title: 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.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Orthopaedics and Rheumatology, University Hospital Marburg, Baldingerstrasse, 35043, Marburg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Struewer
authors.forename: Philip P
authors.initials: PP
authors.lastname: Roessler
authors.forename: Karl F
authors.initials: KF
authors.lastname: Schuettler
authors.forename: Volker
authors.initials: V
authors.lastname: Ruppert
authors.forename: Thomas
authors.initials: T
authors.lastname: Stein
authors.forename: Nina
authors.initials: N
authors.lastname: Timmesfeld
authors.forename: Juergen R J
authors.initials: JR
authors.lastname: Paletta
authors.forename: Turgay
authors.initials: T
authors.lastname: Efe
mesh_terms: D000818:Animals; D059549:Anterior Cruciate Ligament Reconstruction / Q000379:methods*; D015415:Biomarkers / Q000032:analysis; D001696:Biomechanical Phenomena; D001842:Bone and Bones; D002478:Cells, Cultured; D018920:Coculture Techniques; D005347:Fibroblasts; D008297:Male; D010006:Osteoblasts; D010012:Osteogenesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013710:Tendons; D016474:Weight-Bearing
chemical_list: D015415:Biomarkers