Effect of 3D scaffolds funcionalized with anti-fibronectin aptamers on blood clot formation and osteogenesis. Ex vivo and in vivo study in rats

Author(s)

Natacha Malu Miranda da Costa

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-05-31

Examining board members

Daniela Bazan Palioto Bulle; Rafael Rodrigues Lima; Michel Reis Messora; Jamil Awad Shibli

• Advisor: Daniela Bazan Palioto Bulle; Paulo Tambasco de Oliveira

Abstract

In dentistry, the use of three-dimensional scaffolds (SCA) has been used in maxillofacial bone regeneration. The adsorption of bioactive molecules on SCA surface possibly optimizes osteogenesis. Thus, in this research were evaluated, ex vivo, in vitro (osteoblasts - OSB) and in vivo (calvaria defect at 5 and 15 days) the effect of SCA functionalization (chitosan +20% β-TCP) with anti-fibronectin aptamers (APT) on osteogenesis. SCA functionalization was performed with 20 µg of APT by simple adsorption. The physiological coagulum (PhC) was developed for 16 hours on the SCA with or without APT in critical defects created in rat calvaria, characterizing the ex vivo study. Then, the OSB were cultured on the PhC formed on SCA with and without APT. In vitro assays were performed on SCAs without PhC. The OSB and PhC morphology was verified by SEM. The cellular characterization of PhC was performed by flow cytometry (CF). Cell viability, mineralized matrix accumulation and ALP and BSP expression were investigated by MTT, ARS and IF, respectively. Additionally, the Alpl, Runx2, Bsp, Ocn and Tgf-β1 gene expression was verified by RT-PCR. In the in vivo experiment, Micro-CT, morphometry by Masom\'s trichrome staining, IF, transcriptomic analysis by RNAseq and RT-PCR were performed at 5 and 15 days in the SCA and SCA+APT groups. The ex vivo results showed high ALP and IBSP labelind in the APT group. FC revealed greater detection of CD90, CD45 and CD44 in PhC formed in the same group. In SEM, a dense fibrina enriched network was observed, composed of different cell types in PhC formed on SCA with APT. RT-PCR showed more Tgf-β1 gene expression in the SCA+APT+PhC+OSB group. In relation to in vitro assays (without the PhC) there was more formation of mineralized matrix in SCA+APT+OSB group in 10 days. The same was observed in IF, showing more immunostaining of IBSP in SCA+APT+OSB, while ALP was more prevalent in the SCA+OSB group. In in vivo analyses, Micro-CT revealed greater bone volume and less bone porosity at 5 days in SCA+APT. In addition, there was more collagen tissue formation, some bone formation points and increased ALP expression in the APT group at 5 days, while RUNX2 expression was more predominant in SCA+APT at 15 days. In RNAseq showed more differentially expressed genes in 15 days. Gene ontology (GO) showed differences in biological functions associated with cell adhesion and cell membrane ion channels. At 15 days, this difference was associated with the immune response, ECM components, antioxidant activity and cellular cytoskeleton polymerization (downregulated). These findings were validated by verifying Vim, Hprt1, Clcn4, Cd24, Krtap7-1, Psme2, Tnf and Il-1β gene expression. Taken together, these results showed that APT improves the coagulation pattern, osteoblastic differentiation and early events associated with osteoimmunology. (AU)

• FAPESP's process: 18/16925-7 - 3D scaffolds functionalized with anti-fibronectin aptamers on coagulum features: ex vivo and in vivo studies
• Grantee: Natacha Malu Miranda da Costa
• Support Opportunities: Scholarships in Brazil - Doctorate