Development of 3D cell-free biomimetic scaffold to support endochondral ossification

• Grant number: 22/01219-5
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): July 01, 2022
• Effective date (End): June 30, 2023
• Field of knowledge: Physical Sciences and Mathematics - Chemistry
• Valor Concedido/Desembolsado (R$): 155,772.48 / 98,584.68
• Principal Investigator: Ana Paula Ramos
• Grantee: Lucas Fabrício Bahia Nogueira
• Supervisor: Massimo Bottini
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Research place: Università degli Studi di Roma Tor Vergata, Italy
• Associated to the scholarship: 18/25871-8 - Scaffolds composed by bioactive glasses and injectable matrices for bone regeneration, BP.DR

Abstract

Understanding the bone formation process involves the difficult task to realize how both time and nature acted harmonically in a unique association between calcium phosphate and collagen that gave rise to one of the most remarkable biomaterials we have ever known. The bone consists of a porous hybrid organic/inorganic three-dimensional (3D) matrix, composed mainly by collagen, a variety of non-collagenous proteins and a calcium phosphate mineral phase, which is formed and regulated by the orchestrated action of a complex array of cells that include chondrocytes, osteoblasts, osteocytes, and osteoclasts. The interactions between cells, proteins, and minerals are essential for the constant renewal of the bone tissue, responsible for the maintenance of its functions under a variety of physiological loading conditions, trauma, and fractures. From this perspective, bone formation has been studied from the interactions between the extracellular matrix (ECM) and cellular activity considering two essential processes: intramembranous ossification and endochondral ossification. Intramembranous ossification directly converts mesenchyme to bone, whereas endochondral ossification is a process in which mesenchyme transforms into cartilage, eventually replaced by bone. Endochondral ossification is involved in the natural healing of bone fractures, and it is also beneficial for vascularization of bone substituting-implants due to the secretion of angiogenic factors by hypertrophic chondrocytes. Therefore, the aim of this BEPE project is to further investigate ossification processes triggered by 3D cell-laden collagenous scaffolds in vitro replicating the mechanical and biological properties of bone after implantation. Inspired by the bone composition, we propose to assess the role of non-collagenous macromolecules, such as glycosaminoglycans, on the organization of the collagenous matrix and on cell behavior, specifically, on the mineralization ability of entrapped cells in this biomimetic matrix. Hypertrophic chondrocytes will be cultivated in biomimetic 3D matrices under chondrogenic conditions. After different cultivation times, the chondrocyte phenotype will be determined by gene expression (real-time PCR). Mineralization in the scaffolds will be evaluated in terms of the activity of tissue non-specific alkaline phosphatase (TNAP) and the number of mineralization nodules formed by means of spectroscopic and microscopic techniques. Mineralization will also be evaluated in terms of the secretion of specific extracellular vesicles which are believed to be related to the biochemical pathways that culminate in ECM mineralization. Thus, these vesicles, named matrix vesicles (MVs), will be extracted from chondrocyte cultures both in the presence and absence of the 3D biomimetic matrices that will be produced in this study. The exploration of this 3D biomimetic model in these two conditions should allow the evaluation of the role of MVs in the ECM mineralization process in an environment close to the native bone tissue. Professor Massimo Bottini has expertise both in primary chondrocyte cultures, a scant field in Brazil, and in the study of the role played by MVs during bone formation. Thus, at the end of this project, we expected that the proposed model will promote the understanding of the relationships between osteogenic cells, collagen-based 3D scaffolds, MVs and mineral deposition and, in turn, it will shed light on the biochemical pathways driving the biogenesis of MVs during endochondral ossification. Moreover, the acquisition of expertise in cultivating chondrocytes will open doors for a new research field and the development of biomaterials for endochondral regeneration in our group. (AU)