Nano-particles of calcium sulfate as a ceramic scaffold for delivery of growth factors to enhance bone regeneration
Park, Young Bum
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The goal of these studies was to develop and evaluate biocompatible nano scale calcium sulfate (nCS) and propylene glycol alginate (PGA) based composite scaffolds as a third generation biomaterial device to deliver growth factors for bone and periodontal tissue regeneration. De novo nCS or nCS and PGA composite with growth factors may enhance bone and periodontal tissue regeneration within the normal healing period of tissue and prohibit the retardation of regeneration of tissue due to slow degradation of remnants of grafted materials. The cryo-vacuum method was used to process dihydrate CS into dihydrate nCS, which was then subjected to oven drying to produce hemihydrate. Then the nCS was sterilized by glow discharge treatment (GDT) to make it available for use as a sterilized synthetic graft material for the treatment of bone defects or periodontal defects to induce bone and periodontal tissue regeneration. SEM and TEM images of nCS dihydrate powder showed that the powder consisted of aggregates of closely arranged acicular crystals linked together to form a narrow network. These acicular crystals were approximately 20 - 50 nm in width, 400 - 600 nm in length and approximately 80-100 times smaller than conventional calcium sulfate. SEM images of nCS β-hemihydrate showed that the powder contained multiple cleavage fragments of rod-shaped crystals that were approximately 30-60 nm in width and closely arranged and packed to form aggregates. EDS spectra confirmed the presence of calcium (Ca) and Sulfur (S) within the nCS samples. FTIR and XRD analyses also confirmed the presence of nCS dihydrate, and nCS β-hemihydrate. TGA revealed that the proper temperature to make the hemihydrate form of nCS was at about 125° ~ 145°C. BET results showed that the surface area of the nCS was about 10 times greater than that of the conventional micron-sized form. In addition, Surface microhardness testing showed that the nCS was stronger than conventional CS while the MTT results confirmed the safety and biocompatibility of nCS. The results showed the nCS is safe and biocompatible. Nanotechnology offers exciting alternatives to traditional scaffolds. However, too small nano-scale particles allow only nano-scale pores. It cannot provide space for cell migration and vascularization. Space for cells and blood vessels is one of the most important elements of scaffolds. Addition of macro pores using alginate should not only enhance cell migration, it should also help vascularization for oxygen and nutrient delivery, waste removal, protein transport. Initial SEM micrographs after 12 hours show that HPDL and HOB cells do attach to nano calcium sulfate scaffolds. After 3 days, cells exhibited a spindle shape or polygonal shape and the pseudopodia of cells became far longer and extended in different directions. The MTT assay results after 3 days culture confirmed that PDL and HOB cells do adhere to nano calcium sulfate samples and proliferate. The MTT assay revealed that CAPSET® and nano calcium sulfate scaffolds exhibited similar levels of cell proliferation. Comparison of the MTT assay with and without centrifuge showed that nano calcium sulfate undergoes a faster structural deformation, compared to that of CAPSET®. SEM micrographs of different proportions of alginate and nano calcium sulfate show cells attached to 95:5 and 90:10. Cell proliferation rate was found to be highest for the 95:5 scaffolds. Histological analysis and degradation rate of Ca-45 showed that nCS and PGA 10% group almost completely degrade within 4 week and had macropores in situ. Nano sized calcium sulfate and PGA composite scaffolds may provide a more efficient scaffold than nano sized calcium sulfate alone or standard sized calcium sulfate (CAPSET®) scaffolds for bone and periodontal tissue engineering. The critical animal in vivo models were used to evaluate the de novo nCS and nCA+PGA composite effects on the bone and periodontal tissue regeneration. Results showed that nCS or nCS composite with BMP-2 has the best effect on bone formation within short healing period without delay of regeneration of bone tissue. nCS or nCS+PGA with PDGF revealed that there were more mineralized tissues formed, however it is not significant. In conclusion, nCS without growth factors may inhibit cell migration and vascularization due to nano-scale too small pores. Therefore, macropores formation in periodontal tissue using PGA may be considered to enhance periodontal tissue regeneration. In bone Tissue regeneration, BMP-2 such a very strong bone inducing growth factors can be used with nCS with or without small amount of PGA.