title: Dense collagen matrix accelerates osteogenic differentiation and rescues the apoptotic response to MMP inhibition.
abstract: Bone is distinguished from other tissues by its mechanical properties, in particular stiffness. However, we know little of how osteoblasts react to the stiffness of their microenvironment; in this study we describe their response to a dense (>10 wt.%) collagenous 3D environment. Primary pre-osteoblasts were seeded within a novel form of native collagen, dense collagen, and cultured for up to 14 days in the presence and absence of osteogenic supplements: analysis was via Q-PCR, histology, fluorescent in situ zymography, MMP loss-of-function and tensile testing. Differentiation as measured through the up-regulation of Bsp (247-fold), Alp (14.2-fold), Col1A1 (4.5-fold), Mmp-13 (8.0-fold) and Runx2 (1.2-fold) transcripts was greatly accelerated compared to 2D plastic at 7 and 14 days in the same medium. The scale of this enhancement was confirmed through the use of growth factor stimulation on 2D via the addition of BMP-6 and the Hedgehog agonist purmorphamine. In concert, these molecules were capable of the same level of osteo-induction (measured by Bsp and Alp expression) as the dense collagen alone. Mineralisation was initially localised to remodelled pericellular regions, but by 14 days embedded cells were discernible within regions of apatite (confirmed by MicroRaman). Tensile testing of the matrices showed that this had resulted in a significant increase in Young's modulus at low strain values, consistent with a stiffening of the matrix. To determine the need for matrix remodelling in the mineralisation event the broad spectrum MMP Inhibitor Ilomastat was used. It was found that in its presence mineralisation could still occur (though serum-specific) and the apoptosis associated with MMP inhibition in hydrated collagen gels was abrogated. Analysis of gene expression indicated that this was due to the up-regulation of Mmp-13 in the presence of Ilomastat in dense collagen (400-fold), demonstrating a powerful feedback loop and a potential mechanism for the rescue from apoptosis. Osteoid-like matrix (dense collagen) is therefore a potent stimulant of osteoblast differentiation in vitro and provides an environment that enables survival and differentiation in the presence of MMP inhibition.
authors.affiliation: Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London, WC1X 8LD, UK. Electronic address: paul.buxton@ucl.ac.uk.
authors.forename: P G
authors.initials: PG
authors.lastname: Buxton
authors.affiliation: Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London, WC1X 8LD, UK; Materials Biology Interactions Group, Swiss Federal Laboratories for Materials Testing and Research (EMPA), Lerchenfeldstr 5, CH-9014 St. Gallen, Switzerland. Electronic address: m.bitar@eastman.ucl.ac.uk.
authors.forename: M
authors.initials: M
authors.lastname: Bitar
authors.affiliation: Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London, WC1X 8LD, UK. Electronic address: k.gellynck@eastman.ucl.ac.uk.
authors.forename: K
authors.initials: K
authors.lastname: Gellynck
authors.affiliation: Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London, WC1X 8LD, UK. Electronic address: m.parkar@eastman.ucl.ac.uk.
authors.forename: M
authors.initials: M
authors.lastname: Parkar
authors.affiliation: UCL Institute of Orthopaedics and Musculoskeletal Science, Royal National Orthopaedic Hospital, Stanmore, London, UK. Electronic address: rehkrab@ucl.ac.uk.
authors.forename: R A
authors.initials: RA
authors.lastname: Brown
authors.affiliation: Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London, WC1X 8LD, UK. Electronic address: a.young@eastman.ucl.ac.uk.
authors.forename: A M
authors.initials: AM
authors.lastname: Young
authors.affiliation: Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London, WC1X 8LD, UK. Electronic address: j.c.knowles@eastman.ucl.ac.uk.
authors.forename: J C
authors.initials: JC
authors.lastname: Knowles
authors.affiliation: Department of Mining and Materials Engineering, McGill University, Montreal, Quebec, Canada H3A 2B2. Electronic address: showan.nazhat@mcgill.ca.
authors.forename: S N
authors.initials: SN
authors.lastname: Nazhat
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D003094:Collagen / Q000378:metabolism*; D016616:Cryoultramicrotomy; D003470:Culture Media; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D008813:Mice, Inbred ICR; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010012:Osteogenesis / Q000187:drug effects
chemical_list: D003470:Culture Media; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D003094:Collagen