Fibrin: a natural biodegradable scaffold in vascular tissue engineering

Arterial occlusive disease remains a major health issue in the developed world and a rapidly growing problem in the developing world. Although a growing number of patients are now being effectively treated with minimally invasive techniques, there remains a tremendous pressure on the vascular community to develop a synthetic small-diameter vascular graft with improved long-term patency rates. The field of tissue engineering offers an exciting alternative in the search for living organ replacement structures. Several methodologies have emerged for constructing blood vessel replacements with biological functionality. Common strategies include cell-seeded biodegradable synthetic scaffolds, cell self-assembly, cell-seeded gels and xenogeneic acellular materials. A wide range of materials are being investigated as potential scaffolds for vascular tissue engineering applications. Some are commercialised and others are still in development. Recently, researchers have studied the role of fibrin gel as a three-dimensional scaffold in vascular tissue engineering. This overview describes the properties of fibrin gel in vascular tissue engineering and highlights some recent progress and difficulties encountered in the development of cell fibrin scaffold technology.     

Fibrin gel as alternative scaffold for respiratory tissue engineering

Fibrin gel has proven a valuable scaffold for tissue engineering. Complex geometries can be produced by injection molding; it offers effective cell seeding and can be produced autologous. In order to evaluate its suitability for respiratory tissue engineering, we examined proliferation, functionality, and differentiation of respiratory epithelial cells on fibrin gel in comparison to culture on collagen-coated, microporous membranes. Respiratory epithelial cells formed a confluent layer by day 4, and proliferation showed no significant difference with respect to surface. Measurement of the transepithelial electrical resistance reflected the development of a confluent epithelial cell layer and the subsequent initiation of adequate ion-transfer processes. Appearance of ciliae could be detected at similar time points, and ciliary beating could be observed for cells on both surfaces. Histology and immunohistochemistry of cells grown on fibrin gel revealed the onset of adequate differentiation. As no significant differences in respiratory epithelial cells’ proliferation, function, and differentiation could be observed between cells grown on fibrin gel compared to cells on a collagen-coated, microporous surface, we concluded that fibrin gel might prove a suitable scaffold for respiratory tissue engineering and merits further investigation to overcome the limitations associated with scaffolds currently in use.     

Hydrogels for tissue engineering: scaffold design variables and applications

Polymer scaffolds have many different functions in the field of tissue engineering. They are applied as space filling agents, as delivery vehicles for bioactive molecules, and as three-dimensional structures that organize cells and present stimuli to direct the formation of a desired tissue. Much of the success of scaffolds in these roles hinges on finding an appropriate material to address the critical physical, mass transport, and biological design variables inherent to each application. Hydrogels are an appealing scaffold material because they are structurally similar to the extracellular matrix of many tissues, can often be processed under relatively mild conditions, and may be delivered in a minimally invasive manner. Consequently, hydrogels have been utilized as scaffold materials for drug and growth factor delivery, engineering tissue replacements, and a variety of other applications.     

Fibrin as a cell carrier in cardiovascular tissue engineering applications

In cardiovascular tissue engineering approaches, efficient seeding methods are essential. To achieve this and to save time, cells can be encapsulated in gels. Combining the advantages of a gel as a cell carrier with the advantages of a fiber-based scaffold, providing structural integrity to the developing tissue, might offer several advantages. In this study, seeding by using fibrin as a cell carrier is compared to the conventional static seeding method with regard to tissue development. Seeding with fibrin resulted in less loss of soluble collagen into the medium and a more mature extracellular matrix in a shorter period of time. The use of fibrin degradation inhibitors was shown to inhibit extracellular matrix formation, although it did not hamper cell proliferation. The use of fibrin as a cell carrier to seed cells into a fiber-based scaffold may represent a promising, timesaving approach in cardiovascular tissue engineering applications.     

Development and characterization of a porous micro-patterned scaffold for vascular tissue engineering applications

The fabrication of functional small diameter blood vessel analogs has implications in vascular disease treatment. Current 3D models of the medial vessel layer lack micron-scale topographical cues that have shown promise in vitro by recapitulating native vascular smooth muscle cell (VSMC) behavior. A major obstacle to fabricating 3D scaffolds is maintaining adequate nutrient diffusion to cells. We have developed and characterized porous micro-patterned poly-caprolactone (PCL) scaffolds using a novel technique that integrates soft lithography, melt molding and particulate leaching of polylactic-co-glycolic acid (PLGA) micro/nanoparticles. Scanning electron microscopy showed that PLGA-leached scaffolds have circular pores significantly smaller than the size scale of the grooved surface pattern (48 microm grooves; 5 microm deep; 12 microm spacing). Diffusion of media through PLGA-leached scaffolds was six-fold greater than through non-porous scaffolds, indicating successful introduction of through-pores into PCL by the PLGA leaching technique. VSMC alignment on micro-patterned PLGA-leached scaffolds was similar to that on micro-patterned non-porous scaffolds, indicating no loss in cellular organization on PLGA-leached scaffolds. In contrast, cells seeded on micro-patterned sodium bicarbonate-leached scaffolds remained un-aligned. The ability to micro-pattern cells on porous scaffolds may facilitate the transfer of micro-technology from simple 2D substrates to complex 3D architectures, allowing for tight control over cellular organization in fabricated tissues.     

Development of a 'mechano-active' scaffold for tissue engineering

In this study. we investigate the potential for manipulating bone cell mechanotransducers in tissue engineering. Membrane ion channels such as voltage operated calcium channels (VOCC) have been shown to be a critical component of the bone cell transduction pathway with agonists and inhibitors of this pathway having profound effects on the load signal. By encapsulating a calcium channel agonist with slow release within a poly(L-lactide) (PLLA) scaffold, we can generate a 'mechano-active' scaffold for use in skeletal tissue engineering. PLLA scaffolds with and without a calcium channel agonist, BAY K8644, were seeded with primary human bone cells or the human MG63 bone cell line and cultured for 13 weeks followed by mechanical stimulation with a four-point bending model. Our results show that addition of the agonist for slow release is sufficient to enhance the load-related responses in bone cells within the scaffolds. Specifically, collagen type I expression and the ratio of alkaline phosphatase to protein are elevated in response to cyclical mechanical stimulation of approximately 1000 microstr which is then further enhanced in the mechano-active' scaffolds. As the agonists only act when the calcium channels are open by attenuating the calcium flux, the stimulation is specifically targeted to scaffolds subjected to load either in vitro or ultimately in vivo. Our results suggest that manipulating the VOCC and attenuating the opening of the calcium channels may be an effective technique to amplify matrix production via mechanical stimulation which may be applied to bone tissue engineering and potentially engineering of other load-bearing connective tissues.     

Biocompatibility of PCL/PLGA-BCP porous scaffold for bone tissue engineering applications

In this study, biomimic porous polycaprolactone/poly (lactide-co-glycolide) loading biphasic tricalcium phosphate (PCL/PLGA-BCP) scaffolds were fabricated successfully by solvent evaporation method. The distribution of biphasic tricalcium phosphate (BCP) in polycaprolactone/poly (lactide-co-glycolide) (PCL/PLGA) scaffold was confirmed by micro-computed tomography (micro-CT) scanning, scanning electron microscope (SEM) observation and Energy-dispersive X-ray Spectroscopy (EDS) analysis. The hydrophilicity of the scaffolds was confirmed by contact angle measurement. In in vitro experiments, proliferation of human bone marrow mesenchymal stem cell (hBMSCs) and its osteoblastic differentiation on scaffold were assessed for 1, 2 and 3 weeks using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence observation, hematoxylin & eosin (H&E) staining and real-time polymerase chain reaction (RT-PCR). In in vivo experiments, ossification was observed using micro-CT analysis and histological staining.     

Cross-linking of extruded collagen fibers--a biomimetic three-dimensional scaffold for tissue engineering applications

The repair of tissue defects remains a challenging clinical problem. Extruded collagen fibers comprise a promising scaffold for anterior cruciate ligament and tendon reconstruction; however the engineering of these fibers has still to be improved to bring this material to clinical practice. In this study, for the first time we investigated the influence of a wide range of cross-linking approaches (chemical, physical, and biological) on the properties of these fibers. Ultrastructural evaluation revealed a closely packed interfiber structure independent of the cross-linking method employed. The thermal properties were dependent on the cross-linking method employed and closely matched native tissues. The stress-strain curves were found to depend on the water content of the fibers, which was influenced by the cross-linking method. An inversely proportional relationship between both dry and wet fiber diameter and stress at break was found, which indicates that tailored-made biomaterials can be produced. Overall, the chemical stabilizations were more potent than both physical and biological approaches. Bifunctional agents such as hexamethylene diisocyanate and ethylene glycol diglycidyl ether or agents that promote matrix formation such as glutaraldehyde produced fibers with properties similar to those of native or synthetic fibers to suit a wide range of tissue engineering applications.     

Development of the human umbilical vein scaffold for cardiovascular tissue engineering applications

Biologic function and the mechanical performance of vascular grafting materials are important predictors of graft patency. As such, "functional" materials that improve biologic integration and function have become increasingly sought after. An important alternative to synthetic materials is the use of biomaterials derived from ex vivo tissues that retain significant biologic and mechanical function. Unfortunately, inconsistent mechanical properties that result from tedious, time consuming, manual dissection methods have reduced the potential usefulness of many of these materials. We describe the preparation of the human umbilical vein (HUV) for use as an acellular, three-dimensional, vascular scaffold using a novel, automated dissection methodology. The goal of this investigation was to determine the effectiveness of the autodissection methodology to yield an ex vivo biomaterial with improved uniformity and reduced variance. Mechanical properties, including burst pressure, compliance, uniaxial tension testing, and suture holding capacity, were assessed to determine the suitability of the HUV scaffold for vascular tissue engineering applications. The automated methodology results in a tubular scaffold with significantly reduced sample to sample variation, requiring significantly less time to excise the vein from the umbilical cord than manual dissection methods. Short-term analysis of the interactions between primary human vascular smooth muscle cells and fibroblasts HUV scaffold have shown an excellent potential for cellular integration by native cellular remodeling processes. Our work has shown that the HUV scaffold is mechanically sound, uniform, and maintains its biphasic stress-strain relationship throughout tissue processing. By maintaining the mechanical properties of the native blood vessels, in concert with promising cellular interactions, the HUV scaffold may lead to improved grafts for vascular reconstructive surgeries.     

Fused deposition modeling of novel scaffold architectures for tissue engineering applications.

Fused deposition modeling, a rapid prototyping technology, was used to produce novel scaffolds with honeycomb-like pattern, fully interconnected channel network, and controllable porosity and channel size. A bioresorbable polymer poly(epsilon-caprolactone) (PCL) was developed as a filament modeling material to produce porous scaffolds, made of layers of directionally aligned microfilaments, using this computer-controlled extrusion and deposition process. The PCL scaffolds were produced with a range of channel size 160-700 microm, filament diameter 260-370 microm and porosity 48-77%, and regular geometrical honeycomb pores, depending on the processing parameters. The scaffolds of different porosity also exhibited a pattern of compressive stress-strain behavior characteristic of porous solids under such loading. The compressive stiffness ranged from 4 to 77 MPa, yield strength from 0.4 to 3.6 MPa and yield strain from 4% to 28%. Analysis of the measured data shows a high correlation between the scaffold porosity and the compressive properties based on a power-law relationship.     

自组装肽纳米纤维支架及其在组织工程的应用

自组装肽纳米纤维支架(SAPNS)是近年来被广泛研究的一种生物支架材料,其在三维细胞培养、组织修复与再生等领域得到了越来越多的应用。本综述在介绍SAPNS研究现状的基础上,按外、中、内3个胚层来源组织的顺序阐述了SAPNS在组织工程中的应用进展、存在问题及展望。     

Development of a fibrin composite-coated poly(epsilon-caprolactone) scaffold for potential vascular tissue engineering applications

Poor cell adhesion, cytotoxicity of degradation products and lack of biological signals for cell growth, survival, and tissue generation are the limitations in the use of a biodegradable polymer scaffold for vascular tissue engineering. We have fabricated a hybrid scaffold by integrating physicochemical characteristics of poly(epsilon-caprolactone) (PCL) and biomimetic property of a composite of fibrin, fibronectin, gelatin, growth factors, and proteoglycans to improve EC growth on the scaffold. Solvent cast porous films of poly(epsilon-caprolactone) was prepared using PEG as a porogen. Porosity varied between 5 and 200 microm, and FTIR spectroscopy confirmed structural aspects of PCL. Films kept in PBS for 60 days showed tensile strength and elongation matching native blood vessel. Slow degradation of the scaffold was demonstrated by gravimetric analysis and molecular weight determination. Human umbilical vein endothelial cell (HUVEC) adhesion and proliferation on bare films were minimal. FTIR spectroscopy and environmental scanning electron microscopy (ESEM) of PCL-fibrin hybrid scaffold confirmed the presence of fibrin composite on PCL film. HUVEC was subsequently cultured on hybrid scaffold, and continuous EC lining was observed in 15 and 30 days of culture using ESEM. Results suggest that the new hybrid scaffold can be a suitable candidate for cardiovascular tissue engineering.     

Novel biodegradable electrospun membrane: scaffold for tissue engineering

Nonwoven fibrous matrixes have been widely used as scaffolds in tissue engineering, and modification of microstructure of these matrices is needed to organize cells in three-dimensional space with spatially balanced proliferation and differentiation required for functional tissue development. The objective of this study was fabrication of nanofibrous matrix from novel biodegradable poly(p-dioxanone-co-L-lactide)-block-poly(ethylene glycol) (PPDO/PLLA-b-PEG) copolymer, and to examine cell proliferation, morphology of cell-matrix interaction with the electrospun nanofibrous matrix. The electrospun structure composed of PPDO/PLLA-b-PEG fibers with an average diameters of 380 nm, median pore size 8 microm, porosity more than 80% and mechanical strength 1.4 MPa, is favorable for cell-matrix interaction and supports the active biocompatibility of the structure. NIH 3T3 fibroblast cell seeded on the structure tend to maintain phenotypic shape and guided growth according to nanofiber orientation. Good capability of the nanofibrous structure for supporting the cell attachment and proliferation are observed. This novel biodegradable scaffold will be applicable for tissue engineering based upon its unique architecture, which acts to support and guide cell growth.     

Collagen-based biomaterials as 3D scaffold for cell cultures: applications for tissue engineering and gene therapy

Many substances are used in the production of biomaterials: metals (titanium), ceramics (alumina), synthetic polymers (polyurethanes, silicones, polyglycolic acid (PGA), polylactic acid (PLA), copolymers of lactic and glycolic acids (PLGA), polyanhydrides, polyorthoesters) and natural polymers (chitosan, glycosaminoglycans, collagen). With the rapid development in tissue engineering, these different biomaterials have been used as three-dimensional scaffolds and cell transplant devices. The principal biochemical and biological characteristics of the collagen-based biomaterials are presented, including their interactions with cells (fibroblasts), distinct from those of synthetic polymers, and their potential use in gene therapy through the formation of neo-organs or organoids.     

Gelatin-chondroitin-hyaluronan tri-copolymer scaffold for cartilage tissue engineering

The mechanism by which the cell synthesizes and secretes extracellular matrix (ECM) and is, in turn, regulated by the ECM is termed dynamic reciprocity. The aim of the present work was to produce a gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer to mimic natural cartilage matrix for use as a scaffold for cartilage tissue engineering. The scaffold produced had a uniform pore size of about 180 microm and adequate porosity of 75%. Porcine chondrocytes were seeded onto the tri-copolymer scaffold and cultured in Petri dishes or spinner flasks for 2, 3, 4, or 5 weeks. Chondrocytes were uniformly distributed in the scaffold in the spinner flask cultures, but less so in the Petri dish cultures. Secretion of ECM was found under histology examination. In spinner flask cultures, chondrocytes retained their phenotype for at least 5 weeks, as shown immunohistochemically, and synthesized type II collagen. These results show that gelatin/chondroitin sulfate/hyaluronan tri-copolymer has potential for use as a cartilage tissue engineering scaffold.     

Minimal surface scaffold designs for tissue engineering

Triply-periodic minimal surfaces are shown to be a more versatile source of biomorphic scaffold designs than currently reported in the tissue engineering literature. A scaffold architecture with sheetlike morphology based on minimal surfaces is discussed, with significant structural and mechanical advantages over conventional designs. These sheet solids are porous solids obtained by inflation of cubic minimal surfaces to sheets of finite thickness, as opposed to the conventional network solids where the minimal surface forms the solid/void interface. Using a finite-element approach, the mechanical stiffness of sheet solids is shown to exceed that of conventional network solids for a wide range of volume fractions and material parameters. We further discuss structure-property relationships for mechanical properties useful for custom-designed fabrication by rapid prototyping. Transport properties of the scaffolds are analyzed using Lattice-Boltzmann computations of the fluid permeability. The large number of different minimal surfaces, each of which can be realized as sheet or network solids and at different volume fractions, provides design flexibility essential for the optimization of competing design targets.     

Cellulose-based scaffold materials for cartilage tissue engineering

Non-woven cellulose II fabrics were used as scaffolds for in vitro cartilage tissue engineering. The scaffolds were activated in a saturated Ca(OH)(2) solution and subsequently coated with a calcium phosphate layer precipitated from a supersaturated physiological solution. Chondrocyte cell response and cartilage development were investigated. The cell adherence was significantly improved compared to untreated cellulose fabrics, and the proliferation and vitality of the adhered chondrocytes were excellent, indicating the biocompatibility of these materials. A homogeneous distribution of the seeded cells was possible and the development of cartilageous tissue could be proved. In contact with a physiological chondrocyte solution, calcium is expected to be leached out from the precipitated layer, which might lead to a microenvironment that triggers the development of cartilage in a way similar to cartilage repair in the vicinity of subchondral bone.     

Biocompatibility of subcutaneously implanted marine macromolecules cross-linked bio-composite scaffold for cartilage tissue engineering applications

There is an intense interest in developing innovative biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Present study demonstrated the in vivo biocompatibility and toxicity of a macromolecules cross-linked biocomposite scaffold composed of hydroxyapatite, alginate, chitosan and fucoidan abbreviated as HACF. The in vivo biocompatibility and toxicity of HACF scaffold were tested by comparing them with those of a biocompatible surgical metal implant (SMI) in a subcutaneous rat model. Following the implantation, animals were sacrificed and the scaffolds were resected at 1st, 4th, and 8th weeks; the surrounding tissue along with the implant was removed to evaluate its biocompatibility. The effects of implanted biomaterial scaffolds on vital organ systems such as liver, kidney, etc., have been studied by hematology and serum biochemistry. The activities of pro-inflammatory marker enzymes such as COX, 5-LOX, 15-LOX, and NOS were normal in rats implanted with HACF scaffold. Hematological parameters, antioxidant and lipid peroxidation status were also found to be normal in implanted rats same as that of control and SMI. The modulatory effect of implanted scaffold over inflammatory and stress signaling cascades were confirmed by the normalized mRNA expressions of NF-κB, TNF-α and IL-6. The histopathological analysis of liver, kidney and tissue support our results. Taken together, these results demonstrated that HACF biocomposite scaffold signifies its suitability for further research as a scaffold material for cartilage tissue engineering applications.     

A bilayer scaffold prepared from collagen and carboxymethyl cellulose for skin tissue engineering applications

Abstract

Treatment of chronic skin wound such as diabetic ulcers, burns, pressure wounds are challenging problems in the medical area. The aim of this study was to design a bilayer skin equivalent mimicking the natural one to be used as a tissue engineered skin graft for use in the treatments of problematic wounds, and also as a model to be used in research related to skin, such as determination of the efficacy of transdermal bioactive agents on skin cells and treatment of acute skin damages that require immediate response. In this study, the top two layers of the skin were mimicked by producing a multilayer construct combining two different porous polymeric scaffolds: as the dermis layer a sodium carboxymethyl cellulose (NaCMC) hydrogel on which fibroblasts were added, and as the epidermis layer collagen (Coll) or chondroitin sulfate-incorporated collagen (CollCS) on which keratinocytes were added. The bilayer construct was designed to allow cross-talk between the two cell populations in the subsequent layers and achieves paracrine signalling. It had interconnected porosity, high water content, appropriate stability and elastic moduli. Expression of vascular endothelial growth factor (VEGF), basic-fibroblast growth factor (bFGF) and Interleukin 8 (IL-8), and the production of collagen I, collagen III, laminin and transglutaminase supported the attachment and proliferation of cells on both layers of the construct. Attachment and proliferation of fibroblasts on NaCMC were lower compared to performance of keratinocyte on collagen where keratinocytes created a dense and a stratified layer similar to epidermis. The resulting constructs succesfully mimicked in vitro the natural skin tissue. They are promising as grafts for use in the treatment of deep wounds and also as models for the study of the efficacy of bioactive agents on the skin.     

Preparation and characterization of a three-dimensional printed scaffold based on a functionalized polyester for bone tissue engineering applications

At present there is a strong need for suitable scaffolds that meet the requirements for bone tissue engineering applications. The objective of this study was to investigate the suitability of porous scaffolds based on a hydroxyl functionalized polymer, poly(hydroxymethylglycolide-co-ε-caprolactone) (pHMGCL), for tissue engineering. In a recent study this polymer was shown to be a promising material for bone regeneration. The scaffolds consisting of pHMGCL or poly(ε-caprolactone) (PCL) were produced by means of a rapid prototyping technique (three-dimensional plotting) and were shown to have a high porosity and an interconnected pore structure. The thermal and mechanical properties of both scaffolds were investigated and human mesenchymal stem cells were seeded onto the scaffolds to evaluate the cell attachment properties, as well as cell viability and differentiation. It was shown that the cells filled the pores of the pHMGCL scaffold within 7 days and displayed increased metabolic activity when compared with cells cultured in PCL scaffolds. Importantly, pHMGCL scaffolds supported osteogenic differentiation. Therefore, scaffolds based on pHMGCL are promising templates for bone tissue engineering applications.