Magnesium Modifies the Structural Features of Enzymatically Mineralized Collagen Gels Affecting the Retraction Capabilities of Human Dermal Fibroblasts Embedded within This 3D System

Federica Boraldi,Daniela Quaglino,Giulia Annovi,Angelica Bartolomeo,Romain Debret

doi:10.3390/ma9060477

Abstract

Mineralized collagen gels have been developed as in vitro models to better understand the mechanisms regulating the calcification process and the behavior of a variety of cell types. The vast majority of data are related to stem cells and to osteoblast-like cells, whereas little information is available for dermal fibroblasts, although these cells have been associated with ectopic calcification and consequently to a number of pathological conditions. Therefore, we developed and characterized an enzymatically mineralized collagen gel in which fibroblasts were encapsulated within the 3D structure. MgCl2 was also added during gel polymerization, given its role as (i) modulator of ectopic calcification; (ii) component of biomaterials used for bone replacement; and (iii) constituent of pathological mineral deposits. Results demonstrate that, in a short time, an enzymatically mineralized collagen gel can be prepared in which mineral deposits and viable cells are homogeneously distributed. MgCl2 is present in mineral deposits and significantly affects collagen fibril assembly and organization. Consequently, cell shape and the ability of fibroblasts to retract collagen gels were modified. The development of three-dimensional (3D) mineralized collagen matrices with both different structural features and mineral composition together with the use of fibroblasts, as a prototype of soft connective tissue mesenchymal cells, may pave new ways for the study of ectopic calcification.

Highlights

Formation of hard connective tissue, such as dentin, bone and cementum, involves calcium phosphate deposition within a collagen matrix
Collagen gel mineralization has been either obtained by incorporating hydroxyapatite [11,12], by seeding cells cultured for several days in the presence of pro-osteogenic supplements as β-glycerophosphate and ascorbic acid [13,14,15], or by preparing collagen/calcium phosphate multilayers, where mineralization is the result of enzymatic reactions [16] and cells are only spread on the matrix surface
The novelty of the present study is that collagen gels were prepared in the absence of crosslinking agents and cells were not prepared separately from the mineralized collagen gel, but added prior to polymerization, causing their embedding within the 3D structure

Summary

Introduction

Formation of hard connective tissue, such as dentin, bone and cementum, involves calcium phosphate deposition within a collagen matrix. Collagen gel mineralization has been either obtained by incorporating hydroxyapatite [11,12], by seeding cells cultured for several days in the presence of pro-osteogenic supplements as β-glycerophosphate and ascorbic acid [13,14,15], or by preparing collagen/calcium phosphate multilayers, where mineralization is the result of enzymatic reactions [16] and cells are only spread on the matrix surface. Stem cells (i.e., dental pulp stem cells, bone marrow mesenchymal stem cells or adipose stem cells) as well as osteoblasts or smooth muscle cells have been widely investigated, whereas other cell types (i.e., fibroblasts) have captured only little attention, even though they regulate connective tissue biosynthesis and organization and are associated with ectopic calcification, and to a large number of pathologic conditions [17]

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