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The biology of the extracellular matrix: novel insights

Hubmacher, Dirk; Apte, Suneel S.

Current Opinion in Rheumatology: January 2013 - Volume 25 - Issue 1 - p 65–70
doi: 10.1097/BOR.0b013e32835b137b

Purpose of review Extracellular matrix (ECM) has both structural and regulatory roles. This update reviews the representative recent developments in diverse aspects of ECM biology relevant to inflammation, tissue destruction, fibrosis, and regeneration.

Recent findings Biological regulation by ECM is emerging as a major research area, driven by several new directions. Sensing of mechanical cues provided by ECM was found to be crucial in regulating cell differentiation. Transforming growth factor-β (TGF-β) is a pivotal agent in fibrosis and inflammation. A combination of structural biology and cell biology provided novel insights on the mechanisms of its activation by cellular traction and ECM. Improved understanding of how fibrillin microfibrils and associated proteins regulated TGF-β sequestration and activation was achieved by analysis of inherited connective tissue disorders having TGF-β dysregulation as an underlying pathologic mechanism. Insights on microRNA-mediated ECM regulation suggest a key role for miR-29, for which potential therapeutic roles are emerging. Advances in understanding the ECM turnover by proteinases provided novel insights on cell regulation and identified useful disease biomarkers.

Summary As a crucial modulator of cell behavior, ECM has exceptionally strong relevance and translational implications for human disease, opening novel opportunities for mechanistic understanding of disease pathogenesis as well as treatment.

Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA

Correspondence to Suneel S. Apte, Department of Biomedical Engineering (ND20), Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Tel: +1 216 445 3278; fax: +1 216 444 9198; e-mail:

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Extracellular matrix (ECM), the material around and between cells, is a composite material with a highly regulated tissue-specific composition. In tissues with an obvious mechanical role such as cartilage, bone, and tendons, it is a quantitatively major component that confers gross mechanical properties. ECM composition and organization in such tissues reflect evolutionary adaptation to mechanical load. In epithelia, basement membranes are specialized ECM assemblies that provide a supporting substratum for epithelial sheets and maintain cell polarity. Here, ECM has an important role as an organizer. Indeed, by incorporating specific molecular components in varying concentrations and geometries, a range of tissue-specific structural demands can be met. Furthermore, even a single tissue type is often regionally specialized; a recent study [1] of anatomically distinct cartilages, for example, highlighted their differing compositions. The structural significance of ECM is clearly evident from inherited connective tissue disorders such as osteogenesis imperfecta and the Ehlers–Danlos syndromes.

Reduced to the simplest mechanical elements, ECM comprises several secreted proteins that form macromolecular structures as their functional embodiments (fibrils, microfibrils, or fibers). This category includes collagens, fibronectin, elastin, and fibrillins. Enzymes that modify these molecules posttranslationally, such as lysyl oxidase, which forms intermolecular cross-links, and proteinases, which cleave peptide bonds, such as the matrix metalloproteinases (MMPs), are also ECM components. Another category of molecules does not directly contribute to the formation or function of structural complexes, but modulates cell–matrix interactions and cell functions. These are termed matricellular proteins, for example, thrombospondins and tenascins. The glycosaminoglycan (GAG) hyaluronan is a major nonproteinaceous component of ECM, and several ECM core proteins are modified by linkage of various types of GAG chains to form proteoglycans. Hydration of these carbohydrate-rich components exerts a swelling pressure against the surrounding fibrous network, providing tissue turgidity and compressibility and facilitating molecular transport. For a recent systems-level bioinformatics view of ECM composition and function, Cromar et al. [2] defined 357 proteins constituting the core of the ECM and 524 gene products with related functions.

Box 1

Box 1

ECM constitutes the cellular microenvironment for all cells outside the circulation and is recognized as a major regulatory or instructive influence on cell behavior. Most cells are surrounded by, and attached to, a dynamic pericellular matrix with considerable regulatory potential. There is considerable interest in the diverse ways in which ECM directly signals to cells or modulates soluble signals. One mechanism is signaling via matrix adhesion molecules and receptors such as integrins and discoidin-domain receptors [3]. Another is through its role in sequestration and activation of growth factors, such as those of the transforming growth factor-β (TGF-β) superfamily [4] and by modulating morphogen gradients. In tissue engineering, there is considerable current interest in how mechanical or physical properties of ECM such as elasticity or stiffness influence cell behavior [5].

Of numerous intriguing developments in this vast field in the last year, we selected some in areas of rheumatology relevance as well as other fields for this update.

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In recent years, there has been considerable interest in aggrecan breakdown in cartilage matrix by ADAMTS proteinases, collectively referred to as aggrecanases. Aggrecan, a large aggregating proteoglycan, is highly hydrated and thus is the principal compression-resisting cartilage ECM component. Its loss is recognized as a crucial early step in arthritis. Wylie et al. [6] investigated the distribution and activation of ADAMTS5, a crucial aggrecanase in osteoarthritis, in a mouse explant model of inflammatory arthritis. They suggested that synovial ADAMTS5, as well as proprotein convertase-mediated activation of secreted ADAMTS5 bound to ECM, could contribute to cartilage destruction [6]. ADAMTS5 was also found to contribute to versican proteolysis in fibroblast pericellular matrix, and reduced versican turnover in Adamts5-deficient fibroblasts led to a striking myofibroblast conversion [7▪].

In contrast to the prevailing notion of enzymes as key mediators of ECM breakdown, Antipova and Orgel [8▪] published a provocative study showing that antibodies to the collagen-binding, small, leucine-rich proteoglycan biglycan induced nonenzymatic decomposition of collagen fibrils. The proposed mechanism was antibody-induced dissociation of biglycan from collagen fibrils, disrupting their structure. This could result in increased susceptibility of collagen to enzymatic breakdown, and altered cell–matrix interactions such as exposure of cryptic sites in the fibrils. Differential protein profiling of synovial fluid from rheumatoid arthritis (RA) vs. osteoarthritis patients demonstrated a greater relative abundance of fibronectin, cartilage acidic protein-1, and cartilage oligomeric matrix protein (COMP) in osteoarthritis fluid, whereas RA fluid had a higher content of MMPs and neutrophil-associated proteins [9].

ECM is a source of diagnostic and prognostic biomarkers, as proteolysis releases intact or fragmented ECM components as soluble ligands. Along these lines, a panel of monoclonal antibodies was developed against COMP fragments, and ELISA was done to monitor the serum levels in patients and mouse models of osteoarthritis and RA. The new ELISA was reported as a sensitive biomarker for cartilage catabolism [10]. Collagen V is a fibril-forming collagen that associates with collagen I, the dominant collagen type in most tissues. A new assay detecting an MMP-cleaved collagen V fragment was reported as a biomarker of ECM turnover, and patients with ankylosing spondylitis were shown to have higher levels of tissue breakdown [11]. The same group reported that liver fibrosis in rats was associated with a biomarker for MMP-2/MMP-9 mediated proteolysis of collagen VI, which is functionally associated with skeletal muscle and whose anomalies result in myopathies. This biomarker could also be useful for inherited and inflammatory myopathies. MMP-2 levels were increased by the promoter SNP G1575A, and this was associated with increased risk of cardiovascular disease in patients with systemic lupus erythematosus [12].

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MicroRNAs (miRs) regulate biologic processes by suppression of translation or induction of degradation of mRNAs. miRs typically regulate clusters of genes involved in a process. There is currently a strong focus on the miR-29 family, which suppresses major ECM genes, including collagens, elastin, and fibrillins [13]. miR-29b overexpression was associated with aging vasculature [13], as well as aneurysm development in a mouse model of Marfan syndrome (MFS) [14]. Its inhibition prevented early aneurysm development in this model, as well as in a mouse elastase infusion model of abdominal aortic aneurysm [15▪▪]. In contrast, miR29 antagonism could upregulate these major ECM components [13]. Reduced miR-29b was noted in a mouse model of pulmonary fibrosis and associated with upregulation of several ECM genes [16]. A development with translational potential was miR-29 inhibition to enhance elastin expression in patients with elastin haploinsufficiency [17]. There is now compelling evidence to support miR-29 antagonism or agonism in specific clinical settings.

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The microenvironment of stem cells (niche) is a key determinant of pluripotency, self renewal, and asymmetric cell divisions from which arise differentiated progeny. Two recent publications showcased the role of ECM. Nakamura-Ishizu et al. [18▪] found that tenascin-C (Tn-C)-deficient mice failed to reconstitute bone marrow after ablation and had reduced ability to support transplanted donor hematopoietic stem cells (HSCs). Expression of Tn-C in supporting niche cells (stromal and endothelial cells) was dramatically upregulated during hematopoietic recovery after myeloablation [18▪]. Tn-C was found to signal HSCs in an integrin-α9-dependent manner, which led to changes in cyclins and cyclin-dependent kinase inhibitors [18▪]. Fujiwara et al. [19▪▪] showed that stem cells in the hair follicle bulge deposited nephronectin in their basement membrane to regulate mesenchymal differentiation toward smooth muscle. Tissue inhibitor of metalloproteinases-3 (TIMP-3) associates with ECM and targets several ECM-degrading metalloproteinases. It is highly expressed by osteoid cells in the endosteal region of bone marrow and its overexpression led to alteration of bone-marrow-derived lineages [20]. However, whether this effect was dependent on the inhibition of metalloproteinases was not elucidated.

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The ECM protein, periostin, was found to be a crucial determinant of metastatic success of cancer stem cells [21▪▪]. Infiltrating tumor cells were reported to induce periostin production by resident fibroblasts at the metastatic site as a prerequisite for colonization [21▪▪]. Furthermore, a matrix proteoglycan, versican, produced by myeloid cells was found to be crucial for colonization of lung by breast cancer cells in a mouse experimental model [22]. Versican facilitated mesenchymal-to-epithelial transition of tumor cells by reduction of Smad-2 phosphorylation, resulting in enhanced cell proliferation and metastatic burden. The authors proposed targeting versican as a novel way of treating metastatic lung cancer.

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TGF-βs have complex roles in fibrosis, inflammation, and cellular metaplasia. They are tethered to the ECM via latent TGF-β-binding proteins (LTBPs), which are anchored to fibrillin microfibrils [23]. Using cells isolated from fibrillin-1 and fibrillin-2 knockout mice, it was shown that fibronectin was required for ECM incorporation of LTBP1, whereas LTBP3 and LTBP4 deposition required fibrillin-1 [24]. ADAMTS proteins are also known to be functionally related to fibrillin microfibrils and, potentially, to TGF-β regulation [25]. Saito et al. [26] recently identified the ability of ADAMTSL6β to improve microfibril assembly in a mouse model of MFS and demonstrated a concomitant reduction in TGF-β signaling.

TGF-β is activated by integrin-mediated physical forces, molecular displacement or proteolysis. An elegant biophysical study [27▪▪] showed that distorting the large latent complex via single-molecule force spectroscopy resulted in the release of active TGF-β1. The cells seem to pull at the latency-associated peptide within the large latent complex/LTBP, ‘squeezing out’ active TGF-β against a counterforce provided by LTBPs attached to ECM. This study, together with the recent determination of the crystal structure of the latent complex of LTBP1 and TGF-β1 [28▪▪], represented a landmark in understanding integrin-mediated TGF-β activation. The enhanced activation of TGF-β in MFS, which results from fibrillin-1 mutations, has been instrumental in devising novel therapies [4]. Thus, other mechanisms of activation are also of interest. The metalloprotease ADAMTS1 and granzyme B were recently identified as novel mediators of TGF-β activation. Granzyme B cleaved the small leucine-rich proteoglycan biglycan and decorin to release TGF-β [29], whereas the ADAMTS1 thrombospondin type 1 motif was shown to activate TGF-β via a nonproteolytic displacement mechanism [30▪].

Perlecan is a ubiquitous heparan sulfate proteoglycan, found in basement membranes and cartilage, which binds multiple regulatory factors, for example, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and IL-2, to concentrate them and form morphogen gradients. Recent analysis of perlecan knockout mice revealed that cartilage perlecan was essential for vascularization of the perichondrium; specifically, cartilage perlecan promoted the activation of VEGF/vascular endothelial growth factor receptor (VEGFR) by binding to the VEGFR of endothelial cells [31].

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Analysis of the integrin repertoire of human mesenchymal stem cells and its modulation during stem cell differentiation on matrices of varying stiffness showed that osteogenic or adipogenic differentiation generally altered the integrin pattern [32]. In tumors, stiffness provided by ECM is now accepted as crucial in regulating cell behavior. Using a creative approach, Pathak and Kumar [33] described cell behavior on so-called micro-polyacrylamide channels, in which matrix stiffness and pore size (confinement) were independently controlled. They found that cells confined to narrow channels, as probably found in dense fibrillar ECM, migrated faster compared with wider channels and changed their typical biphasic response to ECM stiffness into a steady increase in migration speed with stiffness. This behavior was linked to nonmuscle myosin II and potential polarization of traction forces in narrow pores [33]. Such external influences seem to have a long-term effect on cell behavior, probably through the induction of stable cellular pathways. Culture of lung fibroblasts on stiff substrates revealed their incipient cell plasticity through conversion to myofibroblasts, but also a phenomenon termed mechanical memory [34], that is, myofibroblast phenotype was retained for several subsequent passages on soft substrates.

The biology of cell–matrix interactions relates directly to tissue and organ engineering. ‘Bio-inspired’ artificial matrices strive to achieve the scale, composition, and material properties of ECM to guide cell differentiation, migration, and survival [35▪]. Achieving nanoscale scaffolds is also emerging as crucial. A modified electrospinning technique resulted in a porous nanofibrous biomaterial conducive for cell invasion and nutrient diffusion; when modified with chondroitin sulfate, this scaffold was optimized for cartilage formation [36]. In a study on skin epidermal stem cells, Trappmann et al. [37▪▪] found that on collagen-I-coated, acrylamide-based gels, the differentiation of the cells corresponded to the stiffness of the material. However, on polydimethylsiloxane matrices of the same stiffness range (0.1–2.3 MPa), the proportion of cells expressing a keratinocyte marker did not change with stiffness. They attributed the different responses to differences in the porosity of the materials, which translated into different collagen-anchoring densities. They concluded that cells respond to the mechanical feedback they derive from the ECM rather than the ECM stiffness itself.

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This selection from the recent literature demonstrates the breadth and complexity of ECM research, and emphasizes the functional continuity of ECM with the cell and the considerable crosstalk that results (Fig. 1). The cell–matrix interface, in particular, provides a crucial signaling nexus that regulates all aspects of cell behavior. Whereas the traditional emphasis of the ECM field on its structural relevance has by no means diminished, there is now a strong focus in several new areas. Collectively, this research offers a platform on which to build a strong translational pipeline, such as by modifying chemical signals to cells, and bioengineering of artificial organs using mechanical and adhesive cues.



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Conflicts of interest

Neither of the authors has any conflict of interest relevant to this manuscript.

Funding for this work was provided by the National Institutes for Health (Awards AR53890, HL107147 and EY021151 to Suneel S. Apte).

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Papers of particular interest, published within the annual period of review, have been highlighted as:

  • ▪ of special interest
  • ▪▪ of outstanding interest

Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 151).

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1. Onnerfjord P, Khabut A, Reinholt FP, et al. Quantitative proteomic analysis of eight cartilaginous tissues reveals characteristic differences as well as similarities between subgroups. J Biol Chem 2012; 287:18913–18924.
2. Cromar GL, Xiong X, Chautard E, et al. Toward a systems level view of the ECM and related proteins: a framework for the systematic definition and analysis of biological systems. Proteins 2012; 80:1522–1544.
3. Leitinger B. Transmembrane collagen receptors. Annu Rev Cell Dev Biol 2011; 27:265–290.
4. Doyle JJ, Gerber EE, Dietz HC. Matrix-dependent perturbation of TGFbeta signaling and disease. FEBS Lett 2012; 586:2003–2015.
5. Schwarz US, Gardel ML. United we stand – integrating the actin cytoskeleton and cell–matrix adhesions in cellular mechanotransduction. J Cell Sci 2012; 125:3051–3060.
6. Wylie JD, Ho JC, Singh S, et al. Adamts5 (aggrecanase-2) is widely expressed in the mouse musculoskeletal system and is induced in specific regions of knee joint explants by inflammatory cytokines. J Orthop Res 2012; 30:226–233.
7▪. Hattori N, Carrino DA, Lauer ME, et al. Pericellular versican regulates the fibroblast–myofibroblast transition: a role for ADAMTS5 protease-mediated proteolysis. J Biol Chem 2011; 286:34298–34310.

This article demonstrates how modifying the composition of pericellular matrix has a profound influence on cell behavior.

8▪. Antipova O, Orgel JP. Nonenzymatic decomposition of collagen fibers by a biglycan antibody and a plausible mechanism for rheumatoid arthritis. PLoS One 2012; 7:e32241.

This provocative article suggests a way in which autoantibodies can contribute to tissue breakdown.

9. Mateos J, Lourido L, Fernandez-Puente P, et al. Differential protein profiling of synovial fluid from rheumatoid arthritis and osteoarthritis patients using LC-MALDI TOF/TOF. J Proteomics 2012; 75:2869–2878.
10. Lai Y, Yu XP, Zhang Y, et al. Enhanced COMP catabolism detected in serum of patients with arthritis and animal disease models through a novel capture ELISA. Osteoarthritis Cartilage 2012; 20:854–862.
11. Veidal SS, Larsen DV, Chen X, et al. MMP mediated type V collagen degradation (C5M) is elevated in ankylosing spondylitis. Clin Biochem 2012; 45:541–546.
12. Bahrehmand F, Vaisi-Raygani A, Kiani A, et al. Matrix metalloproteinase-2 functional promoter polymorphism G1575A is associated with elevated circulatory MMP-2 levels and increased risk of cardiovascular disease in systemic lupus erythematosus patients. Lupus 2012; 21:616–624.
13. Boon RA, Seeger T, Heydt S, et al. MicroRNA-29 in aortic dilation: implications for aneurysm formation. Circ Res 2011; 109:1115–1119.
14. Merk DR, Chin JT, Dake BA, et al. miR-29b participates in early aneurysm development in Marfan syndrome. Circ Res 2012; 110:312–324.
15▪▪. Maegdefessel L, Azuma J, Toh R, et al. Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development. J Clin Invest 2012; 122:497–506.

This article offers a potential therapeutic approach to the treatment of aortic aneurysms. Studies in a mouse model were done alongside human aneurysm samples.

16. Cushing L, Kuang PP, Qian J, et al. miR-29 is a major regulator of genes associated with pulmonary fibrosis. Am J Respir Cell Mol Biol 2011; 45:287–294.
17. Zhang P, Huang A, Ferruzzi J, et al. Inhibition of microRNA-29 enhances elastin levels in cells haploinsufficient for elastin and in bioengineered vessels: brief report. Arterioscler Thromb Vasc Biol 2012; 32:756–759.
18▪. Nakamura-Ishizu A, Okuno Y, Omatsu Y, et al. Extracellular matrix protein tenascin-C is required in the bone marrow microenvironment primed for hematopoietic regeneration. Blood 2012; 119:5429–5437.

This article identified a niche role for Tenascin-C in bone marrow for the first time.

19▪▪. Fujiwara H, Ferreira M, Donati G, et al. The basement membrane of hair follicle stem cells is a muscle cell niche. Cell 2011; 144:577–589.

Fujiwara et al. showed that bulge stem cells, via nephronectin expression, create a smooth muscle cell niche and act as tendon cells for the arrector pili muscle. Their results revealed a functional role for basement membrane heterogeneity in tissue patterning.

20. Shen Y, Winkler IG, Barbier V, et al. Tissue inhibitor of metalloproteinase-3 (TIMP-3) regulates hematopoiesis and bone formation in vivo. PLoS One 2010; 5:e13086.
21▪▪. Malanchi I, Santamaria-Martinez A, Susanto E, et al. Interactions between cancer stem cells and their niche govern metastatic colonization. Nature 2012; 481:85–89.

The authors show that infiltrating tumor cells need to induce stromal POSTN expression in the lung to initiate colonization. POSTN recruits Wnt ligands and thereby increases Wnt signaling in cancer stem cells.

22. Gao D, Joshi N, Choi H, et al. Myeloid progenitor cells in the premetastatic lung promote metastases by inducing mesenchymal to epithelial transition. Cancer Res 2012; 72:1384–1394.
23. Todorovic V, Rifkin DB. LTBPs, more than just an escort service. J Cell Biochem 2012; 113:410–418.
24. Zilberberg L, Todorovic V, Dabovic B, et al. Specificity of latent TGF-ss binding protein (LTBP) incorporation into matrix: role of fibrillins and fibronectin. J Cell Physiol 2012; 227:3828–3836.
25. Hubmacher D, Apte SS. Genetic and functional linkage between ADAMTS superfamily proteins and fibrillin-1: a novel mechanism influencing microfibril assembly and function. Cell Mol Life Sci 2011; 68:3137–3148.
26. Saito M, Kurokawa M, Oda M, et al. ADAMTSL6beta protein rescues fibrillin-1 microfibril disorder in a Marfan syndrome mouse model through the promotion of fibrillin-1 assembly. J Biol Chem 2011; 286:38602–38613.
27▪▪. Buscemi L, Ramonet D, Klingberg F, et al. The single-molecule mechanics of the latent TGF-beta1 complex. Curr Biol 2011; 21:2046–2054.

The authors use atomic force microscopy to quantify the forces exerted by single integrins to activate TGF-β. The mechanism underscored the requirement for LTBPs.

28▪▪. Shi M, Zhu J, Wang R, et al. Latent TGF-beta structure and activation. Nature 2011; 474:343–349.

This landmark crystal structure first provided the structural basis for the activation of integrins by TGF-β. This is an elegantly written and illustrated article.

29. Boivin WA, Shackleford M, Vanden Hoek A, et al. Granzyme B cleaves decorin, biglycan and soluble betaglycan, releasing active transforming growth factor-beta1. PLoS One 2012; 7:e33163.
30▪. Bourd-Boittin K, Bonnier D, Leyme A, et al. Protease profiling of liver fibrosis reveals the ADAM metallopeptidase with thrombospondin type 1 motif, 1 as a central activator of transforming growth factor beta. Hepatology 2011; 54:2173–2184.

The authors describe for the first time a novel activator of TGF-β and a novel function for ADAMTS1.

31. Ishijima M, Suzuki N, Hozumi K, et al. Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation. Matrix Biol 2012; 31:234–245.
32. Frith JE, Mills RJ, Hudson JE, Cooper-White JJ. Tailored integrin-extracellular matrix interactions to direct human mesenchymal stem cell differentiation. Stem Cells Dev 2012; 21:2442–2456.
33. Pathak A, Kumar S. Independent regulation of tumor cell migration by matrix stiffness and confinement. Proc Natl Acad Sci USA 2012; 109:10334–10339.
34. Balestrini JL, Chaudhry S, Sarrazy V, et al. The mechanical memory of lung myofibroblasts. Integr Biol (Camb) 2012; 4:410–421.
35▪. Kim DH, Provenzano PP, Smith CL, Levchenko A. Matrix nanotopography as a regulator of cell function. J Cell Biol 2012; 197:351–360.

This is a detailed review of how cells respond to surface topography and of attempts to reproduce that for manipulating cell behaviour on artificial matrices.

36. Coburn JM, Gibson M, Monagle S, et al. Bioinspired nanofibers support chondrogenesis for articular cartilage repair. Proc Natl Acad Sci U S A 2012; 109:10012–10017.
37▪▪. Trappmann B, Gautrot JE, Connelly JT, et al. Extracellular-matrix tethering regulates stem-cell fate. Nat Mater 2012; 11:642–649.

fibrosis; inflammation; microRNA; TGF-β; tissue engineering

© 2013 Lippincott Williams & Wilkins, Inc.