Doxycycline-doped collagen membranes accelerate in vitro osteoblast proliferation and differentiation

Abstract

Objective: The aim of the study was to evaluate the effect of doxycycline- and dexamethasone-doped collagen membranes on the proliferation and differentiation of osteoblasts.

Background: Collagen barrier membranes are frequently used to promote bone regeneration and to boost this biological activity their functionalization with antibacterial and immunomodulatory substances has been suggested.

Methods: The design included commercially available collagen membranes doped with doxycycline (Dox-Col-M) or dexamethasone (Dex-Col-M), as well as undoped membranes (Col-M) as controls, which were placed in contact with cultured MG63 osteoblast-like cells (ATCC). Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and differentiation by measuring the alkaline phosphatase (ALP) activity using spectrophotometry. Real-time quantitative polymerase chain reaction was used to study the expression of the genes: Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-β1, VEGF, TGF-βR1, TGF-βR2, and TGF-βR3. Scanning electron microscopy was used to study osteoblast morphology. Data were assessed using one-way analysis of variance or Kruskal-Wallis tests, once their distribution normality was assessed by Kolmogorov-Smirnov tests (p > .05). Bonferroni for multiple comparisons were carried out (p < .05).

Results: Osteoblast proliferation was significantly enhanced in the functionalized membranes as follows: (Col-M < Dex-Col-M < Dox-Col-M). ALP activity was significantly higher on cultured osteoblasts on Dox-Col-M. Runx-2, OSX, ALP, OSC, BMP-2, BMP-7, TGF-β1, VEGF, TGF-βR1, TGF-βR2, and TGF-βR3 were overexpressed, and RANKL was down-regulated in osteoblasts cultured on Dox-Col-M. The osteoblasts cultured in contact with the functionalized membranes demonstrated an elongated spindle-shaped morphology.

Conclusion: The functionalization of collagen membranes with Dox promoted an increase in the proliferation and differentiation of osteoblasts.

Keywords: bone regeneration; dexamethasone; doxycycline; membranes; osteoblasts.