Preparation And Properties Of Human Fibrin Gel And Its Composite Scaffolds

Composite scaffolds consisting of fibrin gel and poly-L-lactide (PLLA) porous sponges and poly(l-lactic-co-glycolic acid) (PLGA) microcarriers were fabricated. Fibrinogen was isolated from human plasma by a freeze-thaw circle. Gelation of the fibrinogen was accomplished by mixing with thrombin. PLLA porous sponges were fabricated by thermally induced phase seperation (TIPS). PLGA microspheres were fabricated by emulsion-solvent evaporation. Their surfaces were further modified by fibrinogen. The fibrin gel and PLLA porous sponges or PLGA microcarriers were finally compounded to form the composite scaffolds.Fibrinogen was isolated from human plasma by a freeze-thaw circle. Its structure was characterized by electrophoresis. Gelation of the fibrinogen was accomplished by mixing with thrombin. Absorbance of the fibrinogen/thrombin mixture at 550nm as a function of reaction time was monitored by UV-vis spectroscopy. It was found that the clotting time is significantly influenced by the thrombin concentration and the temperature, while less influenced by the fibrinogen concentration. After free dried, the fibrin gel was characterized by scanning electron microscopy (SEM), revealing fibrous microstructure. Thermal stability analysis by thermal gravimetric analysis found that the crosslinked fibrin gel was degradated from 288℃, which is about 30℃ higher than that of the fibrinogen. The hydrogel has an initial water-uptake ratio of ～50, decreased to 30-40 after incubated in water for 11h depending on the thrombin concentration. The fibrin gels lost their weights in PBS very rapidly, while slowly in DMEM/fetal bovine serum and DMEM. In vitro cell culture found that human fibroblasts could normally proliferate in the fibrin gel with spreading morphology andeven distribution.It is essential that the fibrin gel maintains its long-term shaping stability and mechanical strength for application in cartilage tissue engineering. Utilizing the TIPS technique, PLLA scaffolds with good mechanical properties were prepared, followed with incorporated with fibrin gel. The incorporating efficiency and release of protein were detected with different concentration of fibrinogen. In mechanical characterization experiments, the compressive modulus of the PLLA scaffold incorporated with fibrin gel is up to 7.12MPa. Morphology and distribution of the chondrocytes were studied by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The cell growth behaviors were determined by MTT assay, and glycosaminoglycan (GAG) secretion. The cell number was determined by DNA content. Chondrocytes cultured in the hybrid scaffold showed significantly improved cell spreading and growth. This convenient and effective method provides a useful pathway of scaffold preparation and cell transplantation, which can achieve suitable mechanical properties and good cell performance simultaneously.PLGA microspheres were fabricated by emulsion-solvent evaporation, followed with aminolysis in hexanediamine/n-propanol solution to introduce free amino groups on the surface of microspheres. Fibrinogen was immobilized on the microspheres with a diameter of ～100μm after the amino groups were transferred into aldehyde groups by a treatment of glutaraldehyde. Weight loss of the microspheres increased with the prolongation of aminolyzing time. The NH₂ and fibrinogen contents on the microsphere surface were quantitatively determined by ninhydrin analyses and UV-vis spectroscopy. The PLGA microspheres were mixed with the fibrinogen solution firstly. Gelation of the fibrinogen was accomplished by mixing with thrombin. The absorbance of the PLGA microspheres/fibrinogen/thrombin mixture at 550nm as a function of reaction time was monitored by UV-vis spectroscopy. It was found that the a stable structure can be fabricated with the thrombin concentration was higher than 15U/ml. The gelation time was less influenced by the PLGA microspheresconcentration. SEM and CLSM observations indicated that the microspheres distributed in the fibrin gel and formed the hybrid scaffold. The elastic modulus of the scaffold increased with the increase of microspheres' content. In vitro chondrocyte culture showed that cells could survive, grow and proliferate in this hybrid cell carrier scaffold. Cell viability increased with the prolongation of culture time. CLSM and SEM observations indicated that chondrocytes in the fibrin gel moved to the surfaces of the PLGA microcarriers, and then attached, spread and proliferated. These results show that the PLGA microcarriers /fibrin gel is a promising candidate as a injectable scaffold.