Translational Research for Orthopedic Bone Graft Development.

Maria J C Vilela,Christiane L Salgado,Bruno J A Colaço,Fernando J M Monteiro,José Ventura

doi:10.3390/ma14154130

Abstract

Designing biomaterials for bone-substitute applications is still a challenge regarding the natural complex structure of hard tissues. Aiming at bone regeneration applications, scaffolds based on natural collagen and synthetic nanohydroxyapatite were developed, and they showed adequate mechanical and biological properties. The objective of this work was to perform and evaluate a scaled-up production process of this porous biocomposite scaffold, which promotes bone regeneration and works as a barrier for both fibrosis and the proliferation of scar tissue. The material was produced using a prototype bioreactor at an industrial scale, instead of laboratory production at the bench, in order to produce an appropriate medical device for the orthopedic market. Prototypes were produced in porous membranes that were e-beam irradiated (the sterilization process) and then analysed by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), dynamic mechanical analysis (DMA), cytotoxicity tests with mice fibroblasts (L929), human osteoblast-like cells (MG63) and human MSC osteogenic differentiation (HBMSC) with alkaline phosphatase (ALP) activity and qPCR for osteogenic gene expression. The prototypes were also implanted into critical-size bone defects (rabbits’ tibia) for 5 and 15 weeks, and after that were analysed by microCT and histology. The tests performed for the physical characterization of the materials showed the ability of the scaffolds to absorb and retain water-based solvents, as well as adequate mechanical resistance and viscoelastic properties. The cryogels had a heteroporous morphology with microporosity and macroporosity, which are essential conditions for the interaction between the cells and materials, and which consequently promote bone regeneration. Regarding the biological studies, all of the studied cryogels were non-cytotoxic by direct or indirect contact with cells. In fact, the scaffolds promoted the proliferation of the human MSCs, as well as the expression of the osteoblastic phenotype (osteogenic differentiation). The in vivo results showed bone tissue ingrowth and the materials’ degradation, filling the critical bone defect after 15 weeks. Before and after irradiation, the studied scaffolds showed similar properties when compared to the results published in the literature. In conclusion, the material production process upscaling was optimized and the obtained prototypes showed reproducible properties relative to the bench development, and should be able to be commercialized. Therefore, it was a successful effort to harness knowledge from the basic sciences to produce a new biomedical device and enhance human health and wellbeing.

Highlights

The extracellular matrix (ECM) is very important for cells’ microenvironments and survival
This work had as its main objective the evaluation of a scaled-up productive process for the development of a biomaterial based on collagen and nanohydroxyapatite at the industrial prototype level
The cryogels had a heteroporous morphology with microporosity and macroporosity, which are essential conditions for the interaction between the cells and materials, and to promote bone regeneration

Summary

Introduction

The extracellular matrix (ECM) is very important for cells’ microenvironments and survival. It gives structural support to cells and tissues and provides signaling cues that regulate cells’ behaviour in multicellular organisms, such as cell growth, differentiation, shape and viability [1] It is composed of structural proteins (i.e., collagen), polysaccharides/glycosaminoglycans (GAGs), and adhesion proteins, such as integrins, which make the connection between the ECM and the cells. In order to try to avoid these major disadvantages, alternative materials need to be found that are able to mimic the mechanical properties, structure and functions of the bone tissue [6] To this end, 3D scaffolds are widely used in bone regeneration to promote tissue growth by mimicking the ECM of the native tissues [7,8,9]. We observed the in vitro and in vivo cell/tissue behavior within the different samples, and they showed biocompatibility, high cellular viability, proliferation and osteogenic differentiation, which is an advantage for application as a medical device in bone regeneration

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