Protocol of Co-Culture of Human Osteoblasts and Osteoclasts to Test Biomaterials for Bone Tissue Engineering.

Giorgia Borciani,Chiara Vitale-Brovarone,Nicola Baldini,Gabriela Ciapetti,Giorgia Montalbano

doi:10.3390/mps5010008

Abstract

New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration.

Highlights

The coupling of bone formation by osteoblasts (OBs) and bone resorption by osteoclasts (OCs) is at the base of the bone remodeling process and plays a crucial role in the maintenance of bone volume and skeletal homeostasis, with the crosstalk between OBs and OCs tightly regulated by systemic factors and molecules locally secreted [1]
We developed a co-culture of human osteoblasts (OBs) derived from trabecular bone and human osteoclast precursors (PBMCs) isolated from the buffy coat, to set up a human bone cells system able to recreate in vitro a bone microenvironment
Please note that the indirect co-colture is maintained in culture medium without exogenous osteoclastogenic inducers in order to prompt the differentiation of peripheral blood mononuclear cells (PBMCs) towards OCs only by means of the paracrine signals released by OBs

Summary

Introduction

The coupling of bone formation by osteoblasts (OBs) and bone resorption by osteoclasts (OCs) is at the base of the bone remodeling process and plays a crucial role in the maintenance of bone volume and skeletal homeostasis, with the crosstalk between OBs and OCs tightly regulated by systemic factors and molecules locally secreted [1]. Many aspects can be investigated using the OB/OC co-culture system, since this model allows to recreate in vitro a bone microenvironment that more closely mimics the natural complex interactions between bone cells [6]. In this context, the use of human primary cells is strongly recommended for a reliable bone replicate in vitro, even if the poor availability of donor tissue, the phenotypic heterogeneity caused by donor variability, and the limited supply undermine their large application in the research field [7]. As underlined by Abdallah et al, “access to a standardized source of mature human OCs from peripheral blood mononuclear cells (PBMCs) is needed to analyze their roles in bone regeneration and repair” [11]

Methods

Results

Conclusion