Hydroxyapatite nanoparticles for bone tissue regeneration: Synthesis and biocompatibility

Bone defects that exceed the critical-size threshold typically 1-2 cm in load-bearing sites cannot heal spontaneously and require scaffold-based intervention. Hydroxyapatite (HA, Ca₁₀(PO₄)₆(OH)₂), the principal inorganic constituent of bone, is an established bioceramic for this purpose, yet its performance depends critically on particle size, crystallinity, and surface area. This research reports the synthesis of HA nanoparticles (HA-NPs) by a wet chemical co-precipitation method, followed by systematic evaluation of the effect of calcination temperature (400-900°C) on physicochemical properties and biocompatibility. Calcium nitrate tetrahydrate and diammonium hydrogen phosphate served as precursors at a Ca/P molar ratio of 1.67, matching stoichiometric HA. Synthesised HA-NPs were characterised by XRD, FTIR, TEM, BET surface area analysis, and thermogravimetric analysis (TGA). XRD confirmed phase-pure HA formation at all calcination temperatures below 800°C, with biphasic HA/β-TCP formation observed at 900°C. Crystallite sizes increased from 18.3 nm (400°C) to 52.6 nm (900°C) with concomitant reduction in BET surface area from 87.4 to 24.3 m²/g. HA-NPs calcined at 600°C were selected as the optimal sample, balancing crystallinity and surface area. Biocompatibility was assessed using the MTT assay on MG-63 osteosarcoma cells and normal human osteoblasts (hOBs) over 24 and 72 h at concentrations of 25-400 µg/mL. Cell viability remained above 80% at concentrations up to 200 µg/mL for both cell lines at 72 h, confirming acceptable biocompatibility. Alkaline phosphatase activity and calcium deposition were elevated in hOBs exposed to HA-NPs at 50 µg/mL, indicating osteogenic stimulation. These results support the suitability of co-precipitation-derived HA-NPs for incorporation into bone tissue engineering scaffolds.

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Crystallite Size (nm) and BET surface area (m²/g): 400 vs 500 vs 600Close ×

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Introduction

Bone is one of the few human tissues capable of complete regeneration, but this capacity is finite: defects exceeding a critical size generally defined as 2-3 times the bone diameter will not spontaneously bridge and require clinical intervention [1] . Current gold-standard treatments, including autologous and allogeneic bone grafts, each carry significant drawbacks: autografts involve donor-site morbidity and limited volume, while allografts carry immunogenic and disease-transmission risks [2] . Synthetic calcium phosphate ceramics have long been explored as alternatives, with hydroxyapatite occupying a central position because of its chemical similarity to the mineral phase of bone and its well-documented osteoconductive properties. Natural bone mineral is a carbonated, calcium-deficient hydroxyapatite with crystallite dimensions of 20-40 nm and a high specific surface area that facilitates protein adsorption and cell attachment [3] . Nanoscale HA particles more closely mimic these characteristics than micron-sized HA powders and have been shown to enhance protein adsorption, osteoblast adhesion, and alkaline phosphatase activity compared with their coarser counterparts [4] . Synthesis routes for HA-NPs include hydrothermal processing, sol-gel, emulsion precipitation, and mechanochemical methods, each producing particles with different morphologies, crystallinities, and surface chemistries [5] . Co-precipitation is particularly attractive for scale-up because it is simple, low-cost, and produces particles in the 15-50 nm range when reaction conditions are controlled. The key Calcination serves to remove residual organic matter and improve crystallinity but also causes particle sintering and surface area reduction effects that can compromise biological performance [6] . Systematic optimisation of calcination conditions is therefore essential for producing HA-NPs whose physicochemical and biological properties meet scaffold design requirements. Despite extensive literature on HA synthesis, comparative data on the biocompatibility of HA-NPs calcined across a wide temperature range using both osteosarcoma cell lines and primary human osteoblasts are limited. The present research addresses this gap by characterising HA-NPs calcined at six temperatures and evaluating their cytotoxicity and osteogenic potential through MTT and alkaline phosphatase assays. In vitro protocol Osteogenic differentiation potential of the optimal HA-NP sample (600°C calcination) was assessed in hOBs over a 14day culture period. Cells were seeded in 24-well plates (5 × 10⁴/well) in osteogenic differentiation medium (DMEM supplemented with 10 nM dexamethasone, 50 µg/mL ascorbic acid, and 10 mM β-glycerophosphate) containing HA-NPs at 0, 25, and 50 µg/mL. Medium was replaced every 3 days. Alkaline phosphatase (ALP) activity was quantified on days 7 and 14 using pNPP as substrate: cell lysates were incubated with pNPP (4 mg/mL) in alkaline buffer for 30 min at 37°C; absorbance was read at 405 nm. ALP activity was normalised to total protein (Bradford assay) and expressed as nmol pNPP hydrolysed/min/mg protein. Mineralisation was assessed on day 21 by Alizarin red S staining: cultures were fixed in 10% neutral buffered formalin, stained with 2% Alizarin red S for 20 min, and the bound dye was extracted with 10% cetylpyridinium chloride for quantification at 562 nm. Cell attachment and morphology were examined by scanning electron microscopy (SEM, JEOL JSM-6400) on HA-NP-coated glass coverslips seeded with hOBs after 3 and 7 days.

Results

Table to 52.6 nm at 900°C, consistent with particle sintering at elevated temperatures. BET surface areas correspondingly decreased from 87.4 to 24.3 m²/g. FTIR spectra of all HA-NPs displayed characteristic phosphate ν₃ stretching bands near 1040 cm⁻¹ and bending bands at 600 and 562 cm⁻¹, as well as the hydroxyl stretching band at 3573 cm⁻¹ features absent in the β-TCP reference spectrum [7] . Cell viability remained above 80% in both MG-63 and hOB lines at concentrations up to 200 µg/mL after 72 h, indicating acceptable biocompatibility at therapeutically relevant concentrations. At 400 µg/mL, viability dropped to 73.4% (MG-63) and 76.2% (hOBs), below the ISO 10993-5 threshold of 80% for non-cytotoxicity, suggesting a concentration ceiling for safe use. Osteogenic markers showed a dose-dependent increase up to 50 µg/mL, with ALP activity peaking at 24.3 nmol/min/mg a 96% increase over control and Alizarin red absorbance reaching 0.37 at the same concentration. Both markers declined at 100 and 200 µg/mL, possibly due to partial inhibition of mineralisation kinetics at higher nanoparticle loads.

Discussion

The co-precipitation method yielded phase-pure HA across a calcination range of 400-800°C, confirming the reliability of the synthesis protocol. The systematic relationship between calcination temperature and crystallite size follows the well-established Ostwald ripening and sintering mechanisms: at temperatures above 600°C, surface diffusion rates accelerate, smaller particles dissolve and redeposit on larger ones, and grain boundaries fuse, resulting in larger crystallites and reduced surface area [8] . The onset of β-TCP formation at 900°C, reflected in the Ca/P ratio drop from 1.67 to 1.54, indicates incongruent decomposition that is likely driven by residual ammonium species volatilising and locally disturbing stoichiometry [9] . The 600°C sample was selected as optimal because it balances crystallinity (27.4 nm crystallite size, consistent with mature bone mineral) with adequate surface area (64.1 m²/g) for protein adsorption and cell interaction. Several literature reports confirm that HA calcined near 600°C shows superior osteoblast attachment relative to both lowertemperature (poorly crystalline) and higher-temperature (low-surface-area) samples [10] . The biocompatibility data obtained here are consistent with this assessment: both cell viability above 80% at 200 µg/mL and the peak osteogenic response at 50 µg/mL suggest a therapeutic window well within practical scaffold loading ranges. The 96% increase in ALP activity at 50 µg/mL HA-NPs compared with the undoped osteogenic medium control is noteworthy. ALP is an early osteogenic marker whose elevation indicates commitment of osteoblast precursors toward the mineralising phenotype. The corresponding increase in Alizarin red staining confirms that this commitment was followed by actual calcium deposition, the hallmark of mature osteoblastic function. Future work will need to establish whether similar osteogenic stimulation occurs in three-dimensional scaffold constructs under dynamic perfusion culture conditions that better simulate in vivo bone physiology [11] .

A practical limitation of the present research is the use of an osteosarcoma cell line (MG-63) alongside primary hOBs. While MG-63 cells are widely used as a convenient screening model, their proliferative and metabolic behaviour differ substantially from primary cells. The close agreement between MG-63 and hOB viability responses (within 3-4 percentage points across all concentrations) is reassuring, but confirmatory in vivo research in rodent critical-size defect models remains necessary before clinical translation can be contemplated.

Conclusion

Hydroxyapatite nanoparticles were successfully synthesised by aqueous co-precipitation and characterised as a function of calcination temperature from 400 to 900°C. Phase-pure HA with increasing crystallinity and decreasing surface area was obtained up to 800°C; biphasic HA/β-TCP appeared at 900°C. HA-NPs calcined at 600°C presented the best balance of crystallite size (27.4 nm), surface area (64.1 m²/g), and stoichiometric Ca/P ratio (1.66). Biocompatibility assessment by MTT assay confirmed cell viability above 80% at concentrations up to 200 µg/mL in both MG-63 cells and primary human osteoblasts. Osteogenic assays revealed a significant increase in ALP activity and mineralisation at 50 µg/mL, indicating that the HA-NPs actively stimulate bone-forming activity rather than merely being tolerated passively. These findings provide a solid physicochemical and biological foundation for incorporating these HA-NPs into polymer-ceramic composite scaffolds for critical-size bone defect repair, which will be pursued in subsequent research phases.

Figures and tables from the paper

Calc. temp (°C)	Phase (XRD)	Crystallite Size (nm)	BET surface area (m²/g)	Ca/P ratio (EDX)	Major FTIR bands (cm⁻¹)
400	Phase-pure HA	18.3 ± 0.9	87.4 ± 3.2	1.64	1036, 603, 566, 3571
500	Phase-pure HA	21.7 ± 1.1	78.3 ± 2.8	1.65	1040, 602, 565, 3573
600	Phase-pure HA	27.4 ± 1.3	64.1 ± 2.5	1.66	1043, 600, 562, 3574

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Physicochemical properties of HA-NPs as a function of calcination temperature

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Concentration (µg/mL)	MG-63 viability (%)	hOB viability (%)	ALP activity (nmol/min/mg)*	Alizarin red (OD₅₆₂)*
Control (0)	100.0 ± 1.3	100.0 ± 1.1	12.4 ± 0.9	0.18 ± 0.02
25	97.3 ± 1.6	98.1 ± 1.4	16.8 ± 1.2	0.24 ± 0.03
50	94.7 ± 2.1	95.8 ± 1.7	24.3 ± 1.6	0.37 ± 0.04
100	91.2 ± 2.4	92.4 ± 2.2	22.1 ± 1.4	0.31 ± 0.03
200	84.6 ± 3.1	86.7 ± 2.8	18.7 ± 1.3	0.26 ± 0.03
400	73.4 ± 3.8	76.2 ± 3.4	14.1 ± 1.1	0.19 ± 0.02

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Biocompatibility data: MG-63 and hOB cell viability (%) after 72 h exposure to HA-NPs (600°C)

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Effect of calcination temperature on crystallite size (left) and BET surface area (right) of synthesised hydroxyapatite nanoparticles

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Box plots showing MG-63 cell viability (%) after 24 h exposure to HA-NPs calcined at 600°C at concentrations of 0-400 µg/mL

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