Human Dental Pulp Stem Cell Osteogenic Differentiation Seeded on Equine Bone Block with Graphene and Melatonin

Rosa Mancinelli,Ester Sara Di Filippo,Silvia Giordani,Margherita Tumedei,Valeria Ettorre,Mariangela Marrone,Adriano Piattelli,Stefania Fulle,Antonella Fontana,Michele Baldrighi,Giovanna Iezzi

doi:10.3390/app11073218

Abstract

Equine bone blocks have osteogenic effects promoting bone regeneration with biocompatibility and osteoconductivity capacity. Human dental pulp stem cells (hDPSCs) can differentiate into osteoblasts enhancing biomineralization with such scaffolds. Melatonin is able to improve bone health and mediate bone formation. Collagenated equine bone blocks were coated with ammonia-functionalized graphene-oxide (G-N) at two different concentrations (2 μg/mL, G-N2; and 10 μg/mL, G-N10). The homogeneity of G-N coating was checked by Raman spectroscopy, whereas thermogravimetric analysis (TGA) allowed us to quantify the amount of G-N deposited on the blocks. The aim of this study was to investigate in vitro the effect of G-N-coated collagenated equine bone blocks on the proliferation and differentiation of hDPSCs with the addition of a melatonin. This evaluation was determined after 7, 14, and 21 days of culture by the expression of specific microRNAs, RUNX2 and SMAD5 gene expression, osteocalcin levels, and histological analysis. The results showed that equine blocks G-N2 and G-N10 and melatonin gave an optimal cell adhesion as shown by histological analysis, and an increase in the hDPSCs osteogenic potential as confirmed by microRNA and gene expression with an increase in osteocalcin levels. This study suggests that equine bone blocks coated with G-N2 and G-N10 and melatonin promote the osteogenic process.

Highlights

The clinical treatment of large-size bone defects requires an interdisciplinary approach of bone regeneration techniques to restore the anatomical, physical, and functional conditions after acquired trauma, infection, neoplasm, or surgical resection [1]
We demonstrated that equine bone block enhanced Human dental pulp stem cells (hDPSCs) differentiation and the effect was further enhanced by the simultaneous presence of an equine bone block and 100 μM of melatonin that stimulated early stages of cell differentiation
MiR-29b expression was upregulated at 7 and 14 days of hDPSCs growth medium (GM) culture in the presence of G-N-coated 10 μg/mL (G-N10) with and without melatonin compared to Ctrl; while in differentiation medium (DM) we found an upregulation only for G-N10 plus melatonin compared to Ctrl, at 7 days

Summary

Introduction

The clinical treatment of large-size bone defects requires an interdisciplinary approach of bone regeneration techniques to restore the anatomical, physical, and functional conditions after acquired trauma, infection, neoplasm, or surgical resection [1]. The osteoconduction process is a physiological mechanism based on the new bone formation on a biomaterials and scaffolds surface. This property is associated to the substrain of the bone healing process by a space-maintaining capacity in fractures or damaged hard tissue defects [4]. These components represent key factors able to enhance the integration of the graft, to promote the remodeling of the bone substitute and to induce the new bone formation [5]. Equine bone has been proposed as a bone substitute alone or in combination with other materials [15]

Objectives

Methods

Results

Discussion

Conclusion