Chondrocyte Phenotype Research Articles

Additive manufacturing of an elastic poly(ester)urethane for cartilage tissue engineering

Although a growing knowledge on the field of tissue engineering of articular cartilage exists, reconstruction or in-vitro growth of functional hyaline tissue still represents an unmet challenge. Despite the simplicity of the tissue in terms of cell population and absence of innervation and vascularization, the outstanding mechanical properties of articular cartilage, which are the result of the specificity of its extra cellular matrix (ECM), are difficult to mimic. Most importantly, controlling the differentiation state or phenotype of chondrocytes, which are responsible of the deposition of this specialized ECM, represents a milestone in the regeneration of native articular cartilage. In this study, we fabricated fused deposition modelled (FDM) scaffolds with different pore sizes and architectures from an elastic and biodegradable poly(ester)urethane (PEU) with mechanical properties that can be modulated by design, and that ranged the elasticity of articular cartilage. Cell culture in additive manufactured 3D scaffolds exceeded the chondrogenic potential of the gold-standard pellet culture. In-vitro cell culture studies demonstrated the intrinsic potential of elastic (PEU) to drive the re-differentiation of de-differentiated chondrocytes when cultured in-vitro, in differentiation or basal media, better than pellet cultures. The formation of neo-tissue was assessed as a high deposition of GAGs and fibrillar collagen II, and a high expression of typical chondrogenic markers. Moreover, the collagen II / collagen I ratio commonly used to evaluate the differentiation state of chondrocytes (ratio > 1 being chondrocytes and, ratio < 0 being de-differentiated chondrocytes) was higher than 5. Statement of significanceTissue engineering of articular cartilage requires material scaffolds capable of driving the deposition of a coherent and specific ECM representative of articular cartilage. Materials explored so far account for low mechanical properties (hydrogels), or are too stiff to mimic the elasticity of the native tissue (traditional polyesters). Here, we fabricated 3D fibrous scaffolds via FDM with a biodegradable poly(ester)urethane. The compressive Young`s modulus and elastic limit of the scaffolds can be tuned by designed, mimicking those of the native tissue. The designed scaffolds showed an intrinsic potential to drive the formation of a GAG and collagen II rich ECM, and to drive a stable chondrogenic cell phenotype.

Exploiting Curcumin Synergy With Natural Products Using Quantitative Analysis of Dose-Effect Relationships in an Experimental In Vitro Model of Osteoarthritis.

Introduction: Drug combination is widely used to treat chronic inflammatory diseases. A similar strategy might be worth of interest to design plant-derived natural products to treat inflammatory conditions. Curcumin is a natural phenolic compound which shares anti-inflammatory activity with both flavocoxid, a flavonoid mixture of baicalin and catechin, and β-caryophyllene, a bicyclic sesquiterpene. The aim of this study was to investigate the synergy potential of curcumin with both flavocoxid and β-caryophyllene in human articular chondrocytes triggered with lipopolysaccharide (LPS), in an experimental in vitro model of osteoarthritis.Materials and Methods: Human articular chondrocytes were stimulated with LPS alone or in combination with different treatments. Total RNA was extracted 4 h after treatment to study interleukin 1β (IL-1β), NF-κB, and STAT3 mRNA expression. A drug combination study was designed choosing 5 doses to demonstrate a synergistic effect of compounds, according to Chou and Talalay method. A median-effect equation was applied and finally, the combination index (CI) was used to clarify the nature of the compounds interaction (synergistic versus additive versus antagonistic inhibitory effects); CI < 1, CI = 1, and CI > 1 indicated synergistic, additive, and antagonistic effects, respectively.Results: LPS prompted IL-1β expression. Curcumin, flavocoxid and β-caryophyllene suppressed IL-1β expression with different IC50. A synergistic action for the reduction of the inflammatory phenotype in human chondrocytes was observed for the combination curcumin-flavocoxid with a percentage from 10% to 90%, and for the combination curcumin-β-caryophyllene from 50% to 90%. IC50 doses of either flavocoxid, β-caryophyllene and curcumin alone or in combination were safe and did not affect cell vitality. Moreover, the same IC50 doses reduced the transcription factors NF-κB and STAT3 mRNA expression and interestingly the effects of the combinations were greater than the natural products alone, thus suggesting that the site where the synergy takes place could be at the signal transduction level.Discussion: The results suggest that curcumin synergizes with either flavocoxid or β-caryophyllene, exerting an anti-inflammatory activity and thus strongly suggesting the potential of a dual combination of these compounds for the management of osteoarthritis and unmasking a new feature of these natural products.

Gene expression markers in horse articular chondrocytes: Chondrogenic differentiaton IN VITRO depends on the proliferative potential and ageing. Implication for tissue engineering of cartilage

Chondrocyte dedifferentiation is a key limitation in therapies based on autologous chondrocyte implantation for cartilage repair. Articular chondrocytes, obtained from (metacarpophalangeal and metatarsophalangeal) joints of different aged horses, were cultured in monolayer for several passages (P0 to P8). Cumulative Populations Doublings Levels (PDL) and gene expression of relevant chondrocyte phenotypic markers were analysed during culturing. Overall data confirmed that, during proliferation in vitro, horse chondrocytes undergo marked morphological and phenotypic alterations of their differentiation status. Particularly, the dedifferentiation started early in culture (P0-P1) and was very marked at P3 subculture (PDL 4–6): proliferative phase after P3 could be critical for maintenance/loss of differentiation potential. In elderly animals, chondrocytes showed aspects of dedifferentiation shortly after their isolation, associated with reduced proliferative capacity. Regarding the gene expression of major cartilage markers (Col2, Aggrecan, SOX9) there was a very early reduction (P1) in proliferating chondrocytes independent of age. The chondrocytes from adult donors showed a more stable expression (up to P3) of some (Col6, Fibromodulin, SOX6, TGβ1) markers of mature cartilage; these markers could be tested as parameter to determine the dedifferentiation level. This study can provide parameters to identify up to which “culture step” chondrocytes for implantation with a conserved phenotypic potential can be obtained, and to test the efficiency of biomaterial scaffold or chondroinductive media/signals to maintain/recover the chondrocyte phenotype. Moreover, the determination of levels and time related expression of these markers can be useful during the chondroinduction of mesenchymal stem cells.

Degrading products of chondroitin sulfate can induce hypertrophy-like changes and MMP-13/ADAMTS5 production in chondrocytes

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in articular cartilage and the loss of CS-GAG occurs early in OA. As a major component of perichondral matrix interacting directly with chondrocytes, the active turnover of CS can affect to break the homeostasis of chondrocytes. Here we employ CS-based 3-dimensional (3D) hydrogel scaffold system to investigate how the degradation products of CS affect the catabolic phenotype of chondrocytes. The breakdown of CS-based ECM by the chondroitinase ABC (ChABC) resulted in a hypertrophy-like morphologic change in chondrocytes, which was accompanied by catabolic phenotypes, including increased MMP-13 and ADAMTS5 expression, nitric oxide (NO) production and oxidative stress. The inhibition of Toll-like receptor 2 (TLR2) or TLR4 with OxPAPC (TLR2 and TLR4 dual inhibitor) and LPS-RS (TLR4-MD2 inhibitor) ameliorated these catabolic phenotypes of chondrocytes by CS-ECM degradation, suggesting a role of CS breakdown products as damage-associated molecular patterns (DAMPs). As downstream signals of TLRs, MAP kinases, NF-kB, NO and STAT3-related signals were responsible for the catabolic phenotypes of chondrocytes associated with ECM degradation. NO in turn reinforced the activation of MAP kinases as well as NFkB signaling pathway. Thus, these results propose that the breakdown product of CS-GAG can recapitulate the catabolic phenotypes of OA.

Effects of Sprifermin, IGF1, IGF2, BMP7, or CNP on Bovine Chondrocytes in Monolayer and 3D Culture.

ABSTRACTOne possible approach to treat osteoarthritis (OA) is to counteract cartilage degeneration with anabolic compounds that stimulate chondrocyte proliferation and/or extracellular matrix (ECM) production. Several molecules including sprifermin (recombinant human fibroblast growth factor [FGF18]), insulin‐like growth factor‐1 [IGF1] and ‐2 [IGF2], C‐type natriuretic peptide [CNP], and bone metamorphic protein 7 [BMP7] have been shown to have these characteristics both in vitro and in vivo. However, it is not known how these molecules compare each other regarding their effect on phenotype and stimulation of ECM production in primary chondrocytes. The effects of sprifermin, IGF1, IGF2, CNP, and BMP7 were evaluated on bovine articular chondrocytes, first in monolayer to determine their effective concentrations, and then in three‐dimensional (3D) culture at concentrations of 100 ng/ml for sprifermin; 300 ng/ml for IGF1, IGF2, and BMP7; and 10 nM for CNP. In 3D culture, the effects of a permanent exposure or a cyclic exposure consisting of 24 h incubation per week with the compounds were evaluated. All growth factors increased ECM production and cell proliferation to a similar extent but CNP had almost no effect on bovine chondrocytes. Sprifermin was more effective with cyclic exposure, IGF1, and IGF2 with permanent exposure, and BMP7 showed similar results with both exposures. Regarding the cell phenotype, sprifermin appeared to be the only compound favoring the chondrocyte phenotype; it decreased type I collagen expression and had no hypertrophic effect. Together, these results confirmed that sprifermin is a promising disease‐modifying OA drug. © 2019 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:653–662, 2020

Continuous Low-Intensity Ultrasound Promotes Native-to-Native Cartilage Integration

Failure of the host/graft interface to integrate impedes the success of cartilage repair protocols. Continuous low-intensity ultrasound (cLIUS) at the resonant frequency of 5 MHz is proposed as a treatment modality for promoting native-to-native cartilage integration in vitro. Cylindrical incisions (4 mm) simulating chondral discontinuity were made in bovine cartilage and osteochondral explants, and maintained under cLIUS stimulation (14 kPa [5 MHz, 2.5 Vpp], 20 min, four times/day) for 28 days. Incised cartilage and osteochondral explants were categorized into three study groups; Group I: cLIUS was applied immediately upon incision; Group II: cLIUS was applied after 14 days following incision; Group-III: after 14 days following incision, explants were treated with 0.1% hyaluronidase and 30 U/mL collagenase VII. As a separate study group, incised osteochondral explants were treated immediately with cLIUS at a nonresonant frequency of 2 MHz (14 kPa [2 MHz, 6 Vpp], 20 min, four times/day). Cellular migration was analyzed by scratch assays, and by visualizing migrating cells into the hydrogel core of cartilage/hydrogel constructs. Explants under cLIUS (5 MHz) displayed higher percent apposition along with gap closures when compared with untreated controls and explants treated with cLIUS at 2 MHz. cLIUS (5 MHz)-treated explants were immunopositive for type II collagen. The strength of native-to-native cartilage integration was higher (p = 0.005) in cLIUS-treated cartilage explants at 0.19 ± 0.08 MPa as compared with 0.05 ± 0.03 MPa in untreated controls. Enhanced cartilage phenotype coupled with increased cellular migration were noted under cLIUS (5 MHz), alluding to the observed integration between cartilage interfaces. Collectively, cLIUS at cell resonant frequency promoted integrative cartilage repair, therefore, has the potential to improve cartilage repair outcomes. Impact Statement Lack of integration between the host and graft cartilage interfaces impedes the success of cartilage repair techniques. Continuous low-intensity ultrasound (cLIUS) is documented to induce chondrogenesis and chondrocyte phenotype. However, integrative cartilage repair under cLIUS has not been evaluated. Our results demonstrated integration between cartilage interfaces, increased percent apposition, increased strength of integration, and maintenance of cartilage phenotype under cLIUS (5 MHz). Integrative repair under cLIUS (5 MHz) stemmed from enhanced migration of cells and increased expression of cartilage-specific genes, namely SOX9 and COL2A1. Thus, cLIUS has the potential to improve the outcomes of grafting protocols for cartilage repair.

Effects of rAAV-Mediated sox9 Overexpression on the Biological Activities of Human Osteoarthritic Articular Chondrocytes in Their Intrinsic Three-Dimensional Environment.

Gene therapy for osteoarthritis offers powerful, long-lasting tools that are well adapted to treat such a slow, progressive disorder, especially those therapies based on the clinically adapted recombinant adeno-associated viral (rAAV) vectors. Here, we examined the ability of an rAAV construct carrying a therapeutic sequence for the cartilage-specific SOX9 transcription factor to modulate the phenotype of human osteoarthritic articular chondrocytes compared with normal chondrocytes in a three-dimensional environment where the cells are embedded in their extracellular matrix. Successful sox9 overexpression via rAAV was noted for at least 21 days, leading to the significant production of major matrix components (proteoglycans, type-II collagen) without affecting the proliferation of the cells, while the cells contained premature hypertrophic processes relative to control conditions (reporter rAAV-lacZ application, absence of vector treatment). These findings show the value of using rAAV to adjust the osteoarthritic phenotype when the chondrocytes are confined in their inherently altered environment and the possibility of impacting key cellular processes via gene therapy to remodel human osteoarthritic cartilage lesions.

Optimization of Protocol for Isolation of Chondrocytes from Human Articular Cartilage.

Cartilage tissue engineering has evolved as one of the therapeutic strategies for cartilage defect, which relies on a large number of viable chondrocytes. Because of limited availability of cartilage and low chondrocytes yield from cartilage, the need for an improve isolation protocol for maximum yield of viable cells is a key to achieving successful clinical constructs. This study optimizes and compares different protocols for isolation of chondrocytes from cartilage. We employed enzymatic digestion of cartilage using collagenase II and trypsin. The chondrocytes yield, growth kinetics, aggrecan, and collagen type 2 (COL2) expression were evaluated. Collagen type 1 (COL1) mRNA expression was assessed to monitor the possibility of chondrocytes dedifferentiation. Chondrocyte yield per gram of cartilage was significantly higher (P < 0.05) using collagenase II in Hank's balanced salt solution (HBSS) compared with 0.25% trypsin. The number of chondrocyte yield per gram was higher in cartilage digested with collagenase in HBSS compared with Dulbecco's modified Eagle medium/F12; however, the difference was not statistically significant. Chondrocytes seeded at lower densities had shorter population doubling time compared to those seeded at higher density. Protein and gene expression of chondrocyte phenotype indicates the expression of aggrecan and COL2. The expression of COL1 was significantly increased (P < 0.05) in passage 3 compared with primary chondrocytes. The mRNA expression of chondrocyte phenotype was similar in primary and passaged one cells. Collagenase in HBSS yield the highest number of viable chondrocytes and the isolated cells expressed chondrocyte phenotype. This protocol can be employed to generate large number of viable chondrocytes, particularly with limited cartilage biopsies.

Fabrication of biomimetic hydrogel for chondrocyte delivery

A biomimetic scaffold for tissue engineering is the systematic combination of chemical compositions and biological functions. The purpose of this study was to design and prepare the biomimetic hydrogel scaffolds mimicking cartilage extracellular matrix (ECM) with excellent performance. Hydrogels were fabricated by varying the composition and ratio of type I collagen (Col I) and modified hyaluronic acid (HA-CHO)/chondroitin sulfate (CS-ADH). The gelation of hydrogels occurred with the formation of hydrazone bond at mild conditions. Morphology, swelling ratio and compression property were investigated to characterize the physical properties of composite hydrogels. Chondrocytes were encapsulated in these hydrogels to evaluate their potential as scaffolds for chondrocyte delivery and cartilage tissue engineering. The results showed that hydrogel C8CH (the volume ratio of Col I, HA-CHO and CS-ADH was 4:1:1) had balanced mechanical and swelling properties, good cellular biocompatibility and excellent biological function to maintain chondrocyte phenotype and promote sulfated glycosaminoglycan (sGAG) secretion. Therefore, hydrogel C8CH may serve as a promising scaffold for cartilage repair.

Inactivation of FAM20B causes cell fate changes in annulus fibrosus of mouse intervertebral disc and disc defects via the alterations of TGF-β and MAPK signaling pathways

Intervertebral disc (IVD) disorder is often caused by the defect of annulus fibrosus (AF), especially that of the outer AF. Studies about the mechanisms governing the development of the outer AF are needed for a better understanding of pathogenesis of IVD defects. Glycosaminoglycans (GAGs) are essential components of extracellular matrix (ECM) in AF. FAM20B is a newly identified xylose kinase that catalyzes the biosynthesis of GAGs. In this study, we created Fam20B conditional knockout (cKO) mice in which FAM20B was inactivated in type I collagen-expressing cells, the main type of cells in the outer AF of IVD. The cKO mice showed severe spine deformity and remarkable IVD defects associated with AF malformation. The AF of cKO mice had a lower level of chondroitin sulfate and heparan sulfate, and the outer AF cells lost their normal fibroblast-like morphology and acquired chondrocyte phenotypes, expressing a higher level of Sox 9 and type II collagen along with a reduced level of type I collagen. The level of phospho-Smad 2 and phospho-Smad 3, and that of scleraxis, a downstream target molecule of canonical TGF-β signaling pathway were significantly lower in the AF of cKO mice. The AF in cKO mice also manifested altered levels in the molecules associated with the activations of MAPK pathway; the changes included the increase of phospho-P38 and phospho-ERK and a decrease of phospho-JNK. These results indicate that FAM20B plays an essential role in the development of AF by regulating the TGF-β signaling and MAPK signaling pathways.

Hyaluronan synthase 2 (HAS2) overexpression diminishes the procatabolic activity of chondrocytes by a mechanism independent of extracellular hyaluronan

Osteoarthritis (OA) is a progressive degenerative disease of the joints caused in part by a change in the phenotype of resident chondrocytes within affected joints. This altered phenotype, often termed proinflammatory or procatabolic, features enhanced production of endoproteinases and matrix metallo-proteinases (MMPs) as well as secretion of endogenous inflammatory mediators. Degradation and reduced retention of the proteoglycan aggrecan is an early event in OA. Enhanced turnover of hyaluronan (HA) is closely associated with changes in aggrecan. Here, to determine whether experimentally increased HA production promotes aggrecan retention and generates a positive feedback response, we overexpressed HA synthase-2 (HAS2) in chondrocytes via an inducible adenovirus construct (HA synthase-2 viral overexpression; HAS2-OE). HAS2-OE incrementally increased high-molecular-mass HA >100-fold within the cell-associated and growth medium pools. More importantly, our results indicated that the HAS2-OE expression system inhibits MMP3, MMP13, and other markers of the procatabolic phenotype (such as TNF-stimulated gene 6 protein (TSG6)) and also enhances aggrecan retention. These markers were inhibited in OA-associated chondrocytes and in chondrocytes activated by interleukin-1β (IL1β), but also chondrocytes activated by lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), or HA oligosaccharides. However, the enhanced extracellular HA resulting from HAS2-OE did not reduce the procatabolic phenotype of neighboring nontransduced chondrocytes as we had expected. Rather, HA-mediated inhibition of the phenotype occurred only in transduced cells. In addition, high HA biosynthesis rates, especially in transduced procatabolic chondrocytes, resulted in marked changes in chondrocyte dependence on glycolysis versus oxidative phosphorylation for their metabolic energy needs.

Construction of tissue-engineered cartilage with different biological scaffolds in vitro

This study aims to find out which polymeric scaffolds were more appropriate for the construction of tissue engineering cartilage. Rat costal cartilage chondrocytes were cultured in vitro. The first generation of chondrocytes was used as seed cells and inoculated in chitosan/gelatin and bone matrix gelatin (BMG)/fibrin glue scaffolds. Haematoxylin and eosin staining, toluidine blue staining, safranine O, immunological detection, and scanning electron microscopy (SEM) observations were implemented. After2 weeks, all stains of BMG/fibrin glue showed that chondrocytes were uniformly distributed on the surface and inside of the material, and collagen and type II collagen staining was positive. There were slightly more cells on chitosan/gelatin than on BMG/fibrin glue. However, the number of internal cells was small with non-uniform distribution and the staining result was not as apparent as that of BMG/fibrin glue. As the culture time lengthened, results displayed a large quantity of expression of specific collagen and typed II collagen of chondrocytes, with chitosan/gelatin showing obvious advantages over BMG/fibrin glue material. The chitosan/gelatin contributed to maintaining chondrocyte phenotype and secretion of the extracellular matrix. Tissue engineering cartilage was successfully constructed in vitro. Moreover, such tissue growth, proliferation and matrix secretion of chondrocytes on chitosan/gelatin were superior to those on BMG/fibrin glue scaffold.

Ibuprofen attenuates interleukin-1&amp;lt;bold&amp;gt;&amp;beta;&amp;lt;/bold&amp;gt;-induced inflammation and actin reorganization via modulation of RhoA signaling in rabbit chondrocytes

Ibuprofen, a medication in the nonsteroidal anti-inflammatory drug class, is widely used for treating inflammatory diseases such as osteoarthritis. It has been shown in recent years that ibuprofen has a strong effect on Ras homolog gene family, member A (RhoA) inhibition in multiple cell types. Our previous finding also demonstrated that interleukin-1β (IL-1β) increases filamentous actin (F-actin) of chondrocytes via RhoA pathway. Therefore, we hypothesized that ibuprofen may suppress the IL-1β-induced F-actin upregulation in chondrocytes by inhibiting RhoA pathway. To this end, in this study, articular chondrocytes from New Zealand White rabbits were pretreated with 500μM ibuprofen for 2h, then with 10ng/ml IL-1β for 24h. Results showed that pretreatment with ibuprofen inhibited the IL-1β-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, protected the chondrocyte phenotype from IL-1β stimulation, and inhibited the IL-1β-induced actin remodeling via RhoA signaling modulation. In conclusion, ibuprofen showed not only anti-inflammatory function, but also RhoA inhibition in articular chondrocytes.

Collagen microsphere based 3D culture system for human osteoarthritis chondrocytes (hOACs)

The current study aims to evaluate collagen microencapsulation as an in vitro 3D culture platform for human osteoarthritic chondrocytes (hOACs), and to exemplify its feasibility in screening potential disease modifying factors. We first isolated and expanded hOACs from osteoarthritis (OA) cartilage samples harvested from multiple patients during total knee replacement (TKR) surgery. These cells were microencapsulated into collagen microspheres for subsequent 3D cultures. The change in chondrocyte phenotypes and OA phenotype was evaluated over time, using 2D monolayer culture and traditional 3D pellet culture as controls. The hOACs in the 3D collagen microsphere model resumed their in vivo phenotypes when compared to 2D monolayer. When compared with the 3D pellet model, the 3D hOAC-collagen microsphere model better recapitulated the OA phenotypes. We further demonstrated the responsiveness of the microencapsulated hOACs towards a number of external factors altering the chondrogenic phenotype, corroborating with previous studies. The hOAC encapsulated collagen microspheres better maintained the hOAC phenotype than the traditional 2D monolayer and 3D pellet cultures. The feasibility to use this hOAC-collagen microsphere in vitro model as a screening platform for disease-modifying agents has been demonstrated, contributing to future development of OA therapeutics.

Ethanol Alters Phenotype and Synthesis Activity of Rat Neonatal Articular Chondrocytes Grown in 2- and 3-Dimensional Culture.

We sought to evaluate the effect of different concentrations of ethanol on phenotype and activity of articular chondrocyte synthesis of neonatal rats in 2-dimensional (2D) and 3-dimensional (3D) culture. Chondrocytes were cultured in chondrogenic medium with different concentrations of ethanol: 0.0% v/v (control); 0.05% v/v (8.6 mM); 0.25% v/v (42.9 mM), and 0.5% v/v (85.7 mM). Chondrocytes under 2D culture were subjected to MTT assay, while chondrocytes under 3D culture were processed for paraffin inclusion and stained by periodic acid Schiff (PAS) to evaluate mean chondrocyte diameter and percentages of cells, nucleus, cytoplasm, well-differentiated matrix, and PAS+ areas. The expression of gene transcripts for aggrecan, Sox9, and type II collagen was evaluated by real-time quantitative polymerase chain reaction. There was no difference between groups by the MTT assay. PAS staining revealed that chondrocytes treated with 0.5% v/v ethanol had higher percentages of cytoplasm and nuclear areas, but with a reduction in PAS+ matrix area. The mean diameter of chondrocytes was similar between groups. The expression of aggrecan in the group treated with 0.5% v/v ethanol was lower in comparison to that in the control. In the groups treated with 0.25% v/v and 0.5% v/v ethanol, the percentage of differentiated cartilage was lower in comparison with that in the control. The group treated with 0.05% v/v ethanol was similar to the control in all parameters. Ethanol acted directly on in vitro cultured articular chondrocytes of newborn rats, altering the chondrocyte phenotype and its synthesis activity, and these effects were dose dependent.

Tissue Engineering: An Alternative to Repair Cartilage.

Herein we review the state-of-the-art in tissue engineering for repair of articular cartilage. First, we describe the molecular, cellular, and histologic structure and function of endogenous cartilage, focusing on chondrocytes, collagens, extracellular matrix, and proteoglycans. We then explore in vitro cell culture on scaffolds, discussing the difficulties involved in maintaining or obtaining a chondrocytic phenotype. Next, we discuss the diverse compounds and designs used for these scaffolds, including natural and synthetic biomaterials and porous, fibrous, and multilayer architectures. We then report on the mechanical properties of different cell-loaded scaffolds, and the success of these scaffolds following in vivo implantation in small animals, in terms of generating tissue that structurally and functionally resembles native tissue. Last, we highlight future trends in this field. We conclude that despite major technical advances made over the past 15 years, and continually improving results in cartilage repair experiments in animals, the development of clinically useful implants for regeneration of articular cartilage remains a challenge

Effect of hesperidin on anti-inflammation and cellular antioxidant capacity in hydrogen peroxide-stimulated human articular chondrocytes

Excessive levels of oxidative stress can induce a senescence-associated secretory phenotype in chondrocytes which is characterized by the secretions of inflammatory mediators. Citrus flavonoid hesperidin (Hsd) is known to have anti-inflammatory and antioxidant capacities. Accordingly, we demonstrated the antioxidant property of Hsd against hydrogen peroxide (H2O2)-induced oxidative stress damages to chondrocytes in this study. Primary human chondrocytes were cultured in media supplemented with Hsd (5, 10, 50 and 100 μM) to evaluate the dose-dependent cytotoxicity. Subsequently, cells were stimulated by H2O2 and supplied with Hsd to study chondroprotective effects. Results showed that high-dose of Hsd (50 and 100 μM) has a deleterious effect on normal cells, whilst low Hsd (5 and 10 μM) supplements improved mitochondrial activity, cytotoxicity, proliferation kinetic, survival and cell senescence in H2O2-stimulated chondrocytes. Real-time PCR revealed that Hsd downregulated the mRNA levels of COX-2, IL-1β, TNF-α, MMP-3, MMP-9, and upregulated IL-10, TIMP-1, SOX9. Western blotting further revealed that Hsd modulates Foxo1, Foxo3 and Nrf2 signaling pathways. Furthermore, Hsd improved total antioxidant capacity, and restored superoxide dismutases and glutathione peroxidase activities in H2O2-stimulated chondrocytes. In conclusion, our findings suggest that Hsd has the potential to improve antioxidant capacity in human chondrocytes.

Mechanical stimulation promotes the proliferation and the cartilage phenotype of mesenchymal stem cells and chondrocytes co-cultured in vitro

Mesenchymal stem cells and chondrocytes are an important source of the cells for cartilage tissue engineering. Therefore, the culture and expansion methods of these cells need to be improved to overcome the aging of chondrocytes and induced chondrogenic differentiation of mesenchymal stem cells. The aim of this study was to expand the cells for cartilage tissue engineering by combining the advantages of growing cells in co-culture and under a mechanically-stimulated environment. Rabbit chondrocytes and co-cultured cells (bone mesenchymal stem cells and chondrocytes) were subjected to cyclic sinusoidal dynamic tensile mechanical stimulationusing the FX-4000 tension system. Chondrocyte proliferation was assayed by flow cytometry and CFSE labeling. The cell cartilage phenotype was determined by detecting GAG, collagen II and TGF-β1 protein expression by ELISA and the Col2α1, TGF-β1 and Sox9 gene expression by RT-PCR. The results show that the co-culture improved both the proliferation ability of chondrocytes and the cartilage phenotype of co-cultured cells. A proper cyclic sinusoidal dynamic tensile mechanical stimulation improved the proliferation ability and cartilage phenotype of chondrocytes and co-cultured cells. These results suggest that the co-culture of mesenchymal stem cells with chondrocytes and proper mechanical stimulation may be an appropriate way to rapidly expand the cells that have an improved cartilage phenotype for cartilage tissue engineering.

Silk fibroin/carboxymethyl chitosan hydrogel with tunable biomechanical properties has application potential as cartilage scaffold

Tissue engineering is a promising strategy for cartilage repair and regeneration. However, an ideal scaffolding material that not only mimics the biomechanical properties of the native cartilage, but also supports the chondrogenic phenotype of the seeding cells is in need. In this study, we developed a silk fibroin (SF) and carboxymethyl chitosan (CMCS) composite hydrogel with enzymatic cross-links (horseradish peroxidase and hydrogen peroxide) and β-sheet cross-links (ethanol treatment). Results of Fourier transform infrared (FTIR), thermal gravimetric analysis (TGA), and X-ray diffraction (XRD) verified that SF/CMCS composite hydrogels had a tunable β-sheet structure. Therefore, by increasing the time of ethanol treatment from 0 h to 8 h, a series of parameters including pore size (from 50 to 300 μm), equilibrium swelling (from 78.1 ± 2.6% to 91.9 ± 0.9%), degradation (from 100% to 9% reduction in mass over 56 days), rheological properties (storage modulus from 177 Pa to 88,904 Pa), and mechanical properties (compressive modulus from 13 to 829 kPa) of the hydrogels were adjusted. In particular, the material parameters of the hydrogels with 2 h ethanol treatment appeared most suitable for engineered cartilage. Furthermore, the in vitro cellular experiments showed that the hydrogels supported the adhesion, proliferation, glycosaminoglycan synthesis, and chondrogenic phenotype of rabbit articular chondrocytes. Finally, subcutaneous implantation of the hydrogels in mice showed no infections or local inflammatory responses, indicating a good biocompatibility in vivo. In conclusion, the chemical-physical cross-linking SF/CMCS composite hydrogels, with tunable material properties and degradation rate, good biocompatibility, are promising scaffolds for cartilage tissue engineering.

Inducible knockout of CHUK/IKK\u03b1 in adult chondrocytes reduces progression of cartilage degradation in a surgical model of osteoarthritis

CHUK/IKKα contributes to collagenase-driven extracellular matrix remodeling and chondrocyte hypertrophic differentiation in vitro, in a kinase-independent manner. These processes contribute to osteoarthritis (OA), where chondrocytes experience a phenotypic shift towards hypertrophy concomitant with abnormal matrix remodeling. Here we investigated the contribution of IKKα to OA in vivo. To this end, we induced specific IKKα knockout in adult chondrocytes in AcanCreERT2/+; IKKαf/f mice treated with tamoxifen (cKO). Vehicle-treated littermates were used as wild type controls (WT). At 12 weeks of age, WT and cKO mice were subjected to the destabilization of medial meniscus (DMM) model of post-traumatic OA. The cKO mice showed reduced cartilage degradation and collagenase activity and fewer hypertrophy-like features at 12 weeks after DMM. Interestingly, in spite of the protection from structural articular cartilage damage, the postnatal growth plates of IKKα cKO mice after DMM displayed abnormal architecture and composition associated with increased chondrocyte apoptosis, which were not as evident in the articular chondrocytes of the same animals. Together, our results provide evidence of a novel in vivo functional role for IKKα in cartilage degradation in post-traumatic OA, and also suggest intrinsic, cell-autonomous effects of IKKα in chondrocytes that control chondrocyte phenotype and impact on cell survival, matrix homeostasis, and remodeling.