Sustainable synthesis of marine-derived hydroxyapatite for biomedical applications: a systematic review on extraction methods and bioactivity

Synthesis of eggshell derived hydroxyapatite via chemical precipitation and calcination method

About 94% of waste eggshells are composed of calcium carbonate (CaCO3), which allows for the generation of calcium oxide (CaO), which can be utilized to synthesize hydroxyapatite (HAp). This study uses chemical precipitation and calcination methods to synthesize natural HAp from eggshell waste. In the first stage, the powdered eggshell was calcined at 900 °C to convert the calcium carbonate (CaCO3) in the eggshell into calcium oxide (CaO), the precursor particles of HAp, before being subjected to chemical precipitation. To obtain HAp, the calcined eggshell powder was mixed with deionized water, and the suspension, whose pH was adjusted to 8.5 using phosphoric acid, was allowed to age. The precipitates obtained in the second stage were calcined at various temperatures (500 °C, 700 °C, 900 °C, 1000 °C, and 1100 °C) to produce hydroxyapatite (HAp) with the highest purity. The HAp samples synthesized at these calcination temperatures were characterized using several techniques: phase analysis through X-Ray Diffraction (XRD), chemical analysis via X-Ray Fluorescence (XRF) and microscopy, and thermal analysis using differential thermal analysis and thermogravimetric analysis (DTA-TG). XRD patterns show that the most suitable calcination temperature for HAp is 900 °C, and samples calcined at 900 °C, 1000 °C and 1100 °C contain peaks belonging to biphasic HAp and -tricalcium phosphate (-TCP) phase. The chemical analysis results show that HAp samples are mostly composed of Ca, P and O elements. The calculated Ca/P ratio for HAp samples recalcined at 900 °C is 1.73, which is close to the expected stoichiometric ratio of 1.67. HAp recalcined at 900 °C exhibited characteristic peaks at 571, 632, 962, 1046 and 1090 cm‒1. The intensities of most of the bands belonging to phosphate vibrations of HAp increased at calcination temperatures of 900 °C and above. As a result, the study showed that HAp can be synthesized from eggshell waste by using the precipitation and calcination methods together.

Hydroxyapatite is the main mineral component in bones and teeth, thus being an important material in bone tissue engineering, e.g., for replacement and elimination of defects. Hydroxyapatite is widely used in real-life applications due to excellent biocompatibility and bioactivity. Wet precipitation synthesis of hydroxyapatite is limited by diffusivity. Hence, choice of a diffusion model becomes critical. The purpose of this work is three-fold. It experimentally validates the use of Ginstling-Brounshtein model for hydroxyapatite synthesis. It determines the effect of Ca(OH)2 concentration on the kinetics and reports a modified model to account for this phenomenon. It reports obtained kinetic constants that describe hydroxyapatite synthesis. Particle size was determined using scanning electron microscopy and digital microscopy. Conversion kinetics were monitored using powder X-ray diffraction. Experimental validation was provided. Furthermore, the process was found dependent on the calcium hydroxide concentration and the model was modified to account for this phenomenon. Kinetic constants describing the synthesis of hydroxyapatite were obtained and reported. The model was well consistent with the experimental data and can be used for describing synthesis of hydroxyapatite for various suspension concentrations.

The purpose was to evaluate whether a novel porous hydroxyapatite (HA) scaffold with a 25-30-µm groove structure (pHAMG) may improve bone osteogenesis, angiogenesis, and bone integration of titanium dental implants in animal models. The pHAMG was prepared by chemical precipitation method and its elemental composition and crystal structure were evaluated. The ability of the scaffolds to induce ectopic osteogenesis and the ability of scaffolds combined with titanium dental implants to induce orthotopic peri-implant angiogenesis, osteogenesis, and osteointegration were tested after implantation into the femur muscle pocket in rats and the mandibular defects in beagle dogs, respectively. The elemental composition was evaluated by SEM-EDS; the expression of the relevant osteogenic/inflammation marker and the anti-/pro-inflammation markers was evaluated by immunostaining and immunofluorescence, respectively. In animal experiments with ectopic and peri-implant osteogenesis, pHAMG resulted in significantly larger neovascularization by hematoxylin-eosin staining, as well as deposition of collagen fibers by Masson staining than HA. Meanwhile, microgrooves in pHAMG upregulate more bone morphogenetic protein (BMP) 2 and interleukin-4 (IL-4) and -10 (IL-10) and downregulate more IL-1β and tumor necrosis factor-α (TNF-α) than that in HA. The pHAMG showed greater expression of arginase (Arg)-1 and lower expression of inducible nitric oxide synthase (iNOS) than HA. The novel pHAMG can better repair bone defects in ectopic and orthotopic model. It also transfers macrophages to anti-inflammatory phenotypes, promoting angiogenic and osteogenesis in scaffolds, and bone integration in implants. The novel pHAMG induce greater osteogenesis and angiogenesis which could be utilized in the clinical treatment.

Bone regeneration is a critical area in tissue engineering because of the increasing incidence of bone defects resulting from trauma, degenerative diseases, and congenital disorders. The focus of this study is the synthesis of hydroxyapatite (HAp) from two natural calcium-rich biowastes: Anadara granosa (blood cockle) and Achatina fulica (snail). The shells were calcined at 900 °C to form calcium oxide (CaO) and then converted into HAp via the wet precipitation method using a Ca/P molar ratio of 1.67. The synthesized HAp powders were evaluated for their chemical properties and biological performance. Fourier transform infrared (FTIR) spectroscopy confirmed the presence of phosphate and hydroxyl functional groups, and among the samples, An 100 showed the highest crystallinity. MC3T3-E1 cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay at 24, 48, and 72 hours. At 72 hours, An 100, An 75, and An 50 maintained a viability above 70%, indicating good biocompatibility. In contrast, An 25 and Ac 100 exhibited significant cytotoxicity (p < 0.05). Only the noncytotoxic concentrations were used for the in vitro scratch wound-healing assay, where An 100 demonstrated the most rapid wound closure, indicating increased osteoblast migration. Furthermore, in this study, the elemental composition and structural integrity of Hap was analyzed to understand the factors affecting its stability and performance in biological environments. These findings suggest that naturally derived HAp is a promising, sustainable, and effective biomaterial for bone tissue engineering and has favorable effects on cell viability and migration.

Tissue engineering (TE) applies scientific and clinical techniques to restore and regenerate tissues using scaffolds, stem cells, growth factors and gene therapy, and, for orthopaedic surgery, has evolved to meet a clinical need to develop suitable alternatives to current bone grafting techniques. Collagen-based scaffolds are successfully used as skin graft substitutes but have limited mechanical properties for bone. Using established animal models, the primary objective of this thesis was to evaluate the potential of a collagen-glycosaminoglycan (CG) scaffold previously developed and optimized in our laboratory for bone repair, and to compare its healing response to three composite scaffolds also developed in our laboratory: (i) a collagencalcium phosphate (CCP) scaffold, (ii) a collagen-hydroxyapatite (CHA) scaffold and (iii) a collagen-nano hydroxyapatite (coll-nHA) scaffold. In addition, we analysed the capacity of these scaffolds to act as delivery systems for cells, growth factors and genes. In Chapter 2, a CG scaffold stiffened 7-fold over the standard skin graft collagen scaffold and a CCP scaffold with 25-fold increased stiffness over the skin graft collagen scaffold were tested in a 7mm rat calvarial model. Results demonstrated that both scaffolds, but especially the CCP scaffolds, significantly enhanced healing over untreated controls and both scaffolds elicited an appropriate tissue remodelling immune response demonstrating their potential for bone repair. In Chapter 3, contrary to expectation, the seeding and culture of mesenchymal stem cells (MSCs) onto the same CG and CCP scaffolds, prior to implantation, produced poorer results than the cell-free scaffolds used in Chapter 2. Irnmunohistochemical analysis revealed impaired healing due to the physical barrier of tissue formed during in vitro culture. This study thus improves our understanding of host response in bone tissue engineering. In Chapter 4, a novel collagen hydroxyapatite (CHA) scaffold demonstrated healing rates of a 15mm rabbit radius segmental defect equivalent to autogenous bone graft (ABG) and the CG scaffold soaked with recombinant bone morphogenetic protein-2 (rhBMP2). When the CHA scaffold was soaked in rhBMP2 at a dose exponentially lower than a commercially available rhBMP2 product, it healed the defect with complete anatomical restoration demonstrating both the off-the-shelfpotential of the product as well as its capacity to be used for growth factor delivery. In Chapter 5, as part of a gene therapy approach, nano-hydroxyapatite (nHA) particles developed in our laboratory successfully transfected MSCs with the BMP2 gene. A gene- activated matrix (GAM) was then successfully created by incorporating the transfected MSCs into a scaffold fabricated with the nHA particles and collagen, demonstrating a positive osteogenic response using MSCs in vitro, validating their potential for use in bone tissue regeneration. In conclusion, the application of novel techniques to the existing clinically-approved collagen scaffold has demonstrated considerable healing potential as off-the-shelf bone graft substitutes using established animal models. The results also demonstrate their versatility in bone tissue engineering as this study has shown the ability of these scaffolds to act as delivery devices for stem cells, growth factors and gene therapy and thus their potential to heal large defects where an off-the-shelf approach might not be sufficient.

Using polyacrylamide to control particle size and synthesize porous nano hydroxyapatite

In conjunction with the global trend towards sustainable industry, this review provides a summary of the research endeavors and efforts made in the field of exploiting eggshells in the production of hydroxyapatite (HA). HA is one of the most used biomaterials and has attracted considerable attention over the years towards biomedical applications. As the traditional production of HA from calcium and phosphorus chemical precursors synthetically has bottlenecks of being expensive, complex, time consuming, and results in a low biocompatible product, natural resources have become an attractive alternative option to synthesize HA, with trace elements providing a higher performance. Eggshell, with a growing production annually, is potentially a promising natural resource for HA production. Many studies have used different wet chemical precipitation routes to produce HA with properties comparable to synthetic HA. Thus, this review provides an overview of the various routes that can be used to synthesize HA from eggshells. In this review, the synthesis of HA from eggshells via wet chemical precipitation methods is specifically discussed in term of synthesis parameters and properties of the synthesized HA. This review should aid in choosing the most suitable route for HA production with the optimum parameters for obtaining the desired properties to meet the requirements of biomedical applications such as tissue engineering.

Hydroxyapatite (HA) is a widely studied biomaterial for bone grafting and tissue engineering applications. The crystal structure of HA lends itself to a wide variety of substitutions, which allows for tailoring of material properties. Iron is of interest in ion substitution in HA due to its magnetic properties. The synthesis and characterization of iron-substituted hydroxyapatite (FeHA) have been widely studied, but there is a lack of studies on the sintering behaviors of FeHA materials compared to pure HA. Studying the sintering behavior of a substituted apatite provides information regarding how the substitution affects material characteristics such as stability and bulk mechanical properties, thereby providing insight into which applications are appropriate for the substituted material. In this study both pure HA and FeHA were synthesized, pressed into pellets, and then sintered at temperatures ranging from 900- 1300°C and 600-1100°C, respectively. The study thoroughly examined the comparative sintering behaviors of the two materials using density measurements, mechanical testing, X-ray diffraction, and electron microscopy. It was found that FeHA is considerably less thermally stable than pure HA, with decomposition beginning around 1200°C for pure HA samples, while at 700°C for the FeHA. The FeHA also had a much lower mechanical strength than that of the pure HA. An in vitro cell culture study was conducted by immersing FeHA powder in cell culture media, with HA powder at equivalent doses as a control, verified that FeHA is a biocompatible material. Although the FeHA would be unsuitable for bulk applications, it is a potential material for a variety of biomedical applications including drug delivery, cancer hyperthermia, and bone tissue engineering composites.

Hydroxyapatite (HA) is a biomaterial that has been widely studied for uses such as bone grafting and tissue engineering. Pure and hydroxyapatite nanoparticles with cationic (Na, Ti, and Li) as well as anionic ( CO32− and Si4+) substitutions were synthesised using a wet chemical precipitation method followed by freeze-drying and investigated for their cytotoxicity effect. Inductively coupled plasma (ICP), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy were used to characterise the produced powders. The production of the HA phase was confirmed in all synthesised HA samples. MTT (3-(4.5-dimethylthiazol-2-yl)−2.5-diphenyl tetrazolium bromide) assay was performed using the BJ1 cell line of human normal fibroblasts to investigate the cytotoxicity of the synthesised HA samples. The cytotoxicity test showed that the HA samples were biocompatible, with the greatest results coming from Li-substituted (LiHA2) and Na-substituted (NaHA) hydroxyapatite.

Anchoring Dental Implant in Tissue-Engineered Bone Using Composite Scaffold: A Preliminary Study in Nude Mouse Model

Hydroxyapatite (HA), a mineral component of bones and teeth, has been widely studied for various medical applications. The purpose of this research is to compare the HA from diverse bovine sources and chemical synthetic in the respectively physical and chemical powder properties such as grain size, morphology, crystallinity, phase stability and chemical functional groups. Bovine HA (B-HA) were extracted from the fresh femur bones of adult bovine, calf and bovine bone bio-waste. Synthesized HA (S-HA) were prepared by chemical precipitation method with the pH 6.0 and 12.0 of mother liquor. All of HA samples then were calcined at 800 °C. The TEM observation illustrated that particle shapes and sizes of HA differed depending on their bovine sources. In addition, XRD and FT-IR results implied that pure HA have been successfully obtained in B-HA group while S-HA with high pH value of 12.0 occurred the phrase transformation after thermal treatment.

— Hydroxyapatite (HA) is a biomaterial used to treat bone defects. The utilization of HA in medicine is currently significantly increased this is because HA has high biocompatibility properties when used as a bone graft. Various kinds of bone grafts from HA are currently available in the market. However, its use is often bumped at a high price and is an imported product. This will ultimately increase the burden of financing that must be borne by hospitals and the government . HA sources can be obtained through extraction from natural products, such as eggshells. The purpose of this study was the creation and characterization of hydroxyapatite from quail egg shells using precipitation methods. The research stage consists of 4 stages, Stage 1 is the calcination of quail egg shells so that CaO compounds are obtained. Stage 2 is the synthesis of HA compounds from CaO compounds produced in the previous stage using wet precipitation or deposition methods. Stage 3 is sintering HA compounds produced in the previous stage using a furnace for five hours at a temperature of 900 o C. Stage 4 is the characterization of the HA compounds produced. The characterization is done using XRD and SEM. Hasil yang diperoleh yaitu Efisiensi dari senyawa HA yang dihasilkan sebesar 52,34%. Based on the characterization carried out using XRD it was obtained that the HA compound was successfully synthesized this is characterized by the peak of the quail eggshell difaktogram that is the same as the peak of the standard HA difractogram which is found at an angle of 2θ: 26.00 o C, 31.90 o C, 32.31 o C, 33.02 o C, 34.19 o C, 46.81 o C, 49.59 o C with the crystal phase and the size of the lattice parameters, namely the lattice a = b = 9.4234 A and c = 6.8801 A. But in addition to the peak of HA there are also other peaks that show the existence of the impurities phase at 2θ : 10.01°, 21.85°, and 53.01°. Based on SEM characterization found that the size of the resulting HA particles is not homogeneous, meaning that there is a difference in the smallest particles with a size of 1,497 μm and there are the largest particles with a size of 60.98 μm. If observed the shape of a single particle tends to be round (Shperical). Thus it was concluded that the manufacture and characterization of hydroxyapatite from quail egg shells using precipitation methods has been successfully carried out. Keywords— hydroxyapatite; quail egg shell; precipitation; bone defects

Hydroxyapatite (HAp), the main calcium phosphate and the most inorganic compounds in hard tissues, exhibits sound cytocompatibility and osteogenic activity for clinical bone replacement and tissue engineering. HAp nanostructures depict the more special characteristics than HAp microstructures in various features such as physical, chemical and biological properties. Besides, outstanding characteristics of HAp in bone tissue engineering, recently, inhibitory effects of nanoscaled HAp in different tumor cells proliferation and especially breast cancer was fully detailed and discussed in the literature. Here for the first time, we propose the capability of three needle, spherical, and mesoporous HAp nanoparticles for inhibition of cancer cell proliferation in vitro. The comparison of the three morphologies of HAp nanoparticles was carried out by MTT assay. The results showed that the proliferation of the cancer cell line was reduced by more than 73% after treatment with the Hap nanoparticles for 3 days. The best inhibitory effect was obtained for the needle-shaped HAp nanoparticles that were assigned to their diffusivity into the cell membrane. These results propose that nanoscaled HAp could inhibit cancer cell growth and proliferation, so these nanomaterials can be considered as promising materials in clinical cancer therapy.

Efficacité et performance des substituts osseux pour remplacer les allogreffes et autogreffes