Tissue-specific Extracellular Matrix Research Articles

Chitin-based Materials in Tissue Engineering: Applications in Soft Tissue and Epithelial Organ

Chitin-based materials and their derivatives are receiving increased attention in tissue engineering because of their unique and appealing biological properties. In this review, we summarize the biomedical potential of chitin-based materials, specifically focusing on chitosan, in tissue engineering approaches for epithelial and soft tissues. Both types of tissues play an important role in supporting anatomical structures and physiological functions. Because of the attractive features of chitin-based materials, many characteristics beneficial to tissue regeneration including the preservation of cellular phenotype, binding and enhancement of bioactive factors, control of gene expression, and synthesis and deposition of tissue-specific extracellular matrix are well-regulated by chitin-based scaffolds. These scaffolds can be used in repairing body surface linings, reconstructing tissue structures, regenerating connective tissue, and supporting nerve and vascular growth and connection. The novel use of these scaffolds in promoting the regeneration of various tissues originating from the epithelium and soft tissue demonstrates that these chitin-based materials have versatile properties and functionality and serve as promising substrates for a great number of future applications.

Chitosan-Based Hyaluronic Acid Hybrid Polymer Fibers as a Scaffold Biomaterial for Cartilage Tissue Engineering

An ideal scaffold material is one that closely mimics the natural environment in the tissue-specific extracellular matrix (ECM). Therefore, we have applied hyaluronic acid (HA), which is a main component of the cartilage ECM, to chitosan as a fundamental material for cartilage regeneration. To mimic the structural environment of cartilage ECM, the fundamental structure of a scaffold should be a three-dimensional (3D) system with adequate mechanical strength. We structurally developed novel polymer chitosan-based HA hybrid fibers as a biomaterial to easily fabricate 3D scaffolds. This review presents the potential of a 3D fabricated scaffold based on these novel hybrid polymer fibers for cartilage tissue engineering.

Optimization of anIn VitroThree-Dimensional Microenvironment to Reprogram Synovium-Derived Stem Cells for Cartilage Tissue Engineering

Adult stem cells gradually lose their stemness when plated in monolayer culture after isolation from their in vivo niche. In this study, we hypothesized that the in vitro microenvironment can be optimized by modulating oxygen tension and mitotic signal in a tissue-specific extracellular matrix (ECM) deposited by synovium-derived stem cells (SDSCs) to rejuvenate expanded SDSC proliferation and chondrogenic potential. Passage 3 SDSCs were plated on either SDSC-derived ECM or plastic flask and incubated in either hypoxia (5% O(2)) or normoxia (21% O(2)) with or without the supplementation of 10 ng/mL of basic fibroblast growth factor-2 (FGF-2) for 7 days, followed by pellet culture in a serum-free chondrogenic medium for 14 days. Our data showed that, compared with the mitotic effect of FGF-2 on SDSCs, ECM expansion greatly enhanced SDSC proliferation while retaining SDSC stem cell characteristics. More importantly, ECM pretreatment yielded SDSC pellets with a comparable chondrogenic index to FGF-2 pretreatment, both of which were much higher than SDSC expansion on plastic flask alone. FGF-2 pretreatment led to the highest glycosaminoglycans and DNA content; intriguingly, it also contributed to the highest expression level of hypertrophic marker genes. Surprisingly, the hypertrophic marker genes could be downregulated if the pretreatment was combined with hypoxia or ECM. The combination of hypoxia, FGF-2, and SDSC-derived ECM contributed to the highest cell number in SDSC expansion. Our study indicates that the three-dimensional microenvironment for ex vivo expansion can be optimized to provide high-quality stem cells for stem cell-based cartilage defect repair.

Reconstruction of a Tissue-Specific Extracellular Matrix for Enhancing Nucleus Pulposus Cell Proliferation and Redifferentiation Capacity

Low back pain is often related to intervertebral disc (IVD) degeneration that results from a progressive loss of proteoglycans and water content in the nucleus pulposus (NP). Autologous disc cell-based therapy is a promising approach for IVD regeneration. Unfortunately, the current in vitro expansion of NP cells in monolayer results in dedifferentiation of these cells.

Isolation of Mammary-Specific Extracellular Matrix to Assess Acute Cell-ECM Interactions in 3D Culture

Studies of mammary epithelial cells (MECs) cultured with reconstituted basement membrane proteins derived from EHS tumors have contributed greatly to the understanding of both normal physiology and transformation. Only when plated on such biologically relevant substratum are MECs able to form morphologically correct, differentiated structures, highlighting a critical role for extracellular matrix (ECM) proteins in MEC organization and function. Here, we describe methods modified from the original EHS matrix protocol for isolating tissue-specific ECM from rat mammary glands, and for subsequent use in short-term 3D cell culture models designed to assess acute cell-ECM interactions. Using this protocol, the final matrix is enriched up to 58-fold for ECM proteins such as fibronectin and laminin, while cellular proteins such as GAPDH are reduced 98-fold. We have previously shown that MECs plated in mammary-specific ECM form more elaborate duct-like and alveolar-like structures compared to MECs plated in Matrigel™, demonstrating the biological relevance of tissue-specific ECM. Use of mammary-specific ECM in 3D cell culture models will further our ability to study the intricate interplay between a cell and its microenvironment, and permit identification of modifying factors.

Reconstitution of Marrow-Derived Extracellular Matrix Ex Vivo: A Robust Culture System for Expanding Large-Scale Highly Functional Human Mesenchymal Stem Cells

The difficulty in long-term expansion of mesenchymal stem cells (MSCs) using standard culture systems without the loss of their stem cell properties suggests that a critical feature of their microenvironment necessary for retention of stem cell properties is absent in these culture systems. We report here the reconstitution of a native extracellular matrix (ECM) made by human marrow cells ex vivo, which consists of at least collagen types I and III, fibronectin, small leucine-rich proteoglycans such as biglycan and decorin, and major components of basement membrane such as the large molecular weight proteoglycan perlecan and laminin. Expansion of human MSCs on this ECM strongly promoted their proliferation, retained their stem cell properties with a low level of reactive oxygen species (ROS), and substantially increased their response to BMP-2. The quality of the expanded cells following each passage was further tested by an in vivo transplantation assay. The results showed that MSCs expanded on the ECM for multiple passages still retained the same capacity for skeletogenesis. In contrast, the bone formation capacity of cells expanded on plastic was dramatically diminished after 6-7 passages. These findings suggest that the marrow stromal cell-derived ECM is a promising matrix for expanding largescale highly functional MSCs for eventualuse in stem cell-based therapy. Moreover, this system should also be invaluable for establishment of a unique tissue-specific ECM, which will facilitate control of the fate of MSCs for therapeutic applications.

Extracellular matrix-mediated osteogenic differentiation of murine embryonic stem cells

Embryonic stem cells (ESCs) are pluripotent and have the ability to differentiate into mineralising cells in vitro. The use of pluripotent cells in engineered bone substitutes will benefit from the development of bioactive scaffolds which encourage cell differentiation and tissue development. Extracellular matrix (ECM) may be a suitable candidate for use in such scaffolds since it plays an active role in cellular differentiation. Here, we test the hypothesis that tissue-specific ECM influences the differentiation of murine ESCs. We induced murine ESCs to differentiate by embryoid body formation, followed by dissociation and culture on ECM prepared by decellularisation of either osteogenic cell (MC3T3-E1) or non-osteogenic cell (A549) cultures, or on defined collagen type I matrix. We assessed osteogenic differentiation by formation of mineralised tissue and osteogenic gene expression, and found it to be significantly greater on MC3T3-E1 matrices than on any other matrix. The osteogenic effect of MC3T3-E1 matrix was reduced by heat treatment and abolished by trypsin, suggesting a bioactive proteinaceous component. These results demonstrate that decellularised bone-specific ECM promotes the osteogenic differentiation of ESCs. Our results are of fundamental interest and may help in tailoring scaffolds for tissue engineering applications which both incorporate tissue-specific ECM signals and stimulate stem-cell differentiation.

Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype

Recent studies have shown that extracellular matrix (ECM) substitutes can have a dramatic impact on cell growth, differentiation and function. However, these ECMs are often applied generically and have yet to be developed for specific cell types. In this study, we developed tissue-specific ECM-based coating substrates for skin, skeletal muscle and liver cell cultures. Cellular components were removed from adult skin, skeletal muscle, and liver tissues, and the resulting acellular matrices were homogenized and dissolved. The ECM solutions were used to coat culture dishes. Tissue matched and non-tissue matched cell types were grown on these coatings to assess adhesion, proliferation, maintenance of phenotype and cell function at several time points. Each cell type showed better proliferation and differentiation in cultures containing ECM from their tissue of origin. Although subtle compositional differences in the three ECM types were not investigated in this study, these results suggest that tissue-specific ECMs provide a culture microenvironment that is similar to the in vivo environment when used as coating substrates, and this new culture technique has the potential for use in drug development and the development of cell-based therapies.

Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation

The mechanical properties of the extracellular matrix (ECM) can exert significant influence in determining cell fate. Human mesenchymal stem cells (MSCs) grown on substrates with varying stiffness have been shown to express various cell lineage markers, without the use of toxic DNA demethylation agents or complex cocktails of expensive growth factors. Here we investigated the myogenic and osteogenic potential of various polyacrylamide gel substrates that were coated with covalently bound tissue-specific ECM proteins (collagen I, collagen IV, laminin, or fibronectin). The gel-protein substrates were shown to support the growth and proliferation of MSCs in a stiffness-dependent manner. Higher stiffness substrates encouraged up to a 10-fold increase in cell number over lower stiffness gels. There appears to be definitive interplay between substrate stiffness and ECM protein with regard to the expression of both osteogenic and myogenic transcription factors by MSCs. Of the 16 gel-protein combinations investigated, osteogenic differentiation was found to occur significantly only on collagen I-coated gels with the highest modulus gel tested (80 kPa). Myogenic differentiation occurred on all gel-protein combinations that had stiffnesses >9 kPa but to varying extents as ascertained by MyoD1 expression. Peak MyoD1 expression was seen on gels with a modulus of 25 kPa coated in fibronectin, with similar levels of expression observed on 80-kPa collagen I-coated gels. The modulation of myogenic and osteogenic transcription factors by various ECM proteins demonstrates that substrate stiffness alone does not direct stem cell lineage specification. This has important implications in the development of tailored biomaterial systems that more closely mimic the microenvironment found in native tissues.

Osteoadherin is Upregulated by Mature Osteoblasts and Enhances Their In Vitro Differentiation and Mineralization

During the process of differentiation, osteoblasts commit through strictly controlled checkpoints under the influence of several growth factors, cytokines, and extracellular matrix (ECM) proteins. The mineralized tissue-specific ECM component osteoadherin (OSAD) belongs to the small leucine-rich repeat protein family of proteoglycans. Proteoglycans modulate cellular behavior either through the attached glycosaminoglycan chains or by direct protein-protein interactions via the core protein sequences. Leucine-rich repeats have been shown to directly interact with cell-surface receptors such as epidermal growth factor receptor, blocking its ability to bind its ligand. In the present study, we investigated the influence of OSAD on the behavior and maturation of MC3T3E1 osteoblasts. OSAD overexpression and repression clones were created by stably transfecting with plasmids coding for either mouse OSAD cDNA or small-hairpin RNA, targeted against mouse OSAD. Overexpression of OSAD resulted in an increase of osteoblast differentiation features, such as increased alkaline phosphatase (ALP) activity and increased in vitro mineralization, as well as reduced proliferation and migration. Bone sialoprotein (BSP) levels were unchanged, while upregulation of osteocalcin (OC) and osteoglycin (OGN) was observed. Conversely, repression of OSAD expression resulted in increased cell proliferation and migration. BSP and OC were unaffected, while OGN was downregulated. ALP activity was reduced, though no change in in vitro mineralization was observed. We conclude that OSAD overexpression enhanced the differentiation and maturation of osteoblasts in vitro.

Cell Polarity in the Anulus of the Human Intervertebral Disc

Human intervertebral disc tissue was obtained in a prospective study of cell morphology and gene expression. Experimental studies were approved by the authors' Human Subjects Institutional Review Board. Discs were obtained from surgical specimens or control donors. To determine if there is morphologic and molecular evidence for polarity in cells of the human anulus. In many tissues, cells become polarized as they develop functional specializations, which involve cell-cell and cell-extracellular matrix interactions and polarized targeting mechanisms. The highly specialized lamellar organization of the anulus is well recognized and suggests that this structure may be the result of directed secretion of extracellular matrix components by polarized disc cells. Human disc specimens from donor and surgical patients were examined with light and electron microscopy to assess morphology. Specimens were examined for immunocytochemical localization of PAR3 and claudin-1 and -11, recognized polarity proteins, and additional anulus specimens were examined for expression of polarity-related genes using microarray analysis. In vitro monolayer and 3-dimensional anulus cultures were also studied for gene expression, and additional surgical anulus specimens were used to obtain gene expression data using real time RT-PCR. At the ultrastructural level, anulus cells showed localization of secretory organelles and directed deposition of extracellular matrix in one portion of the cell, with the nucleus positioned in the opposite side of the cell. Expression of the polarity-related genes claudin-11 and PAR3 and PARD6 was confirmed using RT-PCR and microarray studies and immunocytochemical analyses. The percentage of cells positive for PAR3 immunolocalization was significantly greater in the outer anulus (100%) than in either the inner anulus (43.8%) or nucleus pulposus (22.6%). At the macroscopic level, the characteristic anular lamellar morphology implies a specialized architectural formation and organization, which is achieved by the tissue-specific function of polarized cells. Morphologic and molecular studies provided evidence for the presence of polarity in cells in the anulus. These findings advance our understanding of anulus disc cell function in production of highly aligned collagen fibrils and macroaggregates of these collagen fibrils into lamellar collagen bundles. Such disc cell activity is important in development and maintenance of the tissue-specific extracellular matrix of the disc.

Enhanced chondrogenic differentiation of murine embryonic stem cells in hydrogels with glucosamine

Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). We have previously reported that hydrogels provide EBs a supportive environment for in vitro chondrogenic differentiation and three dimensional tissue formation [Hwang NS, et al. The Effects of three dimensional culture and growth factors on the chondrogenic differentiation of murine ES cells. Stem Cells 2006;24:284-91]. In this study, we report chondrogenic differentiation of murine ES cells encapsulated in photopolymerizing poly(ethylene-glycol)-based (PEG) hydrogels in the presence of glucosamine (GlcN), an amino monosaccharide found in chitin, glycoproteins and glycosaminoglycans such as hyaluronic acid, chondroitin sulfate and heparin sulfate. We examined the growth and differentiation of encapsulated EBs in standard chondrogenic differentiation medium containing 0-, 2-, and 10-mm GlcN. Morphometric analysis and examination of gene and protein expression indicated that treatment of hydrogel cultures with 2-mm GlcN for 21 days significantly increased EB size, levels of aggrecan mRNA, and tissue-specific extracellular matrix accumulation. GlcN can induce multiple aspects of cell behavior and optimal GlcN concentrations can be beneficial for directing the differentiation and tissue formation of ES cells.

Vitronectin: A Possible Determinant of Adenovirus Type 19 Tropism for Human Corneal Epithelium

Adenoviruses typically demonstrate specific tissue tropisms, as in the association of Ad19 with epidemic keratoconjunctivitis. We sought to determine factors that might influence the apparent tropism of Ad19 for the cornea. Laboratory investigation. Adenovirus serotypes Ad2, 5, 9, 10, 11, 13, and 19 were compared for their capacity to replicate in human corneal epithelial cells (HCECs) in culture. Organotypically cultured human corneas were infected with Ad19 or Ad2, and viral titers were compared after 7 days. Replication of both viruses was compared in HCECs cultured on various extracellular matrices. Western blot analysis and immunohistochemistry were applied to human donor corneas and HCECs. One week after infection of HCEC monolayer cultures, Ad2 titers were significantly higher than any of the other viruses tested (P <.05). In organotypic corneal cultures, Ad19 titers were significantly higher than Ad2 (P = .0003). Ad2 replication in HCECs equaled or exceeded that of Ad19 on all extracellular matrices except vitronectin, where Ad2 replication was reduced and Ad19 replication enhanced (P <.0001). Vitronectin was detected by immunohistochemistry within the corneal epithelial basement membranes of human donor corneas. Increased alpha(v) integrin expression and greater tyrosine kinase phosphorylation in HCECs cultured on vitronectin were demonstrated by Western blot analysis. In vitro, vitronectin enhances growth of Ad19, possibly by up-regulation of receptor alpha(v) integrins and increased activity of tyrosine kinases necessary for adenoviral internalization. We hypothesize that differential tissue tropisms for adenoviruses may derive in part from tissue-specific extracellular matrix expression.

Prolyl 4-hydroxylase α-related proteins in Drosophila melanogaster: tissue-specific embryonic expression of the 99F8-9 cluster

The extracellular matrix (ECM) is proposed to play critical roles in organ morphogenesis through the stabilization and/or sequestration of signaling factors and adhesion molecules, and by maintaining organ integrity. As a first step toward understanding molecules involved in ECM modification and maturation, we have examined the embryonic expression profiles of ten prolyl 4-hydroxylase α subunit (PH4α)-related genes. Prolyl 4-hydroxylases (PH4) catalyze the formation of 4-hydroxyproline in collagens, the major components of the ECM, and are implicated in the hydroxylation of proline in several other secreted proteins. To date, two α subunit proteins have been described in both humans (PHαI and PHαII) and worms (PHY-1/DPY-18 and PHY-2), whereas only a single Drosophila α subunit has been identified. The ten PH4α-related genes described in this study are clustered in a 183-kb region near the tip of chromosome arm 3R and include the previously described Drosophila α subunit gene. Six of the ten PH4α genes in the cluster have tissue-specific embryonic expression. PH4αSG1 and PH4αSG2 are expressed in the salivary gland, PH4αMP is expressed in mouth-part precursors, PH4αPV is expressed in the proventriculus, and CG9698-E is expressed in the epidermis. PH4αEFB is expressed more broadly, with expression in the anterior and posterior midgut primordia, the fat body, the hemocytes and the epidermis. The expression profiles of these PH4α-related genes suggest that tissue-specific ECM modifications may be critical to organ formation and/or function.

Dynamics of Basement Membrane Formation by Keratinocyte–Fibroblast Interactions in Organotypic Skin Culture

The cutaneous basement membrane zone, composed of numerous macromolecules, plays a multifunctional role in tissue regeneration and maintenance. To elucidate the cellular origin and dynamics of basement membrane formation,de novosynthesis, deposition, and ultrastructural assembly of its components were analyzed in organotypic cultures of adult skin keratinocytes on collagen gels with or without collagen-embedded dermal cells. Collagen IV and laminin-1 deposition occurred only in the presence of mesenchymal cells: patchy at day 4 and continuous after 1 week. Chain-specific mRNA expression started at day 2 in both keratinocytes and fibroblasts. It steadily increased up to day 10, however, with a reciprocal induction pattern, mRNA abundance shifting from keratinocytes to fibroblasts. On the other hand, laminin-5 staining was first observed at day 4, but in keratinocyte both mono- and cocultures. This was followed by nidogen, which was detected in cocultures but also in dermal monocultures. Laminin-5 protein persisted throughout day 21, whereas nidogen steadily increased in intensity. Expression kinetics revealed high levels of laminin-5 transcripts early and in keratinocytes only, whereas nidogen was expressed later and predominantly in fibroblasts. Although basement membrane protein deposition was continuous at day 14, the ultrastructural organization was still fragmentary, eventually normalizing at 3 weeks. These data demonstrate a dynamic interaction and cooperation of epithelial and mesenchymal skin cells in basement membrane formation. This interaction is supposedly mediated via diffusible factors. Our findings further extend the scope of epithelial–mesenchymal interactions stressing that both cell compartments are essential to constitute a tissue-specific extracellular matrix structure.

Recent advances in tissue synthesis in vivo by use of collagen-glycosaminoglycan copolymers

Biologically active analogues of the extracellular matrix (ECM) are synthesized by grafting glycosaminoglycan (GAG) chains onto type I collagen, and by controlling the physicochemical properties of the resulting graft copolymer. Collagen-GAG ECM analogues have previously been shown to induce regeneration of the dermis in humans and the guinea pig, and of the rat sciatic nerve. Current studies have emphasized elucidation of the molecular mechanism through which tissue-specific ECM analogues induce regeneration. The contribution of the GAGs to the biological activity of the skin regeneration template was confirmed by studying the contribution of several GAGs to the inhibition of wound contraction in guinea pigs. The interaction between cells and the porous structure of an ECM analogue was studied with emphasis on the deformation of pores which occurs during wound contraction. The synthesis of scar, as well as of partly regenerated tissue which has a morphology between that appropriate for scar and for normal dermis, was quantitatively assayed for the first time using a laser light scattering technique. An ECM analogue which has been shown to be capable of inducing regeneration of functional sciatic nerve in the rat over a gap larger than 10 mm was incorporated in the design of a biodegradable implant for peripheral nerve regeneration.

Amelogenesis in vitro: a model for studies of epithelial postsecretory processing during tissue-specific extracellular matrix biomineralization

Abstract The extracellular matrix (ECM) of developing mammalian enamel comprises a complex of unusual epithelial-derived proteins, which appear to function in concert to initiate and propagate tissue-specific biomineralization. Following enamel protein synthesis by ameloblast cells within the enamel organ, the subsequent steps of posttranslational modification, secretion, postsecretory processing and eventual removal of these proteins from forming enamel are largely unknown. To address this issue we have designed studies to investigate the hypothesis that enamel proteins are removed from enamel and translocated into the vasculature as relatively high-molecular-weight components. We examined enamel proteins recovered from serumless medium during prolonged organ culture of mouse capstage mandibular first molars. By 21 days in vitro the tooth crown formed and dentine and enamel biomineralization were apparent. At 31 days, explants retained metabolic activity and the enamel matrix showed extensive transformation. Immunologically identified enamel proteins of 26–18 k Da were produced by cultured tooth organs, translocated from tooth explants to the culture medium, recovered from the medium and then compared to control enamel protein from in vivo preparations. Comparable postsecretory processing of the 26-k Da amelogenin protein was observed in vitro and in vivo. We speculate that the pathway reported in the present studies is comparable to the processing of the enamel protein polypeptides of the maturing enamel which occurs in vivo. The in vitro organ culture model described in this report provides an approach with which to investigate the molecular events associated with epithelial-derived postsecretory processing of ECM molecules associated with tissue-specific biomineralization.