Structural changes and biological responsiveness of an injectable and mouldable monetite bone graft generated by a facile synthetic method

Brushite (dicalcium phosphate dihydrate) and monetite (dicalcium phosphate anhydrous) are of considerable interest in bone augmentation owing to their metastable nature in physiological fluids. The anhydrous form of brushite, namely monetite, has a finer microstructure with higher surface area, strength and bioresorbability, which does not transform to the poorly resorbable hydroxyapatite, thus making it a viable alternative for use as a scaffold for engineering of bone tissue. We recently reported the formation of monetite cements by a simple processing route without the need of hydrothermal treatment by using a high concentration of sodium chloride in the reaction mix of β-tricalcium phosphate and monocalcium phosphate monohydrate. In this paper, we report the biological responsiveness of monetite formed by this method. The in vitro behaviour of monetite after interaction and ageing both in an acellular and cellular environment showed that the crystalline phase of monetite was retained over three weeks as evidenced from X-ray diffraction measurements. The crystal size and morphology also remained unaltered after ageing in different media. Human osteoblast cells seeded on monetite showed the ability of the cells to proliferate and express genes associated with osteoblast maturation and mineralization. Furthermore, the results showed that monetite could stimulate osteoblasts to undergo osteogenesis and accelerate osteoblast maturation earlier than cells cultured on hydroxyapatite scaffolds of similar porosity. Osteoblasts cultured on monetite cement also showed higher expression of osteocalcin, which is an indicator of the maturation stages of osteoblastogenesis and is associated with matrix mineralization and bone forming activity of osteoblasts. Thus, this new method of fabricating porous monetite can be safely used for generating three-dimensional bone graft constructs

The fundamentals of tissue engineering: new scaffolds.

The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/beta-Tricalcium phosphate (HA/beta -TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP- an indicator of osteoblast differentiation) was observed on HA/beta -TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds

Osteoblast behaviour on HA/PE composite surfaces with different HA volumes.

A hydroxyapatite (HA) reinforced polyethylene (PE) composite (designated HAPEX), with high mechanical specification and a bioactive HA phase, has been optimised as a bone analogue material. Manufacturing conditions and machining of the materials were carefully controlled to give a reproducible material surface roughness with minimal batch variation. The effect of surface composition was examined in vitro using primary human osteoblasts (HOB). HOBs were cultured in direct contact with the test materials containing 20% and 40% vol. HA. The results showed that 40% HA/PE enhanced cellular activity by increasing proliferation rate and differentiation compared to the 20% vol. HA composite. The cytoskeletal organisation of the cells was also examined and HOBs cultured on 40% HA/PE were flatter and had an enhanced rate of cytoskeletal organisation and an increase in focal contact points compared to the 20% HA/PE

The determination of detection limits for insulin antibody assays

In order to define the detection limit of a radioimmunoassay for insulin-antibody a correction was made for binding in the presence of an excess of unlabelled insulin and assay precision was calculated. One hundred forty control sera were assessed; all were islet cell antibody negative. For each sample, binding of 125-I human insulin was determined both with and without excess unlabelled insulin, subtraction of the latter acting as a correction. The distribution of uncorrected binding was skewed while corrected binding was normally distributed, (mean (SD) = 0.149 (0.298%)) Precision, defined as the mean of the standard deviations of replicates, was 0.263%. Detection limits calculated from the estimate of precision (0.263%) or from the standard deviation of the corrected binding (0.298%) were similar. Two hundred thirty sera from insulin-treated patients were studied. Precision was plotted as a 'precision profile' and the detection limit calculated from the precision for binding of less than 1% [0.261%]; 88% of the sera were positive [cut-off 1.3%, p less than 0.01]. We conclude that corrected binding is normally distributed in antibody-negative sera and that an estimate of assay precision can be used to define the detection limit of the assay.</p

Bioresorbable glass fibres facilitate peripheral nerve regeneration

This is a proof of principle report showing that fibres of Bioglass((R)) 45S5 can form a biocompatible scaffold to guide regrowing peripheral axons in vivo. We demonstrate that cultured rat Schwann cells and fibroblasts grow on Bioglass((R)) fibres in vitro using SEM and immunohistochemistry, and provide qualitative and quantitative evidence of axonal regeneration through a Silastic conduit filled with Bioglass((R)) fibres in vivo (across a 0.5cm interstump gap in the sciatic nerves of adult rats). Axonal regrowth at 4 weeks is indistinguishable from that which occurs across an autograft. Bioglass((R)) fibres are not only biocompatible and bioresorbable, which are absolute requirements of successful devices, but are also amenable to bioengineering, and therefore have the potential for use in the most challenging clinical cases, where there are long inter-stump gaps to be bridged

PMMA bone cement containing a quaternary amine comonomer with potential antibacterial properties

An iodinated quaternary amine dimethacrylate monomer was synthesized and incorporated as a comonomer in acrylic bone cements. Bone cement is used in orthopaedic surgery and imparting antibacterial properties to the cement can be beneficial in the lowering of bacterial infection post surgery. PMMA based bone cements were modified by copolymerising the monomer methylmethacrylate (MMA) with a quaternary amine dimethacrylate by using the redox initiator activator system as used for curing commercial bone cements. The cements were prepared using the commercial PMMA bone cement CMW and the liquid component was modified with the amine to render antimicrobial properties to the cement. The physical, mechanical, and antimicrobial properties of the modified cements were evaluated; in addition, the viability of the cement to function as a orthopaedic cement was also established, especially with an advantage of it being radiopaque, due to the inclusion of the iodine containing quaternary amine. The cytotoxicity of the modified cements were tested using a human cell model and the results indicated that the cells remained metabolically active and proliferated when placed in direct contact with the experimental cement specimens. The cements and their eluants did not evoke any cytotoxic response.</p

Surface topography and HA filler volume effect on primary human osteoblasts in vitro.

HAPEX, a bone analog material, with similar properties to cortical bone, has been studied in vitro with particular reference to the effect of surface topography. The stimulation of a favorable bone response by this composite depends on optimization of the hydroxyapatite (HA) content in relation to the material bioactivity without compromising the mechanical characteristics. In this study we have started to investigate the effects of surface topography on cell attachment and subsequent cellular behavior in relation to proliferation. Different volumes of HA (20% and 40%) were added to a high density polyethylene (HDPE) matrix to produce the test materials. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to examine cell morphology on HAPEX, and the surface characteristics produced by different machining protocols. The measurement of cellular DNA and tritiated thymidine ([3H]-TdR) incorporation has been used to asses cell proliferation upon the materials. The results show that the material surface topography has a large influence on cell proliferation and attachment, and with a controlled material topography the 40% vol HA/HDPE composite gives the greater biological response compared to the 20% vol HA/HDPE composite