Cartilage ECM Research Articles

Calcium signaling in response to fluid flow by chondrocytes in 3D alginate culture

Quantifying the effects of mechanical loading on the metabolic response of chondrocytes is difficult due to complicated structure of cartilage ECM and the coupled nature of the mechanical stimuli presented to the cells. In this study we describe the effects of fluid flow, particularly hydrostatic pressure and wall shear stress, on the Ca(2+) signaling response of bovine articular chondrocytes in 3D culture. Using well-established alginate hydrogel system to maintain spherical chondrocyte morphology, we altered solid volume fraction to change scaffold mechanics. Fluid velocities in the bulk of the scaffolds were directly measured via an optical technique and scaffold permeability and aggregate modulus was characterized to quantify the mechanical stimuli presented to cells. Ca(2+) signaling response to direct perfusion of chondrocyte-seeded scaffolds increased monotonically with flow rate and was found more directly dependent on fluid velocity rather than shear stress or hydrostatic pressure. Chondrocytes in alginate scaffolds responded to fluid flow at velocities and shear stresses 2-3 orders of magnitude lower than seen in previous monolayer studies. Our data suggest that flow-induced Ca(2+) signaling response of chondrocytes in alginate culture may be due to mechanical signaling pathways, which is influenced by the 3D nature of cell shape.

Mesenchymal stem cells on a decellularized cartilage matrix for cartilage tissue engineering

An ideal scaffold for cartilage tissue engineering should be biomimetic in not only its biochemical composition, but also in the morphological structure of the scaffold. In this study, we fabricated a scaffold with an oriented structure using a nanofibrous articular cartilage extracellular matrix (ACECM), in which the ACECM was used to mimic the biochemical composition and oriented structure of articular cartilage. Histology analysis showed that the scaffold contained cartilage ECM (GAGs and collagen II). Scanning electron microscopy (SEM) indicated that the scaffolds were composed of nanofibers and possessed vertical microtubules. Chondrogenic differentiation-induced mesenchymal stem cells (MSCs) were seeded on the scaffold in vitro. SEM showed that MSCs proliferated well and aligned along the vertical microtubules, which mimicked the orientation of deep zone articular cartilage. A cell proliferation assay and live/dead cell staining demonstrated that the ACECM possessed good cell affinity, which favored cell adherence and proliferation. The MSCs that had been labeled with the fluorescent dye PKH26 and seeded on scaffolds were implanted into nude mice. The differentiated cells/ACECM implants formed cartilage-like tissue 4 weeks after implantation, and stained positive for collagen type II and toluidine blue. In addition, the in vivo fluorescent images verified that the MSCs in the implants were the labeled MSCs. These results demonstrated that the oriented ACECM scaffolds hold great promise for use in cartilage tissue engineering applications.

Contrast agent electrostatic attraction rather than repulsion to glycosaminoglycans affords a greater contrast uptake ratio and improved quantitative CT imaging in cartilage

The purpose of this study is to evaluate the effect of contrast agent charge on the contrast agent uptake ratio (CUR) in cartilage and to image the naturally occurring variations in glycosaminoglycan (GAG) content present in bovine articular cartilage. In an exvivo bovine osteochondral plug model, we utilized three charged contrast agents (Gadopentetate/Magnevist [-2], Ioxaglate/Hexabrix [-1], and CA4+ [+4]) and μCT to image cartilage. The X-ray attenuation of the cartilage tissue after equilibration in each contrast agent was also related to the initial X-ray attenuation of each contrast agent in solution to compute the uptake of the respective contrast agent (i.e., the CUR). Use of the cationic contrast agent resulted in significantly higher equilibrium X-ray attenuations in cartilage ECM than either of the anionic contrast agents (Gadopentetate [-2] and Ioxaglate [-1]). The CUR (Mean±SD) as computed in this study was 2.38 (±0.26) for the cationic contrast agent indicating a 2.38 fold increase in computed tomography (CT) attenuation of the cartilage. For the anionic contrast agents, the CUR was 0.62 (±0.26) for Ioxaglate [-1] and 0.52 (±0.17) for Gadopentetate [-2], indicating exclusion of 38% Ioxaglate and 48% Gadopentetate from the cartilage extracellular matrix. The cationic contrast agent exhibited significant correlations between CT attenuation and GAG content whereas Ioxaglate and Gadopentetate did not (R(2)=0.83 for CA4+, R(2)=0.20 for Ioxaglate, and R(2)=0.22 for Gadopentetate). Electrostatic attraction of CA4+ allowed effective imaging of the GAG components of articular cartilage at 50% lower molar concentration than Ioxaglate and 20-fold lower molar concentration than Gadopentetate. The CA4+ contrast agent exhibited a significant correlation between CT attenuation and GAG content in exvivo bovine osteochondral plugs.

Fabrication and cell affinity of biomimetic structured PLGA/articular cartilage ECM composite scaffold

An ideal scaffold for cartilage tissue engineering should be biomimetic in not only mechanical property and biochemical composition, but also the morphological structure. In this research, we fabricated a composite scaffold with oriented structure to mimic cartilage physiological morphology, where natural nanofibrous articular cartilage extracellular matrix (ACECM) was used to mimic the biochemical composition, and synthetic PLGA was used to enhance the mechanical strength of ACECM. The composite scaffold has well oriented structure and more than 89% of porosity as well as about 107 μm of average pore diameter. The composite scaffold was compared with ACECM and PLGA scaffolds. Cell proliferation test showed that the number of MSCs in ACECM and composite scaffolds was noticeably bigger than that in PLGA scaffold, which was coincident with results of SEM observation and cell viability staining. The water absorption of ACECM and composite scaffolds were 22.1 and 10.2 times respectively, which was much higher than that of PLGA scaffolds (3.8 times). The compressive modulus of composite scaffold in hydrous status was 1.03 MPa, which was near 10 times higher than that of hydrous ACECM scaffold. The aforementioned results suggested that the composite scaffold has the potential for application in cartilage tissue engineering.

Microscale Versus Nanoscale Scaffold Architecture for Mesenchymal Stem Cell Chondrogenesis

Nanofiber scaffolds, produced by the electrospinning technique, have gained widespread attention in tissue engineering due to their morphological similarities to the native extracellular matrix. For cartilage repair, studies have examined their feasibility; however these studies have been limited, excluding the influence of other scaffold design features. This study evaluated the effect of scaffold design, specifically examining a range of nano to micron-sized fibers and resulting pore size and mechanical properties, on human mesenchymal stem cells (MSCs) derived from the adult bone marrow during chondrogenesis. MSC differentiation was examined on these scaffolds with an emphasis on temporal gene expression of chondrogenic markers and the pluripotent gene, Sox2, which has yet to be explored for MSCs during chondrogenesis and in combination with tissue engineering scaffolds. Chondrogenic markers of aggrecan, chondroadherin, sox9, and collagen type II were highest for cells on micron-sized fibers (5 and 9 μm) with pore sizes of 27 and 29 μm, respectively, in comparison to cells on nano-sized fibers (300 nm and 600 to 1400 nm) having pore sizes of 2 and 3 μm, respectively. Undifferentiated MSCs expressed high levels of the Sox2 gene but displayed negligible levels on all scaffolds with or without the presence of inductive factors, suggesting that the physical features of the scaffold play an important role in differentiation. Micron-sized fibers with large pore structures and mechanical properties comparable to the cartilage ECM enhanced chondrogenesis, demonstrating architectural features as well as mechanical properties of electrospun fibrous scaffolds enhance differentiation.

Identification of miR-1 as a micro RNA that supports late-stage differentiation of growth cartilage cells

The process of endochondral ossification is strictly regulated by a variety of extracellular and intracellular factors. Recently, it has become recognized that specific miRNAs are involved in this process by regulating the expression of the relevant genes at the post-transcriptional level. In this present study we obtained the first evidence of the involvement of a specific micro RNA (miRNA) in the regulation of the chondrocyte phenotype during late stages of differentiation. By use of the microarray technique, miR-1 was identified as this miRNA, the expression of which was most repressed upon hypertrophic differentiation. Transfection of human chondrocytic HCS-2/8 cells and chicken normal chondrocytes with miR-1 led to repressed expression of aggrecan, the major cartilaginous proteoglycan gene. Therefore, miR-1 was found to be involved in the regulation of the chondrocytic phenotype and thus to play an important role in chondrocytes during the late stage of the differentiation process, maintaining the integrity of the cartilage tissue.

Stimuli-responsive chitosan-starch injectable hydrogels combined with encapsulated adipose-derived stromal cells for articular cartilage regeneration

Tissue engineering strategies have been showing promising early results in articular cartilage lesions repair. Hydrogels based on natural origin polymers as chitosan glycerol-phosphate (CGP) thermo-sensitive formulation that can be implanted in a minimal invasive manner, represent a great promise as injectable scaffold choice for cartilage tissue engineering, but it lacks in mechanical properties. A different formulation, from which a firm texture gels results is, therefore, desirable. In this work we first aim to investigate the suitability of CGP to produce an injectable thermosensitive, pH-dependent solution, when combined with increasing concentrations of starch: 0.5% (I), 1% (II), and 1.5% (III). The data collected from the rheological measurements showed that the addition of starch to the CGP did not alter the transition temperature and confirmed the heating inducing gelation of all solutions, supporting the ability of these novel formulations to be applied as minimal invasive systems. The evaluation of the dynamic mechanical analysis of the hydrogels showed an increase in the storage modulus within increasing starch concentration, clearly demonstrating that best viscoelastic properties were obtained with the novel chitosan-starch based solution. The incorporation of starch also improved the degradation profile. All materials showed to be biocompatible through the cytotoxicity screening in vitro. These data suggested the potential of novel thermo-responsive chitosan-starch hydrogels to be used as injectable vehicles for cell delivery in cartilage tissue engineering applications. In a second phase, the potential of chitosan-β-glycerophosphate (CGP) and chitosan-β-glycerophosphate-1% starch (CST) hydrogels to induce chondrocytic differentiation and cartilage matrix accumulation were evaluated, as well as the influence of starch in the chondrogenesis of encapsulated adipose derived stromal (ADSC) cells. The ADSC were homogeneously encapsulated, remained viable, proliferated, and maintained the expression of typical chondrogenic markers genes, and deposited cartilage ECM molecules. Improved results were obtained within the novel CST constructs. The overall data suggest that chitosan-β-glycerophosphate-starch hydrogels could be considered for chondrogenic differentiation of adipose derived stromal cells for cartilage-engineered regeneration using minimal invasive techniques.

Cartilage ECM molecules are differentially expressed between nasal and articular bovine chondrocytes

Bovine nasal septum is frequently used in studies of cartilage biology (Xu et al. 1996; Watanabe et al. 1994), and is often preferred over articular cartilage as it contains fewer collagen cross-links and gives a higher yield of tissue per animal. The extracellular matrix surrounding the chondrocytes within nasal cartilage differs from that of articular cartilage and so regulation of matrix proteins might be expected to differ between the two cell types (Parkkinen et al. 1993). We sought to compare the expression profiles of various matrix protein genes between these two types of cartilage, and to examine their regulation in chondrocyte monolayer and alginate bead culture.

Cartilage ECM molecules are differentially expressed between nasal and articular bovine chondrocytes

Cartilage ECM molecules are differentially expressed between nasal and articular bovine chondrocytes