Extracellular Matrix Analogs Research Articles

The incorporation and controlled release of platelet‐rich plasma‐derived biomolecules from polymeric tissue engineering scaffolds

AbstractPlatelet‐rich plasma (PRP) has been gaining popularity in recent years as a cost‐effective material capable of stimulating healing in a number of different clinical applications. As the clinical role ofPRPhas been growing so too has its prevalence in the fields of tissue engineering and regenerative medicine, particularly in the field of extracellular matrix (ECM) analogue scaffold fabrication. As polymeric scaffold fabrication techniques strive to create structures that ever more closely replicate the nativeECM's form and function, the need for increased scaffold bioactivity becomes more pronounced.PRP, which has been shown to contain over 300 bioactive molecules, has the potential to deliver a combination of growth factors and cytokines capable of stimulating cellular activity through enhanced chemotaxis, proliferation andECMproduction. The ability to incorporate such a potent bioactive milieu into a polymeric tissue engineering scaffold, which lacks intrinsic cell signaling molecules, may help to promote scaffold integration with native tissues and increase the overall patency of polymericECManalogue structures. This mini‐review briefly discusses the physiological basis ofPRPand its current clinical use, as well as the potential role thatPRPmay play in the future of polymeric tissue engineering scaffold design. Copyright © 2012 Society of Chemical Industry

Design of Bioactive Electrospun Scaffolds for Bone Tissue Engineering

This study aimed to produce polycaprolactone (PCL) and PCL/gelatin fibrous scaffolds by electrospinning to engineer functional bone by the careful reproduction of the native microenviroment of the natural tissue. Polymer solutions were processed by electrospinning technique to fabricate 2D and 3D platforms in the form of random flat membranes and bilayered conduits, through the use of collectors - i.e., grounded metal plates and a rotating mandrel, via a 2-step electrospinning process, to produce a bilayered structure. The results showed that solvent properties and the integration of gelatin could strongly influence the scaffold features in terms of fiber size scale and homogeneity, thus potentially affecting the final biological response. Moreover, 3D bilayered devices ensured the mechanical stability required to guide the forming bone during the regeneration process. Overall, bioactive 2D or 3D electrospun platforms with microstructured and nanostructured properties can be used successfully as extracellular matrix analogues in bone regeneration.

Design of Biomimetic Cell-Interactive Substrates Using Hyaluronic Acid Hydrogels with Tunable Mechanical Properties

Hyaluronic acid (HA) is a natural polysaccharide abundant in biological tissues with excellent potential for constructing synthetic extracellular matrix analogues. In this work, we established a simple and dependable approach to prepare hyaluronic acid-based hydrogels with controlled stiffness and cell recognition properties for use as cell-interactive substrates. This approach relied on a new procedure for the synthesis of methacrylate-modified HA macromers (HA-MA) and, on photorheometry allowing real time monitoring of gelation during photopolymerization. We showed in this way the ability to obtain gels that encompass the range of physiologically relevant elastic moduli while still maintaining the recognition properties of HA by specific cell surface receptors. These hydrogels were prepared from HA macromers having a degree of methacrylation <0.5, which allows to minimize compromising effects on the binding affinity of HA to its cell receptors due to high substitution on the one hand, and to achieve nearly 100% conversion of the methacrylate groups on the other. When the HA hydrogels were immobilized on glass substrates, it was observed that the attachment and the spreading of a variety of mammalian cells rely on CD44 and its coreceptor RHAMM. The attachment and spreading were also shown to be modulated by the elastic properties of the HA matrix. All together, these results highlight the biological potential of these HA hydrogel systems and the needs of controlling their chemical and physical properties for applications in cell culture and tissue engineering.

ECM analog technology a simple tool for exploring cell-ECM dynamics

Cells in a functional tissue display a highly interactive relation with their neighboring cells and associated biochemical milieu. Serious disruption in the existing homeostatic balance in the extra-cellular matrix (ECM) may lead to abnormal response by the cells. With insufficient understanding of Cell-ECM interaction and in absence of simple tools for in vitro cell-culture in 3D, we still have to rely on the data generated by growing cells in 2D. In order to comprehend Cell-ECM dynamics it is important to recreate in vivo like microenvironment in 3D. Senescence and loss of function commonly observed in cells cultured in 2D are expected to be surmounted using such tools. Unlike prevailing belief that 3D culture is required only for tissue engineering (TE) and regenerative medicine, simple and easy to handle tools for ex vivo 3D culture may lead to greater impact. With the potential to improve our understanding about cellular behavior, both in normal and abnormal surroundings, they may eventually influence the diagnosis. Here we discuss some of the tools for cell culture in 3D, made available through novel cell-interactive ECM analog technology.

ChemInform Abstract: Microfluidic Devices as Tools for Mimicking the in vivo Environment

AbstractReview: 170 refs.

Design of Functional Polymer and Composite Scaffolds for the Regeneration of Bone, Menisci, Osteochondral and Peripheral Nervous Tissues

In order to mimic the behaviors of natural tissue, the optimal approach for designing novel biomaterials has to be inspired to nature guidelines. One of the major challenge consists in the development of well-organized structures or scaffolds with controlled porosity in terms of pore size, pore shape and interconnection degree able to guide new tissue formation during the in vivo degradation following the scaffold implantation. Scaffolds endowed with molecular cues together to a controlled degradation profile should contribute to cell proliferation and differentiation, controlled vascularization, promoting the remodeling of neo tissue through a gradual transmission of bio-chemicals and biophysical signals as performed by the extracellular matrix (ECM). Here, different polymers and composites have been investigated to design scaffolds with peculiar micro and/or nanometric morphological features in order to satisfy all these requirements: a) bioactive scaffolds, with tailored porosity and high pores interconnectivity were developed by integrating PLA fibres, Calcium Phosphates particles or Hyaff11 phases into a Poly(ε-caprolactone) (PCL) matrix by the combination of filament winding technology and phase inversion/salt leaching technique as mineralised ECM analogue for bone regeneration; b) custom made PCL/hydroxyapatite scaffolds were designed by imaging and rapid prototyping technologies for the osteochondral defect. c) Ester of Hyaluronic Acid reinforced with degradable fibres were processed by composite technology, phase inversion and salt leaching technique, to obtain scaffolds for meniscus regeneration. d) PCL and gelatin nanofibres were obtained by highly customized fibre deposition via electrospinning to guide the nerve outgrowth in nerve regeneration. All the proposed approaches offer the chance of realizing tailor-made platforms with micro/nanoscale architecture and chemical composition suitable for the regeneration of the extracellular matrix of a large variety of natural tissues (i.e, bone, menisci, osteochondral and peripheral nervous tissues).

Incorporation of biomimetic matrix molecules in PEG hydrogels enhances matrix deposition and reduces load‐induced loss of chondrocyte‐secreted matrix

Poly(ethylene glycol) (PEG) hydrogels offer numerous advantages in designing controlled 3D environments for cartilage regeneration, but offer little biorecognition for the cells. Incorporating molecules that more closely mimic the native tissue may provide key signals for matrix synthesis and may also help in the retention of neotissue, particularly when mechanical stimulation is employed. Therefore, this research tested the hypothesis that exogenous hyaluronan encapsulated within PEG hydrogels improves tissue deposition by chondrocytes, while the incorporation of Link-N (DHLSDNYTLDHDRAIH), a fragment of link protein that is involved in stabilizing hyaluronan and aggrecan in cartilage, aids in the retention of the entrapped hyaluronan as well as cell-secreted glycosaminoglycans (GAGs), particularly when dynamic loading is employed. The incorporation of Link-N as covalent tethers resulted in a significant reduction, ~60%, in the loss of entrapped exogenous hyaluronan under dynamic stimulation. When chondrocytes were encapsulated in PEG hydrogels containing exogenous hyaluronan and/or Link-N, the extracellular matrix (ECM) analogs aided in the retention of cell-secreted GAGs under loading. The presence of hyaluronan led to enhanced deposition of collagen type II and aggrecan. In conclusion, our results highlight the importance of ECM analogs, specifically hyaluronan and Link-N, in matrix retention and matrix development and offer new strategies for designing scaffolds for cartilage regeneration.

Microfluidic devices as tools for mimicking the in vivo environment

One of the major branches of microfluidic development is cell engineering. A number of devices for cell cultivation, lysis, single-cell analysis and cell-based toxicity tests have been reported in the literature. The variety of structures that can be created leads to devices more closely mimicking the in vivo environment than classic cell cultures. Studies on this topic will have an effect on the evaluation of methods that can replace animals in biomedical research. The aim of this review is to present latest advancements of “lab-on-a-chip” for cell cultivation and engineering. The authors focus on the achievements leading to in vivo-like methods. The materials and fabrication methods in silicon, glass, PDMS and other polymers were briefly characterized. Microfluidic devices were applied for mimicking the in vivo environment at various levels of mammalian body organization—from the surroundings of single cells to interactions between functional organs. Solutions for “human-on-a-chip”, perfusion cell cultures, extracellular matrix analogues, microscaffolds, spheroid formation and co-cultures were reviewed in this paper. The presented solutions have the potential to become new cellular models for toxicology, drug development and biomedical research.

Molecularly designed alginate hydrogels susceptible to local proteolysis as three-dimensional cellular microenvironments

The development of sophisticated three-dimensional (3-D) cell culture microenvironments that recreate some of the complexity of the natural extracellular matrix (ECM) remains a challenging task. Here, the modification of alginate through partial crosslinking with a matrix metalloproteinase (MMP) cleavable peptide (proline–valine–glycine–leucine–isoleucine–glycine, PVGLIG) is described, and its use in the preparation of injectable, in situ crosslinkable hydrogel-like matrices is proposed. PVGLIG-grafted alginates were synthesized by carbodiimide chemistry and characterized. Their biological performance was evaluated by comparing the response of 3-D cultured mesenchymal stem cells (MSCs) to alginate hydrogels containing only cell-adhesion peptides (RGD-alginate) or both peptides (PVGLIG/RGD-alginate). After 1week, cells remained essentially round within RGD-alginate, while they exhibited an elongated morphology within PVGLIG/RGD-alginate hydrogels, forming cellular networks. This suggests that cells were able to structurally reorganize the matrix, through enzymatic hydrolysis of PVGLIG residues, overcoming biophysical hydrogel resistance. As shown by gelatine-zymography, MSC presented higher activity of MMP-2 when cultured within alginate functionalized with MMP-sensitive peptide, suggesting that the cell’s proteolytic phenotype was modulated by the matrix composition. Additionally, PVGLIG/RGD-alginate hydrogels were clearly degraded in cell culture. Taken together, the results demonstrate that the co-incorporation of MMP-labile peptides in cell-adhesive RGD-alginate hydrogels improved their performance as ECM analogues, providing a more dynamic and physiological 3-D cellular microenvironment.

The Use of Natural Polymers in Tissue Engineering: A Focus on Electrospun Extracellular Matrix Analogues

Natural polymers such as collagens, elastin, and fibrinogen make up much of the body’s native extracellular matrix (ECM). This ECM provides structure and mechanical integrity to tissues, as well as communicating with the cellular components it supports to help facilitate and regulate daily cellular processes and wound healing. An ideal tissue engineering scaffold would not only replicate the structure of this ECM, but would also replicate the many functions that the ECM performs. In the past decade, the process of electrospinning has proven effective in creating non-woven ECM analogue scaffolds of micro to nanoscale diameter fibers from an array of synthetic and natural polymers. The ability of this fabrication technique to utilize the aforementioned natural polymers to create tissue engineering scaffolds has yielded promising results, both in vitro and in vivo, due in part to the enhanced bioactivity afforded by materials normally found within the human body. This review will present the process of electrospinning and describe the use of natural polymers in the creation of bioactive ECM analogues in tissue engineering.

An optical method to quantify the density of ligands for cell adhesion receptors in three-dimensional matrices.

The three-dimensional matrix that surrounds cells is an important insoluble regulator of cell phenotypes. Examples of such insoluble surfaces are the extracellular matrix (ECM), ECM analogues and synthetic polymeric biomaterials. Cell-matrix interactions are mediated by cell adhesion receptors that bind to chemical entities (adhesion ligands) on the surface of the matrix. There are currently no established methods to obtain quantitative estimates of the density of adhesion ligands recognized by specific cell adhesion receptors. This article presents a new optical-based methodology for measuring ligands of adhesion receptors on three-dimensional matrices. The study also provides preliminary quantitative results for the density of adhesion ligands of integrins alpha(1)beta(1) and alpha(2)beta(1) on the surface of collagen-based scaffolds, similar to biomaterials that are used clinically to induce regeneration in injured skin and peripheral nerves. Preliminary estimates of the surface density of the ligands of these two major collagen-binding receptors are 5775 +/- 2064 ligands microm(-2) for ligands of alpha(1)beta(1) and 17 084 +/- 5353 ligands microm(-2) for ligands of alpha(2)beta(1). The proposed methodology can be used to quantify the surface chemistry of insoluble surfaces that possess biological activity, such as native tissue ECM and biomaterials, and therefore can be used in cell biology, biomaterials science and regenerative medical studies for quantitative description of a matrix and its effects on cells.

Tissue scaffolds for skin wound healing and dermal reconstruction

One of the major applications of tissue-engineered skin substitutes for wound healing is to promote the healing of cutaneous wounds. In this respect, many important clinical milestones have been reached in the past decades. However, currently available skin substitutes for wound healing often suffer from a range of problems including wound contraction, scar formation, and poor integration with host tissue. Engineering skin substitutes by tissue engineering approach has relied upon the creation of three-dimensional scaffolds as extracellular matrix (ECM) analog to guide cell adhesion, growth, and differentiation to form skin-functional and structural tissue. The three-dimensional scaffolds can not only cover wound and give a physical barrier against external infection as wound dressing, but also can provide support both for dermal fibroblasts and the overlying keratinocytes for skin tissue engineering. A successful tissue scaffold should exhibit appropriate physical and mechanical characteristics and provide an appropriate surface chemistry and nano and microstructures to facilitate cellular attachment, proliferation, and differentiation. A variety of scaffolds have been fabricated based on materials ranging from naturally occurring ones to those manufactured synthetically. This review discusses a variety of commercial or laboratory-engineered skin substitutes for wound healing. Central to the discussion are the scaffolds/materials, fabrication techniques, and their characteristics associated with wound healing. One specifically highlighted emerging fabrication technique is electrospinning that allows the design and fabrication of biomimetic scaffolds that offer tremendous potential applications in wound healing of skin.

Engineering integrin signaling for promoting embryonic stem cell self-renewal in a precisely defined niche

We present development and use of a 3D synthetic extracellular matrix (ECM) analog with integrin-specific adhesion ligands to characterize the microenvironmental influences in embryonic stem cell (ESC) self-renewal. Transcriptional analysis of 24 integrin subunits followed by confirmation at the translational and functional levels suggested that integrins α 5β 1, α vβ 5, α 6β 1 and α 9β 1 play important roles in maintenance of stemness in undifferentiated mouse ESCs. Using the well-defined matrix as a tool to activate integrins α 5β 1 plus α vβ 5, α 6β 1 and α 9β 1, individually and in combination, differential integrin activation was demonstrated to exert exquisite control over ESC fate decisions. Simultaneous ligation of these four integrin heterodimers promoted self-renewal, as evidence by prolonged SSEA-1, Oct4 and Nanog expression, and induced Akt1 kinase signaling along with translational regulation of other stemness-related genes. The biofunctional network we have designed based on this knowledge may be useful as a defined niche for regulating ESC pluripotency through selective cell–matrix interactions, and the method we present may be more generally useful for probing matrix interactions in stem cell self-renewal and differentiation.

Functionalized self‐assembling peptide hydrogel enhance maintenance of hepatocyte activity in vitro

There is a major challenge in maintaining functional hepatocytes in vivo as these cells rapidly lose their metabolic properties in culture. In this work we have developed a bioengineered platform that replaces the use of the collagen I – in the traditional culture sandwich technique – by a defined extracellular matrix analogue, the self-assembling peptide hydrogel RAD16-I functionalized with biologically active motifs. Thus, after examining side by side the two culture systems we have found that in both cases hepatocytes acquired similar parenchymal morphology, presence of functional bile canaliculi structures, CYP3A2 induction by dexamethasone, urea production, secretion of proteins such as apolipoprotein (class A1, E, J), α1-microglobulin, α1-macroglobulin, retinol binding protein, fibronectin, α1-inhibitor III and biotin-dependent carboxylases. Interestingly, by assessing in more detail some other hepatic markers, one of the functionalized matrix analogues – carrying the 67 kD laminin receptor ligand – enhanced the gene expression of albumin, HNF4-α, MDR2 and tyrosine aminotransferase. We conclude that the use of a synthetic culture system with designed matrix functionalization has the advantage in controlling specific cellular responses.

Science of Nanofibrous Scaffold Fabrication: Strategies for Next Generation Tissue-Engineering Scaffolds

Native extracellular matrix (ECM) provides structural support to the multicellular organism on a macroscopic scale and establishes a unique microenvironment (niche) to tissue- and organ-specific cell types. Both these functions are critical for optimal function of the organism. These natural ECMs comprise predominantly fibrillar proteins, collagen and elastin and are synthesized as monomers but undergo hierarchical organization into well-defined nanoscaled structural units. The interaction between the cells and ECM is dynamic, reciprocal and essential for tissue development, maintenance of function, repair and regeneration processes. Tissue-engineering scaffolds are synthetic, biomimetic ECM analogues that have great promise in regenerative medicine. Ongoing efforts in mimicking the native ECM in terms of composition and dimension have resulted in three strategies that permit the generation of scaffolds in nanometer dimensions. Although excellent reviews regarding the applications of these strategies in tissue engineering are available, a comprehensive review of the science behind these fabrication techniques does not exist. This review intends to fill this critical gap in the existing knowledge in the fast-expanding field of nanofibrous scaffolds. A thorough understanding of the fabrication processes would enable us to better exploit available technologies to produce superior tissue-engineering scaffolds.

Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability

The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN's central cell-binding domains (FN III9–10) affects its integrin specificity. Through production of FN III9–10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin α 5β 1 and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of α 5β 1 integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that α 5β 1 has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for α 5β 1 versus α vβ 3 results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.

Use of Hyaluronan‐Derived Hydrogels for Three‐Dimensional Cell Culture and Tumor Xenografts

The practice of in vitro three-dimensional (3-D) cell culture has lagged behind the realization that classical two-dimensional (2-D) culture on plastic surfaces fails to mirror normal cell biology. Biologically, a complex network of proteins and proteoglycans that constitute the extracellular matrix (ECM) surrounds every cell. To recapitulate the normal cellular behavior, scaffolds (ECM analogs) that reconstitute the essential biological cues are required. This unit describes the 3-D cell culture and tumor engineering applications of Extracel, a novel semisynthetic ECM (sECM), based on cross-linked derivatives of hyaluronan and gelatin. A simplified cell encapsulation and pseudo-3-D culturing (on top of hydrogels) protocol is provided. In addition, the use of this sECM as a vehicle to obtain tumor xenografts with improved take rates and tumor growth is presented. These engineered tumors can be used to evaluate anticancer therapies under physiologically relevant conditions.

Defining the Role of Matrix Compliance and Proteolysis in Three-Dimensional Cell Spreading and Remodeling

Recent studies have identified extracellular matrix (ECM) compliance as an influential factor in determining the fate of anchorage-dependent cells. We explore a method of examining the influence of ECM compliance on cell morphology and remodeling in three-dimensional culture. For this purpose, a biological ECM analog material was developed to pseudo-independently alter its biochemical and physical properties. A set of 18 material variants were prepared with shear modulus ranging from 10 to 700Pa. Smooth muscle cells were encapsulated in these materials and time-lapse video microscopy was used to show a relationship between matrix modulus, proteolytic biodegradation, cell spreading, and cell compaction of the matrix. The proteolytic susceptibility of the matrix, the degree of matrix compaction, and the cell morphology were quantified for each of the material variants to correlate with the modulus data. The initial cell spreading into the hydrogel matrix was dependent on the proteolytic susceptibility of the materials, whereas the extent of cell compaction proved to be more correlated to the modulus of the material. Inhibition of matrix metalloproteinases profoundly affected initial cell spreading and remodeling even in the most compliant materials. We concluded that smooth muscle cells use proteolysis to form lamellipodia and tractional forces to contract and remodel their surrounding microenvironment. Matrix modulus can therefore be used to control the extent of cellular remodeling and compaction. This study further shows that the interconnection between matrix modulus and proteolytic resistance in the ECM may be partly uncoupled to provide insight into how cells interpret their physical three-dimensional microenvironment.

Surface controlled biomimetic coating of polycaprolactone nanofiber meshes to be used as bone extracellular matrix analogues

The aim of this work was to develop novel electrospun nanofiber meshes coated with a biomimetic calcium phosphate (BCP) layer that mimics the extracellular microenvironment found in the human bone structure. Poly (ε-caprolactone) (PCL) was selected because of its well-known medical applications, its biodegradability, biocompatibility and its susceptibility to partial hydrolysis by a straightforward alkaline treatment. The deposition of a calcium phosphate layer, similar to the inorganic phase of bone, on PCL nanofiber meshes was achieved by means of a surface modification. This initial surface modification was followed by treatment with solutions containing calcium and phosphate ions. The process was finished by a posterior immersion in a simulated body fluid (SBF) with nearly 1.5 × the inorganic concentration of the human blood plasma ions. After some optimization work, the best conditions were chosen to perform the biological assays. The influence of the bone-like BCP layer on the viability and adhesion, as well as on the proliferation of human osteoblast-like cells, was assessed. It was shown that PCL nanofiber meshes coated with a BCP layer support and enhance the proliferation of osteoblasts for long culture periods. The attractive properties of the coated structures produced in the present work demonstrated that those materials have potential to be used for applications in bone tissue engineering. This is the first time that nanofiber meshes could be coated with a biomimetic bone-like calcium phosphate layer produced in a way that the original mesh architecture can be fully maintained.

Effects of extracellular matrix analogues on primary human fibroblast behavior

In vitro cell culture is a vital research tool for cell biology, pharmacology, toxicology, protein production, systems biology and drug discovery. Traditional culturing methods on plastic surfaces do not accurately represent the in vivo environment, and a paradigm shift from two-dimensional to three-dimensional (3-D) experimental techniques is underway. To enable this change, a variety of natural, synthetic and semi-synthetic extracellular matrix (ECM) equivalents have been developed to provide an appropriate cellular microenvironment. We describe herein an investigation of the properties of four commercially available ECM equivalents on the growth and proliferation of primary human tracheal scar fibroblast behavior, both in 3-D and pseudo-3-D conditions. We also compare subcutaneous tissue growth of 3-D encapsulated fibroblasts in vivo in two of these materials, Matrigel™ and Extracel™. The latter shows increased cell proliferation and remodeling of the ECM equivalent. The results provide researchers with a rational basis for selection of a given ECM equivalent based on its biological performance in vitro and in vivo, as well as the practicality of the experimental protocols. Biomaterials that use a customizable glycosaminoglycan-based hydrogel appear to offer the most convenient and flexible system for conducting in vitro research that accurately translates to in vivo physiology needed for tissue engineering.