The development of liposome encapsulated calcium phosphates for bone regeneration
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Osteoporosis, a degenerative bone disorder, is one of the leading causes of morbidity in the elderly. Proper nutrition plays a role in the prevention and treatment of osteoporosis. Intake of calcium and Vitamin D are some of the most important nutritional factors, and supplementation remains the gold standard and first line of treatment for low bone mineral density and osteoporosis. Supplementation can prevent bone loss and reduce fracture risk. This work set about to produce, characterise and encapsulate for direct delivery to the bone various micro and nano sized calcium based mineral compounds which may be beneficial to bone health, using the precipitation method and biomimetic processes. Calcium phosphate mineral was produced and characterised, including hydroxyapatite (Hap), dicalcium phosphate dihydrate (DCPD), as well as multiphase and substituted calcium phosphates using biomimetic process. Standard simulated body Fluid (SBF) solution was modified, creating a high carbonate solution which better mimics the bone environment, and produces precipitates more similar to bone than traditional low carbonate SBF, as confirmed using Fourier Tansform Infrared Spectroscopy (FTIR) and X-Ray Diffraction (XRD). The use of liposomes as a delivery vesicle for the calcium mineral was evaluated usmg FTIR, XRD, Electron Dispersive Spectroscopy, Mass Spectrometry Transmission and Scanning Electron Microscopy, and X-Ray Mapping. The calcium mineral from aqueous solutions and prepared HAp and DCPD was incorporated into the liposome. Functional groups were synthesised based on a published structure used to target the bone marrow macrophage, and incorporation into liposomes was confitmed using Nuclear Magnetic Resonance Spectroscopy and FTIR. Preliminary cell culture studies showed no direct effect on osteoblast like Mg63 or Saos-2 cells or osteoclast resorption, measured by bone collagen release. Macrophage response was explored using U93 7 cell line. Expression of TNF-a and IL-1 , markers of inflammation, increased with liposome treatments compared to the negative control but decreased compared the positive control. The Mg63 cells given U937 supematants showed liposomes increased OPG production, but this was regardless of mineralisation. The calcium based mineral compounds were produced, characterised, successfully encapsulated using liposomes and functionalised to improve uptake at the bone site . This shows the potential to deliver calcium to the bone, however further work to inhibit inflammation and increase the calcium dose to elicit greater cell response is required before this approach can be developed as a treatment option.
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