Kemik Doku Onarımı Için Hidroksiapatit Peptit Amfifil Bazlı Nanokompozit Doku İskelelerinin Geliştirilmesi

Abstract

A part of this study was supported by 112M442 TÜBİTAK Project and Soner Çakmak was also supported by Hacettepe University Scientific Research Coordination Unit, under The International Cooperation Programme with Project Number of 13 G 602 003 during his studies in USA. In the present study, biomaterials with different contents were designed, fabricated and tested in vitro by cell culture studies for bone and osteochondral tissue engineering. For this purpose bone tissue engineering studies were carried out with biphasic peptide amphiphile (PA)/hydroxyapatite (HA) scaffolds, whereas triphasic silk-HA/silk/PA scaffolds were used for osteochondral tissue engineering studies. For bone tissue engineering studies, HA matrices in nanofiber form were fabricated by electrospinning method, while arginine-glycine-aspartic acid (RGD) bearing PA hydrogels (PA-RGD) were prepared by self assembling technique. The chemical structure of HA nanofiber matrix was verified by Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray diffraction (XRD) analyses and it was determined that this structure had high crystallinity and exhibited better biodegradability when compared to pure HA, depending on its CaCO3 content. Scanning and transmission electron microscopy (SEM and TEM) analyses demonstrated that HA nanofibers had an average diameter of 437 ± 64 nm. AFM analysis showed that PA-RGD hydrogels underwent self assembly and exhibited nanofiber organization similar to extracellular matrix (ECM) structure after gelation. The average diameter of peptide nanofibers was calculated as 100 ± 64 nm by using SEM images. Circular dichroism and ATR-FTIR analyses showed that nanofiber organization occurred via β-sheet secondary structure and RGD was located on the nanofiber surface. For in vitro studies, pre-osteoblastic MC3T3-E1 cells were encapsulated in PA-RGD gels and following this step; nanocomposite structures were fabricated by embedding HA nanofiber matrix within this gel. It was seen that cell proliferation was increased on all of the three scaffolds; HA, PA-RGD and PA-RGD/ HA scaffolds, after the 7th day and on the 18th day, the highest proliferation was detected in PA-RGD gel by cell proliferation analysis. Fluorescence microscopy and SEM analyses demonstrated that cells exhibited osteoblastic morphology on both scaffolds. Depending on the results of alkaline phosphatase (ALP) activity analysis, it was seen that the osteogenic differentiation was enhanced by the addition of HA to the scaffold structure. By real time polymerase chain reaction (RT-PCR) analysis, the expression of Alp gene of cells was investigated as an early marker, while osteocalcin (Ocn) and bone sialoprotein (Bsp) gene expressions of cells were analyzed as late markers of osteogenic differentiation. Although no differences in Alp and Ocn expressions were detected among the samples throughout the cell culture period, significantly higher expression of Bsp on PA-RGD/HA nanocomposite on the 18th day showed that bone mineralization, which is an important indication of osteogenic differentiation, was supported more by this scaffold. Consequently, it was evaluated that PA-RGD/HA nanocomposite scaffold could be successfully used in bone tissue engineering applications. In the osteochondral tissue engineering study as the second part of the present work, silk scaffolds from 6% (w/v) solution was used as the bone part and arginine-glycine-aspartic acid-serine (RGDS) containing PA hydrogel (PA-RGDS) was used as the cartilage part, while silk scaffolds from 4% (w/v) solution was designed as the bone-cartilage interface. In addition to that, for the bone part of the scaffold human bone marrow derived mesenchymal stem cells (hBMSCs) was used, whereas human chondrocytes (hAC) were used as cell source for the cartilage part and the trophic effects of these two cells on each other were investigated under co-culture conditions. The pore sizes of the silk scaffolds with 6% (w/v) and 4% (w/v) silk contents were calculated as 416 ± 87 and 194 ± 67 µm, respectively and it was seen that the silk scaffolds had suitable properties for bone and interface tissues due to their biocompatibility and interconnected pore structures. Human BMSCs were cultured on silk scaffolds in osteogenic medium and chondrocytes were cultured on PA-RGDS in chondrogenic medium for two weeks. Following this culture period, these two cell seeded scaffolds were combined together by placing the 4% (w/v) silk scaffold in between and the resulting structure was cultured for an additional two weeks in the osteochondral cocktail medium. The trophic effects of chondrocytes, which were cultured in the coctail medium, on hBMSCs were revealed as enhanced expression of runt related transcription factor (RUNX2), ALP, collagen type I (COLL I), BSP, osteopontin (OPN) and increased calcium content. When the expression levels of SOX9, agregan (AGC) and collagen type II (COLL II) were evaluated, it was seen that the chondrogenic differentiation of chondrocytes in PA-RGDS hydrogels occurred significantly earlier than pellet cultures. However, gene expressions in hydrogels were found to be similar to that of pellet cultures after the 7th day of incubation. No significant trophic effect of coculture on chondrocytes was determined and this finding showed that TGF-β1 had stronger influence on the chondrocyte differentiation over the co-culture. By histology stainings, it was seen that the hBMSCs in co-culture intensely filled the pores of the scaffold and it was seen that these cells also produced higher amounts of denser mineralized structures detected by Alizarin red and von Kossa stainings. Besides, densely production of cartilage ECM by the chondrocytes in co-culture on PA-RGDS hydrogels was shown by Alcian blue staining. The findings of this study has shown that hBMSCs could able to form an enabled bone structure by co-culture technique without the use of expensive growth factors and chondrocytes could produce cartilage matrix even in the absence of TGF-β1. As a result, it was concluded that the triphasic scaffold designed in this study was a promising biomaterial which could be used for the treatment of osteochondral tissue defects in clinic.