3D生物列印策略製作生物高分子支架於骨再生應用

Title of thesis : 3D bioprinting strategies based on biopolymers scaffolds for bone regeneration
Author : Taufik Abdullah Mappa
Thesis advised by : Yung-Kang Shen
School of Dental Technology,
College of Oral Medicine, Taipei Medical University.
Background: Multidisciplinary collaboration in tissue engineering for bone regeneration, reconstruction, or improved function in the regenerative medicine field uses methods to promote cell growth by manipulating various biomaterials. In advanced technology, bone graft substitutes are most popular because it was made through bone tissue engineering using bone support cells and growth factors to stimulate cells seeded on a scaffold of natural or synthetic biomaterials. This study focused on the bone gets a traumatic injury also as well as occurs a directly immune response due to the immune cells accumulate at the injury spot as regulating the multiple inflammatory growth factors. In the human body, cortical and cancellous bones have a different structure looked at from macro and micro-architecture, leading to the difference bone strength in the functional segment. Cancellous as internal bone tissue that provides a porous like spongy structure around 50–90% porosity volume if compared to the cortical bone obtained 5–15% porosity. Through this specification, we can regeneration the bone uses 3D scaffolds to stimulate osteoblast, osteogenic, osteoclasts, and osteocytes by the proliferation of bone cells. Bone graft substitutes have success in bone transplantation surgery through the application of three-dimensional (3D) construct required for bone function reconstruction. The primary function of scaffolds is to build a structural and mechanical support for the interactions of cells, providing a microenvironment that is responsive for cells osteogenesis to attach, task, and produce bone extracellular matrix (ECM) on the surface. The combination of several biopolymers like a sodium alginate and gelatin to make the 3D scaffolds using micro-extrusion bioprinting is the advanced bone graft substitute methods. The cells and biomaterials are extruded via a nozzle that produces shear and extensional stresses. The MG63 cells adaptability into the alginate-gelatin hydrogels provide advantages like good printability and cell viability, then same study trying to find the ideal characterization thru types of biopolymers and the cells including this research. Objective: to investigate the mechanical, rheological, printability, and cell viability characterizations of 3D bioprinting strategies based on biopolymers scaffolds for bone regeneration. Methods: A regenHU 3D Discovery 8.23.8.26 was assembled and used to print bio-inks based hydrogels through micro-extrusion bioprinting methods. The pattern of scaffolds designed by 3D Global Biotech Inc. Bioinks as gelatin from porcine skin (Sigma, gel strength 300 type A, G2500, USA) combined with sodium alginate (Sigma-Aldrich, W201502, China) were synthesized by the heat-treatment at a temperature of 65oC (CORNING, PC-420D, USA) was stirrer (Chemist, MS-1400D, Taiwan) during 2 h. The AGH (Alginate Gelatin based Hydrogel) was resulted hydrogel have denoted according to the concentration of gelatin added, namely is AGH 4%, AGH 3%, AGH 2%, and AGH 1%, then centrifuge the hydrogel bio-inks for 5 minutes at 21 oC with 2500 rpm and kept at 4 oC. Dissolved calcium chloride dihydrate (CaCl2) (Sigma, C7902, Japan) into the PBS solution until it reached 50 mM as a crosslinking agent. The hydrogel bio-inks were puted at room temperature for 4 h and ready to used. The AGH bio-inks have compared to AGF127 6% w/v (Alginate Gelatin mixed Pluronic F127) as a competitor. The characterization of bio-inks was analyzed through mechanical test, rheological various, surface property analysis, printability evaluation and cell viability assessment. The cell response of the bio-inks will evaluate by live/dead staining containing the MG-63 cells (4 x105 cells mL-1). Results: The bio-inks of AGH 2% compared with AGF127 6% have investigated through several tests such as surface property measurements, rheological test, printability evaluation and cell viability analysis. The results performed that AGH 2% has superior material characteristics as hydrogels including mechanical, rheological and cell viability test which compared to AGF127 6%. However, between AGH 2% and AGF127 6% have almost the same printability evaluation results, it is not damaged when printing through 3D bioprinting even though both have different bio-ink properties. Discussions: The difference in characteristics of AGH 2% and AGF127 6% was caused by the addition of Pluronic F127 as a comparison. We found that, by adding 6% of Pluronic F127 the bio-ink was solid, due to the effect of temperature from materials. The solid characteristics of bio-ink require high pressure during printability and impact the number of extruded cells. Meanwhile, AGH 2% which is a hydrogel was sufficient with low pressure. The printability of AGH 2% used optimal pressure at 1.0 bar with print speed 4 mm/s. In this experiment indicated that viscosity increased at low shear rates, it meant that high viscosity may poor printability but through controlling the optimum of print speed and air pressure can mantain the filament form not collapse. There was minimal cell death caused by before printing process, and the viability of MG63 in after printing construct was decreases of amounts cell for AGH 2% and AGF127 6% samples. But AGH 2% sample has keep on 60% of the cell viability for day 1, day 4, and day 7 in after printing process. Conclusions: thru this research could be as a reference to described about AGH 2% can be used as biomaterials in the manufacture of 3D scaffolds as bone graft substitution. Bio-ink which is hydrogel easily forms filaments with the application of low air pressure, good printability, and compatible for cell viability.

Keywords: 3D bioprinting, biopolymer, bio-inks, scaffold, bone regeneration.