Ps-41: Sustain Release of Insulin from Porous Scaffold for Cartilage Tissue Regeneration

Objective: Cartilage defects are very difficult to heal due to its limited ability of self-repair and regeneration. Therefore cartilage tissue engineering using porous scaffolds, chondrocytes or human mesenchymal stem cell and bioactive instructive cues has been evolved promising approach to treat cartilage defects. Collagen as a natural biomaterial is extensively investigated for preparation of porous scaffolds for cartilage tissue engineering applications. Growth factor and therapeutics are employed for maintenance of cell viability, proliferation and promotion of tissue regeneration. Insulin administration has demonstrated its ability to prolong the survival of chondrocytes and prevent the formation of necrosis in 3D collagen hydrogel construct. Controlled and prolonged delivery of the insulin using PLGA micro beads into porous collagen matrix has been demonstrated to be useful in cartilage tissue engineering. Our purpose was to prepare a controlled insulin releasing scaffold with controlled pore structure as a bioactive 3D culture system for cartilage tissue engineering. Materials and Methods: Insulin was microencapsulated in PLGA microbeads using w-o-w double emulsion technique. The recovered microbeads were washed with water and freeze-dried in a freeze drier. The collagen- microbead hybrid porous scaffold was prepared by a freeze-drying method using pre-prepared ice particulates of a diameter range of 150 μm-250 μm as porogen. collagen solution was mixed with the prepared microbead suspension at a ratio of 9:1 to prepare microbead dispersed collagen solution.The manipulation was carried out at 4oC and the mixture was magnetically stirred.The final mixture was molded in a frame template. Then the mold was freeze-dried and cross-linked EDC and NHS in ethanol for 24 hours at room temperature. The cross-linked scaffold was washed and freeze dried to prepare hybrid scaffold of collagen-microbead. Bovine articular chondrocytes were cultured in tissue culture flasks DMEM containing 10% fetal bovine serum, 4500 mg/L glucose, 4 mM glutamine, 100 U/mL penicillin, 100μg/mL streptomycin, 0.1 mM nonessential amino acids, 0.4 mM proline, 1 mM sodium pyruvate and 50 μg/mL ascorbic acid. The confluent monolayer of the cells was harvested using trypsin/EDTA treatment and seeded into the scaffolds by dispensing 80 μL of cell suspension (7.5×105 cells/scaffold). The cell-scaffold constructs were incubated for 3 hours in a CO2 incubator to allow the seeded cells to adhere over the scaffolds. Results: The scaffolds had controlled pore structure and the large pores were replica of the ice particulates used during fabrication process. The large pores were connected to each other with interconnected pores. The hybrid scaffold exhibited a homogeneous spatial distribution of micro beads throughout the pore walls. The mechanical strength of the scaffolds was determined using a compression test. The result indicated the control and collagen-micro bead hybrid scaffolds had high mechanical strength (150,155KPa respectively). The release profile from microbeads showed an initial burst release (33% in day 1) followed by a rise in cumulative insulin (upto 3 weeks) and a very slow release phase (4th week). The scaffolds showed high cell seeding efficiencies and the seeding efficiencies of the scaffolds were 87.12 ± 1.13% (control) and 86.99 ± 1.38% (experiment). Conclusion: The collagen-micro bead hybrid scaffold demonstrated a high mechanical strength and a stable release of insulin for 4 weeks. The released insulin demonstrated its effect on cultured chondrocytes for their survival and proliferation. The bioactive hybrid scaffold should be useful for maintenance of prolonged survival and proliferation of cultured chondrocytes towards the application in cartilage tissue engineering.