Construction Of Injectable Polylactide Cell Microcarriers/Chitosan Hydrogel Composite Scaffold

A novel injectable scaffold consisting of polylactide cell microcarriers and chitosan hydrogel was fabricated. Polylactide (PLA) microspheres were fabricated by emulsion-solvent evaporation. Their surfaces were modified to improve cytocompatibility. Crosslinkable and water-soluble chitosan was synthesized, which could be crosslinked to form chitosan hydrogel under mild conditions. The microcarriers and the chitosan hydrogel were finally compounded to form the novel injectable scaffold.PLA microspheres were fabricated by emulsion-solvent evaporation. Their particle size and size distribution could be controlled by stirring rate, PLA concentration and dispersant concentration. Moreover, PLA porous microspheres were fabricated by solution induced phase separation based on emulsion-solvent evaporation. There were lots of small pores on the surfaces of the porous microspheres, and irregular pores throughout the whole inner structure. The particle size, size distribution and inner pore size all could be controlled by stirring rate, PLA concentration, dispersant concentration and non-solvent/solvent ratio.Biomacromolecules, such as collagen type I and chitosan, were immobilized on the surfaces of the PLA microspheres with a diameter of 180~280μm via the combination of aminolysis and grafting-coating to improve the cytocompatibility. The weight of microspheres decreased with the prolongation of aminolyzed time, while the ammo content increased initially, then reached a constant. Using glutaraldehyde as a couple reagent, collagen type I and chitosan were grafted and coated on the surfaces of the PLA microspheres. In vitro chondrocytes culture showed that compared with the unmodified PLA microspheres, collagen and chitosan coated PLA microspheres possessed better cytocompatibility. Chondrocytes could adhesion, spread and proliferate on the surfaces, in particular on those coated with collagen.Using water-soluble carbodiimide (EDAC) as condensation reagent, methyl acrylic acid (MA) and lactic acid (LA) were grafted on the chitosan chain to obtain a crosslinkable and water-soluble chitosan derivative (CML). The molecular structure of CML was confirmed by FTIR, ~1HNMR and elemental analysis. The grafting ratios of MA and LA increased with the increase of EDAC content. CML (23%MA and 52%LA) was readily soluble in pure water and did not precipitate till pH 9. And the viscosity of its solution was very low, only ~40cp (1% CML and 50rpm).Gelation of the CML was realized by thermal treatment at body temperature under the initiation of a redox system, ammonium persulfate (APS)/N,N,N',N'-tetramethylethylenediamine(TMEDA). Dynamic investigation of hydrogel formation showed that the reaction rate mainly depended on the initiator concentration. The gelation time could be mediated in a wide range, e.g. from 6min to 20min, by reaction temperature and/or initiator's concentration. In vitro lysozyme degradation of chitosan hydrogel was related to crosslinking degree. At lower crosslinking degree, the hydrogel has been degraded completely at day 8. But the elastic modulus of the hydrogel was smaller than that of the normal human cartilage.3T3 fibroblast culture showed that the cytotoxicity of the hydrogel extractant was attributed to the existence of the initiators. The cytotoxicity of the hydrogel extractant was dependent on the cell seeding number and the initiator's concentration. With large enough number of cells (>2.5xlO4) and low initiator' concentration (5mM), the cytotoxicity introduced by the initiator is very minimal and neglectable. 3T3 and chondrocytes encapsulated in the hydrogel could survive but could not proliferate. The cell viability increased initially to a highest value with the prolongation of culture time, then decreased. Although the hydrogel could cause acute inflammation and foreign body reaction, no tissue necrosis and malignancy were evidenced in vivo, demonstrating that the material has better histocompatibility. These features have endowed the chitosan with great opportunity as injectable biomaterials, which may find wide applications in the rapid developed fields such as tissue engineering and orthopaedics.To ensure that PLA microspheres can be suspended in 1% CML solution and can be injected easily, konjac glucomannan (KGM) as thickening agent was added into 1 % CML solution to increase its viscosity. According to the suspension test, gelation time and elastic modulus, when the KGM content was 0.6%, the mixture met with the requirement of PLA microspheres' suspension and also was found no side effect on the gelation time. The addition of KGM could increase the elastic modulus of the hydrogel.PLA microcarriers were mixed with KGM/CML solution firstly, and then under the initiation of APS/TMEDA, PLA microcarriers/chitosan hydrogel scaffold was formed. The elastic modulus of the scaffold increased with the increase of microspheres' content. When the microcarriers' content was 5%, the elastic modulus of the scaffold was 0.12~1.15MPa, a value closed to that normal human cartilage(0.25~2.5MPa). In vitro chondrocyte culture showed that cells could survive, grow and proliferate in this scaffold. Cell viability increased with the prolongation of culture time, and then reached a constant after 9d. The morphology of cells in hydrogel was round, but on the microcarriers surface spread cells showed pebble-like shape. CLSM and SEM observation indicated that chondrocytes in hydrogel moved to the surfaces of the PLA microcarriers, and then attached, spread and proliferated. And the increase of cell viability in this case was attributed to the cell proliferation on the PLA microcarriers. The combination of the PLA microcarriers and the chitosan hydrogel possessed synergistic effect. Liquid chitosan hydrogel precursor could be used as the carrier to inject PLA microcarriers into body. After gelation, hydrogel could help PLA microcarriers to shape and to prevent microcarriers from moving in vivo. On the other hand, addition of the PLA microcarriers could improve the mechanical strength of chitosan hydrogel. These features indicate that this novel PLA microcarriers/chitosan hydrogel scaffold has potential application for injectable scaffold in tissue engineering.