Synthesis and characterization of a new class of injectable, thermoresponsive and biodegradable hydrogels as cell substract for cell differentiation

Author(s)

Daniel Vinícius Mistura

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia Mecânica
• Defense date: 2018-12-06

Examining board members

Eliana Aparecida de Rezende Duek; Marcos Akira D'Avila; João Sinézio de Carvalho Campos; Arnaldo Rodrigues dos Santos Junior; Marcia Adriana Tomaz Duarte

• Advisor: Eliana Aparecida de Rezende Duek

Abstract

The class of hydrogels has been extensively studied, mainly as injectable materials with minimally invasive applications for biomaterials. This material can present characteristics of thermosensitivity close to the body temperature (LCST) in addition to rapid gelation, which allows its use as carrier of drugs, cells or cell growth factors when injected. The current challenge is to make these hydrogels biodegradable and with mechanical properties suitable for their use. The main objective of this work was to synthesize poly (N-isopropylacrylamide) hydrogels (PNIPAAm) from a new class of macromer composed of 2-hydroxyethyl methacrylate (HEMA), poly (L-co-D, L lactic acid), trimethylene carbonate (TMC) and Acrylic Acid (AAc), so that it is inherent to the synthesized material characteristics such as: biodegradation and mechanical properties compatible with those required in tissue engineering applications. In addition, another goal for the hydrogel was to evaluate its role as a substrate in the process of differentiating embryonic stem cells into cardiomyocyte-like cells, with potential applications in cardiac tissue regeneration. Thus, the synthesis occurs in two steps, first step, open ring polymerization of cyclic esters and cyclic carbonate to synthesize de macromere, in a second step, synthesize the hydrogels by free radical polymerization. By this routes, nine syntheses were made, with 2, 4 and 6% of acrylic acid and 10,15 and 20% of macromere, forming the combinations (constituted by NIPAAm, Macromer and AAc, respectively) 88/10/2, 83/15/2, 78/20/2, 86/10/4, 81/15/4, 76/20/4, 84/10/6, 79/15/6 e 74/20/6. After synthesis the material was characterized the hydrogels and macromes by chemical, thermical and rheological; was evaluated in vitro degradation and cell viability and was determined best material characteristics to induce embryonic stem cells into cardiomyocyties. NMR (1H and 13C) showed chemical bonds intensities of polymerized monomers, verifying macromer polymerization and posterior hydrogel copolymerization, FTIR results showed characteristics bands for the chemical bonds, and formation of expected products for polymerization. DSC showed hydrogels with glass transition temperatures (Tg¬) above 100ºC for all compositions and LCST behavior by calorimetry indicating values below 37ºC, indication for biological applications. Swelling studies evidenced all compositions with water absorption between 36 and 171%, rheological tests showed firstly quickly gelation for all compositions, and LCST behavior corroborating DSC, secondly, viscosity in 2 and 6% of acrylic acid superior of water (1 Pa.s) and with 4% composition below than water, this composition was used for in vivo tests. In vitro degradation showed marker loss of polymeric mass in all compositions, cell differentiation with qPCR confirming gene maintenance and immunofluorescence showing the maintenance of cell phenotype. Finally, in vivo studies in 86/10/4 and 81/15/4 compositions, showed low inflammatory response with gradual reduction and quickly resolution. The induced tissue reorganization showed an acute inflammation. This reorganization was inducing by hydrogel compartmentalization indicating material integration to the tissue, suggesting application as a biomaterial (AU)

• FAPESP's process: 14/12428-8 - Synthesis and characterization of a new class of injectables, termosensitives and biodegradables hydrogels as substrate for cell differentiation
• Grantee: Daniel Vinícius Mistura
• Support Opportunities: Scholarships in Brazil - Doctorate