Wet extrusion of fibronectin-fibrinogen cables for application in tissue engineering

A method for the wet extrusion of human plasma-derived fibronectin-fibrinogen cables is described. Solutions of fibronectin and fibrinogen with and without sodium alginate and carboxymethylcellulose (CMC) are tested. The rheological properties of the protein solutions changed from Newtonian to shear thinning non-Newtonian in the presence of small quantities of these additives, the apparent viscosity increased, and the extrusion properties of the protein solutions improved. Cables were prepared using a capillary with a diameter of 1 mm and overall length of 18 mm. Cable diameter was reduced to about 0.5 mm by drawing using a series of rollers. Cables prepared with sodium alginate were found to have suitable properties, and those made with CMC were sticky and difficult to handle. Solutions containing no sodium alginate required a minimum total protein concentration of about 70 mg/mL for extrusion. Extruded cables were prepared with solutions containing 140 mg/mL total protein with 12.9 mg/mL alginate (high protein), and 46 mg/mL total protein with 47.6 mg/mL of sodium alginate (high alginate). The mechanical strength of the extruded cables was within the range suitable for application in tissue engineering. Extrusion of the protein solutions into cables was achieved in a coagulation bath. Cables with a mechanical strength of approximately 30 N/mm(2), suitable for wound repair and nerve regeneration applications, were prepared with a coagulation bath containing 0.25 M HCl, 2% CaCl(2) at a pH of <0.9. These cables also had a large average elongation at break of 52%, and showed an increase in cable length after breakage (permanent set) of 20%, demonstrating the potential for drawing the cables down to a fine diameter.     

Photopolymerized water-soluble chitosan-based hydrogel as potential use in tissue engineering

Novel biodegradable hydrogels by photocrosslinking macromers based on chitosan derivative are reported. Photocrosslinkable macromers, a water-soluble (methacryloyloxy) ethyl carboxyethyl chitosan were prepared by Michael-addition reaction between chitosan and ethylene glycol acrylate methacrylate. The macromers were characterized by Fourier transform infrared spectroscopy, (1)H NMR and (13)C NMR. Hydrogels were fabricated by exposing aqueous solutions of macromers with 0.1% (w/v) photoinitiator to UV light irradiation, and their swelling kinetics as well as degradation behaviors was evaluated. The results demonstrated that the degradation rates were affected strongly by crosslinking density. The hydrogel was compatible to Vero cells, not exhibiting significant cytotoxicity. Cell culture assay also demonstrated that the hydrogels were good in promoting the cell attachment and proliferation, showing their potential as tissue engineering scaffolds.     

The use of mesenchymal stem cells in tissue engineering

Mesenchymal stem cells (MSCs) are of great interest to both clinicians and researchers for their great potential to enhance tissue engineering. Their ease of isolation, manipulability, and potential for differentiation are specifically what have made them so attractive. These multipotent cells have been found to differentiate into cartilage, bone, fat, muscle, tendon, skin, hematopoietic-supporting stroma and neural tissue. Their diverse in vivo distribution includes bone marrow, adipose, periosteum, synovial membrane, skeletal muscle, dermis, pericytes, blood, trabecular bone, human umbilical cord, lung, dental pulp, and periodontal ligament. Despite their frequent use in research, no standardized criteria exist for the identification of mesenchymal stem cells; The International Society for Cellular Therapy has sought to change this with a set of guidelines elucidating the major surface markers found on these cells. While many studies have shown MSCs to be just as effective as unipotent cells for certain types of tissue regeneration, limitations do exist due to their immunosuppressive properties. This paper serves as a review pertaining to these issues, as well as others related to the use of MSCs in tissue engineering.     

Preparation and properties of starch acetate fibers for potential tissue engineering applications

This article reports the development of fibers from starch acetates that have mechanical properties and water stability better than most polysaccharide‐based biomaterials and protein fibers used in tissue engineering. In this research, starch acetates with three different degrees of substitution (DS) have been used to develop fibers for potential use as tissue engineering scaffolds. Varying the DS of starch acetate will provide fibers with different mechanical properties, hydrophilicity, and degradation behavior. Fibers made from DS 2.3 and 2.8 starch acetates have mechanical properties and water stability required for tissue engineering applications. The starch acetate fibers support the adhesion of fibroblasts demonstrating that the fibers would be suitable for tissue engineering and other medical applications. Biotechnol. Bioeng. 2009;103: 1016–1022. © 2009 Wiley Periodicals, Inc.     

Preparation of vinylated polysaccharides and photofabrication of tubular scaffolds as potential use in tissue engineering

Polysaccharides, such as heparin, hyaluronan, and chitosan, were partially derivatized with a styryl or a methacryloyl group by condensation at a carboxyl or an amino group of the polysaccharides with 4-vinylaniline or 4-vinylbenzoic acid. The degree of substitution depended on the reaction conditions. These compounds with low degrees of derivatization produced water-swollen hydrogels only at relatively high concentrations (30-40 wt %) in the presence of a carboxylated camphorquinone upon visible light irradiation. A high degree of derivatization of heparin increased the gel yield and concomitantly reduced the degree of swelling. The copolymerization of these vinylated polysaccharides with styrenated gelatin considerably reduced the degree of swelling. Tubular photoconstructs were prepared by photocopolymerization of vinylated polysaccharide and vinylated gelatin. The mixing of diacrylated poly(ethylene glycol) with vinylated polysaccharide improved the burst strength of photogels against the gradual infusion of water. These photocurable polysaccharides may be used as photocured scaffolds in tissue-engineered devices.     

The compressive material properties of the plantar soft tissue

The plantar soft tissue is the primary means of physical interaction between a person and the ground during locomotion. Dynamic loads greater than body weight are borne across the entire plantar surface during each step. However, most testing of these tissues has concentrated on the structural properties of the heel pad. The purpose of this study was to determine the material properties of the plantar soft tissue from six locations beneath: the great toe (subhallucal), the 1st, 3rd and 5th metatarsal heads (submetatarsal), the lateral midfoot (lateral submidfoot) and the heel (subcalcaneal). We obtained specimens from these locations from 11 young, non-diabetic donors; the tissue was cut into 2 cm x 2 cm blocks and the skin was removed. Stress relaxation experiments were conducted and the data were fit using the quasi-linear viscoelastic (QLV) theory. To determine tissue modulus, energy loss and the effect of test frequency, we also conducted displacement controlled triangle waves at five frequencies ranging from 0.005 to 10 Hz. The subcalcaneal tissue was found to have an increased relaxation time compared to the other areas. The subcalcaneal tissue was also found to have an increased modulus and decreased energy loss compared to the other areas. Across all areas, the modulus and energy loss increased for the 1 and 10 Hz tests compared to the other testing frequencies. This study is the first to generate material properties for all areas of the plantar soft tissue, demonstrating that the subcalcaneal tissue is different than the other plantar soft tissue areas. These data will have implications for foot computational modeling efforts and potentially for orthotic pressure reduction devices.     

The use of electric fields in tissue engineering: A review

The use of electric fields for measuring cell and tissue properties has a long history. However, the exploration of the use of electric fields in tissue engineering is only very recent. A review is given of the various methods by which electric fields may be used in tissue engineering, concentrating on the assembly of artificial tissues from its component cells using electrokinetics. A comparison is made of electrokinetic techniques with other physical cell manipulation techniques which can be used in the construction of artificial tissues.Key words: tissue engineering, electric field, microenvironment, electrokinetics, dielectrophoresis, polarity     

A material decoy of biological media based on chitosan physical hydrogels: application to cartilage tissue engineering

The cartilage tissue has a limited self-regenerative capacity. Tissue-engineering represents a promising trend for cartilage repair. The present study was aimed to develop a biomaterial formulation by combining fragments of chitosan hydrogel with isolated rabbit or human chondrocytes. We first reported the properties of the constructs elaborated with rabbit chondrocytes and pure chitosan physical hydrogels with defined molecular weight, acetylation degree and polymer concentration. Morphological data showed that chondrocytes were not penetrating the hydrogels but tightly bound to the surface of the fragments and spontaneously formed aggregates of combined cell/chitosan. A significant amount of neo-formed cartilage-like extracellular matrix (ECM) was first accumulated in-between cells and hydrogel fragments and furthermore was widely distributed within the neo-construct. The optimal biological response was obtained with hydrogel fragments concentrated at 1.5% (w/w) of polymer made from a chitosan with a degree of acetylation between 30 and 40%. Such hydrogels were then mixed with human chondrocytes. The phenotype of the cells was analyzed by using chondrocytic (mRNA expression of mature type II collagen and aggrecan as well as secretion of proteoglycans of high molecular weight) and non chondrocytic (mRNA expression of immature type II collagen and type I collagen) molecular markers. As compared with human chondrocytes cultured without chitosan hydrogel which rapidly dedifferentiated in primary culture, cells mixed with chitosan rapidly loose the expression of type I and immature type II collagen while they expressed mature type II collagen and aggrecan. In these conditions, chondrocytes maintained their phenotype for as long as 45 days, thus forming cartilage-like nodules. Taken together, these data suggest that a chitosan hydrogel does not work as a scaffold, but could be considered as a decoy of cartilage ECM components, thus favoring the binding of chondrocytes to chitosan. Such a biological response could be described by the concept of reverse encapsulation.     

Quantifying nonlinear anisotropic elastic material properties of biological tissue by use of membrane inflation

Determination of material parameters for soft tissue frequently involves regression of material parameters for nonlinear, anisotropic constitutive models against experimental data from heterogeneous tests. Here, parameter estimation based on membrane inflation is considered. A four parameter nonlinear, anisotropic hyperelastic strain energy function was used to model the material, in which the parameters are cast in terms of key response features. The experiment was simulated using finite element (FE) analysis in order to predict the experimental measurements of pressure versus profile strain. Material parameter regression was automated using inverse FE analysis; parameter values were updated by use of both local and global techniques, and the ability of these techniques to efficiently converge to a best case was examined. This approach provides a framework in which additional experimental data, including surface strain measurements or local structural information, may be incorporated in order to quantify heterogeneous nonlinear material properties.     

Mesothelial progenitor cells and their potential in tissue engineering

The mesothelium consists of a single layer of flattened mesothelial cells that lines serosal cavities and the majority of internal organs, playing important roles in maintaining normal serosal integrity and function. A mesothelial 'stem' cell has not been identified, but evidence from numerous studies suggests that a progenitor mesothelial cell exists. Although mesothelial cells are of a mesodermal origin, they express characteristics of both epithelial and mesenchymal phenotypes. In addition, following injury, new mesothelium regenerates via centripetal ingrowth of cells from the wound edge and from a free-floating population of cells present in the serosal fluid, the origin of which is currently unknown. Recent findings have shown that mesothelial cells can undergo an epithelial to mesenchymal transition, and transform into myofibroblasts and possibly smooth muscle cells, suggesting plasticity in nature. Further evidence for a mesothelial progenitor comes from tissue engineering applications where mesothelial cells seeded onto tubular constructs have been used to generate vascular replacements and grafts to bridge transected nerve fibres. These findings suggest that mesothelial cell progenitors are able to switch between different cell phenotypes depending on the local environment. However, only by performing detailed investigations involving selective cell isolation, clonal analysis together with cell labelling and tracking studies, will we begin to determine the true existence of a mesothelial stem cell.     

Rapid prototyping in tissue engineering: challenges and potential

Tissue engineering aims to produce patient-specific biological substitutes in an attempt to circumvent the limitations of existing clinical treatments for damaged tissue or organs. The main regenerative tissue engineering approach involves transplantation of cells onto scaffolds. The scaffold attempts to mimic the function of the natural extracellular matrix, providing a temporary template for the growth of target tissues. Scaffolds should have suitable architecture and strength to serve their intended function. This paper presents a comprehensive review of the fabrication methods, including conventional, mainly manual, techniques and advanced processing methods such as rapid prototyping (RP) techniques. The potential and challenges of scaffold-based technology are discussed from the perspective of RP technology.     

Molecular properties in cell adhesion: a physical and engineering perspective.

The past several years have seen accelerating growth in research directed towards the understanding and control of cell adhesion processes, from a spectrum of disciplinary approaches including molecular cell biology, biochemistry, biophysics and bioengineering. Consequently, our understanding of the mechanisms involved in cell adhesion has increased substantially. Corresponding quantitative analysis and modeling of the key molecular properties governing their action in regulating dynamic cell attachment and detachment events is crucial for advancing conceptual insight along with technological applications.     

Nanofibers from blends of polyvinyl alcohol and polyhydroxy butyrate as potential scaffold material for tissue engineering of skin

Nanofibers were prepared by electrospinning from pure polyvinyl alcohol (PVA), polyhydroxybutyrate (PHB), and their blends. Miscibility and morphology of both polymers in the nanofiber blends were studied by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC), revealing that PVA and PHB were miscible with good compatibility. DSC also revealed suppression of crystallinity of PHB in the blend nanofibers with increasing proportion of PVA. The hydrolytic degradation of PHB was accelerated with increasing PVA fraction. Cell culture experiments with a human keratinocyte cell line (HaCaT) and dermal fibroblast on the electrospun PHB and PVA/PHB blend nanofibers showed maximum adhesion and proliferation on pure PHB. However, the addition of 5 wt % PVA to PHB inhibited growth of HaCaT cells but not of fibroblasts. On the contrary, adhesion and proliferation of HaCaT cells were promoted on PVA/PHB (50/50) fibers, which inhibited growth of fibroblasts.     

The molecular and fibrillar structure of collagen and its relationship to the mechanical properties of connective tissue

The conformation of type I collagen molecules has been refined using a linked-atom least-squares procedure in conjunction with high-quality X-ray diffraction data. In many tendons these molecules pack in crystalline arrays and a careful measurement of the positions of the Bragg reflections allows the unit cell to be determined with high precision. From a further analysis of the X-ray data it can be shown that the highly ordered overlap region of the collagen fibrils consists of a crystalline array of molecular segments inclined by a small angle with respect to the fibril axis. In contrast, the gap region is less well ordered and contains molecular segments that are likely to be inclined by a similar angle but in a different vertical plane to that found in the overlap region. The collagen molecule thus has a D-periodic crimp in addition to the macroscopic crimp observed visually in the collagen fibres of many connective tissues. The growth and development of collagen fibrils have been studied by electron microscopy for a diverse range of connective tissues and the general pattern of fibril growth has been established as a function of age. In particular, relationships between fibril size distribution, the content and composition of the glycosaminoglycans in the matrix and the mechanical role played by the fibrils in the tissue have been formulated and these now seem capable of explaining many new facets of connective tissue structure and function.     

Evaluation of a cell-based osteogenic formulation compliant with good manufacturing practice for use in tissue engineering

Molecular Biology Reports - Proper bony tissue regeneration requires mechanical stabilization, an osteogenic biological activity and appropriate scaffolds. The latter two elements can be combined...     

The use of mesenchymal stem cells in tissue engineering: A global assessment

Mesenchymal stem cells (MSCs) are of great interest to both clinicians and researchers for their great potential to enhance tissue engineering. Their ease of isolation, manipulability and potential for differentiation are specifically what have made them so attractive. These multipotent cells have been found to differentiate into cartilage, bone, fat, muscle, tendon, skin, hematopoietic-supporting stroma and neural tissue. Their diverse in vivo distribution includes bone marrow, adipose, periosteum, synovial membrane, skeletal muscle, dermis, pericytes, blood, trabecular bone, human umbilical cord, lung, dental pulp and periodontal ligament. Despite their frequent use in research, no standardized criteria exist for the identification of mesenchymal stem cells; The International Society for Cellular Therapy has sought to change this with a set of guidelines elucidating the major surface markers found on these cells. While many studies have shown MSCs to be just as effective as unipotent cells for certain types of tissue regeneration, limitations do exist due to their immunosuppressive properties. This paper serves as a review pertaining to these issues, as well as others related to the use of MSCs in tissue engineering.Key words: mesenchymal stem cells, tissue engineering, regenerative medicine     

Biocompatible and biodegradable ultrafine fibrillar scaffold materials for tissue engineering by facile grafting of L-lactide onto chitosan

A chitosan derivative was prepared with good yields using a "one pot" approach by grafting L-lactide oligomers via ring opening polymerization. Side chains are primarily attached to hydroxyl groups located on carbons 3 and 6 of the glucosamine ring, while the amine group remains nonfunctionalized. By increasing the L-lactide to chitosan ratio, side chain length is controlled. This allows the manipulation of the biodegradation rate and hydrophilicity of the tissue engineering scaffold material. This general synthetic route renders functionalized chitosan soluble in a broad range of organic solvents, facilitating formation of ultrafine fibers via electrospinning. Cytotoxicity tests using fibroblasts (L929 cell line) performed on electrospun L-lactide modified chitosan fibers showed that the specimen with the highest molar ratio of L-lactide (1:24) investigated in this study is the most promising material for tissue engineering purposes, while less stable formulations might still find application in drug delivery vehicles.