Construction of a functional biodegradable bone tissue engineering scaffold for enhanced biomineralization

In case of accidents, injuries or diseases, a various tissue deformations may occur. Tissue regenerations can take a long time and sometimes recovery is not possible completely. The metals, polymers and ceramics are widely used in biomedical field to assist the body to address these problems. Tissue engineering aims to produce tissues by mimicing natural structures with using natural and synthetic polymeric scaffolds. This study aims to obtain composite scaffolds for bone tissue regeneration using silk fibroin and poly(l-lactide) as natural and synthetic polymers. These scaffolds were produced by freeze drying method and insulation cover was used to observe the effect of freezing conditions on properties of scaffolds. After lyophilization process, the scaffolds were treated with methanol to induce beta sheet regions from random coil structures in silk fibroin protein. To induce biomineralization, various peptides can be used. In this study, OSN peptide, which is obtained from osteocalcin protein, was used to promote hydroxyapatite (HAp) crystallization. The scaffolds were characterized with water uptake, biodegradation, biomineralization and cell proliferation tests. The water uptake capacities of scaffolds were found to be higher than 400% for both SF:PLLA (1:3 w/w) and SF:PLLA (3:1 w/w) scaffolds. The samples with high silk fibroin ratio had higher water uptake capacity (almost two fold) compared to other ones. The degradation analysis was carried out for two month. The protease XIV enyzme (0.1U/mL) was used to see its effect on fibroin degradation. After two months, the remaining weight of SF:PLLA (1:3 w/w) and SF:PLLA (3:1 w/w) scaffolds were higher than 50% with and without enzyme. As expected, sample with high SF showed more degradation in the presence of protease enzyme. The effect of methanol treatment on formation of beta sheet structures from random coils were investigated with FTIR analysis by observing shifted peaks at amide I and amide II regions for SF:PLLA (1:3 w/w) and only amide I region for SF:PLLA (3:1 w/w) scaffolds. After methanol treatment, the insoluble structures were obtained with increasing beta sheet formations in fibroin proteins and used for further analysis. The mineralization process was followed for a month and weight changes of scaffolds were reported. Two events simultaneously occur in solution: The formation of apatite crystals and degradation of polymers and weight of scaffolds was affected from both. The scaffolds that were incubated in 3xmSBF solution showed weight loss at the end of a month. On the other hand, the scaffolds incubated in 1xmSBF showed weight gains after a month. The effect of OSN peptides could not be clearly observed from weight change data. The SEM images were taken and the atomic ratios of Ca/P were determined by EDS after 2, 3 and 4th weeks of mineralization process. According to the SEM images, mineral formations can be observed after 2nd week in scaffolds with and without peptides in 1xmSBF and 3xmSBF solutions. The Ca/P ratios were calculated in scaffolds incubated in 1xmSBF solution. The calcification appeared because of the more calcium accumulation after 3rd and 4th weeks of mineralization. The Ca/P ratios of SF:PLLA (3:1 w/w) scaffolds with peptides after 4th week of mineralization and SF:PLLA (1:3 w/w) scaffolds with peptides after 2nd week of mineralization were found to be closer to the stoichiometric value of HAp (1.67). The FTIR spectra were obtained for both scaffolds without peptides incubated in 3xmSBF solution. The presence of characteristic groups of PLLA made diffucult to distinguish the groups of HAp crystals. The few absorption peaks shifted because of the apatite growth on polymer surfaces. The CO32-, PO43-, type A and B carbonate (CO3HAp) and OH- groups that belongs to HAp crystals were obtained on scaffolds especially after 1th week of mineralization according to the FTIR spectra. To investigate the proliferation of human osteoblast cells (hFOB) on composite scaffolds, MTS analysis was applied at 1, 4 and 7 day. The SF:PLLA (3:1 w/w) scaffolds showed higher cell numbers than SF:PLLA (1:3 w/w) scaffolds. Additionally, the SF:PLLA (1:3 w/w) with peptides had more hFOB cells on polymer surfaces than without peptides scaffolds. On the other hand, the SF:PLLA (3:1 w/w) scaffolds with peptides showed less cell proliferation than without peptides forms. In conclusion, the composite scaffolds with two different concentrations were produced by lyophilization process to mimic bone tissue structures and a peptide to direct the mineralization process was added for further improvement. The biodegradability of the scaffolds seemed to be appropriate for bone tissue engineering. Peptide addition affected the mineralization and cell proliferation behavior of the scaffolds but not always in positive manner. For future prospects, the labeled-peptides can be used to obtain the distribution of peptides on the surfaces of polymers. HPLC analysis can be applied to optimize the concentration of immobilized peptides. In addition, the von Kossa staining can be used to observe calcium deposition. In sum, a more comprehensive study is needed to understand and optimize the effect of peptides on the scaffolds surface.