# Enhanced Bioactivity of Cu-Doped Bioactive Glass Coatings on Human Freeze-Dried Cortical Bone: An In Vitro Study

## TL;DR

This study shows that copper-doped bioactive glass coatings on bone grafts improve bone regeneration and blood vessel growth in lab tests.

## Contribution

The novel Cu-BG coating enhances osteogenic and pro-angiogenic properties of allogeneic bone grafts using pulsed-laser deposition.

## Key findings

- Cu-BG coatings showed total liquid absorption and a homogeneous layer with micrometric grain-like aggregates.
- Cu-BG samples significantly increased SP7, SPP1, BGLAP, and VEGF gene expressions in osteoblast-like and stem cells.
- The coating demonstrated biocompatibility and no cytotoxic effects on Saos-2 cells.

## Abstract

Bone grafting is one of the most used surgical techniques to favor bone regeneration and repair in orthopedic procedures. Despite autografting continuing to be considered the gold standard, allogeneic bone tissues remain a viable alternative albeit in the last decades, only a few studies have been carried out to translate enhanced allogeneic bone grafts into clinical solutions. In this in vitro study, cortical allogeneic bone samples were coated with copper-doped bioactive glass 45S5 (Cu-BG) by means of the pulsed-laser deposition technique to combine the mechanical support and osteoconductive properties of human bone with the osteogenic and pro-angiogenic features of the bioactive material. Contact angle (CA), scanning electron microscopy (SEM), and atomic force microscopy (AFM) measurements were carried out to quantitatively compare the impact on the bone surface properties of the morphological changes induced by the presence of the coating. Specifically, the obtained results have shown a total absorption of the drop on the coated samples. The coating on the bone tissue surface consisted of a homogeneous Cu-BG background layer with micrometric grain-like aggregates on it—a morphological feature that can facilitate osteoblast adhesion and proliferation. Cytotoxicity and cell viability were carried out on Saos-2 osteoblast-like cells, demonstrating the biocompatibility of the novel composite bone tissue and the absence of cytotoxic residuals. Moreover, human bone marrow-derived mesenchymal stem cells (hBMSCs) were seeded on Cu-BG and not-coated (NC) samples to evaluate the bioactivity and their differentiation toward the osteogenic phenotype. Our findings showed the pro-osteogenic and pro-angiogenic potential of Cu-BG coatings, although dynamic changes were observed over time. At seven days, the Cu-BG samples exhibited significantly higher expressions of SP7, SPP1, and BGLAP genes, indicating an enhanced early osteogenic commitment. Moreover, VEGF expression was significantly increased in Cu-BG compared to the control. These results pave the way for the development of an innovative class of bone-based products distributed by tissue banks.