Extracellular Matrices Scaffolds Research Articles

Amyloid fibril-based thixotropic hydrogels for modeling of tumor spheroids in vitro

Biomaterials mimicking extracellular matrices (ECM) for three-dimensional (3D) cultures have gained immense interest in tumor modeling and in vitro organ development. Here, we introduce a new class of amyloid fibril-based peptide hydrogels as a versatile biomimetic ECM scaffold for 3D cell culture and homogenous tumor spheroid modeling. We show that these amyloid fibril-based hydrogels are thixotropic and allow cancer cell adhesion, proliferation, and migration. All seven designed hydrogels support 3D cell culture with five different cancer cell lines forming spheroid with necrotic core and upregulation of the cancer biomarkers. We further developed the homogenous, single spheroid using the drop cast method and the data suggest that all hydrogels support the tumor spheroid formation but with different necrotic core diameters. The detailed gene expression analysis of MCF7 spheroid by microarray suggested the involvement of pro-oncogenes and significant regulatory pathways responsible for tumor spheroid formation. Further, using breast tumor tissue from a mouse xenograft model, we show that selected amyloid hydrogels support the formation of tumor spheroids with a well-defined necrotic core, cancer-associated gene expression, higher drug resistance, and tumor heterogeneity reminiscent of the original tumor. Altogether, we have developed an easy-to-use, rapid, cost-effective, and scalable platform for generating in vitro cancer models for the screening of anti-cancer therapeutics and developing personalized medicine.

ECM scaffolds mimicking extracellular matrices of endochondral ossification for the regulation of mesenchymal stem cell differentiation

Endochondral ossification (ECO) is an important process of bone tissue development. During ECO, extracellular matrices (ECMs) are essential factors to control cell functions and induce bone regeneration. However, the exact role of ECO ECMs on stem cell differentiation remains elusive. In this study, ECM scaffolds were prepared to mimic the ECO-related ECM microenvironments and their effects on stem cell differentiation were compared. Four types of ECM scaffolds mimicking the ECMs of stem cells (SC), chondrogenic (CH), hypertrophic (HY) and osteogenic (OS) stages were prepared by controlling differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) at different stages. Composition of the ECM scaffolds was dependent on the differentiation stage of MSCs. They showed different influence on osteogenic differentiation of MSCs. HY ECM scaffold had the most promotive effect on osteogenic differentiation of MSCs. CH ECM and OS ECM scaffolds showed moderate effect, while SC ECM scaffold had the lowest effect on osteogenic differentiation of MSCs. Their effects on chondrogenic or adipogenic differentiation were not significantly different. The results suggested that the engineered HY ECM scaffold had superior effect for osteogenic differentiation of MSCs. Statement of significance ECM scaffolds mimicking endochondral ossification-related ECM microenvironments are pivotal for elucidation of their roles in regulation of stem cell functions and bone tissue regeneration. This study offers a method to prepare ECM scaffolds that mimic the ECMs from cells at hypertrophic, osteogenic, chondrogenic and stem cell stages. Their composition and impacts on osteogenic differentiation of MSCs were compared. The hypertrophic ECM scaffold had the highest promotive effect on osteogenic differentiation of MSCs. The results advance our understanding about the role of ECO ECMs in regulation of stem cell functions and provide perspective for bone defect repair strategies.

The Antimicrobial Effectiveness and Cytotoxicity of the Antibiotic-Loaded Chitosan: ECM Scaffolds

Background: The development of multifunctional wound dressings with the ability to control hemostasis, limit infection and promote rapid wound healing and constructive tissue remodeling has been a challenge for many years. In view of these challenges, a hybrid scaffold platform was developed that combined two different extracellular matrices (ECM): ECM from decellularized mammalian tissue and ECM (chitosan) from crustaceans. Both types of ECM have well established clinical benefits that support and promote wound healing and control hemostasis. This scaffold platform could also be augmented with antibiotics to provide bactericidal activity directly to the wound site. Methods: Four different scaffold formulations were developed containing chitosan supplemented with either 20% or 50% urinary bladder matrix (UBM) hydrogel or 1% (w/v) or 10% (w/v) UBM–ECM particulates. 100% chitosan scaffolds were used as controls. The scaffolds were augmented with either minocycline or rifampicin. Escherichia Coli and Staphylococcus Aureus were used to assesses antimicrobial efficacy and duration of activity, while neutral red uptake assays were performed to establish direct and indirect cytotoxicity. Results: Results showed that scaffold handling properties, scaffold integrity over time and the efficacy and release rate of loaded antibiotics could be modified by altering scaffold composition. Moreover, antibiotics were easily released from the scaffold and could remain effective for up to 24 h by modifying the scaffold composition. Variable results with cytotoxicity testing show that further work is required to optimize the scaffold formulations but these proof of principle experiments suggest that these scaffolds have potential as bioactive wound dressings.

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine.

Photooxidatively crosslinked acellular tumor extracellular matrices as potential tumor engineering scaffolds

Acellular tumor extracellular matrices (ECMs) have limitations when employed as three-dimensional (3D) scaffolds for tumor engineering. In this work, methylene blue-mediated photooxidation was used to crosslink acellular tumor ECMs. Photooxidative crosslinking greatly increased the stiffness of acellular tumor ECM scaffolds but barely altered the Amide III band of the secondary structure of polypeptides and proteins. MCF-7, HepG2 and A549 cells cultured on photooxidatively crosslinked acellular tumor ECM scaffolds exhibited greater cell number per scaffold, more IL-8 and VEGF secretion, and increase migration and invasion abilities than cells cultured on uncrosslinked acellular tumor ECM scaffolds. The three tumor cell lines cultured on the stiffer photooxidatively crosslinked acellular matrices acquire mesenchymal properties (mesenchymal shift) and dedifferentiated phenotypes. Furthermore, the malignant phenotypes induced in vitro when cultured on the crosslinked scaffold promoted the in vivo tumor growth of BALB/c nude mice. Finally, the dedifferentiated cancer cells, including MCF-7, HepG2 and A549 cells, were less sensitive to chemotherapeutics. Thus, photooxidatively crosslinked acellular tumor ECMs have potentials as 3D tumor engineering scaffolds for cancer research. Statement of SignificanceNatural material scaffolds have been successfully used as 3D matrices to study the in vitro tumor cell growth and mimic the in vivo tumor microenvironment. Acellular tumor ECMs are developed as 3D scaffolds for tumor engineering but have limitations in terms of elastic modulus and cell spheroid formation. Here we use methylene blue-mediated photooxidation to crosslink acellular tumor ECMs and investigate the influence of photooxidative crosslinking on structural, mechanical and biological characteristics of acellular tumor ECM scaffolds. It is the first study to evaluate the feasibility of photooxidatively crosslinked acellular tumor ECMs as 3D scaffolds for cancer research and the results are encouraging. Moreover, this study provides new research areas in regard to photodynamic therapy (PDT) for Cancer.

The Challenge in Using Mesenchymal Stromal Cells for Recellularization of Decellularized Cartilage.

Some decellularized musculoskeletal extracellular matrices (ECM)s derived from tissues such as bone, tendon and fibrocartilaginous meniscus have already been clinical use for tissue reconstruction. Repair of articular cartilage with its unique zonal ECM architecture and composition is still an unsolved problem, and the question is whether allogenic or xenogeneic decellularized cartilage ECM could serve as a biomimetic scaffold for this purpose.Hence, this survey outlines the present state of preparing decellularized cartilage ECM-derived scaffolds or composites for reconstruction of different cartilage types and of reseeding it particularly with mesenchymal stromal cells (MSCs).The preparation of natural decellularized cartilage ECM scaffolds hampers from the high density of the cartilage ECM and lacking interconnectivity of the rather small natural pores within it: the chondrocytes lacunae. Nevertheless, the reseeding of decellularized ECM scaffolds before implantation provided superior results compared with simply implanting cell-free constructs in several other tissues, but cartilage recellularization remains still challenging. Induced by cartilage ECM-derived scaffolds MSCs underwent chondrogenesis.Major problems to be addressed for the application of cell-free cartilage were discussed such as to maintain ECM structure, natural chemistry, biomechanics and to achieve a homogenous and stable cell recolonization, promote chondrogenic and prevent terminal differentiation (hypertrophy) and induce the deposition of a novel functional ECM. Some promising approaches were proposed including further processing of the decellularized ECM before recellularization of the ECM with MSCs, co-culturing of MSCs with chondrocytes and establishing bioreactor culture e.g. with mechanostimulation, flow perfusion pressure and lowered oxygen tension. Graphical Abstract Synopsis of tissue engineering approaches based on cartilage-derived ECM.

Extracellular matrix-derived hydrogels for dental stem cell delivery.

Decellularized mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human apical papilla derived mesenchymal stem cells (SCAP) for spinal cord regeneration. Bone, spinal cord, and dentine ECM hydrogels exhibited distinct structural, mechanical, and biological characteristics. All three hydrogels supported SCAP viability and proliferation. However, only spinal cord and bone derived hydrogels promoted the expression of neural lineage markers. The specific environment of ECM scaffolds significantly affected the differentiation of SCAP to a neural lineage, with stronger responses observed with spinal cord ECM hydrogels, suggesting that site-specific tissues are more likely to facilitate optimal stem cell behavior for constructive spinal cord regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 319-328, 2017.

Composite scaffolds for cartilage tissue engineering based on natural polymers of bacterial origin, thermoplastic poly(3‐hydroxybutyrate) and micro‐fibrillated bacterial cellulose

AbstractCartilage tissue engineering is an emerging therapeutic strategy that aims to regenerate damaged cartilage caused by disease, trauma, ageing or developmental disorder. Since cartilage lacks regenerative capabilities, it is essential to develop approaches that deliver the appropriate cells, biomaterials and signalling factors to the defect site. Materials and fabrication technologies are therefore critically important for cartilage tissue engineering in designing temporary, artificial extracellular matrices (scaffolds), which support 3D cartilage formation. Hence, this work aimed to investigate the use of poly(3‐hydroxybutyrate)/microfibrillated bacterial cellulose (P(3HB)/MFC) composites as 3D‐scaffolds for potential application in cartilage tissue engineering. The compression moulding/particulate leaching technique employed in the study resulted in good dispersion and a strong adhesion between the MFC and the P(3HB) matrix. Furthermore, the composite scaffold produced displayed better mechanical properties than the neat P(3HB) scaffold. On addition of 10, 20, 30 and 40 wt% MFC to the P(3HB) matrix, the compressive modulus was found to have increased by 35%, 37%, 64% and 124%, while the compression yield strength increased by 95%, 97%, 98% and 102% respectively with respect to neat P(3HB). Both cell attachment and proliferation were found to be optimal on the polymer‐based 3D composite scaffolds produced, indicating a non‐toxic and highly compatible surface for the adhesion and proliferation of mouse chondrogenic ATDC5 cells. The large pores sizes (60 ‐ 83 µm) in the 3D scaffold allowed infiltration and migration of ATDC5 cells deep into the porous network of the scaffold material. Overall this work confirmed the potential of P(3HB)/MFC composites as novel materials in cartilage tissue engineering. © 2016 Society of Chemical Industry

Crosslinking of extracellular matrix scaffolds derived from pluripotent stem cell aggregates modulates neural differentiation

At various developmental stages, pluripotent stem cells (PSCs) and their progeny secrete a large amount of extracellular matrices (ECMs) which could interact with regulatory growth factors to modulate stem cell lineage commitment. ECMs derived from PSC can be used as unique scaffolds that provide broad signaling capacities to mediate cellular differentiation. However, the rapid degradation of ECMs can impact their applications as the scaffolds for in vitro cell expansion and in vivo transplantation. To address this issue, this study investigated the effects of crosslinking on the ECMs derived from embryonic stem cells (ESCs) and the regulatory capacity of the crosslinked ECMs on the proliferation and differentiation of reseeded ESC-derived neural progenitor cells (NPCs). To create different biological cues, undifferentiated aggregates, spontaneous embryoid bodies, and ESC-derived NPC aggregates were decellularized. The derived ECMs were crosslinked using genipin or glutaraldehyde to enhance the scaffold stability. ESC-derived NPC aggregates were reseeded on different ECM scaffolds and differential cellular compositions of neural progenitors, neurons, and glial cells were observed. The results indicate that ESC-derived ECM scaffolds affect neural differentiation through intrinsic biological cues and biophysical properties. These scaffolds have potential for in vitro cell culture and in vivo tissue regeneration study. Statement of significanceDynamic interactions of acellular extracellular matrices and stem cells are critical for lineage-specific commitment and tissue regeneration. Understanding the synergistic effects of biochemical, biological, and biophysical properties of acellular matrices would facilitate scaffold design and the functional regulation of stem cells.The present study assessed the influence of crosslinked embryonic stem cell-derived extracellular matrix on neural differentiation and revealed the synergistic interactions of various matrix properties.While embryonic stem cell-derived matrices have been assessed as tissue engineering scaffolds, the impact of crosslinking on the embryonic stem cell-derived matrices to modulate neural differentiation has not been studied.The results from this study provide novel knowledge on the interface of embryonic stem cell-derived extracellular matrix and neural aggregates. The findings reported in this manuscript are significant for stem cell differentiation toward the applications in stem cell-based drug screening, disease modeling, and cell therapies.

Extracellular matrix deposition of bone marrow stroma enhanced by macromolecular crowding

Decellularized extracellular matrices (ECM) from in vitro cell cultures can serve as in vivo-like matrix scaffolds for modulating cell-ECM interactions. Macromolecular crowding (MMC), the supplementation of synthetic or naturally occurring molecules resulting in excluded volume effects (EVE), has been demonstrated to provide valuable options for recapitulating the physiological environment of cells during matrix secretion. Human mesenchymal stem cell (MSC)-derived ECM was produced upon supplementation of standard culture medium with three different macromolecules of various size (10–500 kDa). Matrix secretion, ECM morphology and composition were compared for matrices obtained from crowded and non-crowded MSC cultures. In the context of generating functional stem cell niches, the MSC-derived bone marrow mimetic ECM scaffolds were tested for their supportive effect to maintain and expand human hematopoietic stem and progenitor cells (HSPC) in vitro. MMC in combination with metabolic stimulation of MSC was found to result in tissue-specific, highly organized ECM capable of retaining glycosaminoglycans and growth factors to effectively build in vitro microenvironments that support HSPC expansion.

Biological effects of Spirulina (Arthrospira) biopolymers and biomass in the development of nanostructured scaffolds.

Spirulina is produced from pure cultures of the photosynthetic prokaryotic cyanobacteria Arthrospira. For many years research centers throughout the world have studied its application in various scientific fields, especially in foods and medicine. The biomass produced from Spirulina cultivation contains a variety of biocompounds, including biopeptides, biopolymers, carbohydrates, essential fatty acids, minerals, oligoelements, and sterols. Some of these compounds are bioactive and have anti-inflammatory, antibacterial, antioxidant, and antifungal properties. These compounds can be used in tissue engineering, the interdisciplinary field that combines techniques from cell science, engineering, and materials science and which has grown in importance over the past few decades. Spirulina biomass can be used to produce polyhydroxyalkanoates (PHAs), biopolymers that can substitute synthetic polymers in the construction of engineered extracellular matrices (scaffolds) for use in tissue cultures or bioactive molecule construction. This review describes the development of nanostructured scaffolds based on biopolymers extracted from microalgae and biomass from Spirulina production. These scaffolds have the potential to encourage cell growth while reducing the risk of organ or tissue rejection.

Neural differentiation from pluripotent stem cells: The role of natural and synthetic extracellular matrix.

Neural cells differentiated from pluripotent stem cells (PSCs), including both embryonic stem cells and induced pluripotent stem cells, provide a powerful tool for drug screening, disease modeling and regenerative medicine. High-purity oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs) have been derived from PSCs recently due to the advancements in understanding the developmental signaling pathways. Extracellular matrices (ECM) have been shown to play important roles in regulating the survival, proliferation, and differentiation of neural cells. To improve the function and maturation of the derived neural cells from PSCs, understanding the effects of ECM over the course of neural differentiation of PSCs is critical. During neural differentiation of PSCs, the cells are sensitive to the properties of natural or synthetic ECMs, including biochemical composition, biomechanical properties, and structural/topographical features. This review summarizes recent advances in neural differentiation of human PSCs into OPCs and NPCs, focusing on the role of ECM in modulating the composition and function of the differentiated cells. Especially, the importance of using three-dimensional ECM scaffolds to simulate the in vivo microenvironment for neural differentiation of PSCs is highlighted. Future perspectives including the immediate applications of PSC-derived neural cells in drug screening and disease modeling are also discussed.

The construction of 3D-engineered tissues composed of cells and extracellular matrices by hydrogel template approach

The three-dimensional (3D)-engineered tissues composed of only cells and extracellular matrices (ECM) were constructed by the hydrogel template approach. The disulfide-crosslinked poly( γ-glutamic acid) hydrogels were prepared as a template hydrogel. These template hydrogels were easily decomposed under physiological conditions using reductants such as cysteine, glutathione and dithiothreitol by cleavage of disulfide crosslinkage to thiol groups. The decomposed polymers are soluble in cell culture medium. The cleaving of disulfide bond was determined by UV–vis and FT-IR spectroscopies. We successfully prepared the 3D-engineered tissues (thickness/diameter, 2 mm/1 cm) composed of mouse L929 fibroblast cells and ECM by the decomposition of only the template hydrogel with cysteine after 10 days 3D-cell culture on/in the template hydrogel. The size and thickness of the 3D-engineered tissues was completely transferred from the template hydrogel. The cultured L929 cells viability in the obtained engineered tissues was confirmed by a culture test, WST-1 method and LIVE/DEAD staining assay. The engineered tissue was self-standing and highly dense composite of the cultured cells and collagen produced by the cells. This hydrogel template approach may be useful as a new class of soft-tissue engineering technology to substitute a synthetic polymer scaffold to the ECM scaffold produced from the cultured cells.

Effect of storage upon material properties of lyophilized porcine extracellular matrix derived from the urinary bladder

Xenogeneic extracellular matrices (ECMs) have been developed as off-the-shelf biologic scaffolds that have been effectively used in preclinical and clinical applications for tissue reconstruction. Such materials must be suitable for terminal sterilization and capable of storage for extended periods of time without significant changes in material properties and bioactivity. Material properties of interest for ECM scaffolds include hydrostatic permeability index (PI), uniaxial maximum load and elongation, maximum tangential stiffness (MTS), suture retention strength (SRS), and ball-burst strength (BBS). The present study evaluated these material properties for lyophilized forms of an ECM scaffold derived from the porcine urinary bladder, termed urinary bladder matrix (UBM), that was terminally sterilized by e-beam irradiation at 22 kGy and stored at room temperature (RT; 20-24 degrees C) or refrigerated temperature (REFT; 4-8 degrees C) for up to 12 months. UBM devices showed no change in SRS, BBS, and hydrostatic PI after the evaluation period. Lyophilized devices stored at RT showed an increase in maximum load and MTS while devices stored at REFT showed an increase in maximum elongation after 1 year of storage (p < 0.05). These results indicate that structural changes in the UBM device may slowly occur as a function of prolonged storage and storage temperature.

Analytically derived material properties of multilaminated extracellular matrix devices using the ball-burst test

Xenogeneic extracellular matrices (ECMs) have been shown to be effective as naturally occurring scaffolds for soft-tissue repair. As acellular tissue substitutes at the time of surgical implantation, ECMs are subjected to the mechanical forces and micro-environmental conditions representative of the anatomical location in which they are placed. Ideally such natural scaffolds would possess mechanical properties that allow for normal tissue function in and around the implant site. The ball-burst test was used to simulate biaxial forces and to determine the strength of the ECM scaffold under a relevant physiological loading condition. The ball-burst test, in itself, does not quantify intrinsic mechanical properties and therefore a methodology was developed to determine the maximum stress resultant tangent modulus (MSRTM) or the maximum stress tangent modulus (MSTM), stress to failure ( σ f ) , failure stress resultant ( N f ) , ball-burst pressure ( P ) , and maximum elongation ( λ max ) from the raw ball-burst data obtained at a constant-rate of transverse. The analytical methodology was compared to finite element simulations and showed good correlation with the analytical solution presented. The proposed approximations were used to compute biaxial failure properties for a variety of multilaminate ECM devices with varying number of layers, disinfection and sterilization, and organ origin.

Scaffolds for tissue fabrication

Tissue engineering is an interdisciplinary and multidisciplinary field. It has shown great promise in generating living alternatives for harvested tissues and organs for transplantation and reconstructive surgery. Materials and fabrication technologies are critically important for tissue engineering in designing temporary, artificial extracellular matrices (scaffolds), which support three-dimensional tissue formation. This review briefly introduces the concept of tissue engineering, and illustrates the relationship between tissue engineering and materials science and engineering. Important scaffold design principles are described. The most frequently used materials and fabrication technologies for scaffolds are reviewed. Some exciting new developments in scaffold materials and fabrication technologies are also discussed.

Retention of endothelial cell adherence to porcine-derived extracellular matrix after disinfection and sterilization.

Extracellular matrices (ECM) derived from porcine tissue are associated with rapid and extensive repopulation with host cells when used as scaffolds for in vivo tissue repair. Cell adhesion to substrates used for tissue engineering has been studied extensively but the factors that mediate this phenomenon in ECM scaffolds following treatment with oxidants and sterilants have not been examined. Cell adhesion assays were used to examine human microvascular endothelial cell (HMEC) attachment to ECM graft materials harvested from small intestinal submucosa (SIS) and urinary bladder matrix (UBM) following decellularization and sterilization procedures designed to render the ECM safe for clinical use. HMECs were able to attach directly to these ECM scaffolds via several attachment proteins present within the ECM, including type I collagen, type IV collagen, and fibronectin. The ability of the SIS ECM and UBM ECM to support the growth and proliferation of HMEC was also examined. HMEC were able to grow to single-layer confluence on both surfaces of SIS and UBM sheets. The endothelial cells were also able to penetrate the SIS and UBM at later time points if they were seeded on the abluminal side of the ECM sheets. The ability of the processed ECM to support HMEC attachment and proliferation is similar to that reported for unprocessed ECM and may therefore play a role in the rapid remodeling response observed when these matrices are implanted in vivo as scaffolds for wound repair.

Fibronectin peptides mediate HMEC adhesion to porcine-derived extracellular matrix

Extracellular matrices (ECM) derived from porcine tissues have been shown to support the successful repair and remodeling of injured tissues when evaluated in animal models. Cell–matrix interactions, including ligand–integrin associations that facilitate endothelial cell adhesion, are clearly important in the tissue remodeling process. The goal of the present study was to identify the peptide sequences within the ubiquitous protein fibronectin (FN) that may be important in the initial interactions between the host endothelial cells and the ECM scaffold. Human microvascular endothelial cells (HMEC) were seeded upon porcine ECM after having been subjected to pretreatment with peptide ligands derived from tissue FN and were allowed to attach for 20min. Non-adherent cells were removed and the remaining, tritium-labeled cells attached to the ECM were counted. Results showed that cyclo-RGD and REDV, but not LDV or PHSRN, play a role in mediating the attachment of HMEC to porcine ECM.