Compendium of the most significant studies with reference to properties, intake, effects.

George SM, Nayak C, Singh I, Balani K. Multifunctional Hydroxyapatite Composites for Orthopedic Applications: A Review. ACS Biomater Sci Eng. 2022 Aug 8;8(8):3162-3186. doi: 10.1021/acsbiomaterials.2c00140. 

Abstract. Being a bioactive material, hydroxyapatite (HAp) is regarded as one of the most attractive ceramic biomaterials for bone and hard-tissue replacement and regeneration. Despite its substantial biocompatibility, osteoconductivity, and compositional similarity to that of bone, the employment of HAp is still limited in orthopedic applications due to its poor mechanical (low fracture toughness and bending strength) and antibacterial properties. These significant challenges lead to the notion of developing novel HAp-based composites via different fabrication routes. HAp, when efficaciously combined with functionally graded materials and antibacterial agents, like Ag, ZnO, Co, etc., form composites that render remarkable crack resistance and toughening, as well as enhance its bactericidal efficacy. The addition of different materials and a fabrication method, like 3D printing, greatly influence the porosity of the structure and, in turn, control cell adhesion, thereby enabling biological fixation of the material. This article encompasses an elaborate discussion on different multifunctional HAp composites developed for orthopedic applications with particular emphasis on the incorporation of functionally graded materials and antibacterial agents. The influence of 3D printing on the fabrication of HAp-based scaffolds, and the different in vitro and in vivo studies conducted on these, have all been included here. Furthermore, the present review not only provides insights and broad understanding by elucidating recent advancements toward 4D printing but also directs the reader to future research directions in design and application of HAp-based composite coatings and scaffolds.

Šupová, M. (2015). Substituted hydroxyapatites for biomedical applications: A review. Ceramics international, 41(8), 9203-9231.

Abstract. This review summarizes recent and very recent work on preparing substituted hydroxyapatites. Ease of atomic doping or substitution in apatite opens this mineral up for a wide range of biomedical applications. It can be used for repairing and replacing diseased and damaged parts of musculoskeletal systems, and also as a drug or gene delivery agent, as a bioactive coating on metallic osseous implants, biomagnetic particles and fluorescent markers. First, the physicochemical properties of bioapatites are described and discussed. Then a general summary on substitution reaction for hydroxyapatite is made. Special attention is paid to describing anionic, cationic and multisubstituted hydroxyapatites used for various biomedical applications. Finally, conclusions are drawn and future perspectives are discussed.

Fiume E, Magnaterra G, Rahdar A, Verné E, Baino F. Hydroxyapatite for Biomedical Applications: A Short Overview. Ceramics. 2021; 4(4):542-563. https://doi.org/10.3390/ceramics4040039

Abstract. Calcium phosphates (CaPs) are biocompatible and biodegradable materials showing a great promise in bone regeneration as good alternative to the use of auto- and allografts to guide and support tissue regeneration in critically-sized bone defects. This can be certainly attributed to their similarity to the mineral phase of natural bone. Among CaPs, hydroxyapatite (HA) deserves a special attention as it, actually is the main inorganic component of bone tissue. This review offers a comprehensive overview of past and current trends in the use of HA as grafting material, with a focus on manufacturing strategies and their effect on the mechanical properties of the final products. Recent advances in materials processing allowed the production of HA-based grafts in different forms, thus meeting the requirements for a range of clinical applications and achieving enthusiastic results both in vitro and in vivo. Furthermore, the growing interest in the optimization of three-dimensional (3D) porous grafts, mimicking the trabecular architecture of human bone, has opened up new challenges in the development of bone-like scaffolds showing suitable mechanical performances for potential use in load bearing anatomical sites.

Mestres G, Espanol M, Xia W, Persson C, Ginebra MP, Ott MK. Inflammatory response to nano- and microstructured hydroxyapatite. PLoS One. 2015 Apr 2;10(3):e0120381. doi: 10.1371/journal.pone.0120381.

Abstract. The proliferation and activation of leukocytes upon contact with a biomaterial play a crucial role in the degree of inflammatory response, which may then determine the clinical failure or success of an implanted biomaterial. The aim of this study was to evaluate whether nano- and microstructured biomimetic hydroxyapatite substrates can influence the growth and activation of macrophage-like cells. Hydroxyapatite substrates with different crystal morphologies consisting of an entangled network of plate-like and needle-like crystals were evaluated. Macrophage proliferation was evaluated on the material surface (direct contact) and also in extracts i.e. media modified by the material (indirect contact). Additionally, the effect of supplementing the extracts with calcium ions and/or proteins was investigated. Macrophage activation on the substrates was evaluated by quantifying the release of reactive oxygen species and by morphological observations. The results showed that differences in the substrate’s microstructure play a major role in the activation of macrophages as there was a higher release of reactive oxygen species after culturing the macrophages on plate-like crystals substrates compared to the almost non-existent release on needle-like substrates. However, the difference in macrophage proliferation was ascribed to different ionic exchanges and protein adsorption/retention from the substrates rather than to the texture of materials.

Montone, A. M. I., Papaianni, M., Malvano, F., Capuano, F., Capparelli, R., & Albanese, D. (2021). Lactoferrin, quercetin, and hydroxyapatite act synergistically against Pseudomonas fluorescens. International Journal of Molecular Sciences, 22(17), 9247.

Abstract. Pseudomonas fluorescens is an opportunistic, psychotropic pathogen that can live in different environments, such as plant, soil, or water surfaces, and it is associated with food spoilage. Bioactive compounds can be used as antimicrobials and can be added into packaging systems. Quercetin and lactoferrin are the best candidates for the development of a complex of the two molecules absorbed on bio combability structure as hydroxyapatite. The minimum inhibiting concentration (MIC) of single components and of the complex dropped down the single MIC value against Pseudomonas fluorescens. Characterization analysis of the complex was performed by means SEM and zeta-potential analysis. Then, the synergistic activity (Csyn) of single components and the complex was calculated. Finally, the synergistic activity was confirmed, testing in vitro its anti-inflammatory activity on U937 macrophage-like human cell line. In conclusion, the peculiarity of our study consists of optimizing the specific propriety of each component: the affinity of lactoferrin for LPS; that of quercetin for the bacterial membrane. These proprieties make the complex a good candidate in food industry as antimicrobial compounds, and as functional food.

Venkatesan J, Kim SK. Nano-hydroxyapatite composite biomaterials for bone tissue engineering--a review. J Biomed Nanotechnol. 2014 Oct;10(10):3124-40. doi: 10.1166/jbn.2014.1893.

Abstract. In recent years, significant development has been achieved in the construction of artificial bone with ceramics, polymers and metals. Nano-hydroxyapatite (nHA) is widely used bioceramic material for bone graft substitute owing to its biocompatibility and osteoconductive properties. nHA with chitin, chitosan, collagen, gelatin, fibrin, polylactic acid, polycaprolactone, poly(lactic-co-glycolic) acid, polyamide, polyvinyl alcohol, polyurethane and polyhydroxybutyrate based composite scaffolds have been explored in the present review for bone graft substitute. This article further reviews the preparative methods, chemical interaction, biocompatibiity, biodegradation, alkaline phosphatase activity, mineralization effect, mechanical properties and delivery of nHA-based nanocomposites for bone tissue regeneration. The nHA based composite biomaterials proved to be promising biomaterials for bone tissue engineering.

Satou R, Iwasaki M, Kamijo H, Sugihara N. Improved Enamel Acid Resistance Using Biocompatible Nano-Hydroxyapatite Coating Method. Materials (Basel). 2022 Oct 14;15(20):7171. doi: 10.3390/ma15207171.

Abstract. In this study, we attempted to develop a dental caries prevention method using a bioapatite (BioHap), an eggshell-derived apatite with nanoparticle size and biocompatibility, with a high-concentration fluoride tooth surface application method. The enamel acid resistance after the application of the proposed method was compared with that of a conventional topical application of fluoride using bovine tooth enamel as an example. The tooth samples were divided into three groups based on the preventive treatment applied, and an acid challenge was performed. The samples were evaluated for acid resistance using qualitative and quantitative analytical methods. The BioHap group demonstrated reduced enamel loss and improved micro-Vickers hardness, along with a thick coating layer, decreased reaction area depth, and decreased mineral loss value and lesion depth. The combination of BioHap with high-concentration fluoride led to the formation of a thick coating layer on the enamel surface and better suppression of demineralization than the conventional method, both qualitatively and quantitatively. The proposed biocompatible nano-hydroxyapatite coating method is expected to become a new standard for providing professional care to prevent dental caries.

Coelho, C.C., Grenho, L., Gomes, P.S. et al. Nano-hydroxyapatite in oral care cosmetics: characterization and cytotoxicity assessment. Sci Rep 9, 11050 (2019). https://doi.org/10.1038/s41598-019-47491-z

Abstract. Nano-hydroxyapatite has been used as an oral care ingredient, being incorporated in several products for the treatment of dental hypersensitivity and enamel remineralisation. Despite its promising results, regulatory and safety concerns have been discussed and questioned by the European Scientific Committee on Consumer Safety (SCCS) regarding the usage of hydroxyapatite nanoparticles in oral care products. In this work, a commercially available nano-hydroxyapatite was characterized and its cytocompatibility towards human gingival fibroblasts was evaluated, as well as its irritation potential using the in vitro HET-CAM assay. All the conditions chosen in this study tried to simulate the tooth brushing procedure and the hydroxyapatite nanoparticles levels normally incorporated in oral care products. The commercial hydroxyapatite nanoparticles used in this study exhibited a rod-like morphology and the expected chemical and phase composition. The set of in vitro cytotoxicity parameters accessed showed that these nanoparticles are highly cytocompatible towards human gingival fibroblasts. Additionally, these nanoparticles did not possess any irritation potential on HET-CAM assay. This study clarifies the issues raised by SCCS and it concludes that this specific nano-hydroxyapatite is cytocompatible, as these nanoparticles did not alter the normal behaviour of the cells. Therefore, they are safe to be used in oral care products.

Ramis, J. M., Coelho, C. C., Córdoba, A., Quadros, P. A., & Monjo, M. (2018). Safety assessment of nano-hydroxyapatite as an oral care ingredient according to the EU cosmetics regulation. Cosmetics, 5(3), 53.

Abstract. Hydroxyapatite nanoparticles (HAP-NP) are incorporated in oral care products such as toothpastes and mouthwashes to treat dental sensitivity or to promote enamel remineralisation. Despite the good performance of HAP-NP in this application, it is important to ensure its safety for consumers. For that reason, the Scientific Committee on Consumer Safety (SCCS) evaluated the safety of HAP-NP as an oral care ingredient, but the issued opinion was not completely conclusive and the SCCS recommended that additional tests should be performed. Here, we used a commercially available human gingival epithelium (HGE) as a non-animal alternative and MTT cell viability, LDH activity, and IL-1alpha production were evaluated after 3.1% HAP-NP treatment for 10 min, 1 h, and 3 h. Moreover, the absorption of HAP-NP in the gingival tissue was assessed by transmission electron microscopy (TEM) analysis. Finally, the dissolution behaviour of HAP-NP in simulated gastric fluid was also investigated. No deleterious effect was observed for HGE tissues incubated with HAP-NP for all time-points and parameters evaluated. Moreover, a complete dissolution of 3.1% HAP-NP in simulated gastric fluid was observed after 7.5 min at 37 °C. In conclusion, our results evidence the safety of HAP-NP for oral care products with the use of an in vitro replacement alternative for human gingival epithelium and a simulated gastric fluid assay

Hydroxyapatite is a mineral consisting mainly of calcium is present in the human body and milk in good quantities and is found in bones and teeth as a significant inorganic component where it acts as a shield against caries. In a process using ethanol and water, calcium apatite Ca₅(PO₄)₃(OH) is mineralised to obtain a very fine powder consisting of nano particles.

Chemical Composition and Structure

Hydroxyapatite has the chemical formula Ca₅HO₁₃P₃, with a crystalline structure that provides high stability and biological compatibility. Its composition is similar to that of human mineral tissue, making it ideal for applications that involve strengthening bones and teeth.

Physical Properties

It appears as a white powder, insoluble in water, with a highly compact crystalline structure. Its ability to adhere to the surface of teeth and bones allows it to promote remineralization and regeneration of mineral tissue, thereby protecting enamel and repairing small damages.

Production Process

Hydroxyapatite can be produced through synthetic methods or extracted from natural sources. Synthetic production involves reacting calcium phosphate and calcium hydroxide solutions, which are then purified and micronized to create the powdered form used in cosmetics and toothpaste.

1. Chemical Synthesis: This method involves the combination of calcium and phosphate salts in aqueous solutions. Commonly used chemicals include calcium chloride and monosodium phosphate. The chemical reaction leads to the formation of hydroxyapatite, which is then filtered, washed, and dried.

2. Precipitation: In this process, reagents are added to a solution of calcium and phosphate salts to promote the formation of hydroxyapatite in the form of a precipitate. This precipitate is collected, washed, and treated to remove impurities.

3. Sol-gel: An innovative method that uses a colloidal solution (sol) to form a hydroxyapatite gel. This gel is then dried and sintered to obtain a solid and porous material.

4. Biomass-derived Hydroxyapatite: This eco-friendly method utilizes biological sources, such as animal bones or algae, which are processed to extract hydroxyapatite. The biomass is first demineralized and then chemically treated to obtain the desired mineral.

5. Sintering: Once hydroxyapatite is obtained, the material can undergo a sintering process, which involves heating at high temperatures to improve its stability and mechanical properties.

These methods allow for the production of high-purity hydroxyapatite suitable for medical, dental, and industrial applications.

What it is used for and where it is used

• Calcium supplementation. Hydroxyapatite can be used as a source of calcium in dietary supplements. This is especially useful for maintaining bone health and preventing conditions such as osteoporosis.
• Bone repair. Hydroxyapatite is used in orthopedic and dental surgery for bone repair and replacement. It is particularly useful in treating critically large bone defects where natural healing is not possible. It is also used in the creation of scaffolds that mimic the architecture of human bone, providing a structure on which new bone cells can grow.
• Caries treatment. Hydroxyapatite is used in toothpastes and other oral hygiene products to help prevent cavities. It can also be used in dental surgery to repair and replace teeth.
• Drug delivery. Hydroxyapatite can act as a carrier for drug delivery, particularly for drug delivery to bone. Hydroxyapatite's structure allows it to carry a variety of substances, and it has been explored for targeted drug delivery.
• Tissue engineering. Hydroxyapatite is used in the development of tissue engineering scaffolds. These scaffolds provide a structure on which cells can grow, helping to form new tissue.
• Protein or drug carriers. Hydroxyapatite has been used as a carrier for proteins or drugs because of its bioactivity and biocompatibility. It can adsorb and release active molecules, which is beneficial for various biomedical applications.

Medical

Hydroxyapatite is classified as bioactive, i.e. with the ability to aid bone growth in surgical applications: orthopaedic, dental, maxillofacial and spine surgery. See 'Hydroxyapatite (Biomedical applications)' .  Hydroxyapatite fillers have demonstrated important advantages when compared to other fillers in terms of both volume of product required and duration of action (1). It has interesting biocompatibility properties as a scaffold in tissue engineering and degrades into non-toxic products. It is an effective supplement of calcium and other nutrients due to its compositional characteristics.

Potential carrier for bioactive compounds in the development of edible coating systems (2).

Dentistry

Hydroxyapatite nanoparticles are used to achieve a post-brushing teeth whitening effect (3) and the reason is that hydroxyapatite has an extraordinary affinity and similarity to the apatite crystal of tooth enamel and is considered biologically safe and cytocompatible. It is also used in products for the treatment of dental hypersensitivity and enamel remineralisation. 

Other uses

• Chromatographic purification to separate and purify proteins, enzymes, nucleic acids and plasmids.
• Removal of toxic metals from water
• Biochemical research, isolation of natural and denatured DNA.

Applications

• Oral Care: Hydroxyapatite is widely used in toothpaste for its remineralizing properties, helping to repair tooth enamel and prevent cavities.

• Skincare: In cosmetics and anti-aging treatments, Hydroxyapatite is used for its regenerative abilities, helping to stimulate collagen production and reduce wrinkles.

• Medical Applications: It is also employed in bone regeneration procedures, such as dental implants and orthopedic treatments, due to its biocompatibility and ability to integrate with bone tissue.

Health and Safety Considerations

Safety in Use
Hydroxyapatite is considered extremely safe and biocompatible. It is non-toxic and does not cause allergic reactions or sensitization, making it ideal for use in toothpaste, skincare products, and medical applications.

Allergic Reactions
Allergic reactions to Hydroxyapatite are virtually nonexistent, as it is a natural material compatible with human tissues. However, as with any ingredient, it is always advisable to perform a patch test before use on the skin.

Toxicity and Carcinogenicity
 It has been extensively studied and used in dental health and medical fields without reports of adverse effects.

Environmental and Safety Considerations
As a natural mineral, Hydroxyapatite is biodegradable and poses minimal environmental risks. Its ecological impact is minimal, especially when used in cosmetic and health products.

Regulatory Status
Hydroxyapatite is approved for use in cosmetics and toothpaste by the European Union and the Food and Drug Administration (FDA) in the United States. It is considered safe and is used in a variety of formulations, particularly in oral care products.

For more information:

Hydroxyapatite studies

Typical commercial product characteristics Hydroxyapatite

• Molecular Formula      Ca₅HO₁₃P₃        Ca₁₀(PO₄)₆(OH)₂, HAp
• Linear Formula      3Ca₃(PO₄)₂ · Ca(OH)₂
• Molecular Weight      502.3
• Massa esatta    501.675964
• CAS  1306-06-5
• UNII    91D9GV0Z28
• EC Number   215-145-7
• DSSTox Substance ID  DTXSID10872538
• IUPAC  pentacalcium;hydroxide;triphosphate
• InChl=1S/5Ca.3H3O4P.H2O/c;;;;;3*1-5(2,3)4;/h;;;;;3*(H3,1,2,3,4);1H2/q5*+2;;;;/p-10
• InChl Key      XYJRXVWERLGGKC-UHFFFAOYSA-D
• SMILES   [OH-].[O-]P(=O)([O-])[O-].[O-]P(=O)([O-])[O-].[O-]P(=O)([O-])[O-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2]
• MDL number  MFCD00010904
• PubChem Substance ID    57646735
• RTECS  MY8434000
• NACRES  NA.21   

Synonyms

• Durapatite
• Monite
• Calcium hydroxide phosphate (5:1:3)

References_____________________________________________________________________

(1) Tansavatdi K, Mangat DS. Calcium hydroxyapatite fillers. Facial Plast Surg. 2011 Dec;27(6):510-6. doi: 10.1055/s-0031-1298783.

(2) Malvano, F., Montone, A. M. I., Capparelli, R., Capuano, F., & Albanese, D. (2021). Development of a Novel Active Edible Coating Containing Hydroxyapatite for Food Shelf-life Extension. Chemical Engineering Transactions, 87, 25-30.

(3) Shang R, Kaisarly D, Kunzelmann KH. Tooth whitening with an experimental toothpaste containing hydroxyapatite nanoparticles. BMC Oral Health. 2022 Aug 8;22(1):331. doi: 10.1186/s12903-022-02266-3.

Oliveira HL, Da Rosa WLO, Cuevas-Suárez CE, Carreño NLV, da Silva AF, Guim TN, Dellagostin OA, Piva E. Histological Evaluation of Bone Repair with Hydroxyapatite: A Systematic Review. Calcif Tissue Int. 2017 Oct;101(4):341-354. doi: 10.1007/s00223-017-0294-z

Abstract. The aim of this study was to evaluate the morphological bone response in animal experiments by applying hydroxyapatite grafts in critical and non-critical size bone defects. Current report followed the guidelines established by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Animal experiments were selected by assessing repair of bone defects with hydroxyapatite as bone graft and with blood clot only as control. Eight articles were identified in specialized literature and included in the meta-analysis. Statistical analysis was carried out with a random-effect model (p = 0.05). Subgroup analyses were further performed to investigate bone repair in critical and non-critical bone defects. Comprehensive analysis of bone repair outcome showed a statistically significant difference between hydroxyapatite and blood clot control (p < 0.05). Subgroup analyses showed statistically significant difference for critical bone defects (p < 0.05). No statistically significant difference was reported in non-critical bone defects (p > 0.05). Although animal studies revealed a high risk of bias and results should be interpreted with caution, the literature suggests that non-critical bone defects may heal spontaneously and without the need of a bone graft. Conversely, when critical-size defects are present, the use of hydroxyapatite bone graft improves the bone repair process.

Gotz W, Papageorgiou SN. Molecular, Cellular and Pharmaceutical Aspects of Synthetic Hydroxyapatite Bone Substitutes for Oral and Maxillofacial Grafting. Curr Pharm Biotechnol. 2017;18(1):95-106. doi: 10.2174/1389201017666161202103218.

Kolmas J, Groszyk E, Kwiatkowska-Różycka D. Substituted hydroxyapatites with antibacterial properties. Biomed Res Int. 2014;2014:178123. doi: 10.1155/2014/178123. Epub 2014 May 11. 

Abstract Reconstructive surgery is presently struggling with the problem of infections located within implantation biomaterials. Of course, the best antibacterial protection is antibiotic therapy. However, oral antibiotic therapy is sometimes ineffective, while administering an antibiotic at the location of infection is often associated with an unfavourable ratio of dosage efficiency and toxic effect. Thus, the present study aims to find a new factor which may improve antibacterial activity while also presenting low toxicity to the human cells. Such factors are usually implemented along with the implant itself and may be an integral part of it. Many recent studies have focused on inorganic factors, such as metal nanoparticles, salts, and metal oxides. The advantages of inorganic factors include the ease with which they can be combined with ceramic and polymeric biomaterials. The following review focuses on hydroxyapatites substituted with ions with antibacterial properties. It considers materials that have already been applied in regenerative medicine (e.g., hydroxyapatites with silver ions) and those that are only at the preliminary stage of research and which could potentially be used in implantology or dentistry. We present methods for the synthesis of modified apatites and the antibacterial mechanisms of various ions as well as their antibacterial efficiency.