Chitosan-based Hyaluronan Hybrid Polymer Fiber for Novel Scaffold in Ligamenttissue Engineering

INTRODUCTION: Severe ligament and tendon injuries are frequently treated with autograft reconstruction. However, biological grafts are not ideal replacements. The use of autograft will result in donor site morbidity. Furthermore, the grafts initially undergo necrosis after the implantation. This is the main reason why the biological grafts often do not provide adequate mechanical strength until a long term remodeling process has completed. Resorbable scaffolds seeded with cells are potential alternatives to biological grafts. Currently, most of these constructs for tissue engineering have been based on collagen scaffolds. On the other hand, the major limitation is that collagen scaffolds are allogeneic. Also, collagen scaffolds suffer from batch-to-batch variability, making consistent reproduction of these constructs difficult. To solve this problem, we developed novel hybrid polymer fibers consisted from chitosan and hyaluronan (HA). It was reported that chitosan could promote the healing of skin wound. Moreover, chitosan is not immunogenic. HA has been shown to improve healing of a variety of tissues by means of trophic effects through binding, delivery of growth factors, cell adhesion, and anti-inflammatory effects. The purpose of this study is to investigate material properties, fibroblasts adhesion behavior and production of extracellular matrix of novel chitosan-based HA hybrid polymer fiber. METHODS: Chitosan polymer fibers and chitosan-based HA hybrid polymer fibers (Chitosan/0.05HA: Chitosan:HA = 8%:0.05%, Chitosan/0.1HA: Chitosan:HA = 8%:0.1%) were originally developed in our laboratory. These novel polymer fibers were developed by wet spinning method using calcium chloride solution. Polyglactin 910 (Vicryl, Ethicon Co, NJ) was used as a control polymer fiber. Material Properties: Tensile tests for five samples of each material were performed at a cross-head speed of 20 mm/min. The cross sectional area was determined with the non-contact method using microscope and video dimension analyzer. Cell Adhesion: Fibroblasts were isolated from the patellar tendon of a Japanese white rabbit under sterile conditions. Cells were maintained in culture using standard procedures and used at second passage. The fibroblasts suspension was concentrated to 1.4 × 10 cells/ml using a hemocytemeter. Cell adhesion study was performed according to our previous study (1985, Int. J. Biol. Macromol, 7, 100-104). Briefly, the fibrous samples were cut into 7 mm pieces and packed in Teflon tube (30 mm length, 7 mm inner diameter). Then 0.1 ml of fibroblasts suspension was loaded on the column. The cells were allowed to adhere in a humidified incubator for 1 hour. Each column was gently rinsed with 1 ml of phosphate-buffered saline and the number of unattached cells was quantified by the microscopic observation of rinsed solution. Five samples in each polymer fibers were used for cell adhesion test. Production of extracellular matrix: A 3-D scaffold was fabricated by piling up the fiber sheet of each polymer. Suspension of the fibroblasts (8.0 × 10 cells/ml) was loaded on the 3-D scaffolds. Cell morphology and production of extracellular matrix were observed by light microscopy 14 days after culture. Scanning electron microscopy (SEM) observation was performed 4 days after culture. Specimens were also evaluated immunohistochemically for collagen type I and collagen type III production. Statistical comparison was performed using one-way ANOVA and Fisher's PLSD test. Significant level was set at 0.05. RESULTS: Material Properties: HA coating significantly increased tensile strength of the polymer fibers (Table 1). Strain at failure in new fibers was significantly lower than in the control fibers. However, new materials were significantly weaker than control polyglactin fibers. Cell Adhesion: The adhesivity of fibroblasts was significantly higher in the new fibers than in the control. Moreover, HA significantly increased the number of cells adhered to the polymer fibers (Fig. 1). Production of extracellular matrix: Immunostaining for type I collagen was prominent around the polymer fibers 14 days after culture (Fig. 2). SEM examination revealed fibroblast growth on the polymer fibers. It was observed that there was the production of extracellular matrix around the fibroblasts (Fig. 3). DISCUSSION: A number of studies have shown the feasibility of musculoskeletal tissue engineering using hydrogels and porous scaffolds. However, these materials do not have sufficient mechanical properties. To achieve enough mechanical strength, we have originally developed a polymer fiber as a scaffold material for ligament tissue engineering. Material properties and cell adhesivity of chitosan polymer fibers were increased by HA coating. This result may be attributable for adhesion molecules of HA. Tensile strength of the novel polymer fibers was lower than the control. On the other hand, strain at failure was lower than in the control. This strain characteristic of the novel hybrid polymer fibers may be beneficial for ligament scaffold. Fibroblast cultured on the polymer fibers produced collagen type I. These findings showed chitosan-based HA hybrid polymers had great potential as a desirable biomaterial for scaffold in ligament tissue engineering. Further in vivo study should be conducted.