Extracellular Matrix Analogs Research Articles

In vivo and in vitro applications of collagen-GAG scaffolds

Tissue engineering scaffolds are used extensively as three-dimensional analogs of the extracellular matrix (ECM). Collagen-glycosaminoglycan (CG) scaffolds have long been utilized as ECM analogs for the regeneration of skin and are currently being considered for the regeneration of nerve, conjunctiva, and a host or orthopedic tissues. Recently a series of CG scaffolds with a uniform pore microstructure has been developed with a range of sizes of equiaxed pores. Experimental characterization and theoretical modeling techniques have been used to describe the pore microstructure, specific surface area, tensile and compressive mechanical properties, cell attachment, and permeability of these variants. Here we describe the fabrication, and characterization, and modeling of a series of CG and mineralized CG scaffolds. We then discuss their use in vivo to induce tissue regeneration following injury and in vitro as standardized 3D materials to study the influence of microstructural and mechanical features on cell behaviors such as motility and contraction.

Prediction of extracellular matrix stiffness in engineered heart valve tissues based on nonwoven scaffolds

The in vitro development of tissue engineered heart valves (TEHV) exhibiting appropriate structural and mechanical characteristics remains a significant challenge. An important step yet to be addressed is establishing the relationship between scaffold and extracellular matrix (ECM) mechanical properties. In the present study, a composite beam model accounting for nonwoven scaffold-ECM coupling and the transmural collagen concentration distribution was developed, and utilized to retrospectively estimate the ECM effective stiffness in TEHV specimens incubated under static and cyclic flexure conditions (Engelmayr Jr et~al. in Biomaterials 26(2):175-187 2005). The ECM effective stiffness was expressed as the product of the local collagen concentration and the collagen specific stiffness (i.e., stiffness/concentration), and was related to the overall TEHV effective stiffness via an empirically determined scaffold-ECM coupling parameter and measured transmural collagen concentration distributions. The scaffold-ECM coupling parameter was determined by flexural mechanical testing of polyacrylamide gels (i.e., ECM analogs) of variable stiffness and associated scaffold-polyacrylamide gel composites (i.e., engineered tissue analogs). The transmural collagen concentration distributions were quantified from fluorescence micrographs of picro-sirius red stained TEHV sections. As suggested by a previous structural model of the nonwoven scaffold (Engelmayr Jr and Sacks in J Biomech Eng 128(4):610-622, 2006), nonwoven scaffold-ECM composites did not follow a traditional rule of mixtures. The present study provided further evidence that the primary mode of reinforcement in nonwoven scaffold-ECM composites is an increase in the number fiber-fiber bonds with a concomitant increase in the effective stiffness of the spring-like fiber segments. Simulations of potential ECM deposition scenarios using the current model indicated that the present approach is sensitive to the specific time course of tissue deposition, and is thus very suitable for studies of ECM formation in engineered heart valve tissues.

Hydrogel‐filled polylactide porous scaffolds for cartilage tissue engineering

Polymer porous scaffolds and hydrogels have been separately employed as analogues of the native extra-cellular matrix (ECM). However, both of these two kinds of materials have their own advantages and shortcomings. In this work, an attempt to combine the advantages of these two kinds of materials is carried out. Poly-L-lactide (PLLA) scaffolds with good mechanical properties were prepared by thermally induced phase separation, which were then filled with hydrogel aiming at entrapment of cells within a support of predefined shape. Agar, which has a function to promote chondrogenesis, was selected to entrap chondrocytes, acting as analogues of native ECM. A straight forward merit of this construct is that both mechanical strength and macroscopic shape, and analogous ECM can be simultaneously achieved. The morphology and distribution of the chondrocytes were studied by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The cell growth behaviors were determined by MTT assay and collagen and glycosaminoglycan (GAG) secretion. After culture for 7 and 14 days, the cells in the construct were round and surrounded by the hydrogel. The MTT viability and the cell secretion in the chondrocytes/agar/scaffold construct were also higher than that of the chondrocytes/scaffold construct (control). Gelatin was further introduced into the construct, yielding improved GAG secretion and cytoviability. After implantation in the subcutaneous dorsum of nude mice for 4 weeks, cartilage-like specimens maintaining their original rectangular shapes were harvested. Histological examination showed that new cartilage was regenerated and a large quantity of collagen and GAG were secreted, while the cells in the control PLLA scaffold turned to be fibroblast-like with less secretion of extracellular matrices. The method provides a useful pathway of scaffold preparation and cell transplantation, which can achieve suitable mechanical properties and good cell performance simultaneously.

Mechanical characterization of collagen–glycosaminoglycan scaffolds

Tissue engineering scaffolds are used extensively as three-dimensional analogs of the extracellular matrix (ECM). However, less attention has been paid to characterizing the scaffold microstructure and mechanical properties than to the processing and bioactivity of scaffolds. Collagen–glycosaminoglycan (CG) scaffolds have long been utilized as ECM analogs for the regeneration of skin and are currently being considered for the regeneration of nerve and conjunctiva. Recently a series of CG scaffolds with a uniform pore microstructure has been developed with a range of sizes of equiaxed pores. Experimental characterization and theoretical modeling techniques have previously been used to describe the pore microstructure, specific surface area, cell attachment and permeability of these variants. The results of tensile and compressive tests on these CG scaffolds and of bending tests on the individual struts that define the scaffold network are reported here. The CG scaffold variants exhibited stress–strain behavior characteristic of low-density, open-cell foams with distinct linear elastic, collapse plateau and densification regimes. Scaffolds with equiaxed pores were found to be mechanically isotropic. The independent effects of hydration level, pore size, crosslink density and relative density on the mechanical properties was determined. Independent control over scaffold stiffness and pore size was obtained. Good agreement was observed between experimental results of scaffold mechanical characterization and low-density, open-cell foam model predictions for uniform scaffolds. The characterized scaffold variants provide a standardized framework with defined extracellular environments (microstructure, mechanics) for in vitro studies of the mechanical interactions between cells and scaffolds as well as in vivo tissue engineering studies.

Development of a novel collagen–GAG nanofibrous scaffold via electrospinning

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen–GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen–GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen–GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen–GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds.

An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture

Fabrication of nanofibrous scaffolds with well-defined architecture mimicking native extracellular matrix analog has significant potentials for many specific tissue engineering and organs regeneration applications. The fabrication of aligned collagen nanofibrous scaffolds by electrospinning was described in this study. The structure and in vitro properties of these scaffolds were compared with a random collagen scaffold. All the collagen scaffolds were first crosslinked in glutaraldehyde vapor to enhance the biostability and keep the initial nano-scale dimension intact. From in vitro culture of rabbit conjunctiva fibroblast, the aligned scaffold exhibited lower cell adhesion but higher cell proliferation because of the aligned orientation of fibers when compared with the random scaffold. And the alignment of the fibers may control cell orientation and strengthen the interaction between the cell body and the fibers in the longitudinal direction of the fibers.

β 1 integrins modulate p66 ShcA expression and EGF-induced MAP kinase activation in fetal lung cells

ShcA proteins mediate Erk1/Erk2 activation by integrins and epidermal growth factor (EGF), and are expressed as p46 ShcA, p52 ShcA, and p66 ShcA. Although p52 ShcA and p46 ShcA mediate Erk1/Erk2 activation, p66 ShcA antagonizes Erk activation. p66 ShcA is spatially regulated during lung development, leading us to hypothesize that integrin signaling regulates p66 ShcA expression and, consequently, EGF signaling. Fetal lung mesenchymal cells were isolated from E16 Swiss–Webster mice, stimulated with oligopeptide extracellular matrix analogs or anti-integrin antibodies, and subjected to ShcA Western analyses and EGF-stimulated Erk1/Erk2 kinase assays. p66 ShcA expression was decreased by anti-α 1 integrin antibody and DGEA collagen analog, and increased by anti-β 1, anti-α 4, and anti-α 5 integrin antibodies and RGDS fibronectin analog. Paradoxically, β 1 integrin stimulation increased EGF-induced Erk activation while increasing expression of the inhibitory p66 ShcA isoform. This paradox was resolved by demonstrating that Erk inhibition attenuates integrin-mediated p66 ShcA induction. These results suggest that p66 ShcA is up-regulated as inhibitory feedback on integrin-mediated Erk activation.

Expression of alpha-smooth muscle actin by and contraction of cells derived from synovium.

Cells derived from synovium have drawn interest as donor cells for articular cartilage tissue engineering because they have been implicated in certain cartilage repair processes in vivo and the chondrogenic potential of the cells has been demonstrated in vitro. Studies have demonstrated that several other types of musculoskeletal connective tissue cells--including chondrocytes, fibrochondrocytes, ligament fibroblasts and osteoblasts, and mesenchymal stem cells can express the gene for the contractile actin isoform, alpha-smooth muscle actin (SMA), and can contract analogs of extracellular matrix in vitro. Although the physiological roles of SMA-enabled contraction of these cells have yet to be established, cell-mediated contraction of scaffolds employed for tissue engineering can alter the pore diameter of the matrix and distort its overall shape, and thus needs to be addressed. Toward this goal, the objective of this study was to investigate the expression of SMA by synovial cells and to evaluate their contraction of collagen-glycosaminoglycan (GAG) scaffolds. Synovial membranes obtained from the knees (stifle joints) of six adult dogs were evaluated for the presence of SMA by immunohistochemistry. Cells isolated from the synovial tissue were expanded through seven passages in monolayer culture, with samples from each passage allocated for Western blot analysis of SMA. Cells from passage 4 were seeded into porous type I collagen-GAG matrices and cultured for 4 weeks. Synovial cell-mediated contraction of the scaffolds was determined by measuring the diameters of the cell-seeded scaffolds and nonseeded controls every other day. Synovium-derived cells cultured as micropellets or in collagen-GAG matrices were incubated in chondrogenic medium with and without fetal bovine serum and evaluated for chondrogenesis by type II collagen immunohistochemistry. Immunohistochemistry revealed the presence of SMA in some cells (less than 10% of the cells) in the intimal layer of synovium from four of the five animals analyzed. Western blot analysis demonstrated a regular increase in the amount of SMA in the synovium-derived cells with passage number. Synovial cell-mediated contraction of the collagen-GAG scaffolds reached a value of 43% of the original diameter after 4 weeks, comparable to that found with other musculoskeletal cell types. Incubation of micropellet cultures of synovium-derived cells with chondrogenic medium revealed trace amounts of type II collagen production by immunohistochemistry. The findings of this study indicate that control of SMA-enabled contraction may be important when employing synovial cells for cartilage repair procedures, and warrant further investigation into the physiological role of SMA expression in synovial cells.

Contractile forces generated by articular chondrocytes in collagen-glycosaminoglycan matrices

The objective of the study was to directly measure the force of contraction of adult articular chondrocytes and to examine their contractile behavior in collagen-glycosaminoglycan analogs of extracellular matrix by live cell imaging in vitro. The contractile forces generated by passages 2 and 3 adult canine articular chondrocytes were measured using a cell force monitor. The contractile behavior of the cells was also directly imaged as they were cultured in collagen-glycosaminoglycan scaffolds. Passage 2 cells seeded in a collagen-glycosaminoglycan scaffold were capable of generating a force of 0.3 nN/cell. Chondrocytes subcultured through a third passage generated a force twice that level, paralleling the increase in the α-smooth muscle actin (SMA) content of the cells as demonstrated by Western blot analysis. Treatment of passage 3 cells with staurosporine reduced the force of contraction by approximately one-half, reflecting the effects of this agent in reducing the SMA content of the cells and disrupting the microfilaments. These values compare with 1 nN previously reported for lapine dermal fibroblasts of passage 5–7, using the same apparatus. Direct live cell imaging documented the contractile behavior of the articular chondrocytes in the collagen-glycosaminoglycan matrix in the time frame in which the force was directly measured in the cell force monitor. This imaging demonstrated how the cells acted individually and in unison to buckle the collagen struts making up the matrix. Adult articular chondrocytes are capable of generating a SMA-enabled force of contraction sufficient to model extracellular matrix molecules.

Effects of epidermal growth factor on fibroblast migration through biomimetic hydrogels.

We have previously reported on the development and use of synthetic hydrogel extracellular matrix (ECM) analogues that can be used to study the mechanisms of migration. These biomimetic hydrogels consist of bioinert poly(ethylene glycol) diacrylate derivatives with proteolytically degradable peptide sequences included in the backbone of the polymer and adhesion peptide sequences grafted into the network. Cells adhere to the hydrogel via interaction between the grafted adhesion ligands and receptors on the cell surface. The cells migrate through the three-dimensional system by secreting the appropriate proteolytic enzymes, which are involved in cell migration and are targeted to the peptide sequences incorporated in the backbone of the polymer. It was observed that cell migration has a biphasic dependence on adhesion ligand concentration, with optimal migration at intermediate ligand levels. In this study, we demonstrate that we can covalently attach epidermal growth factor (EGF) to PEG and graft them into the hydrogels. It was observed that EGF when tethered maintained mitogenic activity. It was also observed that fibroblast migration significantly increased in the presence of the grafted EGF through the collagenase-sensitive hydrogels. In addition, the increase in migration was found to be independent from the proliferative response of the cells. These synthetic ECM analogues allow one to systematically control identities and concentrations of biomolecules and are useful tools to study mechanisms of cell migration.

Expression of smooth muscle actin in connective tissue cells participating in fracture healing in a murine model

The role of α-smooth muscle actin (SMA)-expressing fibroblasts in the contraction of skin wounds has been known for three decades. Recent studies have demonstrated that osteoblasts can also express the gene for this contractile muscle actin isoform and can contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. These findings provided rationale for the hypothesis that SMA-expressing cells contribute to fracture healing by drawing the bone ends together. To begin to test this hypothesis, immunohistochemistry was employed to evaluate the distribution of connective tissue cells expressing SMA in a mouse model of successful fracture healing. The results demonstrated that the majority of the cells comprising the mesenchymal tissue interposed between the fracture ends contained SMA after 7 and 21 days, supporting the working hypothesis. Most of the osteoblasts lining the surfaces of newly forming bone and the chondrocytes comprising the cartilaginous callus also expressed this contractile actin isoform. The maximal SMA expression extended from 7 to 21 days postfracture. The finding of high levels of SMA expression in connective tissue cells participating in fracture healing suggests that SMA-enabled contraction may be playing a role in the healing process. These results warrant further study of the specific SMA-dependent cell behavior.

Cell migration through defined, synthetic ECM analogs.

We have developed synthetic hydrogel extracellular matrix (ECM) analogues that can be used to study mechanisms involved in cell migration, such as receptor-ligand interactions and proteolysis. The biomimetic hydrogels consist of bioinert polyethylene glycol diacrylate derivatives with proteolytically degradable peptide sequences included in the backbone of the polymer and adhesive peptide sequences grafted to the network. Hydrogels have been developed that degrade as cells secrete proteolytic enzymes. Adhesive peptide sequences grafted to the hydrogel provide ligands that can interact with receptors on the cell surface to mediate adhesion and spreading. In this study, we have characterized the effects of adhesive ligand density on fibroblast migration through collagenase-degradable and plasmin-degradable hydrogels and on smooth muscle cell migration through elastase-degradable hydrogels. In all three cases, we found that cell migration has a biphasic dependence on adhesion ligand concentration, with optimal migration at intermediate ligand levels. Furthermore, both adhesive and proteolytically degradable sequences were required for cell migration to occur. These synthetic ECM analogues may be useful for 3-D mechanistic studies of many aspects of cell migration

-Smooth-muscle Actin in and Contraction of Porcine Dental Pulp Cells

The finding of expression of a muscle actin isoform, alpha-smooth-muscle actin (SMA), by the stromal cells of several tissues prompted this study of SMA expression by cells derived from the porcine dental pulp. The SMA content of the cells increased with time in culture. These SMA-containing cells were found to have the capability to contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro.

α-Smooth-muscle Actin in and Contraction of Porcine Dental Pulp Cells

The finding of expression of a muscle actin isoform, α-smooth-muscle actin (SMA), by the stromal cells of several tissues prompted this study of SMA expression by cells derived from the porcine dental pulp. The SMA content of the cells increased with time in culture. These SMA-containing cells were found to have the capability to contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro.

Lapine and Canine Bone Marrow Stromal Cells Contain Smooth Muscle Actin and Contract a Collagen-Glycosaminoglycan Matrix

Lapine and canine marrow stromal cells were found to contain a contractile actin isoform, alpha-smooth muscle actin (SMA), by immunohistochemistry and Western blot analysis. The SMA was found to be incorporated into stress fibers that were prominently displayed by the cells in monolayer culture. The cell content of this actin isoform increased with passage number. The contractility of SMA-expressing stromal cells was demonstrated by their contraction of collagen-glycosaminoglycan analogs of extracellular matrix into which they were seeded. The demonstration that marrow-derived stromal cells express the SMA gene may explain recent findings of this expression in musculoskeletal connective tissue cells including osteoblasts, chondrocytes, and fibrochondrocytes that may be derived from this mesenchymal stem cell. The implications of these findings for tissue engineering strategies employing marrow stromal cells are also discussed.

Growth Factor Regulation of Smooth Muscle Actin Expression and Contraction of Human Articular Chondrocytes and Meniscal Cells in a Collagen–GAG Matrix

Recent work has demonstrated that human articular chondrocytes and meniscus cells can express the gene for a contractile actin isoform, α-smooth muscle actin (SMA), in vivo. The objective of the present study was to evaluate the effects of two growth factors, transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF)-BB, on the SMA content of these cells and their contraction of a collagen–glycosaminoglycan (GAG) analog of extracellular matrix in vitro. TGF-β1 was found to markedly increase SMA content of the cells and PDGF-BB decreased SMA expression, with both findings achieving statistical significance. A notable finding was the increased contraction of the collagen–GAG matrix induced by TGF-β1 and the decrease in contraction resulting from PDGF-BB treatment, indicating a causal relationship between expression of SMA and the contractility of the cells. A novel cell force monitor, employed to estimate the force exerted per cell, demonstrated a higher force exerted by the TGF-β1-treated cells. The findings demonstrate that the expression of SMA by articular chondrocytes and meniscal cells and their associated contractile behavior can be regulated by selected growth factors. This work provides a foundation for the rational investigation of the mechanisms underlying SMA-enabled contraction of these cell types and the control of this behavior in tissue engineering.

Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering

Photopolymerizable polyethylene glycol (PEG) derivatives have been investigated as hydrogel tissue engineering scaffolds. These materials have been modified with bioactive peptides in order to create materials that mimic some of the properties of the natural extracellular matrix (ECM). The PEG derivatives with proteolytically degradable peptides in their backbone have been used to form hydrogels that are degraded by enzymes involved in cell migration, such as collagenase and elastase. Cell adhesive peptides, such as the peptide RGD, have been grafted into photopolymerized hydrogels to achieve biospecific cell adhesion. Cells seeded homogeneously in the hydrogels during photopolymerization remain viable, proliferate, and produce ECM proteins. Cells can also migrate through hydrogels that contain both proteolytically degradable and cell adhesive peptides. The biological activities of these materials can be tailored to meet the requirements of a given tissue engineering application by creating a mixture of various bioactive PEG derivatives prior to photopolymerization.

The migration of cells from the ruptured human anterior cruciate ligament into collagen-glycosaminoglycan regeneration templates in vitro

Guided tissue regeneration of the ruptured anterior cruciate ligament (ACL) offers the potential benefits of retaining the complex footprints of the ACL and the proprioceptive nerve fibers of the tissue. For this approach to be successful, ACL cells must retain the ability to migrate into an adjacent regeneration template, or scaffold, after ligament rupture. Ruptured ACLs were obtained from the knees of four men, ages 25–35, at the time of ACL reconstruction. Explants of ACL tissue were taken from three locations along the longitudinal axis of the remnant: the rupture site, the middle of the remnant, and far from the rupture site. These three areas have been found to be distinct histologically, with the region far from the rupture site having a histologic appearance similar to the intact ligament. Explants from each area were cultured in conventional tissue culture dishes (2-D culture) and on porous collagen-glycosaminoglycan (CG) scaffolds. Two-dimensional outgrowth was measured 3 times a week, and the 3-D explant/scaffold constructs were examined at 1, 2, 3 and 4 weeks to assess outgrowth of cells into the scaffold. The cell number density and expression of α-smooth muscle actin (SMA) were determined at each time point. The decrease in the diameter of the scaffolds and non-seeded controls were determined as a function of time in culture. The outgrowth of cells onto the tissue culture dishes was observed to begin as early as 3 days and as late as 21 days, with outgrowth first detected at an average of 6.8±2.0 days after explantation. In general, there was a larger area of outgrowth at the 2-week time point from explants with higher cell number density and higher blood vessel density. The 2-week area of outgrowth also correlated with the percentage of SMA-positive cells in the explant. In the experimental constructs with CG scaffolds, fibroblasts were noted to migrate from the human ACL explants into the templates at the earliest time point recorded (1 week). The migration and proliferation of cells from the explants in the CG matrices resulted in an increase in the cell density in the scaffolds with time. There was a significant effect of the location from which the explant was taken on cell density in the scaffold, with a higher density of cells migrating from the explants from the rupture site of the ACL specimens. The percentage of cells staining positive for the SMA isoform varied from 0 to 50% of cells in the scaffold. Scaffolds co-cultured with explants showed a reduction in diameter that was significantly affected by time in culture and the location in the ACL from which the explant was taken. The percentage contraction attributed to the cells was 15% at 2 weeks, and increased to 27% for the injury-zone explant at 4 weeks. There was a significant correlation of the cell-mediated contraction of the matrices at 4 weeks with the cell density in the scaffolds, but not with the number of SMA-positive cells in the scaffolds. These data demonstrate that cells in the human ACL retain their ability to migrate into an adjacent CG scaffold in vitro, weeks after complete rupture. Moreover, the ACL-derived cells can express a contractile actin isoform and can contract a CG analog of extracellular matrix.

The presence of smooth muscle actin in fibroblasts in the torn human rotator cuff

The rotator cuff frequently sustains athletic and occupational injury, often resulting in chronic pain and disability. However, despite the high incidence of such shoulder problems, the pathophysiology of rotator cuff injury and healing has not yet been fully elucidated. The notable finding of this study was the presence of a contractile actin isoform, α-smooth muscle actin (SMA), in nonvascular cells in all of the seven torn human rotator cuff specimens evaluated immunohistochemically. Up to 95% of cells in any one region, and over 95% of elongated cells found in association with crimped collagen, contained SMA. Most of the cells staining positive for SMA in these sections had morphological features of the fibroblast, though a small number were chondrocyte-like. Treatment of cells growing out from human rotator cuff explants with TGF-β1 significantly increased the amount of SMA evaluated by Western blot analysis. PDGF-BB and IFN-γ had no effect on the cell content of SMA. This is the first documentation of the presence of SMA-positive cells in the human rotator cuff tendon. SMA has been found in a number of other healing connective tissues including skin, ligament, meniscus, cartilage, and other types of tendon. Of importance are previous findings that SMA-positive cells can contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. The results of the present study thus suggest that SMA-containing cells could contribute to the retraction of the torn ends of a ruptured rotator cuff and play an important role in healing.

Smooth muscle actin expression by human articular chondrocytes and their contraction of a collagen–glycosaminoglycan matrix in vitro

Recent studies have demonstrated that human articular chondrocytes can express the gene for a contractile muscle actin, α-smooth muscle actin (SMA), in situ. One objective of this work was to evaluate the SMA-content of isolated human articular chondrocytes using Western blot analysis and to correlate the amount of SMA in the cells with passage number and the number of days in culture. A second objective was to determine if articular cartilage-derived cells expressing the gene for SMA in vitro also continue to express type II collagen. A final aim of the current study was to determine if SMA-containing cartilage-derived cells were capable of contracting a collagen–glycosaminoglycan analog of extracellular matrix in vitro. Articular chondrocytes were isolated from 13 patients undergoing total joint arthroplasty. Cells were serially passaged through passage 7. Samples were allocated for Western blot analysis of SMA. Cells in monolayer culture were also stained immunohistochemically for SMA and type II collagen. Cells from passage 3 and 7 were seeded into a porous type I collagen–glycosaminoglycan matrix and the diameter of the scaffolds measured every other day for 21 days. Immunohistochemistry of the articular cartilage samples revealed SMA in the articular chondrocytes in situ with a greater percentage of cells staining positive in the superficial half (60±1.2%; mean ± SEM) of the cartilage than in the basal half (28±1.3%). There was an increasing amount of SMA in the cells in monolayer culture with passage number and a meaningful correlation of the SMA content with the days in culture (linear regression analysis; R 2=0.72). Double staining for SMA and type II collagen showed that type II collagen-expressing cells in monolayer could also express SMA. SMA-containing cells were found to contract the collagen–glycosaminoglycan matrix, with the cells containing more SMA (passage 7 cells) displaying more matrix contraction than those with a lesser amount of SMA (passage 3 cells). The results indicate that control of the expression of SMA may be important when employing articular chondrocytes, expanded in monolayer culture, for implantation alone or in a cell-seeded matrix for cartilage repair procedures.