Toxicity of Natural Hydroxyapatite

Abstract

Hydroxyapatite (HA) has been extensively researched in bone regeneration procedures for its close similarity with natural bone in composition and also due to its osteoconductive and bone healing properties. Natural hydroxyapatite (NHA) is dissimilar to its synthetic counterpart. It has a slight difference in the calcium phosphate ratio and contains carbonate groups and some trace elements, which makes it a more viable material as a substitute for bone. Biowaste is a huge environmental concern. NHA is generated from biowaste of mostly poultry and marine origin. Hence, its proven biocompatibility would advocate the translation of this knowledge to clinical practice for bone regenerative procedures. In vitro biocompatibility of NHA from various sources has been reported. Also, in vivo studies, including implantation studies, have been carried out to certify the biological safety of NHA. Various authors have stated that the preparation technique (which influences features of NHA), degradation characteristics, and resulting tissue response of NHA are also satisfactory. This chapter elaborates on the toxicity assessment in vitro, and in vivo and hence the biocompatibility of NHA obtained from various sources.

1. Introduction

Hydroxyapatite (HA) resembles bone minerals in terms of bioactivity, mechanical characteristics, and composition; hence, it is frequently employed as a bone substitute [1, 2]. Recently, hydroxyapatite nanoparticles have been used for the purpose of drug delivery, where they serve as vehicles for pharmaceutical molecules, bioimaging molecules, and other therapeutic agents. It is asserted that these particle drug delivery methods have improved bioavailability, predictable treatment outcomes, higher efficacy and safety, and the potential for controlled release.

Although NHA is demonstrating considerable promise in the biomedical field, careful consideration of its possible toxicity is necessary before it can be considered a viable option for medical application in the broader human population [3, 4].

2. Safety evaluation of NHA

The safety evaluation of NHA includes reports on its in vitro response in cell-culture followed by in vivo response to short term and long term presence of NHA in the various animal models [1, 3, 4, 5]. The sequence of toxicity evaluation tests for NHA reported in literature has been represented in Figure 1.

Figure 1.

The sequence of toxicity evaluation tests carried out for NHA reported in literature.

2.1 In vitro toxicity evaluation of NHA

2.1.1 Cytotoxicity

It is crucial to assess in vitro cytotoxicity of medical devices to understand their impact on cell functions. Various authors have studied cytotoxicity of NHA in vitro prior to its applications in bone regeneration [3, 4, 5]. MTT assay (based on cell culture) is considered as the most preferred methods to carry out in cytotoxicity assessment [5]. Only the viable cells will have mitochondrial succinate dehydrogenase to convert the MTT to formazan crystals. Various cell lines, preferably osteoblast based are utilized to carry out MTT assay to clearly identify response of NHA on osteoblast cell lineage. DNA quantification assay is also carried out to analyze the DNA changes in these cells if they correspond with healthy cells.

Oyster (Crassostrea angulate) shells based on their microporous scaffold-like form have been used to create NHA scaffolds. Osteoblast lineage cells when seeded in these scaffolds followed by their proliferation was compared to synthetic HA scaffolds using MTT assay. NHA scaffolds yielded better results than synthetic HA scaffolds [4].

NHA was extracted using fishbone of Tuna (Thunnus thynnus) and from sword fish (Xiphias gladius) [3]. The frozen bones were cleaned, dried, calcined (at 600 and 950°C) and milled. Both the NHA extracts were incorporated in mouse calvarial cell culture. Following MTT assay was found to be nontoxic for 100% extract concentration.

Osteoblast (MG63) and fibroblast (NIH3T3) cells were cultured in Mercenaria mercenaria seashells extract. Following MTT assay was found to be noncytotoxic [6].

Shamsuria [7] assessed the in vitro cytotoxicity of natural corals in human osteoblast cells following extraction in complete culture medium. After a 72-h incubation time, the viability of the osteoblast CRL-1543 was evaluated and compared to a negative control using the neutral red assay using a spectrophotometer set at 540 nm. The outcomes demonstrated that the materials were not cytotoxic. Following 72 h of incubation, hydroxyapatite revealed 123% viable cells, Natural Coral revealed 99.43%, and Polyhydroxybutarate revealed 176.75%. The cytotoxicity test in this investigation primarily focused on the chemicals that leached out of the biomaterial. The outcomes demonstrated the materials’ lack of toxicity and their ability to support biofunctional cell growth.

Prado et al. [8] evaluated NHA powders using fish waste (Micropogonias furnieri). It was incubated (0.05 g/ml concentration) in osteoblast cell lines placed in αMEM culture medium with 10% fetal bovine serum and 1% antibiotic. In vitro cytotoxicity was analyzed based on its incorporation in the cells using Alamar Blue assay. Pre-osteoblast cells on 3rd and 5th day demonstrated reduced cytotoxicity. Also DNA quantification revealed reduced concentration and quantitative RT-PCR did not reveal any significant difference between the control and test group.

Panda et al. [9] prepared NHA powder obtained from fish scales (fresh water fish-Labeo rohita and Catla catla). MTT assay was carried out using Mesenchymal stem cells derived from cord blood. Cell viability was found to be enhanced on the 5th day. DNA quantification assay revealed increase in DNA content.

Lee et al. [10] fabricated chitosan based micro- and nano-hydroxyapaptite as scaffolds for bone tissue engineering. In vitro cell viability and cell proliferation assay on osteoblast cell lines revealed the NHA to be biocompatible and promote cell growth.

Venkatesan et al. [11] isolated NHA from salmon fish bone. Cytotoxicity was assessed in cultured MSCs using MTT assay. Up to 100 μg/ml NHA was found to noncytotoxic to MSCs. Cell interactions observed using optical microscopy revealed no changes in MSCS incorporated with NHA.

Pon-on et al. [12] synthesized calcium phosphate of HA powder from the fish scales of Probarbus jullieni. Fish scale derived NHA was found to be biocompatible with osteoblast cell lines. The cells exhibited greater adhesion and spreadability on these fish scale-based NHA as compared to synthetic HA.

Shi et al. 2018 used mouse preosteoblast MC3T3-E1 in an alkaline phosphatase activity investigation and the MTT cell viability assay to assess the cytocompatibility of calcined nHAP [13]. According to the results of a cell experiment, nHAP derived from rainbow trout and salmon bones had superior biological compatibility than nHAP derived from cod bone and chemically synthesized HAP (cHAP). The inclusion of CO3²⁻ and Mg²⁺ in the nHAP generated from rainbow trout and salmon bones is most likely to blame for this difference in element composition. As a result, the natural waste fish bone product (nHAP) derived from rainbow trout and salmon bones has a promising possibility for utilization as an alternative for biomaterial in bone tissue engineering and may be employed as hydroxyapatite.

2.1.2 Hemocompatibility test

Lu [14] carried out hemocompatibility test for nHA-chitosan composite in rabbit blood diluted with saline solution and assessed using spectrophotometer. It revealed good hemocompatibility.

2.2 In vivo toxicity evaluation of NHA

2.2.1 Implantation test

The most crucial factor in determining biocompatibility of a biomaterial is its response locally and systemically once implanted in in vivo models [1, 5]. It needs to be assessed for integration, or biodegradation within the animal models (ISO 10993-6, 2016) [1].

In vivo study on rat calvarial defects revealed chronic inflammatory infiltrate in response to nHA (from marine biowaste generated from Micropogonias furnieri), after the 7th and 15th day which was absent after 30 days of the implantation [8].

Strombus gigas (conch) shells and Tridacna gigas (Giant clam) shells were converted to NHA bone implants using hydrothermal method [15]. This was placed in femoral defects in rats to assess whether they can be used as block grafts for load-bearing areas. The study revealed good biocompatibility with definitive osseointegration for bulk shell derived NHA.

NHA derived from dolphin (Neophocaena asiaeorientalis) back bone was combined with poly-l-lactic acid (PLA) and compared with standard PLA [16]. Both were subcutaneously implanted to assess toxicity in vivo. There was no severe immunological response to this NHA composite in the subcutaneous tissues of the Sprague-Dawley rats.

Nandi et al synthesized NHA scaffolds from sea corals [17]. The in vivo tests were done in tibial defects in New Zealand white rabbits. Eighteen rabbits were divided into three groups. In the first group only coralline NHA implant was placed. In the second group insulin growth factor-1 was incorporated into the coralline NHA implant prior to placement. In the third group bone morphogenetic protein-2 was incorporated into the coralline NHA implants. After 90 days of implantation bone histology slides revealed healthy bone formation in all the groups however the quality of bone was the best in group two.

2.2.2 Acute systemic toxicity

Lu et al. [14] utilized hydroxyapatite from pig bones and combined with chitosan to form a composite bone graft material. The acute systemic toxicity was assessed in 30 Kumming rats after injecting the extract of the composite material. The mice in the experimental group did not show any signs of toxicity.

Lee [16] assessed a composite of NHA derived from dolphin backbone with PLA in Sprague-Dawley rats. After eight months systemic toxicity was assessed based on response in the liver and kidneys to the NHA based implant. There was no significant difference in the hepato- and nephro-toxicity indicators between the test and control groups after eight months.

2.2.3 Genotoxicity

Genotoxicity involves assessment of any mutations or chromosomal abnormalities in response to the medical device being tested [1, 5]. Genotoxicity testing is usually not carried out if the chemical characterization of extracts from the device has been already carried out. Also, if there is sufficient literature stating that the components of the device have been tested for genotoxicity these tests are not repeated for the device [1].

Yamamura et al. [18] synthesized hydroxyapatite from Whitemouth croaker (Micropogonias furnieri) fish waste. They carried out genotoxicity assessment after 30 days in the vital organs in Wistar rats following subcutaneous implantation of nHA in of the animals. NHA obtained was found to have no cytotoxicity and genotoxicity.

There are no reports till date on intracutaneous reactivity and skin sensitization assay.

2.2.4 Pyrogenicity test

Lu [14] examined the pyrogenicity of nHA-Chitosan composite in New Zealand rabbits. Rectal temperature was recorded ten minutes after injecting the extract in the ear vein. No significant variation in the body temperature of the rabbit was found.

2.2.5 Carcinogenicity assessment

Lu [14] assessed teratogenicity/mutagenicity of nHA-Chitosan using rabbit calvarial defect models. The graft material was placed in the defects and these were reassessed after six months and no genetic changes were found.

2.3 Performance and efficacy evaluation

Implantation test is crucial to determine the performance and efficacy of the medical device [1]. Biocompatibility and efficacy of the bone grafts can be evaluated simultaneously through critical size defects in animal models ISO10993-6 (2016) (FDA, 2016) [1]. As per the ISO guidelines these tests need to be carried out in identical test and control groups with minimum 10 implantation sites.

Similarly Prado et al. [8] evaluated the biological performance and biocompatibility of NHA simultaneously in rat calvarial defects. Runx-2 expression following immunohistochemistry was detected after 7th, 15th and 30th day indicative that the nHA might be osteoinductive in nature.

Oryan et al [19] evaluated salmon fish bone and demineralized bone matrix implanted in the radial bone defect model (murine study). Radiographic, histopathological and biochemical evaluation was found to be favorable. There was evidence of new bone formation on routine histopathological examination on 35th and 56th day postoperatively.

3. Toxicology aspects in major applications of natural hydroxyapatite

3.1 In membrane protein interactions

Xu et al. [20] studied the interaction of nanoscale hydroxyapatite with cytochrome c, a heme protein in the inner mitochondrial membrane, and hemoglobin, an iron-containing metalloprotein, in zebrafish embryonic development. Experimental results showed that the interaction was formed by intramolecular charge and hydrogen bond interactions. The two functional proteins are cross-linked by charge and hydrogen bond interactions between hydroxyapatite particles aggregated colloidally. Therefore, this study found hydroxyapatite can accumulate in larger particles around membrane proteins and is toxic to zebrafish embryo development.

3.2 In dentine surface coating

Nano-sized hydroxyapatite can be used as a coating material for the remineralization of caries dentin specimens [21]. This study found that dentin specimens could be effectively coated with hydroxyapatite and there was no evidence of toxicity. In addition to its nontoxic effects, hydroxyapatite also actively contributed to the viability of L929 fibroblasts and showed antibacterial effects against certain bacteria responsible for caries.

3.3 In bone marrow stem cells

Remya et al. [22], has studied the molecular toxicity of the hydroxyapatite nanoparticles using mouse bone marrow mesenchymal stem cells. The MTT assay showed that hydroxyapatite did not cause any kind of cytotoxicity up to 800 μg/ml. Moreover, when the oxidative stress induced apoptosis and the levels of reactive oxygen species generation were studied, it was giving a result which was not significantly different from the control group, which concludes the nontoxic or safe nature of hydroxy apatite especially in mesenchymal stem cells.

3.4 In bone tissue engineering scaffolds

Paras et al., 2020 performed a toxicological assessment of highly porous nanohydroxyapatite-based scaffolds [23]. In vitro genotoxicity was measured using the Comet assay and evaluated for systemic subchronic toxicity by oral administration of nano calcium hydroxyapatite covered with a small scaffold layer for 120 days. These nanohydroxyapatite-based scaffolds had minimal risk, as evidenced by genotoxicity and systemic toxicity studies. No genotoxic effects were seen even at high concentrations of 50 mg nanohydroxyapatite scaffolds. In addition, daily oral administration of nanohydroxyapatite-based scaffolds at high concentrations over extended periods did not induce significant adverse changes in the internal organs of the test animals. This subacute, chronic toxicity study shows that this may be a promising advance in mimicking natural bone in terms of structural and mechanical properties.

3.5 Intravenously as a drug vehicle

In a study conducted by Liu et al. 2005 [24], hydroxyapatite solutions were injected intravenously into rabbits at different concentrations, and the response and viability of the rabbits were observed to study the effect of nanohydroxyapatite on living organs. Nanohydroxyapatite had no cumulative toxicity to rabbits and is considered to be safe. It can be intravenously administered with hydroxyapatite as a drug carrier at a low dose lower than the average lethal dose.

3.6 In bone grafting applications

Several studies demonstrated the ability of hydroxyapatite to generate a positive environment for facilitating new bone tissue growth and regeneration. It results in perfect incorporation of the graft without undergoing any development of severe immune response. Calcium hydroxyapatite-based bioceramics show excellent biocompatibility, corrosion resistance, and excellent compressive strength, making them good candidates for implants.

Natural hydroxyapatite is the most successful in bone graft applications due to its low toxicity and resemblance to the mineral bone, with properties that promote osseointegration and new bone formation processes. Rincón-López et al. 2018; showed that alternative allogeneic transplant materials derived from naturally occurring heterologous hydroxyapatite have been developed [25]. Heterologous bone graft materials can be used as fully crystalline, naturally porous, bovine-derived hydroxyapatite (all collagen protein removed) in a particle size range of 250–450 μm. Organic matter is previously been removed, but the bone microstructure is preserved. Hydroxyapatite has been used in a variety of biomedical applications because it is synthetically or naturally produced and has the ability to form a bone-like apatite layer primarily at the interface between bone tissues.

4. Conclusion

Various studies support the biocompatibility of natural hydroxyapatite and have concluded that it is appropriate for bone substitution. The biological waste fish bone products is suitable for the production of hydroxyapatite as part of bio-waste treatment, and the nHAP derived using rainbow trout and salmon remains has significant promise for usage as an implant product alternative in tissue engineering of bones. Bone tissue engineering has come up as a novel field in regenerative medicine and biomaterials have three-dimensional essential functions of cell adhesion, diffusion, proliferation, differentiation, and tissue formation. The application of hydroxyapatite nanoparticles in the biopolymer matrix enhances the mechanical strength and nano topographical characteristics which resembles the nanostructures of natural bone. Based on the reported studies, the integration of beneficial effects of natural absorbent polymers and nanoscale sized bioactive ceramic components has been found to be important for usage in bone regeneration. Natural hydroxyapatite is known for its biocompatibility, and bioactivity which is called as the ability to form direct chemical bonds with surrounding tissues, osteoconductive, nontoxicity, noninflammatory and nonimmunogenic properties. Therefore, natural hydroxyapatite is one of the ideal materials for bone tissue engineering due to its biocompatibility and mechanical strength.

Conflict of interest

The authors declare no conflict of interest.

Appendix and nomenclature

NHA	natural hydroxyapatite