Chondrocyte-derived Extracellular Matrix Research Articles

In situ ornamenting poly(ε-caprolactone) electrospun fibers with different fiber diameters using chondrocyte-derived extracellular matrix for chondrogenesis of mesenchymal stem cells

Biomimetic instructive tissue engineering scaffolds are critical for achieving successful tissue regeneration. In the present study, we developed a novel scaffold via ornamenting poly(ε-caprolactone) (PCL) electrospun fibers with a chondrocyte-derived extracellular matrix (ECM)-coating, which was applied for chondrogenesis of mesenchymal stem cells (MSCs). PCL fibrous films with different fiber diameters (1282±121 nm, 549±61 nm and 285±38 nm) were first prepared via electrospinning. Rabbit articular chondrocytes (rACs) were cultured on PCL fibrous scaffolds, followed by a decellularization treatment to generate decellularized ECM (dECM)-coated PCL scaffolds (dECM/PCL). Rabbit bone marrow-derived MSCs (rMSCs) were then seeded onto these scaffolds and adhesion, proliferation and chondrogenic differentiation were evaluated. dECM/PCL scaffolds displayed distinct surface microstructural features with varying fiber diameters and fibrous mesh-like ECM with more developed collagen fibers was observed on nanofibers. On dECM/PCL scaffolds, rMSCs tended to spread more at 24 h post-seeding and proliferated better within 7 d compared to those on uncoated PCL scaffolds. Based on analysis of gene expression, rMSCs underwent the best chondrogenic differentiation on dECM/PCL scaffolds of 549-nm fibers. Collectively, such dECM/PCL composite scaffolds are very promising for cartilage tissue regeneration.

The Chondro-Osseous Continuum: Is It Possible to Unlock the Potential Assigned Within?

Endochondral ossification (EO), by which long bones of the axial skeleton form, is a tightly regulated process involving chondrocyte maturation with successive stages of proliferation, maturation, and hypertrophy, accompanied by cartilage matrix synthesis, calcification, and angiogenesis, followed by osteoblast-mediated ossification. This developmental sequence reappears during fracture repair and in osteoarthritic etiopathology. These similarities suggest that EO, and the cells involved, are of great clinical importance for bone regeneration as it could provide novel targeted approaches to increase specific signaling to promote fracture healing, and if regulated appropriately in the treatment of osteoarthritis. The long-held accepted dogma states that hypertrophic chondrocytes are terminally differentiated and will eventually undergo apoptosis. In this mini review, we will explore recent evidence from experiments that revisit the idea that hypertrophic chondrocytes have pluripotent capacity and may instead transdifferentiate into a specific sub-population of osteoblast cells. There are multiple lines of evidence, including our own, showing that local, selective alterations in cartilage extracellular matrix (ECM) remodeling also indelibly alter bone quality. This would be consistent with the hypothesis that osteoblast behavior in long bones is regulated by a combination of their lineage origins and the epigenetic effects of chondrocyte-derived ECM which they encounter during their recruitment. Further exploration of these processes could help to unlock potential novel targets for bone repair and regeneration and in the treatment of osteoarthritis.

Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells

The extracellular matrix (ECM) surrounding cells contains a variety of proteins that provide structural support and regulate cellular functions. Previous studies have shown that decellularized ECM isolated from tissues or cultured cells can be used to improve cell differentiation in tissue engineering applications. In this study we evaluated the effect of decellularized chondrocyte-derived ECM (CDECM) on the chondrogenesis of human placenta-derived mesenchymal stem cells (hPDMSCs) in a pellet culture system. After incubation with or without chondrocyte-derived ECM in chondrogenic medium for 1 or 3 weeks, the sizes and wet masses of the cell pellets were compared with untreated controls (hPDMSCs incubated in chondrogenic medium without chondrocyte-derived ECM). In addition, histologic analysis of the cell pellets (Safranin O and collagen type II staining) and quantitative reverse transcription-PCR analysis of chondrogenic markers (aggrecan, collagen type II, and SOX9) were carried out. Our results showed that the sizes and masses of hPDMSC pellets incubated with chondrocyte-derived ECM were significantly higher than those of untreated controls. Differentiation of hPDMSCs (both with and without chondrocyte-derived ECM) was confirmed by Safranin O and collagen type II staining. Chondrogenic marker expression and glycosaminoglycan (GAG) levels were significantly higher in hPDMSC pellets incubated with chondrocyte-derived ECM compared with untreated controls, especially in cells precultured with chondrocyte-derived ECM for 7 d. Taken together, these results demonstrate that chondrocyte-derived ECM enhances the chondrogenesis of hPDMSCs, and this effect is further increased by preculture with chondrocyte-derived ECM. This preculture method for hPDMSC chondrogenesis represents a promising approach for cartilage tissue engineering.

Effects of topical chondrocyte-derived extracellular matrix treatment on corneal wound healing, following an alkali burn injury.

Numerous treatments have been used in the management of corneal chemical burns; however, no optimal treatment for corneal chemical burns currently exists. The present study investigated the effects of topical chondrocyte-derived extracellular matrix (CD-ECM) treatment on corneal wound healing, using an alkali burn mouse model. Topical treatment with CD-ECM was shown to reduce corneal opacity following an alkali burn. A histological examination observed the presence of regenerated epithelial cells and a small number of inflammatory cells in the corneas of CD-ECM-treated mice. The majority of the inflammatory cells present in the corneas of the phosphate-buffered saline (PBS)-treated mice were neutrophils that expressed matrix metalloproteinase (MMP)-9. The amount of neutrophils was significantly reduced in the corneas of the CD-ECM-treated mice. Furthermore, the expression levels of interleukin (IL)-8 were significantly reduced in the CD-ECM treatment group, but not in the mice that received the PBS treatment. The results of the present study indicate that CD-ECM treatment may accelerate wound healing in a model of alkali burn-induced corneal injury. The therapeutic mechanism may be associated with accelerated reepithelialization and reduced recruitment of MMP-9-expressing neutrophils, through inhibiting the production of IL-8.

Preparation and characterization of bone marrow mesenchymal stem cell‐derived extracellular matrix scaffolds

The aim of this study was to assess the feasibility of creating extracellular matrix (ECM) scaffolds from mesenchymal stem cells. Bone marrow mesenchymal stem cell (BMSC)-derived ECM (BMSC-dECM) scaffolds were fabricated by lyophilization after crosslinking, without using a decellularization process. Acellular porcine chondrocyte-derived ECM (AC-dECM) scaffolds were used as a control. The surface morphology, internal structure, water uptake ratio, mechanical properties, and biocompatibility of the scaffolds, as well as the in vitro behavior of cells grown on the scaffolds were examined and compared between the two scaffold types. For the BMSC-dECM scaffolds, the average pore size was 304.4 ± 108.2 μm, average porosity was 93.3% ± 4.5%, average compressive modulus was 6.8 ± 1.5 kPa, and average water uptake ratio exceeded 20. The BMSC-dECM scaffolds supported the in vitro attachment and proliferation of cells, with these aspects likely being comparable to those of the AC-dECM scaffolds. The findings of this preliminary study highlight the potential utility of BMSC-derived ECM scaffolds for future cartilage tissue-engineering applications.

Anti-neovascular effect of chondrocyte-derived extracellular matrix on corneal alkaline burns in rabbits.

We investigated the effect of a chondrocyte-derived extracellular matrix (CDECM) on experimental corneal alkaline burns in rabbits. Corneal neovascularization (NV) was induced by applying an 8-mm filter paper soaked in 1N NaOH to the right central corneas of rabbits for 1minute. Ten days later, the rabbits were randomly divided into three groups: the alkaline burn group, the CDECM transplantation group, and the human amniotic membrane (HAM) transplantation group. The left eyes were used as controls. CDECM and HAM were transplanted onto the corneal surface to completely cover the resected area and were subsequently sutured. On the 10th day after transplantation, the structural changes of the cornea were analyzed histologically. We examined the effects of CDECM on clinical NV features and on the expression of corneal NV markers. The alkaline burn produced significant NV and increased the corneal thickness. On day 10 after transplantation, the thickness, NV and opacity of the cornea were markedly decreased in the CDECM group (p < 0.001). However, the HAM transplantation group did not exhibit improvements in these clinical parameters, and there were no significant differences relative to the burn group. In addition, the use of CDECM improved the healing of the cornea following the alkaline burn by disrupting the corneal epithelial proliferation and reducing the fibrotic changes of the stroma. The hallmarks of NV were significantly induced in the subepithelium by the alkaline burn, and these levels were also suppressed by CDECM. The CDECM suppressed corneal NV by inhibiting nuclear factor-kappa B (NF-κB) activation by blocking the PKC and Akt signaling pathways. CDECM transplantation was markedly effective in healing alkali-burned corneas by modulating the translocation of NF-κB to the nucleus, thereby representing a promising material for the noninvasive treatment of ocular surface disease.

Inhibition of blood vessel formation by a chondrocyte-derived extracellular matrix

In this study, the chondrocyte-derived extracellular matrix (CECM) was evaluated for its activity to inhibit vessel invasion in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) and rabbit chondrocytes were plated on a bio-membrane made of CECM or human amniotic membrane (HAM). The adhesion, proliferation, and tube formation activity of HUVECs and chondrocytes were examined. The CECM and HAM powders were then mixed individually in Matrigel and injected subcutaneously into nude mice to examine vessel invasion in vivo after 1 week. Finally, a rabbit model of corneal neovascularization (NV) was induced by 3-point sutures in the upper cornea, and CECM and HAM membranes were implanted onto the corneal surface at day 5 after suture injury. The rabbits were sacrificed at 7 days after transplantation and the histopathological analysis was performed. The adhesion and proliferation of HUVECs were more efficient on the HAM than on the CECM membrane. However, chondrocytes on each membrane showed an opposite result being more efficient on the CECM membrane. The vessel invasion in vivo also occurred more deeply and intensively in Matrigel containing HAM than in the one containing CECM. In the rabbit NV model, CECM efficiently inhibited the neovessels formation and histological remodeling in the injured cornea. In summary, our findings suggest that CECM, an integral cartilage ECM composite, shows an inhibitory effect on vessel invasion both in vitro and in vivo, and could be a useful tool in a variety of biological and therapeutic applications including the prevention of neovascularization after cornea injury.

Effect of porcine chondrocyte-derived extracellular matrix on the pterygium in mouse model

To investigate the effect of porcine chondrocyte-derived extracellular matrix (PCDECM) on an experimental mouse model of human pterygial epithelial cells. Cultured human pterygial epithelial cells (hPECs) were stained with pan-cytokeratin (CK), CK3/2p, vimentin, and CK13 antibodies to characterize the cells. A pterygium mouse model was developed by injecting 1X10⁴ hPECs into the nasal subconjunctival space in athymic nude mice. PCDECM (25 mg/mL, 10 μL) was injected into the nasal subconjunctival space in the right eye 7, 10 and 14 days after the epithelial cell injection (PCDECM group). Image analysis was performed using ImageJ® to compare the lesion size. A histopathological analysis of the cornea was conducted to evaluate the state of the epithelium and the expression of pterygial epithelial cell markers. The isolated pterygial cells were positive for pan-CK, CK3/2p and vimentin, and they were negative for CK13 under immunofluorescence microscopy. On day 17 after epithelial cell injection, the size of the lesion compared to the entire cornea was increased to 37.1 % in the control group. However, in the PCDECM group, the lesion covered only 26.3 % of the entire cornea. The corneas of the pterygium mice showed an epithelium of irregular thickness, proliferation of the stroma, extracellular matrix breakdown and overexpression of pterygium-positive markers. However, these changes were significantly suppressed by the application of PCDEDM. Our findings suggest that PCDECM seems to suppress pterygial epithelial cell growth and it could be used as a promising biomaterial for the noninvasive treatment of pterygium.

Cartilage tissue engineering using chondrocyte-derived extracellular matrix scaffold suppressed vessel invasion during chondrogenesis of mesenchymal stem cells in vivo

Loss of chondrogenic phenotypes of tissue engineered cartilage using mesenchymal stem cells (MSCs) in vivo are thought to be influenced by environmental factors like vessel invasion in particular. This study investigated effect of a chondrocyte-derived extracellular matrix (CD-ECM) scaffold on the hypertrophic changes and vessel invasion into tissue engineered cartilage using rabbit MSCs in comparison with a synthetic polyglycolic acid (PGA) scaffold. Rabbit MSCs in CD-ECM or PGA scaffold were differentiated for 1 week in vitro and implanted in the back of nude mice for 6 weeks in vivo. Gross observation showed red stains, indicative of vessel invasion, increased along with the loss of chondrogenic phenotype in safranin-O stains, which was more prominent in the PGA constructs. The area showing loss of chondrogenic phenotypes in safranin-O stain was correlated well with the mineralized area in the von kossa stain and the area with vessel-like structures in the gomori aldehyde fuchsin stain at 6 weeks in terms of their size and distribution. Also, vessel invasion took place more deeply and intensively into the constructs, in accordance with the expression of angiogenic markers (CD31, VEGF-A and HIF-1α) and a macrophage marker (CD68). This phenomenon progressed much more rapidly in the PGA constructs than in the CDECM constructs, and correlated well with the loss of chondrogenic phenotypes. In conclusion, this study showed that tissue engineered cartilage using the CD-ECM scaffold maintained better chondrogenic phenotypes in vivo and showed lower levels of hypertrophic changes and vessel invasion.

Maintenance of cartilaginous gene expression on extracellular matrix derived from serially passaged chondrocytes during in vitro chondrocyte expansion

The loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier for the application of cartilage tissue engineering. The use of matrices mimicking the in vivo extracellular matrix (ECM) microenvironment is anticipated to be an efficient method to suppress chondrocyte phenotype loss. In this study, we developed several types of ECM derived from serially passaged chondrocytes for use as cell-culture substrata and compared their effects on chondrocyte functions. Primary bovine chondrocytes and serially passaged chondrocytes (at passages 2 and 6) were cultured on tissue-culture polystyrene. After culture, the cellular components were selectively removed from the ECM deposited by the cells. The remaining ECM proteins were used as cell-culture substrata. The composition of the deposited ECM depended on the culture stage of the serially passaged chondrocytes used for the ECM production. The deposited ECM supported the adhesion and proliferation of chondrocytes. The effects of the ECM on the chondrocyte dedifferentiation during in vitro passage culture differed dramatically depending on the phenotype of the chondrocytes used to produce the ECM. The primary chondrocyte-derived ECM delayed the chondrocyte dedifferentiation during in vitro passage culture and is a good candidate for chondrocyte subculture for tissue engineering.

Cultured cell-derived extracellular matrix scaffolds for tissue engineering

Cell-derived extracellular matrix (ECM) scaffolds have received considerable interest for tissue engineering applications. In this study, ECM scaffolds derived from mesenchymal stem cell (MSC), chondrocyte, and fibroblast were prepared by culturing cells in a selectively removable poly(lactic-co-glycolic acid) (PLGA) template. These three types of ECM scaffolds were used for in vitro cultures of MSC and fibroblasts to examine their potential as scaffolds for cartilage and skin tissue engineering. The MSC were cultured in MSC- and chondrocyte-derived ECM scaffolds. The ECM scaffolds supported cell adhesion, promoted both cell proliferation and the production of ECM and demonstrated a stronger stimulatory effect on the chondrogenesis of MSC compared with a conventional pellet culture method. Histological and immunohistochemical staining indicated that cartilage-like tissues were regenerated after the MSC were cultured in ECM scaffolds. Fibroblasts were cultured in the fibroblast-derived ECM scaffolds. Fibroblasts proliferated and produced ECM to fill the pores and spaces in the scaffold. After 2 weeks of culture, a uniform multilayered tissue was generated with homogenously distributed fibroblasts. Cell-derived ECM scaffolds have been demonstrated to facilitate tissue regeneration and will be a useful tool for tissue engineering.

Assembly of a chondrocyte-like pericellular matrix on non-chondrogenic cells Role of the cell surface hyaluronan receptors in the assembly of a pericellular matrix

In this study, we have examined the capacity of various cell types, which express cell surface hyaluronan receptors, to organize a chondrocyte-like pericellular matrix when given chondrocyte-derived extracellular matrix macromolecules exogenously. The assembly of a pericellular matrix was visualized by a particle exclusion assay. Without the addition of exogenous macromolecular components, none of the cell types studied exhibited significant pericellular matrices extending from their plasma membranes. However, upon the addition of high molecular weight hyaluronan in combination with aggregating cartilage proteoglycan monomers, large pericellular matrices were formed within two hours of incubation. No pericellular matrices were formed if these macromolecular components were added separately at equivalent concentrations or if the components were added in the presence of hyaluronan hexasaccharide, a competitive inhibitor of hyaluronan interaction with cell surface hyaluronan receptors. Fully assembled pericellular matrices could also be displaced by the subsequent addition of hyaluronan hexasaccharides. Nonliving, glutaraldehyde-fixed cells, which retained functional hyaluronan receptors, maintained the capacity to assembly pericellular matrices with exogenous components, in serum-containing or serum-free medium. Cells that were incubated with exogenous matrix macromolecules for 24 h, followed by a chase incubation in medium minus the exogenous macromolecules, continued to maintain the matrix for up to 6 h on live cells and more than 24 h on glutaraldehyde-fixed cells. Cell types that did not express hyaluronan receptors were not capable of organizing such pericellular matrices when incubated with these exogenous components. These findings suggest that cells expressing hyaluronan receptors have a significant capacity to organize their immediate extracellular environment via hyaluronan-hyaluronan receptor interactions. Possible physiological functions for this type of matrix organizing capacity are discussed.