Howard W.T. Matthew

Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review

Once damaged, articular cartilage has very little capacity for spontaneous healing because of the avascular nature of the tissue. Although many repair techniques have been proposed over the past four decades, none has sucessfully regenerated long-lasting hyaline cartilage tissue to replace damaged cartilage. Tissue engineering approaches, such as transplantation of isolated chondrocytes, have recently demonstrated tremendous clinical potential for regeneration of hyaline-like cartilage tissue and treatment of chondral lesions. As such a new approach emerges, new important questions arise. One of such questions is: what kinds of biomaterials can be used with chondrocytes to tissue-engineer articular cartilage? The success of chondrocyte transplantation and/or the quality of neocartilage formation strongly depend on the specific cell-carrier material. The present article reviews some of those biomaterials, which have been suggested to promote chondrogenesis and to have potentials for tissue engineering of articular cartilage. A new biomaterial, a chitosan-based polysaccharide hydrogel, is also introduced and discussed in terms of the biocompatibility with chondrocytes.

Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization

Natural bone growth greatly depends on the precedent vascular network that supplies oxygen and essential nutrients and removes metabolites. Likewise, it is crucial for tissue-engineered bone to establish a vascular network that temporally precedes new bone formation, and spatially originates from within the graft. In order to recapitulate physiological skeletal development, we have developed a complex bone graft to repair rat bone defects. We have demonstrated that endothelial cells and osteoblasts (identified by cell morphology, quantification of specific marker antigens, calcium deposition and capillary-like growth) were able to differentiate and expand from donor rat bone marrow mononuclear cell populations. The biocompatibilities of poly-epsilon-caprolactone (PCL)-hydroxyapatite (HA) composites used for graft fabrication were evaluated at different component ratios to identify the optimal and support of cellular viability and functions for endothelial cells and osteoblasts. Using point-injection and low-pressure techniques, seeded endothelial cells and osteoblasts were able to assemble into microvascular networks and form bony matrix in grafts. The exogenous origination of these cells and their contribution to the vascularization and osteogenesis was confirmed using sex-mismatch implantation and Y chromosome tracking. By pre-seeding with endothelial cells, the resulting vascularization was able to promote osteogenesis, prevent ischemic necrosis and improve the mechanical properties in engineered bone tissue. Taken together, the results indicated that the integration of complex cell populations with composite scaffold materials provided an effective technique to improve osteogenesis in engineered bone graft. These findings suggest that hybrid grafts have great potential for clinical use to treat large bone defects.

Vascular cell responses to polysaccharide materials: in vitro and in vivo evaluations

Chitosan has shown promise as a structural material for a number of tissue engineering applications. Similarly the glycosaminoglycans (GAGs) and their analogs have been known to exert a variety of biological activities. In this study we evaluated the potential of GAG-chitosan and dextran sulfate (DS)-chitosan complex materials for controlling the proliferation of vascular endothelial (EC) and smooth muscle cells (SMC). GAG-chitosan complex membranes were generated in vitro and seeded with human ECs or SMCs for culture up to 9d. In addition, porous chitosan and GAG-chitosan complex scaffolds were implanted subcutaneously in rats to evaluate the in vivo response to these materials. The results indicated that while chitosan alone supported cell attachment and growth, GAG-chitosan materials inhibited spreading and proliferation of ECs and SMCs in vitro. In contrast, DS-chitosan surfaces supported proliferation of both cell types. In vivo, heparin-chitosan and DS-chitosan scaffolds stimulated cell proliferation and the formation of a thick layer of dense granulation tissue. In the case of heparin scaffolds the granulation layer was highly vascularized. These results indicate that the GAG-chitosan materials can be used to modulate the proliferation of vascular cells both in vitro and in vivo.

Biomaterials and scaffolds in reparative medicine

Abstract: Most approaches currently pursued or contemplated within the framework of reparative medicine, including cell‐based therapies, artificial organs, and engineered living tissues, are dependent on our ability to synthesize or otherwise generate novel materials, fabricate or assemble materials into appropriate 2‐D and 3‐D forms, and precisely tailor material‐related physical and biological properties so as to achieve a desired clinical response. This paper summarizes the scientific and technological opportunities within the fields of biomaterials science and molecular engineering that will likely establish new enabling technologies for cellular and molecular therapies directed at the repair, replacement, or reconstruction of diseased or damaged organs and tissues.

Promotion of osteogenesis in tissue-engineered bone by pre-seeding endothelial progenitor cells-derived endothelial cells

In addition to a biocompatible scaffold and an osteogenic cell population, tissue‐engineered bone requires an appropriate vascular bed to overcome the obstacle of nutrient and oxygen transport in the 3D structure. We hypothesized that the addition of endothelial cells (ECs) may improve osteogenesis and prevent necrosis of engineered bone via effective neovascularization. Osteoblasts and ECs were differentiated from bone marrow of BALB/c mice, and their phenotypes were confirmed prior to implantation. Cylindrical porous polycaprolactone (PCL)‐hydroxyapatite (HA) scaffolds were synthesized. ECs were seeded on scaffolds followed by seeding of osteoblasts in the EC‐OB group. In the OB group, scaffolds were only seeded with osteoblasts. The cell‐free scaffolds were denoted as control group. A 0.4‐cm‐long segmental femur defect was established and replaced with the grafts. The grafts were evaluated histologically at 6 weeks postimplantation. In comparison with the OB group, the EC‐OB group resulted in a widely distributed capillary network, osteoid generated by osteoblasts and absent ischemic necroses. Pre‐seeding scaffold with ECs effectively promoted neovascularization in grafts, prevented the ischemic necrosis, and improved osteogenesis. The integration of bone marrow‐derived ECs and osteoblasts in porous scaffold is a useful strategy to achieve engineered bone.

Effect of porosity and pore size on microstructures and mechanical properties of poly-ε-caprolactone-hydroxyapatite composites

The influence of variant pore-size and porosity on the microstructure and the mechanical properties of poly-epsilon-caprolactone (PCL) and hydroxyapatite (HA) composite were examined for an optimal scaffold in bone tissue engineering. Three various amounts of sodium chloride (NaCl, as porogens) with two distinct particle size ranges (212-355 mum and 355-600 mum) were blended into PCL and HA mixture, followed by a leaching technique to generate PCL-HA scaffolds with various pores and porosity. The porosities of the scaffolds were correlated with the porogen size and concentration. The morphological properties of the resulting scaffolds were assessed by micro-computerized tomography (muCT), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX). Extensive PCL-HA pore interconnections with thinner pore walls were present in scaffolds with higher concentration (4:1 w/w) and larger particulate of porogen used in the fabrication process. Embedding of HA particles in the scaffold resulted in rough surfaces on the composites. Instron actuator testing indicated that the tensile strengths and Youngs moduli of scaffolds were influenced by both the porosities and pore sizes of the scaffold. It was apparent that increasing the concentration of porogen compromised the mechanical properties; and a larger porogen particle size led to increased tensile strength but a reduction in Youngs modulus. Overall, the data indicated that modification of the concentration and particle size of porogen altered the porous features and mechanical strength of HA-PCL scaffolds. This provided means to manipulate the properties of biocompatible cell-supporting scaffolds for use as bone graft substitutes.

Improving the mechanical properties of chitosan-based heart valve scaffolds using chitosan fibers

Chitosan is being widely studied for tissue engineering applications due to its biocompatibility and biodegradability. However, its use in load-bearing applications is limited due to low mechanical properties. In this study, we investigated the effectiveness of a chitosan fiber reinforcement approach to enhancing the mechanical properties of chitosan scaffolds. Chitosan fibers were fabricated using a solution extrusion and neutralization method and incorporated into porous chitosan scaffolds. The effects of fiber/scaffold mass ratio, fiber mechanical properties and fiber length on scaffold mechanical properties were studied. The results showed that incorporating fibers improved scaffold strength and stiffness in proportion to the fiber/scaffold mass ratio. A fiber-reinforced, heart valve scaffold achieved leaflet tensile strength values of 220±17 kPa, comparable to the radial values of human pulmonary valve leaflets. Additionally, the effects of 2 mm fibers were found to be up to threefold greater than 10 mm fibers at identical mass ratios. Heparin crosslinking of fibers produced a reduction in fiber strength, and thus failed to produce additional improvements to fiber-reinforced scaffold properties. Despite this reduction in fiber strength, heparin-modified fibers still improved the mechanical properties of reinforced scaffolds, but to a lesser extent than unmodified fibers. The results demonstrate that chitosan fiber reinforcement can be used to achieve porous chitosan scaffold strength approaching that of tissue, and that fiber length and mechanical properties are important parameters in defining the degree of mechanical improvement.

Chitosan fibers with improved biological and mechanical properties for tissue engineering applications

The low mechanical properties of hydrogel materials such as chitosan hinder their broad utility for tissue engineering applications. Previous research efforts improved the mechanical properties of chitosan fiber through chemical and physical modifications; however, unfavorable toxicity effects on cells were reported. In this paper, we report the preparation of chitosan fibers with improved mechanical and biocompatibility properties. The structure-property relationships of extruded chitosan fibers were explored by varying acetic acid (AA) concentration, ammonia concentration, annealing temperature and degree of heparin crosslinking. Results showed that optimizing AA concentration to 2vol% improved fiber strength and stiffness by 2-fold. Extruding chitosan solution into 25wt% of ammonia solution reduced fiber diameters and improved fiber strength by 2-fold and stiffness by 3-fold, due to an increase in crystallinity as confirmed by XRD. Fiber annealing further reduced fiber diameter and improved fiber strength and stiffness as temperature increased. Chitosan fibers crosslinked with heparin had increased diameter but lower strength and stiffness properties and higher breaking strain values. When individual parameters were combined, further improvement in fiber mechanical properties was achieved. All mechanically improved fibers and heparin crosslinked fibers promoted valvular interstitial cells (VIC) attachment and growth over 10 day cultures. Our results demonstrate the ability to substantially improve the mechanical properties of chitosan fibers without adversely affecting their biological properties. The investigated treatments offer numerous advantages over previous physical/chemical modifications and thus are expected to expand the utility of chitosan fibers with tunable mechanical properties in various tissue engineering applications.

Effects of plasma exposure on cultured hepatocytes: Implications for bioartificial liver support

In order to examine their potential for use in a bioartificial liver, hepatocytes maintained in a collagen sandwich configuration were cultured for 9 days in heparinized rat plasma. The cells exhibited a progressive accumulation of cytoplasmic lipid droplets which proved to be mainly triglyceride (TG). The rate of TG accumulation correlated with the free fatty acid (FFA) content of the plasma. Removal of FFA and TG from plasma by ether extraction significantly reduced the rate and extent of TG accumulation. A smaller reduction in the rate and extent of TG accumulation was observed when cells were maintained in an oxygen enriched environment. The lipid accumulation suppressed urea synthesis, but clearance of the drug diazepam, although constitutively depressed in plasma, appeared unaffected by the accumulation. The functional and morphological effects of plasma exposure could be fully reversed after at least 6 days of plasma exposure by returning the cells to culture medium.

Enhanced oxygen delivery to primary hepatocytes within a hollow fiber bioreactor facilitated via hemoglobin-based oxygen carriers.

The production of a fully functional bioartificial liver assist device (BLAD) would greatly enhance available treatment options for patients suffering from acute liver failure. Currently, inadequate oxygen provision to hepatocytes seeded within hollow fiber bioreactors hampers development of a viable hollow fiber-based BLAD. Experimentally, oxygen provision to primary rat hepatocytes cultured within hollow fiber bioreactors was measured, it was observed that supplementation with an oxygen carrier (bovine red blood cells at ∼ 2% human hematocrit) did not significantly improve oxygenation compared to the absence of an oxygen carrier. Therefore, an oxygen transport model of an individual hollow fiber within the bioreactor was developed and simulated (up to ∼ 10% human hematocrit) to more fully examine the effect of oxygen carrier supplementation on oxygenation within the bioreactor. The modeling analysis, supported via the experimental results, was utilized to predict optimal bioreactor operating conditions for the delivery of in vivo-like oxygen gradients to cultured hepatocytes in clinically relevant settings.