Paul Buxton

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 Youngs 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.

Muscling in on stem cells

Skeletal muscle is one of the few adult tissues that possesses the capacity for regeneration (restoration of lost functional tissue) as opposed to repair. This capacity is due to the presence of ‘muscle stem cells’ known as satellite cells. Detailed investigation of these cells over the past 50 years has revealed that both these and other cells within the skeletal muscle complex are capable of regenerating both muscle and other cell types as well. Here, we review this information, and suggest that skeletal muscle is an exciting reservoir of cells for regenerating skeletal muscle itself, as well as other cell types.

Reactive calcium-phosphate-containing poly(ester-co-ether) methacrylate bone adhesives: Chemical, mechanical and biological considerations

A poly(propylene glycol-co-lactide) dimethacrylate adhesive with monocalcium phosphate monohydrate (MCPM)/beta-tricalcium phosphate (beta-TCP) fillers in various levels has been investigated. Water sorption by the photo-polymerized materials catalyzed varying filler conversion to dicalcium phosphate (DCP). Polymer modulus was found to be enhanced upon raising total calcium phosphate content. With greater DCP levels, faster release of phosphate and calcium ions and improved buffering of polymer degradation products were observed. This could reduce the likelihood of pH-catalyzed bulk degradation and localized acid production and thereby may prevent adverse biological responses. Bone-like MG-63 cells were found to attach, spread and have normal morphology on both the polymer and composite surfaces. Moreover, composites implanted into chick embryo femurs became closely apposed to the host tissue and did not appear to induce adverse immunological reaction. The above results suggest that the new composite materials hold promise as clinical effective bone adhesives.

Cell attachment and response to photocured, degradable bone adhesives containing tricalcium phosphate and purmorphamine.

The aim of this study was to quantify and provide evidence as to how addition of tricalcium phosphate (β-TCP) and the Hedgehog agonist purmorphamine to a degradable bone adhesive affects cell attachment/proliferation and Hedgehog pathway activation. Fourier transform infrared spectroscopy demonstrated that high levels (75 wt.%) of β-TCP addition reduced the photocure rate of the chosen poly(propylene glycol-co-lactide) dimethacrylate (PPLM) bone adhesive, but this problem was overcome by increased light exposure. In phosphate-buffered saline the total surface mass loss of set 15 mm diameter PPLM films was ∼3.2 mg in 12 weeks, irrespective of thickness (200 or 400 μm) or β-TCP level (50 or 75 wt.%). With 400 μm samples there was additional bulk material loss. Proliferation of pre-osteoblast cells (MC3T3-E1) on the set adhesive surfaces was enhanced by decreased sample thickness or filler content increase. Degradation evidence suggested that both effects were due to reduced acidic polymeric degradation products. Activation of the Hedgehog pathway was quantified by measuring Gli expression in Light II reporter cells. The 0.01 and 0.1 wt.% purmorphamine in composite discs (400 μm, 75 wt.% β-TCP) enhanced Gli expression of attached cells 2- and 5-fold, respectively, without influencing their number. Pre-storage of the composite samples in culture medium had no detrimental effect on this response. Furthermore, sample storage medium gave no enhanced Gli expression in cells on tissue culture plastic. This suggests drug release levels were very low. Purmorphamine and β-TCP incorporation in PPLM adhesives might, therefore, provide prolonged enhancement of in vivo bone repair without systemic drug side-effects.

Small molecule stimulation enhances bone regeneration but not titanium implant osseointegration.

The osteogenic and osseointegrative potential of a small molecule was examined to assess its usefulness in regenerative procedures. Purmorphamine was used to stimulate bone growth and repair in an in vitro cell-based assay and an in vivo chick embryo CAM-assay with and without the presence of an implant. Purmorphamine adhered to precipitated hydroxyapatite coating, could activate the sonic hedgehog pathway and thereby stimulated osteodifferentiation. Porous calcium phosphate beads were used to deliver this small molecule in vivo and showed that purmorphamine increased the trabecular bone to bone area significantly. The assay showed purmorphamine failed to induce any significant difference in osseointegration on titanium coated PTFE implants. This suggests that, while a small molecule can enhance osteogenesis and might be useful in regenerative procedures, it failed to enhance the osseointegration of a Ti coated implant, suggesting that this sort of stimulation might be useful for enhancing bone regeneration where bone loss due to disease exists, but not for enhancing early stability of an implant.