Cell cytoskeletal changes effected by static compressive stress lead to changes in the contractile properties of tissue regenerative collagen membranes.

Static compressive stress can influence the matrix, which subsequently affects cell behaviour and the cell's ability to further transform the matrix. This study aimed to assess response to static compressive stress at different stages of osteoblast differentiation and assess the cell cytoskeleton's role as a conduit of matrix-derived stimuli. Mouse bone marrow mesenchymal stem cells (MSCs) (D1 ORL UVA), osteoblastic cells (MC3T3-E1) and post-osteoblast/pre-osteocyte-like cells (MLO-A5) were seeded in hydrated and compressed collagen gels. Contraction was quantified macroscopically, and cell morphology, survival, differentiation and mineralisation assessed using confocal microscopy, alamarBlue® assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and histological stains, respectively. Confocal microscopy demonstrated cell shape changes and favourable microfilament organisation with static compressive stress of the collagen matrix; furthermore, cell survival was greater compared to the hydrated gels. The stage of osteoblast differentiation determined the degree of matrix contraction, with MSCs demonstrating the greatest amount. Introduction of microfilament disrupting inhibitors confirmed that pre-stress and tensegrity forces were under the influence of gel density, and there was increased survival and differentiation of the cells within the compressed collagen compared to the hydrated collagen. There was also relative stiffening and differentiation with time of the compressed cell-seeded collagen, allowing for greater manipulation. In conclusion, the combined collagen chemistry and increased density of the microenvironment can promote upregulation of osteogenic genes and mineralisation; MSCs can facilitate matrix contraction to form an engineered membrane with the potential to serve as a 'pseudo-periosteum' in the regeneration of bone defects.

The influence of a bisphosphonate on bone generation determined using a chick-femur model.

AIM: To determine the direct influence of a bisphosphonate (pamidronate) delivered by one of two carriers, on bone generation in chick-femurs supported by chick egg chorio-allantoic membranes. METHODOLOGY: Twenty chick femurs freshly harvested from fertilized eggs were randomly allocated to two groups: (i) Affi-Gel blue bead carrier (n=10); and (ii) hydroxyapatite bead carrier (n=10). The femurs in each group were further randomly divided into control (n=4) and experimental (n=6) subgroups. Carriers charged with PBS solution or 0.1 M pamidronate were delivered into the bone marrow of each femur at its mid-portion through a needle puncture. Each femur was then grafted onto the chorio-allantoic membrane of a chick egg and incubated for 7 days. Each experimental and control subgroup femur yielded four histological sections at the puncture site, constituting the test and inter-bone controls. In addition, two histological sections were also obtained from 400 to 450 µm away from each end of the experimental puncture site to act as the intra-bone controls. Bone generation was quantified and the ratio of cross-sectional area of bone marrow to circumference of bone (outcome measure) was determined using a software package, Image-Pro(®) Plus. The data were analysed using Mann-Whitney tests and Wilcoxon signed rank tests. RESULTS: The outcome ratio in the test group was significantly (P<0.001) smaller than both the inter-bone and intra-bone control groups. There was evidence of increased bone formation directly over the pamidronate-charged carriers. CONCLUSIONS: The test model established that pamidronate had a positive effect on bone generation over a period of 7 days, regardless of the carrier type.

Dense collagen matrix accelerates osteogenic differentiation and rescues the apoptotic response to MMP inhibition.

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.