Résumé
OBJECTIVE: To prepare recombinant peptide(rAHP, amino acid sequence is VLPVPR) PLGA sustained release microspheres by double emulsion solvent evaporation method. METHODS: The rAHP-PLGA microspheres preparation process was optimized by orthogonal experiments using PLGA copolymers with different LA/GA ratios or different molecular weights as sustained material, and in vitro drug release profiles of the microspheres were also investigated. RESULTS: The microspheres were smaller (P < 0.05) and more porous with lower molecular weight of PLGA. The optimal preparation process of rAHP-PLGA microspheres was as follows; PLGA concentration was 12 g · 100 mL-1, the stirring rate of the first emulsion was 1000 r · min-1, the volume ratio of inner water phase to oil phase was 1-7.5, and the PVA concentration was 4 g · 100 mL-1. The encapsulation efficiency of the rAHP-PLGA microspheres prepared by the optimum process was more than 90%, the drug loading was above 11% and the average particle diameter-was in the range of 70-90 μn. The cumulative in vitro release percentage in PBS buffer was less than 40% within 2 h, the release speed increased(P < 0.05) with lower molecular weight or lower ratio of LA/GA. The drug release curve was fit to Higuchi equation, suggesting that rAHP released from the microspheres based on diffusion mechanism. CONCLUSION: The rAHP-PLGA microspheres can be prepared easily with good morphology, high entrapment efficiency and certain sustained release capacity.
Résumé
PURPOSE: We investigated the effects of recombinant 9-10th type III repeat of fibronectin (rhFNIII9-10) on the adhesion, proliferation, and the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells(hMSCs). MATERIALS AND METHODS: Adhesion and blocking assay for hMSCs were performed on the plates which had been coated with 100 microgram/ml rhFNIII9-10 or fibronectin. hMSCs seeded on the precoated plates were cultured in the osteogenic media for 3 weeks. MTS(Dimethylthiazole carboxymethoxyphenyl sulfophenyl tetrazolium compound) assay for the cell number, [Methyl-3H] thymidine incorporation study, alkaline phosphatase activity assay, calcium content assay and RT-PCR for alkaline phosphatase, osteopontin, cbfa-1, and type I collagen were performed during the osteogenic differentiation. RESULTS: hMSCs showed significantly increased adhesion to rhFNIII9-10-coated plates and fibronectin-coated plates. A monoclonal antibody to the integrin alpha 5 beta 1 inhibited adhesion to rhFNIII9-10-coated plates and fibronectin-coated plates in dose-dependent manner. hMSCs seeded on the rhFNIII9-10-coated plates showed increased proliferation during the osteogenic differentiation. However, there was no significant difference in the alkaline phosphatase activity, calcium content and expression levels of mRNAs for alkaline phosphatase, osteopontin, cbfa-1, and type I collagen of hMSCs seeded on the rhFNIII9-10-coated plates. CONCLUSION: rhFNIII9-10 stimulates hMSCs adhesion and increases hMSCs proliferation during the osteogenic differentiation. Although osteogenic differentiation is not promoted, adsorption of rhFNIII9-10 onto appropriate biomaterials can enhance integrin-mediated hMSCs adhesion and proliferation. This biomolecular engineering strategy represents a robust approach to increase biofunctional activity and integrin specificity of hMSCs.