Biomaterial science has developed significantly during recent decades. HA attracted researchers’ attention due to its resemblance to bone biological apatite. Many methods can be used for HA synthesis. In the living body, biological mineralization occurs in body fluids medium to form Nanoparticles of unstoichiometric calcium phosphate substituted with several ions specially CO3 − 2 and Mg+ 2 2, 29, this leads to the use of wet chemical precipitation for N-HA synthesis, especially from the bio-waste resource. N-HA which has been synthesized from bio-waste materials revealed better biocompatibility when implanted in living tissues because of its homogeneity with bone tissue both in physical and chemical characteristics30, 31. But using wet chemical precipitation in the synthesis of N-Ha was associated with the production of low crystallinity powder with weak mechanical properties32, 33, the sintering step was done to overcome this snag. The high temperature of sintering stimulates crystal growth, increases particle size, and decreases the porosity of the powder34. FE-SEM result of non-doped HA sample showed large hexagonal particles with poor porosity after the powder was sintered at 1200○C, but for Si-Zn doped HA sample, particles decreased in size and showed different shapes with high interconnect porosity, even though the sample was sintered at the same high temperature (Fig. 2). This attributes that doing n-HA with Zn+ 2 and Si+ 4 led to crystal lattice shrinkage because of the differences in ionic radius between doping ions (Zn+ 2 and Si+ 4 ) and substituted ions 35, 36. In the hexagonal crystal system of HA, Zn+ 2 ions substitute Ca+ 2 ions in the second position (Ca2) and Si+ 4 ions substitute P+ 5 ions13. This causes changes in lattice parameters and leads to changes in crystal size and crystallinity, which can be proved by XRD results (Fig. 6). The crystal size of Si-Zn doped HA sample (46 nm) was smaller than the crystal size of non-doped HA (61 nm). Also, peaks shifting in XRD pattern of Si-Zn doped HA sample were observed (Fig. 5) which confirms, alongside EDX results (Fig. 4), the successful substitution of Zn+ 2 and Si+ 4 in crystal structure and not just the adsorption on its surface. When Zn+ 2 substitutes Ca+ 2, the peak will shift to a higher 2ϴ because Zn+ 2 has a smaller ionic radius. Likewise, Si+ 4 has a larger ionic radius than P+ 5 and the peak will shift to a lower 2ϴ (Fig. 8). The insertion of doping ions into crystal lattice is considered an important factor in the biological behavior of the powder. This insertion provides few but continuous releases for these ions during bone remodeling process37. Doping ions also affect the thermal stability of the synthesized n-HA and resulted in decomposition into other phases even after sintering at high temperatures. The Phase purity of both samples was studied using XRD, results of both doped and non-doped HA showed the presence of a second phase of whitlockite (WH) with chemical formula Ca18Mg2(HPO4)2(PO4)12 (Fig. 5). WH was found to be the second most abundant inorganic compound in bone38. WH has biological importance because its biocompatibility39, biodegradability40and negative surface charge41. WH has the same crystal structure as β-TCP but with the presence of magnesium at two of the calcium positions in rhombohedral crystal lattice42. WH phase appeared due to using chicken eggshells as a precursor in the synthesis owing to that dry chicken egg-shells contain about 3% of its weight as magnesium43.
From a biological point of view, the microstructure and surface topography of N-HA play an essential role in cells interaction, Lebre et al. found that HA with spherical nanoparticles enhances successful tissue remodeling both in vitro and in vivo studies when compared to needle-like particle shape44. Bone regeneration scaffolds should have a good porosity to support bone ingrowth and cell function, extracellular proteins adsorption, and angiogenesis45. Thus, for Si-Zn doped HA sample, the interconnected porosity and the difference in pores size are considered suitable for cell adhesion and angiogenesis through the scaffold when it has implanted in living bone.
Alkaline phosphatases are Zn+ 2 metalized glycoproteins that play an important role during the maturation of newly-formed bone by catalyzing the hydrolysis of phosphmonomesters into inorganic phosphate. Substantially, their role is creating an alkaline environment that enhances the precipitation and mineralization of these inorganic phosphate onto the extracellular matrix16. Doping n-HA with Zn+ 2 provides the release of Zn+ 2 during bone remodeling process which increases osteoblastic response to form new bone, furthermore the antibacterial activity of zinc36. Silicon is considered an essential element for collagen synthesis in bone and other connective tissues46, Silicon has a stimulatory effect on proliferation and differentiation of osteoblasts-like cells47. By inducing VEFG expression, silicon promotes angiogenesis which is considered crucial for bone ingrowth 23. In this study, in vitro analysis results of osteoblasts-like cells (MC3T3-E1) demonstrated that the doped sample (Si-Zn doped HA) has higher cell viability than non-doped HA (Fig. 7), our results support the conclusion of Makarova et al. demonstrating that doping N-HA with Zn and Si improved the biocompatibility, decreased the cytotoxicity, and increased the solubility of the material48.
For examination of Si-Zn doped HA material bioactivity, a suitable bone defect must be established in vivo by using proper animal models. Rabbits have similarities in bone mineral density and fracture toughness with humans49, also are easy to house and handle which makes them the first choice for in vivo tests of new bone biomaterial. A critical bone defect is a kind of defect that can’t heal spontaneously and need to be grafted. In our study, a cylinder critical bone defects (6.5×7 mm2) were drilled in rabbits’ distal fumer for both grafted and control side (Fig. 1), histological examination of control defects showed non-bone formation and the connective tissue filled the defect all over the observation periods (Fig. 9,10) which demonstrate that these defects are critical.
The radiographic screening showed the ability to distinguish Si-Zn doped HA martial in regular X-rays. The radiographic density (RD) of A1 material decreased over time, which gives a sign that it may be replaced by another type of tissue was done (Fig. 8).
The histological study showed the good integration of Si-Zn doped HA material with host bone. The material particles were a scaffold that supported the bone formation in the defect without any sign of immune response (Fig. 9). Critical-sized bone defect is defined as a defect which can’t heal spontaneously50. The need to graft critical-sized bone defects comes from the importance of providing a scaffold that allows cell adhesion and supplies cells nutrition in the sites away from defect borders. This can be achieved by using a porous scaffold. The pores between A1 particles formed an optimum place for woven bone formation and support the revascularization between defect walls. During different observation periods, Si-Zn doped HA material was gradually resorbed and replaced with bone tissue which demonstrated its biocompatibility, bioresorbable, and osteoconductivity.