Biomedical Application of Marine-derived Osteogenic Peptides for Bone Tissue Regeneration

Abstract

Chapter 1 presents a general overview of the marine products on bioactivity field, Chapter 2 gives a more detailed literature overview of various aspects of application for tissue engineering (TE). Topics discussed are the general requirements involved in scaffold design, well‐known and frequently used biomaterials, commonly applied scaffold fabrication methods and the importance of surface topography. Additionally, these sections highlight the specific biomaterials and fabrication method adopted in the various chapters of this thesis. The freezing dry and 3D printer method have been used to fabricate the porous scaffolds and 3D scaffolds. Chapter 3 more specifically relate to bioactive and the mechanism of marine compound in cell interaction. This chapter examined the effects of marine Ciona intestinalis calcitonin-like peptide (CCLP) on osteoblast differentiation and mineralization in the culture system of MC3T3-E1 cells. The primary structures of the CCLP were synthesized automatically using the solid phase method with fluorenyl methoxy carbonyl (Fmoc) resin. Pre-osteoblast MC3T3-E1 cells were cultured with various concentrations of CCLP during the osteoblast differentiation period. To examine osteoblast differentiation, alkaline phosphatase (ALP) and mineralization activity were evaluated. Moreover, the expression of differentiation markers such as ALP, osteocalcin (OSC), and osteopontin (OPN) was measured using RT-PCR and Western blot analysis. The results showed that CCLP did not exhibit any cytotoxic effect on MC3T3-E1 cells even at the highest concentration (30 µM) at 2 and 5 days. CCLP also enhanced MC3T3-E1 cells proliferation, differentiation, and mineralization demonstrated by the increased expression of several osteoblast phenotype markers such as ALP, and Alizarin Red S staining. In addition, the CCLP induced mitogen-activated protein kinase (MAPK) pathway in MC3T3-E1 cells. Chapter 4 a novel method to combine PCL 3D scaffold with marine fish collagen (Col) from the Paralichthys olivaceus skin or sodium alginate (Sa) and intestine gastro-intestinal digests peptide (IGDP) from abalone Haliotis discus hannai for tissue-engineered bone was developed. Four 3D scaffolds of PCL modifications were examined: (1) PCL/Col, (2) PCL/Sa, (3) PCL/IGDP/Col, and (4) PCL/IGDP/Sa, in addition to PCL-only and PCL/IGDP. Mouse mesenchymal stem (MSC) cells were seeded onto the PCL 3D scaffolds of pore size 421 - 463 µm, and after 1, 2, and 4 weeks of in vitro culture, MSC-scaffolds have investigated the proliferation, ALP content, and calcium deposited. Results showed that the surface-treated 3D scaffolds had higher protein adsorption and proliferation than did the PCL-only scaffold and the ALP contents in PCL/IGDP/Col and PCL/IGDP/Sa scaffolds were higher than those seen in the PCL-only, PCL/IGDP, PCL/Col, and PCL/Sa scaffolds. The use of PCL/IGDP/Col and PCL/IGDP/Sa to improve the osteoblastic responses in the cell growth and bone tissue regeneration were evaluated. In the in vivo, the 3D scaffolds were implanted in the rabbit tibia. After 2 weeks, the PCL/IGDP/Col and PCL/IGDP/Sa were harvested and dedicated for measurement of mechanical properties and micro-computed tomography. It was found that the PCL/IGDP/Col were strong osteogenic differentiation in the rabbit tibia. These findings of stimulated biological responses in vitro and in vivo suggest that the PCL/IGDP/Col and PCL/IGDP/Sa 3D scaffold were expected to provide adequate mechanical strength, and therefore were the promising material for use in tissue implants and bone regeneration.