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Cell Growth on Biodegradable Poly(Depsipeptide-Co-Lactide) Matrix Releasing Growth Factors as Scaffold for Tissue Engineering

Yuichi Ohya, Jun Matori, Hideaki Matsunami, Hidetoshi Arimura, and Tatsuro Ouchi. Department of Applied Chemistry, Faculty of Engineering & High Technology Research Center, Kansai University, Yamate 3-3-35, Suita, Osaka 564-8680, Japan

The regeneration of damaged or lost tissue by tissue engineering has become an area of intense interest in biomedical materials science. Guided tissue regeneration (GTR) is an approach whereby cells are cultivated on temporary scaffolds at the site of damaged or lost tissue as a means of regeneration. For this purpose, matrices with an appropriate biodegradation profile, excellent biocompatibility, cell attachment and safe degradation products are strongly desired. Recent studies have provided evidences that some growth factors improve the early healing process and regeneration of lost tissue. The biodegradable matrices are desired to show stable entrapment of growth factors, hydrophilic macromolecules, and sustained release of them during degradation process without deactivation of the growth factors. Poly(a-hydroxy acid), such as polyglycolide (PGA), poly-L-lactide (PLLA) and their copolymers, have been frequently applied as implantable carriers for drug delivery systems and as surgical repair materials due to its good biodegradation properties, relatively high biocompatibility, high mechanical strength, and excellent shaping and molding properties. However, these polymers have no reactive side-chain groups and it has been difficult to introduce functionality to these polymers by the usual chemical modification methods. Moreover, these copolymers are usually hydrophobic. Such hydrophobic character is not suitable for efficient entrapment of growth factors. Polydepsipeptides are copolymers containing amino acids and hydroxyl acids. We previously synthesized biodegradable poly(depsipeptide-co-lactide) with reactive side-chain groups by ring-opening copolymerization of L-lactide (LA) and cyclodepsipeptide consisting of glycolic acid (Glc) and aspartic acid (Asp) or lysine (Lys) to give poly[(Glc-Asp)-co-LA]: PGDLA and poly[(Glc-Lys)-co-LA]: PGKLA (Figure). These poly(depsipeptide-co-lactide) obtained exhibited higher non-enzymatic degradability compared to PLLA, depending on the depsipeptide units content of the copolymer. We also reported the preparation of microspheres with reactive surfaces from the copolymers, the chemical modification of those surfaces, and effective entrapment of hydrophilic drug model by electrostatic interaction. Therefore, these copolymers are good candidates as scaffold for GTR. In fact, we demonstrated the films and sponges prepared from PGDLA and PGKLA showed faster and controllable degradation behavior, and better cell attachment and growth compared with PLLA film and sponges. In this study, we prepared films and sponges from PGDLA and PGKLA entrapping growth factors [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and nerve growth factor (NGF)] and their model proteins. The release behavior of the model proteins from the matrices and cell growth on the growth factor-loaded matrices were investigated to evaluate the possibility of these copolymer as scaffold for GTR. The synthesis of PGDLA and PGKLA was carried out by ring-opening copolymerization of LA with the cyclodepsipeptides consisting of glycolic acid (Glc) and aspartic acid (Asp) or lysine (Lys), cyclo[Glc-Asp(OBzl)] and cyclo[Glc-Lys(Z)], according to the method reported previously. Protein-loaded films were prepared by a W/O emulsion solvent evaporation method. Insulin, lysozyme and lactoferrin were chosen as model proteins for EGF, bFGF and NGF, respectively, considering their molecular weights and isoelectric points and used for release test. The release behavior of model proteins (insulin, lysozyme, or lactoferrin) from films was investigated in vitro. The release rates of model proteins from the copolymer films were faster than that from PLLA film. These results can be explained by the fact that the biodegradation rates of copolymers were faster than that of PLLA. Significant differences between PGDLA and PGKLA, and among the model proteins were not observed. Similar tendency was observed in release behavior of the model proteins from the copolymer sponges. Growth factor-loaded films and sponges were prepared by the same method as described above. Films and sponges without growth factors were also prepared and used as controls. L929 cell growth on the copolymer and PLLA films was investigated after 1 to 14 days. As negative control experiments, cell growth on the growth factor unloaded-matrices was also investigated. As positive control experiments, native growth factors were added to culture medium for the growth factor unloaded-matrices, and cell growth was investigated. Cell proliferations on growth factor-loaded films and sponges showed higher growth rate compared with by growth factor unloaded-matrices, and showed the same level as growth factor adding experiments. These results suggest that the growth factors entrapped in the copolymer matrices kept their activity, and no denaturation of the growth factors occurred. The morphology of cells attached to the copolymer films after 20 h incubation was observed by scanning electron microscopy (SEM). Nerve outgrowth of PC12 cells on PGKLA film entrapping NGF or adding NGF was observed.