The silver (0.5-3 at %) substituted nanosize hydroxyapatites (AgHAs) were synthesized by microwave processing. The X-ray diffraction (XRD) peaks are very broad, indicating that the AgHAs were of nanosize (30 nm). Transmission electron microscopy analysis shows needle-like morphology of AgHA, having length 60-70 nm and width 15-20 nm. The AgHA phase was stable up to 700 degrees C without any secondary phases. The antibacterial effect of AgHA against Escherichia coli and Staphylococcus aureus was observed by spread plate method, even for low concentration of silver ions (0.5%) with 1 x 10(5) cells/mL of respective bacterial culture, after a 48 h incubation period. However, some colonies of E. coli were seen with a high dose of 1 x 10(8) cells/mL after 24 h. The zone of inhibition by disc diffusion test method was found to vary with the amount of silver in the sintered AgHA pellets, for both the bacteria, after 24 h of inoculation. Osteoblast cell attachment in varying density was noticed on AgHA samples with 0.5, 1.0, and 1.5% silver substitution. However, osteoblast spreading was significantly greater on 0.5% AgHA compared to 1.0 or 1.5% substituted AgHA samples. Thus, the low amount of AgHA has a potential of minimizing the risk of bacterial contamination, without compromising the bioactivity, and is expected to display greater biological efficacy in terms of osseointegration.
Regeneration of fractured or diseased bones is the challenge faced by current technologies in tissue engineering. The major solid components of human bone consist of collagen and hydroxyapatite. Collagen (Col) and hydroxyapatite (HA) have potential in mimicking natural extracellular matrix and replacing diseased skeletal bones. More attention has been focused on HA because of its crystallographic structure similar to inorganic compound found in natural bone and extensively investigated due to its excellent biocompatibility, bioactivity and osteoconductivity properties. In the present study, electrospun nanofibrous scaffolds are fabricated with collagen (80 mg/ml) and Col/HA (1:1). The diameter of the collagen nanofibers is around 265 +/- 0.64 nm and Col/HA nanofibers are 293 +/- 1.45 nm. The crystalline HA (29 +/- 7.5 nm) loaded into the collagen nanofibers are embedded within nanofibrous matrix of the scaffolds. Osteoblasts cultured on both scaffolds and show insignificant level of proliferation but mineralization was significantly (p < 0.001) increased to 56% in Col/HA nanofibrous scaffolds compared to collagen. Energy dispersive X-ray analysis (EDX) spectroscopy results proved the presence of higher level of calcium and phosphorous in Col/HA nanocomposites than collagen nanofibrous scaffolds grown osteoblasts. The results of the present study suggested that the designed electrospun nanofibrous scaffold (Col/HA) have potential biomaterial for bone tissue engineering.
Nanofibres and nanocomposites are highly promising recent additions to materials in relation to tissue engineering. Mimicking the architecture of an extracellular matrix is one of the major challenges for tissue engineering. An operationally simple electrospinning technique was used to fabricate polycaprolactone/nanohydroxyapatite/collagen (PCL/nHA/Col) biocomposite nanofibrous scaffolds to provide mechanical support and to direct the growth of human fetal osteoblasts (hFOB) for tissue engineering of bone. Biocomposite nanofibres constructed with PCL, nHA and collagen type I combinations gave fibre diameters around 189 ± 0.026 to 579 ± 272 nm and pore sizes 2–35 µm. Resulting nanofibrous scaffolds were highly porous (>80%) structures and provided a sufficient open pore structure for cell occupancy whilst allowing free transport of nutrients and metabolic waste products; moreover, vascular in-growth was facilitated. The pore organization was determined by the deposition process, including interconnections of the fibre network. The mineralization was significantly increased (55%) in PCL/nHA/Col biocomposite nanofibrous scaffolds after 10 days of culture and appeared as minerals synthesized by osteoblast cells. The unique nanoscale biocomposite system had inherent surface functionalization for hFOB adhesion, migration, proliferation and mineralization to form a bone tissue for the regeneration of bone defects.
The eggshell waste has been value engineered to a nanocrystalline hydroxyapatite (HA) by microwave processing. To highlight the advantages of eggshell as calcium precursor in the synthesis of HA (OHA), synthetic calcium hydroxide was also used to form HA (SHA) following similar procedure and were compared with a commercially available pure HA (CHA). All the HAs were characterized by X-ray powder diffraction (XRD) method, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and specific surface area measurements. Nanocrystalline nature of OHA is revealed through characteristic broad peaks in XRD patterns, platelets of length 33-50 nm and width 8-14 nm in TEM micrograph and size calculations from specific surface area measurements. FT-IR spectra showed characteristic bands of HA and additionally peaks of carbonate ions. The cell parameter calculations suggest the formation of carbonated HA of B-type. The OHA exhibits superior sinterability in terms of hardness and density than both SHA and CHA may be due to larger surface area of its spherulite structure. The in vitro dissolution study shows longer stability in phosphate buffer and cell culture test using osteoblast cells establishes biocompatibility of OHA.
Xenogeneic bone procured from the slaughterhouse waste was deproteinated by heat treatment method intended for use as a bone substitute. The effect of heat treatment was investigated by thermal analysis and by physico-chemical methods such as X-ray powder diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy. The heat treatment temperatures for the bovine bone samples were predetermined by thermogravimetric (TG) analysis. The XRD results revealed that the process of heat treatment promoted the crystallinity of bone samples, particularly at 700 and 900°C. There was no secondary phase transformation detected for heat-deproteinated bone except the presence of the hydroxyapatite (HA) phase, which indicated its phase purity even at a higher temperature. The FTIR spectra of raw bone and bone heated at 300°C indicated the presence of organic macromolecules whereas these disappeared in the samples heated at 500, 700 and 900°C, which suggested the removal of antigenic organic matters around 500°C. The same results were also confirmed quantitatively by calculating the amount of collagen using hydroxyproline estimation. There was no significant change in the TG-thermogram of bone heated at 500, 700 and 900°C, which indicated their thermal stability. These findings implied that the heat treated bone at 500°C had properties similar to carbonated HA with low crystallinity, while 700 and 900°C samples had the same with higher crystallinity. As low temperature treatment does not alter morphological and structural properties, we propose that the 500°C heat treated xenogeneic bone may act as an excellent osteogenic bone substitute.
Strontium‐substituted apatites have provoked increased interest in recent years for their beneficial effects on osteoporotic bone treatment and replacement. In this study, rod‐ and acicular‐shaped, strontium‐substituted calcium deficient hydroxyapatite (CDHA) nanoparticles with (Ca + Sr)/P ratio of 1.61 were synthesized via accelerated microwave processing. The X‐ray powder diffraction analysis indicates the synthesized nanoparticles as apatite phase with diffraction patterns similar to those of hydroxyapatite. The hydrodynamic diameter of the particles were observed to be ~200–500 nm and found to increase with strontium substitution along with an increase in the negative zeta potential by dynamic light scattering method, suggesting the particles to be agglomerates in water. The morphology of the nanoparticles was studied using transmission electron microscopy (TEM), where, pure CDHA showed globular and strontium substituted CDHAs showed rod and acicular shape for 5% and 10% Sr substitution, respectively. The average size of the particles in TEM was measured to be 33 nm × 5 nm, 40 nm × 6 nm, and 55 nm × 8 nm (L × W) for pure and strontium‐substituted CDHAs, respectively. Inductively coupled plasma spectroscopy, energy dispersive X‐ray analysis, and Fourier transform infrared spectroscopy further confirm the substitution of strontium and deficiency of calcium in the synthesized nanoparticles. Thermal stability and in vitro solubility of CDHA nanoparticles were observed to increase with strontium substitution. The MTT [3‐(4, 5‐Dimethylthiazole‐2‐yl)‐2, 5‐diphenyl tetrazolium bromide] assay indicate that the substituted nanoparticles are non‐toxic to human periodontal ligament fibroblast (HPDLF) cells. Cell uptake study by fluorescence microscopy using rhodamine‐123 and actin/DAPI stained HPDLF cells show cellular localization of the nCDHA, nSr5CDHA, nSr10CDHA nanoparticles without any adverse effects. The strontium‐substituted CDHAs showed significant antimicrobial activity against Escherichia coli and Staphylococcus aureus bacteria by colony count method. The 10% Sr substituted CDHA show the maximum microbial reduction of around 56% for E. coli and 35% for S. aureus with 1 × 105 cells/mL of respective bacterial culture.
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