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. 2014 May;38(5):1083-9.
doi: 10.1007/s00264-013-2165-1. Epub 2013 Nov 19.

Influence of cyclical mechanical loading on osteogenic markers in an osteoblast-fibroblast co-culture in vitro: tendon-to-bone interface in anterior cruciate ligament reconstruction

Johannes Struewer  1 , Philip P Roessler, Karl F Schuettler, Volker Ruppert, Thomas Stein, Nina Timmesfeld, Juergen R J Paletta, Turgay Efe
Affiliations

Influence of cyclical mechanical loading on osteogenic markers in an osteoblast-fibroblast co-culture in vitro: tendon-to-bone interface in anterior cruciate ligament reconstruction

Johannes Struewer et al. Int Orthop. 2014 May.

Abstract

Purpose: We aimed to evaluate the influence of cyclical mechanical loading on osteoblasts and fibroblasts, and co-cultures of both in vitro, simulating the conditions of the tendon-to-bone interface in anterior cruciate ligament reconstruction.

Methods: Osteoblast-like cells (OBL) and tendon-derived rodent fibroblasts (TDF) were cultured alone or in co-culture to simulate the tendon-to-bone interface. Cyclical loading was applied for one hour twice a day for three days, with a frequency of 1 Hz and 3 % strain. Alkaline phosphatase (AP), osteocalcin (OC), collagen type 1 (COL1A1), and bone morphogenetic protein 2 (BMP-2) gene expression and protein deposition were detected by real-time polymerase chain reaction (qPCR) and immunocytochemical analysis.

Results: Mechanical loading significantly decreased AP, OC, and COL1A1 gene expression in both OBL and TDF, compared to non-loaded culture. However, mechanical load increased gene expression of the same marker genes including BMP-2 during co-culture. Immunocytochemistry demonstrated increased deposition of corresponding proteins in the same range, independent of culture conditions. Higher depositions of BMP-2 were shown under loading conditions for osteoblast and TDF monocultures. Prolongation of mechanical loading resulted in cell detachment and spheroid formation.

Conclusion: Cyclical mechanical loading caused downregulation of genes involved in osteointegration and osteoinduction, such as OC, ALP, and COL1A1 in monocultures of osteoblasts and fibroblasts; co-cultures lacked this phenomenon. Immunocytochemistry and qPCR analysis showed slight upregulations of marker genes and corresponding proteins. This might be due to the potential stabilising effects of osteoblast-fibroblast cross talk in the co-culture environment, simulating fibrocartilage formation at the tendon-to-bone interface.

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Figures

Fig. 1
Fig. 1
Influence of cyclical mechanical loading on all three culture types on BMP-2 deposition as detected by immunocytochemistry (10× magnification). a Osteoblast-like cells. b Fibroblasts. c Co-culture
Fig. 2
Fig. 2
Influence of prolonged continuous mechanical loading on all three culture types, for one or three days, on BMP-2 deposition as detected by immunocytochemistry. Significant detachment of cells from collagen-coated surfaces were found in all specimens (10× magnification). a Osteoblast-like cells. b Fibroblasts. c Co-culture
See this image and copyright information in PMC

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