Cell-derived Extracellular Matrix Scaffold Research Articles

Bioactive Cell-Derived ECM Scaffold Forms a Unique Cellular Microenvironment for Lung Tissue Engineering.

Chronic lung diseases are one of the leading causes of death worldwide. Lung transplantation is currently the only causal therapeutic for lung diseases, which is restricted to end-stage disease and limited by low access to donor lungs. Lung tissue engineering (LTE) is a promising approach to regenerating a replacement for at least a part of the damaged lung tissue. Currently, lung regeneration is limited to a simplified local level (e.g., alveolar–capillary barrier) due to the sophisticated and complex structure and physiology of the lung. Here, we introduce an extracellular matrix (ECM)-integrated scaffold using a cellularization–decellularization–recellularization technique. This ECM-integrated scaffold was developed on our artificial co-polymeric BETA (biphasic elastic thin for air–liquid interface cell culture conditions) scaffold, which were initially populated with human lung fibroblasts (IMR90 cell line), as the main generator of ECM proteins. Due to the interconnected porous structure of the thin (<5 µm) BETA scaffold, the cells can grow on and infiltrate into the scaffold and deposit their own ECM. After a mild decellularization procedure, the ECM proteins remained on the scaffold, which now closely mimicked the cellular microenvironment of pulmonary cells more realistically than the plain artificial scaffolds. We assessed several decellularization methods and found that 20 mM NH4OH and 0.1% Triton X100 with subsequent DNase treatment completely removed the fibroblasts (from the first cellularization) and maintains collagen I and IV as the key ECM proteins on the scaffold. We also showed the repopulation of the primary fibroblast from human (without chronic lung disease (non-CLD) donors) and human bronchial epithelial (16HBE14o−) cells on the ECM-integrated BETA scaffold. With this technique, we developed a biomimetic scaffold that can mimic both the physico-mechanical properties and the native microenvironment of the lung ECM. The results indicate the potential of the presented bioactive scaffold for LTE application.

Evaluation of an Autologous Bone Mesenchymal Stem Cell-Derived Extracellular Matrix Scaffold in a Rabbit and Minipig Model of Cartilage Repair.

BackgroundThis study aimed to evaluate an autologous bone mesenchymal stem cell (MSC)-derived extracellular matrix (ECM) scaffold in two animal models of cartilage repair.Material/MethodsA rabbit model (n=16) and a minipig model (n=8) of cartilage repair were created with cartilage defects of the knee joints treated with bone marrow stimulation (BMS). In the ECM group, autologous bone MSC-derived ECM scaffolds were implanted into the cartilage defects after bone marrow stimulation. In the BMS group, the cartilage defects were treated by bone marrow stimulation only. The renewal capacity of bone MSCs was measured with a colony-forming unit fibroblast (CFU-F) in vitro assay. The extent of cartilage repair was as-sessed at 6 months after surgery.ResultsIn the rabbit model, the macroscopic appearance of the exudate of the healing wounds in the ECM group showed less fibrosis, and the histology showed more evenly distributed chondrocytes compared with the BMS group. The CFU-F assay showed that the number of bone MSCs in the ECM group was approximately was twice that of the BMS group. In the minipig model, the macroscopic appearance and magnetic resonance imaging (MRI) findings of the ECM group were improved when compared with the BMS group. The repaired tissue in ECM group had similar histological characteristics and biochemical content to normal hyaline cartilage.ConclusionsIn two animal models of knee joint cartilage repair, the use of an ECM scaffold increased the number of bone MSCs and improved the extent of cartilage repair.

Genipin-crosslinked adipose stem cell derived extracellular matrix-nano graphene oxide composite sponge for skin tissue engineering.

Autologous skin grafts, which can cause donor site morbidity, are currently used to treat deep wounds. To improve the regeneration of poorly healing wounds, cell-derived extracellular matrix (ECM) scaffolds are garnering great research interest due to their associated lower risks of pathogen transfer and immune rejection. However, the mechanical properties of cell-derived ECM scaffolds are inferior when compared to those of tissue-derived ECM scaffolds. To overcome this drawback, different amounts (10, 20, 50, and 100 μg mL-1) of graphene oxide (GO) and genipin (1% w/v) were applied to adipose stem cell (ASC)-derived ECM sponges. There are still only a few studies employing cell-derived extracellular matrices as biomimetic scaffolds for biomedical applications. The aim of our study was to develop biocompatible, biodegradable, low immunogenic, and genipin-crosslinked ASC-derived ECM sponges containing a suitable amount of GO for skin-tissue engineering. Sponges were fabricated using cultures of ASCs, cell sheets, and decellularization of an ASC cell sheet, freeze-thawing, and crosslinking in a sequential manner. Scanning electron microscopic analyses of the sponges demonstrated a highly porous microstructure with a pore size of 71.22 ± 19.52 μm. The in vitro degradation rate was found to be significantly higher in the non-crosslinked ECM sponges and pure ECM sponges than in the genipin-crosslinked ECM sponges. During an in vivo study, we investigated the material feasibilities and degradability of the constructed ECM sponges as a suitable skin tissue-engineering scaffold in a xenogenic animal (rat) model for 4 weeks. After subcutaneous implantation, the ECM sponges containing a medium amount of GO showed appropriate biodegradation with a lower inflammatory reaction. Hence, the fabricated ECM sponges might be a suitable xenogenous skin substitute for full-thickness skin defects and in other future soft-tissue engineering applications, such as healing partial tears of the anterior cruciate ligament.

Composite Scaffolds of Mineralized Natural Extracellular Matrix on True Bone Ceramic Induce Bone Regeneration Through Smad1/5/8 and ERK1/2 Pathways.

A stem cell-derived mineralized extracellular matrix (ECM) may be a good strategy to endow scaffolds with a bone microenvironment, thus inducing bone regeneration. However, it also faces some challenges, such as limited number of cells, additional wound for autologous cell isolation, and time consumption of cell expansion. In this study, we designed a novel tissue-derived ECM scaffold fabricated by depositing porcine small intestinal submucosa (SIS) ECM on true bone ceramic (TBC), which was followed by mineralization treatment (mSIS/TBC). In vitro, compared with pure TBC, mSIS/TBC promoted cell proliferation, cell viability, and osteoblastic differentiation of the newly seeded rat bone marrow mesenchymal stem cells (BMSCs), and upregulation of the messenger RNA (mRNA) level of osteogenesis-related genes. Western blot assay revealed that mSIS/TBC enhanced osteoblastic differentiation through activation of phosphorylated Smad1/5/8 and phosphorylated extracellular signal-regulated kinase (ERK), as an underlying mechanism. In vivo, in a rat cranial critical size defect model, mSIS/TBC scaffolds induced greater bone formation than pure TBC scaffolds. Meanwhile, a comparative study on the capacity of bone regeneration was also carried out between mSIS/TBC and BMSC-derived ECM deposited on TBC scaffold in vivo and in vitro. The results demonstrated that mSIS/TBC scaffolds acquired a comparable bone regeneration efficacy to that of BMSC-derived ECM deposited on TBC scaffolds. Collectively, our results demonstrated that mSIS/TBC enhanced bone regeneration by supporting cell proliferation and cell viability, and by activating Smad1/5/8 and ERK1/2 signal pathways of BMSC in vitro and in vivo; thus, mSIS/TBC is an excellent alternative to stem cell-derived ECM scaffold for bone regeneration.

A Drug-Induced Hybrid Electrospun Poly-Capro-Lactone: Cell-Derived Extracellular Matrix Scaffold for Liver Tissue Engineering.

Liver transplant is the only treatment option for patients with end-stage liver failure, however, there are too few donor livers available for transplant. Whole organ tissue engineering presents a potential solution to the problem of rapidly escalating donor liver shortages worldwide. A major challenge for liver tissue engineers is the creation of a hepatocyte microenvironment; a niche in which liver cells can survive and function optimally. While polymers and decellularized tissues pose an attractive option for scaffold manufacturing, neither alone has thus far proved sufficient. This study exploited cell's native extracellular matrix (ECM) producing capabilities using two different histone deacetylase inhibitors, and combined these with the customizability and reproducibility of electrospun polymer scaffolds to produce a "best of both worlds" niche microenvironment for hepatocytes. The resulting hybrid poly-capro-lactone (PCL)-ECM scaffolds were validated using HepG2 hepatocytes. The hybrid PCL-ECM scaffolds maintained hepatocyte growth and function, as evidenced by metabolic activity and DNA quantitation. Mechanical testing revealed little significant difference between scaffolds, indicating that cells were responding to a biochemical and topographical profile rather than mechanical changes. Immunohistochemistry showed that the biochemical profile of the drug-derived and nondrug-derived ECMs differed in ratio of Collagen I, Laminin, and Fibronectin. Furthermore, the hybrid PCL-ECM scaffolds influence the gene expression profile of the HepG2s drastically; with expression of Albumin, Cytochrome P450 Family 1 Subfamily A Polypeptide 1, Cytochrome P450 Family 1 Subfamily A Polypeptide 2, Cytochrome P450 Family 3 Subfamily A Polypeptide 4, Fibronectin, Collagen I, and Collagen IV undergoing significant changes. Our results demonstrate that drug-induced hybrid PCL-ECM scaffolds provide a viable, translatable platform for creating a niche microenvironment for hepatocytes, supporting in vivo phenotype and function. These scaffolds offer great potential for tissue engineering and regenerative medicine strategies for whole organ tissue engineering.

Cell-Derived Extracellular Matrix: Basic Characteristics and Current Applications in Orthopedic Tissue Engineering.

The extracellular matrix (ECM) is a dynamic and intricate microenvironment with excellent biophysical, biomechanical, and biochemical properties, which can directly or indirectly regulate cell proliferation, adhesion, migration, and differentiation, as well as plays key roles in homeostasis and regeneration of tissues and organs. The ECM has attracted a great deal of attention with the rapid development of tissue engineering in the field of regenerative medicine. Tissue-derived ECM scaffolds (also referred to as decellularized tissues and whole organs) are considered a promising therapy for the repair of musculoskeletal defects, including those that are widely used in orthopedics, although there are a few shortcomings. Similar to tissue-derived ECM scaffolds, cell-derived ECM scaffolds also have highly advantageous biophysical and biochemical properties, in particular their ability to be produced in vitro from a number of different cell types. Furthermore, cell-derived ECM scaffolds more closely resemble native ECM microenvironments. The products of cell-derived ECM have a wide range of biomedical applications; these include reagents for cell culture substrates and biomaterials for scaffolds, hybrid scaffolds, and living cell sheet coculture systems. Although cell-derived ECM has only just begun to be investigated, it has great potential as a novel approach for cell-based tissue repair in orthopedic tissue engineering. This review summarizes and analyzes the various types of cell-derived ECM products applied in cartilage, bone, and nerve tissue engineering in vitro or in vivo and discusses future directions for investigation of cell-derived ECM.

Chondrogenic Differentiation Could Be Induced by Autologous Bone Marrow Mesenchymal Stem Cell-Derived Extracellular Matrix Scaffolds Without Exogenous Growth Factor.

We previously found that the combination of an autologous bone mesenchymal stem cell-derived extracellular matrix (aBMSC-dECM) scaffold with bone marrow stimulation could enhance hyaline cartilage regeneration. We suspected that chondrogenic differentiation could be induced by the aBMSC-dECM scaffold. This study aimed to investigate whether aBMSC-dECM scaffolds could promote chondrogenic differentiation without exogenous growth factors. BMSCs were seeded on aBMSC-dECM scaffolds and cultured in vitro with or without transforming growth factor-β3 (E(+) or E(-) group). Atelocollagen scaffolds were used as controls (C(+) or C(-) group). The chondrogenic differentiation was evaluated by histological, biochemical, and real-time polymerase chain reaction assays. After 3 weeks, cartilage-like tissue with a homogeneous structure, a high cartilaginous matrix content (proteoglycan and type II collagen), and high expression levels of cartilage-associated genes (COL2A1, ACAN, and SOX9) were observed in the E(+), E(-), and C(+) groups. In addition, BMSCs in each scaffold (E group or C group) were preconditioned with chondrogenic media in vitro for 1 week, and then implanted in the backs of nude mice for 3 weeks. Three weeks later, cartilage matrix formation (proteoglycan and type II collagen) was achieved only in the E group, confirmed by safranin O staining and immunohistochemical staining for type II collagen. Taken together, these results indicate that aBMSC-dECM scaffolds could induce chondrogenic differentiation. Thus, they could be successful candidate scaffolds for cartilage tissue engineering.

Nitric oxide regulates cell behavior on an interactive cell-derived extracellular matrix scaffold.

During tissue injury and wound healing process, there are dynamic reciprocal interactions among cells, extracellular matrix (ECM), and mediating molecules which are crucial for functional tissue repair. Nitric oxide (NO) is one of the key mediating molecules that can positively regulate various biological activities involved in wound healing. Various ECM components serve as binding sites for cells and mediating molecules, and the interactions further stimulate cellular activities. Human mesenchymal stem cells (hMSCs) can migrate to the wound site and contribute to tissue regeneration through differentiation and paracrine signaling. The objective of this work was to investigate the regulatory effect of NO on hMSCs in an interactive ECM-rich microenvironment. In order to mimic the in vivo stromal environment in wound site, a cell-derived ECM scaffold that was able to release NO within the range of in vivo wound fluid NO level was fabricated. Results showed that the micro-molar level of NO released from the ECM scaffold had an inhibitory effect on cellular activities of hMSCs. The NO impaired cell growth, altered cell morphology, disrupted the F-actin organization, also decreased the expression of focal adhesion related molecules integrin α5 and paxillin. These results may contribute to the elucidation of how NO acts on hMSCs in wound healing process.

新型 BMSC外基质支架的制备 及性状研究

Objective To fabricate a novel biological scaffold using bone marrow stem cells (BMSCs)-derived extracellular matrix and investigate the feasibility of the scaffold used in cartilage tissue engineering.Methods Bone marrows were isolated from immature New Zealand rabbits to further harvest second-or third-generation BMSCs.ECM derived from BMSCs was collected after high-density culture and was fabricated into a three-dimensional porous scaffold using a freeze-dying technique.Physicochemical properties of the scaffolds were determined by histology,immunohistochemistry,scanning electron microscope,porosity and water absorption measurement,and biomechanical study.Third-generation chondrocytes harvested from knee joints of adult New Zealand rabbits were seeded at a density of 5.0 ×104 cells/ml with Dulbecco' s modified Eagle' s medium (DMEM) (control group),scaffold lixivium at volume fraction of 50％ (study group Ⅰ),and scaffold lixivium at volume fraction of 100％ (study group Ⅱ),respectively.At days 1,3,and 7 after co-culture,MTT test was done to assess the absorbance and cytotoxicity of the scaffold.Results Scaffolds appeared white/silver white/ivory color.HE staining and scanning electron microscopy demonstrated that the scaffolds were cross-linked and pore interconnected with pore size of 100-400 μm,porosity of (91.50 ±2.84) ％,water absorption rate of (2 014 ± 224) ％,and compression strength of (6.80 ± 1.50) kPa.MTT test showed that the chondrocytes were proliferated well in each group and that the absorbance value was increased with time.Besides,the absorbance value was significantly different within groups at each time point (P ＜ 0.05).Conclusion Novel three-dimensional porous scaffolds made from BMSCs-derived ECM are acellular,safe and non-toxic,exhibit favorable pore size and porosity and good biocompatibility,and hence can be good scaffolds for cartilage tissue engineering. Key words: Extracellular matrix; Mesenchymal stem cells; Biocompatible materials; Cartilage tissue engineering

Chitosan/silk fibroin-based, Schwann cell-derived extracellular matrix-modified scaffolds for bridging rat sciatic nerve gaps

Extracellular matrix (ECM) plays a prominent role in establishing and maintaining an ideal microenvironment for tissue regeneration, and ECM scaffolds are used as a feasible alternative to cellular and molecular therapy in the fields of tissue engineering. Because of their advantages over tissue-derived ECM scaffolds, cultured cell-derived ECM scaffolds are beginning to attract attention, but they have been scarcely studied for peripheral nerve repair. Here we aimed to develop a tissue engineered nerve scaffold by reconstituting nerve cell-derived ECM with natural biomaterials. A protocol was adopted to prepare and characterize the cultured Schwann cell (SC)-derived ECM. A chitosan conduit and silk fibroin (SF) fibers were prepared, cultured with SCs for ECM deposition, and subjected to decellularization, followed by assembly into a chitosan/SF-based, SC-derived ECM-modified scaffold, which was used to bridge a 10 mm rat sciatic nerve gap. The results from morphological analysis as well as electrophysiological examination indicated that regenerative outcomes achieved by our developed scaffold were similar to those by an acellular nerve graft (namely a nerve tissue-derived ECM scaffold), but superior to those by a plain chitosan/SF scaffold. Moreover, blood and histopathological parameters confirmed the safety of scaffold modification by SC-derived ECM. Therefore, a hybrid scaffold based on joint use of acellular and classical biomaterials represents a promising approach to nerve tissue engineering.

Application of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold on tissue engineering cartilage in vitro

Objective To evaluate the feasibility of the formation of tissue engineering cartilage in vitro by using autologous bone marrow mesenchymal stem cell-derived extracellular matrix (aBMSC-dECM)scaffold.Methods The autologous bone marrow mesenchymal stem cells were harvested from 5 New Zealand white rabbits which are two weeks old.The extracellular matrix was collected after 4 weeks culture and fabricated into aBMSC-dECM scaffold.The scaffold was investigated by a scanning electron microscope and HE staining.The autologous articular chondrocytes were isolated,cultured and seeded into the aBMSC-dECM scaffold.Live-Dead staining was analyzed 48 h after seeding.Gross morphological and volume measurement,HE staining,Safranin-O staining and type Ⅱ collagen immunohistochemistry staining,RT-PCRassay,and compression strength test were operated for the cell-scaffold composite at 1,2,4,and 6 weeks after the cultivation respectively.Atelocollagen scaffold was used as the control group.Results The aBMSC-dECM scaffold was three-dimensional and spongy.The engineered cartilage in aBMSC-dECM scaffold group appeared milk white in color with smooth and glossy surface.Compared with the atelocollagen scaffold group,the volume of engineered cartilage in aBMSC-dECM scaffold group significantly grew with time,the chondrocyte,proteoglycan and type Ⅱ collagen were gradually accumulated,the mRNA of type Ⅱ collage and Aggrecan were constantly expressed,and the compressive strength significantly increased with time.Conclusion The aBMSC-dECM scaffold could enhance the viability and biological function of chondrocytes,and promote engineered cartilage regeneration.It could be a novel candidate scaffold for cartilage tissue engineering. Key words: Mesenchymal stem cells; Cartilage; Tissue engineering; In vitro

Comparison of decellularization techniques for preparation of extracellular matrix scaffolds derived from three‐dimensional cell culture

Extracellular matrix (ECM) scaffolds derived from cultured cells have drawn increasing attention for use in tissue engineering. We have developed a method to prepare cultured cell-derived ECM scaffolds by combining three-dimensional cell culture, decellularization, and selective template removal. Cell-ECM-template complexes were first formed by culture of cells in a poly(lactic-co-glycolic acid) (PLGA) mesh template to deposit their own ECM. The complexes were subsequently decellularized to remove cellular components. Finally, the PLGA template was selectively removed to obtain the ECM scaffolds. Seven decellularization methods were compared for their decellularization effects during scaffold preparation. They were: freeze-thaw cycling (-80°C, six times) with ammonia water (25 mM); 0.1% Triton™ X-100 (TX100) with 1.5M KCl aqueous solution; freeze-thaw cycling alone; ammonia water alone; TX100 extraction; osmotic shock with 1.5M KCl; and freeze-thaw cycling with 3M NaCl. Among these methods, the methods of freeze-thaw cycling with NH(4) OH and TX100 with 1.5M KCl showed the best effect on the removal of cellular components from the complexes, while the other five methods could only partially remove cellular components. The ECM scaffolds prepared by these two methods had similar gross appearances and microstructures. In vivo implantation of the ECM scaffolds prepared by these two methods induced mild host responses. The two decellularization methods were demonstrated to be effective for preparation of cultured cell-derived ECM scaffolds.

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.

Cartilage engineering using cell‐derived extracellular matrix scaffold in vitro

A cell-derived extracellular matrix (ECM) scaffold was constructed using cultured porcine chondrocytes via a freeze-drying method, and its ability to promote cartilage formation was evaluated in vitro. Scanning electron microscope (SEM) revealed that the scaffold had highly uniform porous microstructure. Then, rabbit chondrocytes were seeded dynamically on ECM scaffold and cultured for 2 days, 1, 2, and 4 weeks in vitro for analysis. Polyglycolic acid (PGA) scaffold was used as a control. On gross observation of neocartilage tissue, a silvery white cartilage-like tissue was observed after 1 week of culture in ECM scaffold, while similar morphology was seen only after 4 weeks in PGA scaffold. The volume of neocartilage-like tissue was significantly increased in both ECM and PGA groups. The compressive strength was gradually increased with time in ECM group, while gradually decreased in PGA group. DNA, glycosaminoglycan (GAG) and collagen contents also increased gradually with time in both groups, but showed more significant increase in ECM group. Histological staining for GAG (Safranin O staining) and type II collagen (immunohistochemistry) showed sustained accumulation of ECM molecules with time, which gradually and uniformly filled porous space in ECM scaffold. On the contrary, they accumulated only at the peripheral area of PGA scaffold. These results suggest that a novel cell-derived ECM scaffold can provide a promising environment for generating a high quality cartilage in vitro.

Crosslinking of cell-derived 3D scaffolds up-regulates the stretching and unfolding of new extracellular matrix assembled by reseeded cells

Elevated levels of tissue crosslinking are associated with numerous diseases (cancer stroma, organ fibrosis), and also eliminate the otherwise remarkable clinical successes of tissue-derived scaffolds, instead eliciting a foreign body reaction. Nevertheless, it is not well understood how the initial physical and biochemical properties of cellular microenvironments, stem cell niches, or of 3D tissue scaffolds guide the assembly and remodeling of new extracellular matrix (ECM) that is ultimately sensed by cells. Here, we incorporated FRET-based mechanical strain sensors, either into cell-derived ECM scaffolds or into the fibronectin (Fn) matrix assembled by reseeded fibroblasts, and demonstrated the following. Cell-generated tensile forces change the conformation of Fn in both 3D scaffolds and new matrix over time. The time course by which new matrix fibers are stretched by reseeded cells is accelerated by scaffold crosslinking. Importantly, stretching Fn fibers increases their elastic modulus (rigidity) and alters their biochemical display. Regulated by Fn fiber unfolding, more soluble Fn binds to the native than to the crosslinked scaffolds. Additionally, matrix assembly of fibroblasts is decreased by scaffold crosslinking. Taken together, scaffold crosslinking has a multifactorial impact on the microenvironment that reseeded cells assemble and respond to, with far-reaching implications for tissue engineering and disease physiology.

In Vivo Cartilage Tissue Engineering Using a Cell‐Derived Extracellular Matrix Scaffold

We have observed in our previous study that a cell-derived extracellular matrix (ECM) scaffold could assure the growth of a cartilage tissue construct in vitro. The purpose of the present study was to evaluate the feasibility of a chondrocyte-seeded cell-derived ECM scaffold by implanting it in vivo in nude mouse. A porous cell-derived ECM scaffold was prepared with a freeze-drying protocol using porcine chondrocytes. Rabbit articular chondrocytes were seeded onto the scaffold and cultured for 2 days in vitro, and then implanted into the nude mouse subcutaneously. They were retrieved at 1, 2, and 3 weeks postimplantation. Under macroscopic analysis, the cartilage-like tissue formation matured with time and developed a smooth, white surface. Contrary to the control (in which no cells were seeded), the size of the neocartilage tissue increased slightly by the third week and remained more stable. Total glycosaminoglycan (GAG) content and the GAG/DNA ratio increased significantly with time in the chemical analysis. The histology exhibited a sustained accumulation of newly synthesized sulfated proteoglycans. Immunohistochemistry, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR) clearly identified type II collagen at all time points. Compressive strength of in vivo neocartilage increased from 0.45 +/- 0.06 MPa at 1 week to 1.18 +/- 0.17 MPa at 3 weeks. In conclusion, this study demonstrated that the cell-derived ECM scaffold could provide chondrocytes with favorable in vivo environment to produce a hyaline-like cartilage tissue.