Liposomal Delivery of Demineralized Dentin Matrix for Dental Tissue Regeneration.

Current dental restorations have short longevity, and consequently, there is a need for novel tissue engineering strategies that aim to regenerate the dentin-pulp complex. Dentin matrix contains a myriad of bioactive growth factors and extracellular matrix proteins associated with the recruitment, proliferation, and differentiation of dental pulp progenitor cells. In this study, we show that demineralized dentin matrix (DDM), from noncarious dentine, can be encapsulated into liposomes for delivery to dental tissue to promote regeneration. Liposomes were formulated to encapsulate 0-100 µg/mL DDM, lysed with Triton X, and used in vascular endothelial growth factor (VEGF) and transforming growth factor-ß1 (TGF-ß1) enzyme-linked immunosorbent assays to quantify release. The encapsulation efficiencies were calculated to be 25.9% and 28.8% (VEGF/TGF-ß1) for 50 µg/mL DDM liposomes and 39% and 146.7% (VEGF/TGF-ß1) for 100 µg/mL DDM liposomes. All liposome formulations had no cytotoxic effects on a dental pulp stem cell (DPSC) clone, as shown by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide), Caspase 3/7 assays, and cell counts. The ability of the liposomes to stimulate DPSC chemotactic recruitment was tested by Boyden chamber chemotaxis assays. Unloaded liposomes alone stimulated significant progenitor cell recruitment, while DDM-loaded liposomes further promoted chemotactic recruitment in a dose-dependent manner. DDM liposomes promoted the upregulation of "osteodentin" markers osteocalcin and RUNX2 (Runt-related transcription factor 2) in DPSCs after 9 days of treatment, determined by real-time quantitative PCR. Furthermore, Alizarin Red S staining showed that unloaded liposomes alone induced biomineralization of DPSCs, and DDM liposomes further increased the amount of mineralization observed. DDM liposomes were more effective than free DDM (10 µg/mL) at activating recruitment and osteogenic differentiation of DPSC, which are key events in the endogenous repair of the dentin-pulp complex. The study has highlighted the therapeutic potential of bioactive DDM liposomes in activating dental tissue repair in vitro, suggesting that liposomal delivery from biomaterials could be a valuable tool for reparative dentistry and hard-tissue engineering applications.

Osteopenia in Sparc (osteonectin)-deficient mice: characterization of phenotypic determinants of femoral strength and changes in gene expression.

Sparc null mutants have been generated independently via targeted mutations in exons 4 and 6. Previous studies have identified low-turnover osteopenia in the 129Sv/C57BL/6 exon 4 knockout. Since both Sparc null mutations result in complete absence of Sparc protein, similar phenotypic outcomes are likely. However, genetic background (strain) and/or linkage disequilibrium effects can influence phenotype. Different inactivating mutations should be tested in various mouse strains; similar phenotypic outcomes can then confidently be assigned to the mutated gene. We have evaluated the bone phenotype in the 129Sv/EvSparc(tm1cam) exon 6 knockout at 4 and 9 mo, using physical measurement, mechanical strength tests, and DXA scanning. We have also quantified bone marrow adiposity and circulating leptin levels to assess adipose tissue metabolism. 129Sv/EvSparc(tm1cam) null mice show decreased bone mineral density and bone mineral content and increased mechanical fragility of bone, in line with previous studies. Differences were also noted. Increased body weight and levels of bone marrow adiposity but decreased circulating leptin concentrations were identified at 4, but not 9 mo, and 129Sv/EvSparc(tm1cam) null mice also had shorter femurs. Molecular phenotyping was carried out using mouse HGMP NIA microarrays with cortical femur samples at various ages, using semiquantitative RT-PCR validation. We identified 429 genes highly expressed in normal bone. Six genes (Sparc, Zfp162, Bysl, E2F4, two ESTs) are differentially regulated in 129Sv/EvSparc(tm1cam) cortical femur vs. 129Sv/Ev controls. We confirm low-turnover osteopenia as a feature of the Sparc null phenotype, identifying the usefulness of this mouse as a model for human osteoporosis.

A novel liposomal drug delivery system for PMMA bone cements.

The population in developed countries is ageing and the number of people experiencing joint-related conditions, such as osteoarthritis, is expected to increase. Joint replacements are currently the most effective treatment for severe joint conditions and although many of these procedures are successful, infection developing after the procedure is still an issue, requiring complex and expensive revisions. Whilst incorporating a powdered antibiotic within the bone cement can reduce infection rates, the powder frequently agglomerates, resulting in poor antibiotic release characteristics and compromised mechanical performance of the cement. To overcome these issues, a novel delivery system consisting of antibiotic-loaded nano-sized liposomes was developed for inclusion into polymethyl methacrylate (PMMA) bone cement. This system was tested in a commercial cement (Palacos R) and consistently delivered a higher percentage (22%) of the incorporated antibiotic when compared to the powdered antibiotic cement (9%), meaning less antibiotic needs to be incorporated than with conventional cement. The novel system resulted in a controlled and gradual release of antibiotic over a longer, 30-day period and enhanced the toughness, bending strength and Vickers hardness of the cement, without altering its polymerization or molecular structure. This new material has the potential to significantly reduce infections in cemented joint replacements leading to enhanced patient quality of life and reduced healthcare costs. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1510-1524, 2016.

Ageing and moisture uptake in polymethyl methacrylate (PMMA) bone cements.

Bone cements are extensively employed in orthopaedics for joint arthroplasty, however implant failure in the form of aseptic loosening is known to occur after long-term use. The exact mechanism causing this is not well understood, however it is thought to arise from a combination of fatigue and chemical degradation resulting from the hostile in vivo environment. In this study, two commercial bone cements were aged in an isotonic fluid at physiological temperatures and changes in moisture uptake, microstructure and mechanical and fatigue properties were studied. Initial penetration of water into the cement followed Fickian diffusion and was thought to be caused by vacancies created by leaching monomer. An increase in weight of approximately 2% was experienced after 30 days ageing and was accompanied by hydrolysis of poly(methyl methacrylate) (PMMA) in the outermost layers of the cement. This molecular change and the plasticising effect of water resulted in reduced mechanical and fatigue properties over time. Cement ageing is therefore thought to be a key contributor in the long-term failure of cemented joint replacements. The results from this study have highlighted the need to develop cements capable of withstanding long-term degradation and for more accurate test methods, which fully account for physiological ageing.