Redifferentiation Of Human Articular Chondrocytes Research Articles

The Critical Role of Hypoxia in the Re-Differentiation of Human Articular Chondrocytes.

The preservation of the chondrogenic phenotype and hypoxia-related physiological microenvironment are major challenges in the 2D culture of primary human chondrocytes. To address this problem, we develop a 3D culture system generating scaffold-free spheroids from human chondrocytes. Our results highlight the chondrogenic potential of cultured human articular chondrocytes in a 3D system combined with hypoxia independently of the cartilage source. After 14 days of culture, we developed spheroids with homogenous diameter and shape from hyaline cartilage donors. Spheroids generated in hypoxia showed a significantly increased glycosaminoglycans synthesis and up-regulated the expression of SOX9, ACAN, COL2A1, COMP, and SNAI1 compared to those obtained under normoxic conditions. Therefore, we conclude that spheroids developed under hypoxic conditions modulate the expression of chondrogenesis-related genes and native tissue features better than 2D cultures. Thus, this scaffold-free 3D culture system represents a novel in vitro model that can be used for cartilage biology research.

Chondroitin Sulfate- and Decorin-Based Self-Assembling Scaffolds for Cartilage Tissue Engineering

Cartilage injury and degenerative tissue progression remain poorly understood by the medical community. Therefore, various tissue engineering strategies aim to recover areas of damaged cartilage by using non-traditional approaches. To this end, the use of biomimetic scaffolds for recreating the complex in vivo cartilage microenvironment has become of increasing interest in the field. In the present study, we report the development of two novel biomaterials for cartilage tissue engineering (CTE) with bioactive motifs, aiming to emulate the native cartilage extracellular matrix (ECM). We employed a simple mixture of the self-assembling peptide RAD16-I with either Chondroitin Sulfate (CS) or Decorin molecules, taking advantage of the versatility of RAD16-I. After evaluating the structural stability of the bi-component scaffolds at a physiological pH, we characterized these materials using two different in vitro assessments: re-differentiation of human articular chondrocytes (AC) and induction of human adipose derived stem cells (ADSC) to a chondrogenic commitment. Interestingly, differences in cellular morphology and viability were observed between cell types and culture conditions (control and chondrogenic). In addition, both cell types underwent a chondrogenic commitment under inductive media conditions, and this did not occur under control conditions. Remarkably, the synthesis of important ECM constituents of mature cartilage, such as type II collagen and proteoglycans, was confirmed by gene and protein expression analyses and toluidine blue staining. Furthermore, the viscoelastic behavior of ADSC constructs after 4 weeks of culture was more similar to that of native articular cartilage than to that of AC constructs. Altogether, this comparative study between two cell types demonstrates the versatility of our novel biomaterials and suggests a potential 3D culture system suitable for promoting chondrogenic differentiation.

Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular chondrocytes

Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose-derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three-dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de- and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL-expanded HACs demonstrated better redifferentiation potential than FCS-expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT-PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL-expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation.

TGFβ inhibition during expansion phase increases the chondrogenic re-differentiation capacity of human articular chondrocytes

Autologous chondrocyte implantation is a cell-based treatment to repair articular cartilage defects, relying on the availability of expanded (de-differentiated) chondrocytes. Unfortunately, the expansion process causes several phenotypical changes, requiring re-establishment of the native chondrogenic phenotype to sustain proper repair. Among other proteins, transforming growth factor-β (TGFβ) is known to influence the chondrogenic re-differentiation of human articular chondrocytes (HACs) and their matrix deposition. Thus we investigated the effects of TGFβ-depletion during the expansion phase. HACs were isolated from articular cartilage and expanded in the canonical serum-supplemented medium [fetal calf serum (FCS)] or in a chemically-defined (CD) medium, with or without anti-TGFβ antibody administration. The re-differentiation potential of the cells was assessed by pellet cultures, gene expression analysis and histology. Cell proliferation proceeded more rapidly in CD-medium than in FCS-medium; it was not affected by the use of anti-TGFβ antibody but was further increased by addition of exogenous TGFβ1, via increased p-Smad1/5/8. Conversely, in FCS-medium, addition of anti-TGFβ antibody decreased both proliferation and p-Smad1/5/8 level. Challenging either FCS- or CD-medium with anti-TGFβ antibody during expansion enhanced chondrogenesis in the subsequent pellet cultures. Moreover, TGFβ-depletion during expansion in CD-medium inhibited mRNA expression of hypertrophic markers, collagen type-X (COL10) and matrix metalloproteinase-13 (MMP-13). Interestingly, the TGFβ1 level detected by enzyme-linked immunosorbent sandwich assay (ELISA) during cell expansion was correlated with COL10 mRNA expression after re-differentiation. TGFβ-depletion during expansion improves the re-differentiation capacity of chondrocytes and inhibits hypertrophy. These results indicate the importance of the expansion medium composition to improve chondrogenic re-differentiation and to inhibit hypertrophy.

Redifferentiation of human articular chondrocytes in 2D versus 3D culture

Redifferentiation of human articular chondrocytes in 2D versus 3D culture

P203 Phenotypic characterization and redifferentiation of human articular chondrocytes expanded on microcarriers

P203 Phenotypic characterization and redifferentiation of human articular chondrocytes expanded on microcarriers

Gene expression during redifferentiation of human articular chondrocytes

Objective The aim of the present study was to investigate gene expression during the in vitro redifferentiation process of human articular chondrocytes isolated from clinical samples from patient undergoing an autologous chondrocyte transplantation therapy (ACT). Method Monolayer (ML) expanded human articular chondrocytes from four donors were cultured in a 3D pellet model and the redifferentiation was investigated by biochemistry, histology, immunohistochemistry and microarray analysis. Results The culture expanded chondrocytes redifferentiated in the pellet model as seen by an increase in collagen type II immunoreactivity between day 7 and 14. The gene expression from ML to pellet at day 7 included an increase in cartilage matrix proteins like collagen type XI, tenascin C, dermatopontin, COMP and fibronectin. The late phase consisted of a strong downregulation of extracellular signal-regulated protein kinase (ERK-1) and an upregulation of p38 kinase and SOX-9, suggesting that the late phase mimicked parts of the signaling processes involved in the early chondrogenesis in limb bud cells. Other genes, which indicated a transition from proliferation to tissue formation, were the downregulated cell cycle genes GSPT1 and the upregulated growth-arrest-specific protein (gas). The maturation of the pellets included no signs of hypertrophy or apoptosis as seen by downregulation of collagen type X, Matrix Gla protein and increased expression of caspase 3. Conclusion Our data show that human articular chondrocytes taken from surplus cells of patient undergoing ACT treatment and expanded in ML, redifferentiate and form cartilage like matrix in vitro and that this dynamic process involves genes known to be expressed in early chondrogenesis.