in these joints. In addition, erythromycin inhibited IL-1β and LPS-induced expression of MMPs and iNOS, as well as the positive regulatory loop between IL-1β and Toll-like receptor 4 (TLR4) in articular chondrocytes. Furthermore, erythromycin prevented LPS-induced NF-κB activation, a key mediator of TLR4-mediated cartilage destruction process. Erythromycin has the ability to inhibit catabolic gene expression mediated by IL-1β and TLR4 in chondrocytes in vitro and maintains cartilage matrix levels in experimental inflammatory joint destruction in vivo, suggesting that it possesses a chondroprotective activity.

The effect of scaffold pore size in cartilage tissue engineering.

The effect of scaffold pore size and interconnectivity is undoubtedly a crucial factor for most tissue engineering applications. The aim of this study was to examine the effect of pore size and porosity on cartilage construct development in different scaffolds seeded with articular chondrocytes. We fabricated poly-L-lactide-co-trimethylene carbonate scaffolds with different pore sizes, using a solvent-casting/particulate-leaching technique. We seeded primary bovine articular chondrocytes on these scaffolds, cultured the constructs for 2 weeks and examined cell proliferation, viability and cell-specific production of cartilaginous extracellular matrix proteins, including GAG and collagen. Cell density significantly increased up to 50% with scaffold pore size and porosity, likely facilitated by cell spreading on the internal surface of bigger pores, and by increased mass transport of gases and nutrients to cells, and catabolite removal from cells, allowed by lower diffusion barriers in scaffolds with a higher porosity. However, both the cell metabolic activity and the synthesis of cartilaginous matrix proteins significantly decreased by up to 40% with pore size. We propose that the association of smaller pore diameters, causing 3-dimensional cell aggregation, to a lower oxygenation caused by a lower porosity, could have been the condition that increased the cell-specific synthesis of cartilaginous matrix proteins in the scaffold with the smallest pores and the lowest porosity among those tested. In the initial steps of in vitro cartilage engineering, the combination of small scaffold pores and low porosity is an effective strategy with regard to the promotion of chondrogenesis.

Simvastatin inhibits CD44 fragmentation in chondrocytes

In human osteoarthritic chondrocytes, the hyaluronan receptor CD44 undergoes proteolytic cleavage at the cell surface. CD44 cleavage is thought to require transit of CD44 into cholesterol-rich lipid rafts. The purpose of this study was to investigate whether statins exert a protective effect on articular chondrocytes due to diminution of cholesterol. Three model systems of chondrocytes were examined including human HCS-2/8 chondrosarcoma cells, human osteoarthritic chondrocytes and normal bovine articular chondrocytes. Treatment with IL-1β + Oncostatin M resulted in a substantial increase in CD44 fragmentation in each of the three chondrocyte models. Pre-incubation with simvastatin prior to treatment with IL-1β + Oncostatin M decreased the level of CD44 fragmentation, decreased the proportion of CD44 that transits into the lipid raft fractions, decreased ADAM10 activity and diminished the interaction between CD44 and ADAM10. In HCS-2/8 cells and bovine articular chondrocytes, fragmentation of CD44 was blocked by the knockdown of ADAM10. Inhibition of CD44 fragmentation by simvastatin also resulted in improved retention of pericellular matrix. Addition of cholesterol and farnesyl-pyrophosphate reversed the protective effects of simvastatin. Thus, the addition of simvastatin exerts positive effects on chondrocytes including reduced CD44 fragmentation and enhanced the retention of pericellular matrix.

Gelatin Scaffolds with Controlled Pore Structure and Mechanical Property for Cartilage Tissue Engineering

Engineering of cartilage tissue in vitro using porous scaffolds and chondrocytes provides a promising approach for cartilage repair. However, nonuniform cell distribution and heterogeneous tissue formation together with weak mechanical property of in vitro engineered cartilage limit their clinical application. In this study, gelatin porous scaffolds with homogeneous and open pores were prepared using ice particulates and freeze-drying. The scaffolds were used to culture bovine articular chondrocytes to engineer cartilage tissue in vitro. The pore structure and mechanical property of gelatin scaffolds could be well controlled by using different ratios of ice particulates to gelatin solution and different concentrations of gelatin. Gelatin scaffolds prepared from ≥70% ice particulates enabled homogeneous seeding of bovine articular chondrocytes throughout the scaffolds and formation of homogeneous cartilage extracellular matrix. While soft scaffolds underwent cellular contraction, stiff scaffolds resisted cellular contraction and had significantly higher cell proliferation and synthesis of sulfated glycosaminoglycan. Compared with the gelatin scaffolds prepared without ice particulates, the gelatin scaffolds prepared with ice particulates facilitated formation of homogeneous cartilage tissue with significantly higher compressive modulus. The gelatin scaffolds with highly open pore structure and good mechanical property can be used to improve in vitro tissue-engineered cartilage.

Lithium chloride modulates chondrocyte primary cilia and inhibits Hedgehog signaling.

Lithium chloride (LiCl) exhibits significant therapeutic potential as a treatment for osteoarthritis. Hedgehog signaling is activated in osteoarthritis, where it promotes chondrocyte hypertrophy and cartilage matrix catabolism. Hedgehog signaling requires the primary cilium such that maintenance of this compartment is essential for pathway activity. Here we report that LiCl (50 mM) inhibits Hedgehog signaling in bovine articular chondrocytes such that the induction of GLI1 and PTCH1 expression is reduced ​ by 71 and 55%, respectively. Pathway inhibition is associated with a 97% increase in primary cilia length from 2.09 ± 0.7 μm in untreated cells to 4.06 ± 0.9 μm in LiCl-treated cells. We show that cilia elongation disrupts trafficking within the axoneme with a 38% reduction in Arl13b ciliary localization at the distal region of the cilium, consistent with the role of Arl13b in modulating Hedgehog signaling. In addition, we demonstrate similar increases in cilia length in human chondrocytes in vitro and after administration of dietary lithium to Wistar rats in vivo. Our data provide new insights into the effects of LiCl on chondrocyte primary cilia and Hedgehog signaling and shows for the first time that pharmaceutical targeting of the primary cilium may have therapeutic benefits in the treatment of osteoarthritis.

Evaluation of Surface Properties and &lt;i&gt;In Vitro&lt;/i&gt; Characterization of Surface Modified &lt;i&gt;In Situ&lt;/i&gt; TiO&lt;sub&gt;2&lt;/sub&gt; Nanofibers

In situ TiO2 nanofiber arrays have been successfully produced directly on a Ti-6Al-4V substrate by using thermal oxidation under a limited supply of oxygen. Their morphology, elemental composition, crystal structure, surface roughness and surface wettability were characterized by field-emission scanning electron microscope (FESEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffractometer (XRD), atomic force microscope (AFM) and contact angle goniometer, respectively. The results of material characterization studies revealed that TiO2 nanofibers possessed greater surface roughness and wettability, as well as the degree of crystallinity. In vitro characterization have also been evaluated by using bovine articular chondrocytes on the resulting TiO2 nanofibrous surface at different time points. Cell adhesion was observed qualitatively by using FESEM and cell proliferation was determined quantitatively by using AlamarBlue reduction assay. The results showed that the TiO2 nanofibrous substrate triggers enhanced chondrocytes adhesion, proliferation, and production of extracellular matrix (ECM) fibrils compared to untreated substrate. These results suggest that the oxidation process produces a surface structure to which chondrocytes affinity, and thus this surface would has potential use in implants designed for cartilaginous applications.