Evaluation of strontium containing hydroxyapatite as bone substitute in sheep ’ s tibia

Innov Implant J, Biomater Esthet, São Paulo, v. 5, n. 1, p. 9-14, jan./abr. 2010 ABSTRACT With the advances on biomaterials research have suggested that the best osteoconductivity of hydroxyapatite could be achieved if the crystal was closer to the structure, size and morphology of biological apatite, so the nano-hydroxyapatite (nano-HA) is of current interest. Strontium ions are known to reduce bone resorption, induce osteoblastic activity and thus stimulate bone formation. The aim of this study was to evaluate the biocompatibility and osteoconductive in surgical defects fi lled with spheres of nano-hydroxyapatite containing strontium 1% (nano-SrHA), nano-hydroxyapatite stoichiometric (nano-HA) when compared to the blood clot (control). Four Santa Inês sheeps, weighing on average 32 kg were anesthetized and subjected to three perforation 2 mm in diameter on the medial site of tibia. The surgical defects were fi lled with blood clot, Sr-HA 1% microspheres and HA microspheres. After 30 days the samples were trephyned (6 mm), decalcifi ed, processed for embedding in paraffi n and stained with hematoxilin and eosin (HE) for histological evaluation with light microscopy. All groups showed new bone formation from the periphery to the center of the defects, and the group nano-Sr with a lesser extent among those studied. The mononuclear infl ammatory infi ltrate remained mild in all experimental groups. The presence of giant cells type foreign body was detected only in the HA group. Areas of bone formation were detected in close contact with biomaterials. According to the results spheres containing nano-SrHA 1% and nano-HA can be considered as biocompatible and with osteoconductive properties.

With the advancement of research in biomaterials, it has been suggested that the best osteoconductivity of hydroxyapatite would be achieved if its crystal were closer to the structure, size and morphology of biological apatite, that is why nano-hydroxyapatite (nano-HA) is of great importance. current interest. Strontium ions are known to reduce bone resorption, induce osteoblastic activity and stimulate bone formation. The aim of this study was to evaluate biocompatibility and osteoconduction in surgical defects filled with nano-hydroxyapatite microspheres containing 1% strontium (nano-SrHA), stoichiometric nano-HA microspheres (nano-HA) compared to the clot (control) . Four Santa Inês sheep, weighing an average of 32 kg, were anesthetized and submitted to three 2 mm diameter perforations in the medial face of the tibia. The surgical defects were filled with blood clot, microspheres of Sr-HA 1% and microspheres of HA. After 30 days the samples were drawn (6 mm), decalcified, processed for inclusion in paraffin and stained with hematoxylin and eosin (HE) for histological evaluation with light microscopy. All groups revealed bone neoformation from the periphery to the center of the defect, with the nano-SrHA group being less intense among those studied. Presence of a discrete mononuclear inflammatory infiltrate in all experimental groups. Giant foreign body cells were only observed in the HA group. Areas of bone neoformation were observed in close contact with both biomaterials. According to the results obtained, microspheres of HA and SrHA 1% are biocompatible and have osteoconductive properties.

INTRODUCTION
Localized bone defects can occur as a result of infections, pathological processes, congenital injuries, traumatic injuries or even due to tooth extractions and generally interfere with the installation of implants and prosthetic rehabilitation. In recent years, with the objective of restoring and preserving the alveolar bone morphology, new materials and techniques have been the target of research aimed at providing the development of a set of procedures and biomaterials that optimize the performance of this rehabilitation 10,12 .
Bone grafts are classified according to their origin, as autogenous, allogeneic, xenogenous and synthetic or alloplastic. The limitations and difficulties existing to obtain autogenous bone grafts, such as post-operative patient discomfort, morbidity of the donor site, limitation of the amount of graft, ethical and religious issues, and the possibility of transmitting diseases from allogeneic and xenogenous grafts keeps them stimulated researchers to develop synthetic biomaterials, to assist in the regeneration of lost bone tissue 24 .
Calcium phosphates have been studied as materials used in bone repair for the past 80 years. Of the compounds based on calcium phosphate, the most extensively investigated are hydroxyapatite (HA) and tricalcium phosphate. HA has been widely used as an important bone substitute and is distinguished from other calcium phosphate-based ceramics because it is similar to the inorganic portion of bone tissue, biocompatible, mechanically resistant, bioactive, non-toxic, radiopaque, allowing periodic monitoring through imaging tests, cause little tissue reaction and not be antigenic or carcinogenic, in addition to having a great capacity for protein adsorption on its surface 15 . The synthetic HA generally used is in the form of coarse particles, which have a size and shape of the crystal quite different from the morphology of the biological bone apatites 18 . It has been suggested that the best osteoconductivity of AH would be achieved if its crystal were closer to the structure, size and morphology of biological apatite 8,16,19 . The high stability and flexibility of this apatite structure, allows the wide variety of possible cationic and anionic substitutions, thus having the presence of a number of foreign ions associated with biological apatites 18 . Strontium (Sr) is present in the mineral phase of bones, especially in regions with greater metabolic function 2 ; its content in the new compact bone is three to four times higher than that of an old compact bone, and approximately 2.5 times higher in the new cancellous bone than in the oldest 11 . As it increases the activity of osteoblasts and decreases the activity of osteoclasts, Sr has an anti-resorptive and bone-forming effect in vitro 3 . Calcium phosphate ceramics containing Sr have been shown to increase the proliferation and differentiation of osteoblasts in vitro 23 . A previous study revealed in vivo an increase in the thickness of the bone layer formed at the bone- cement interface and a better osseointegration of SrHA cement, compared to pure HA cement 20 . The aim of this study was to perform a subjective histological evaluation of bone repair in sheep tibias 30 days after implantation of microspheres of nanohydroxyapatite (nano-HA) and nano-HA containing 1% strontium (nano-SrHA) in comparison clot (control).

METHODOLOGY
Four Santa Inês ewes of both genders weighing between 30 and 55 kg and deprived of food 24 hours before the surgical procedure were used in this research. The animals were previously weighed and operated under general anesthesia, and received as preanesthetic medication acepromazine (0.1 mg.kg-1) IV, diazepam (0.2 mg.kg-1) IV and morphine (0.4 mg.kg-1) IM. Induction was performed with propofol (4 mg.kg-1) IV and diazepam (0.1 mg.kg-1) IV (dose effect), and anesthetic maintenance with isoflurane in the appropriate concentration so that autonomic responses did not occur.
After performing the trichotomy and degerming on the medial face of the tibia, the animals were installed on the operating table in lateral decubitus. Then, an incision of approximately 6 cm was made to allow the detachment until exposure of the skeletal plane to make three perforations distant 6-8 cm from the 2 mm diameter tibial crest (2 mm / spear and spherical cutters) SIN -Implant System, São Paulo, SP, Brazil) ( Figure  1A). The bone defects were filled with nano-HA microspheres, clot and nano-SrHA microspheres ( Figure 1B). The internal and external planes were sutured with Vycril 3.0 and Nylon 5.0, respectively. The surgical wound was left uncovered and all animals received as a postoperative protocol to prevent infections and meloxicam pain control at a dose of 0.5 mg.kg-1 for five days and antibiotic enrofloxacin 5 mg.kg-1, before surgery and for five days after all surgical procedures.  After the experimental period of 30 days, the animals were anesthetized, as previously described, and performed: incision, detachment, removal of the samples with a 6 mm internal diameter milling cutter ( Figure 1C and 1D) followed by planar suture. The animals were kept alive and the suture and postoperative protocol adopted was the same as the previous surgical time.
The samples obtained from the 4 animals were fixed for 48 hours in 10% formaldehyde buffered with pH 7.4, washed in running water for 6 hours, demineralized with the Allkimia® bone softener at room temperature, for 48 hours. After the demineralization of the blocks, they were dehydrated, cleared and embedded in paraffin. Transverse sections with a thickness of 5 µm and stained with hematoxylin and eosin (HE) were obtained and analyzed under light microscopy. In the microscopic analysis, the type and intensity of the inflammatory process in response to the procedure, the presence of connective tissue and newly formed bone in the surgical defect were described through subjective analysis.

RESULTS
Histological analysis revealed that in the group containing a clot (control) bone neoformation was observed from the periphery to the center of the bone defect, consisting of large anastomosed bone trabeculae, however, in the central portion of the defect, areas of loose connective tissue and rare inflammatory cells were observed. (Figure 2A).

DISCUSSION
The most important aspect during the development of new biomaterials is the experimental and clinical trial to assess their biocompatibility 17 . The use of sheep for research has increased over time due to similarities with humans in weight, bone and joint structure, and bone regeneration 1,13 . It was observed in this study that the sheep drilling model proves to be an excellent animal model to assess the biocompatibility of bone substitute biomaterials, where in the implantation model (femur), it revealed the possibility of implanting up to 8 biomaterials per animal 14 and also by possibility of keeping the animals alive after the end of the experiment.
Nano-HA is of current interest due to its biological properties compared to HA, in addition to its greater similarity to physiological apatite 8 . Previous studies have shown an increase in the motility of osteoblasts exposed to spherical nano-HA particles 25 and Evaluation of strontium-containing hydroxyapatite as bone substitute in sheep tibiaeme. .1, n.7, p. 153-164, December 18 an increase in the migration of these cells when exposed to spherical nano-HA particles 9 . Nano-HA exhibits excellent adhesion, not only for mineralized and nonmineralized tissues 22 . Based on these previous studies, the present study adopted spheres as the material format.

Callinca Paolla Gomes Machado¹, Andrea Vaz Braga Pintor² and Mônica Diuana Calasans Maia³
The present study demonstrated that HA containing 1% Sr is biocompatible, presenting newly formed bone in close contact with the biomaterial, but revealed less intensity of bone neoformation than nano-HA, results that were not confirmed by a previous study that evaluated a cement of HA containing Sr, which showed better osteoconductivity, biocompatibility and biodegradability than the free HA cement of Sr. In addition, the doses of Sr incorporated in the crystal structure of HA played an active role in vitro and in vivo . From the data from the biocompatibility tests, they observed that the cement containing 5% Sr was more biocompatible, followed by the HA cement containing 10% Sr and finally the Sr 6 free HA cement.. In another study, by these same authors, it was also found that HA cement containing 5% Sr achieved greater resistance to compression in the analyzed samples (5-10%) 7 . These data indicate that there is an ideal dose of Sr to be incorporated into the HA crystal in order to obtain better physico-chemical and biocompatibility properties 5 . However, the results of the intramuscular implant and the implantation experiments in the rabbit femur show that the average dissolution rate of HA cement containing Sr increases with the increase in the dose of Sr 6 . This can be explained by the fact that an HA containing substitutions is considered a calcium deficient HA and, consequently, more soluble and the greater the incorporation of the metal the more calcium deficient it becomes, therefore the concentration of the incorporated metal is a important control parameter to adjust the properties of HA.
An in vitro study evaluated the bioactivity of HA containing Sr in simulated body fluid, and its effect on proliferation, cell morphology, alkaline phosphatase and osteopontin activity in the culture of osteoprecursor cells in vitro. The HA ceramics containing Sr exhibited high bioactivity in simulated body liquid, which was clear by the rapid formation of apatite on its surface. The cell culture test indicated that HA containing Sr has good biocompatibility in human osteoblasts. Compared to HA, Sr-HA promoted adhesion of osteoprecursor cells and cell proliferation, and did not present any deleterious effect on the formation of the extracellular matrix and mineralization. It has also been shown that the presence of Sr stimulates the differentiation of osteoprecursor cells, and increases alkaline phosphatase and expression of osteopontin 23 . This study concluded that Mr promotes osteoblastic action and subsequent bone neoformation. These results different from those obtained can be justified by the concentration of the Sr used in this study (1%). However, more research is needed for a detailed understanding of the cellular and molecular mechanisms of the effects of strontium on bone cells.
Another in vitro study carried out in order to evaluate the osteoblastic and osteoclastic response of HA containing Sr in different concentrations, showed that osteoblastic cells cultured in HA, containing Sr grew showing normal morphology, good proliferation and increased values of differentiation parameters, at the same time. At the same time, the number of osteoclasts was negatively influenced by the presence of Mr. The positive effect of the ion on bone cells was particularly evident in the case of HA deposition containing relatively high Sr (3-7%), values that significantly increased the activity of alkaline phosphatase, osteocalcin, type I collagen and osteoprotegerin / TNF related to cytokine receptors, a considerable reduction in osteoclast proliferation has also been observed 4 .
An in vivo study investigated the bone tissue response of a bone cement of HA containing Sr injected into spongy iliac crest bone of rabbits for 1, 3 and 6 months. Bone affinity to HA cement containing Sr increased from 73.55% ± 3.50% after 3 months to 85.15% ± 2.74% after 6 months (p = 0.01) 21 . These results show that the HA cement containing Sr is biocompatible and osteoconductive, confirming the data obtained in this study despite our experimental period having been only 30 days. In another in vivo study , osseointegration in cancellous bone was achieved with the use of HA cement containing Sr in rabbits, which stimulated bone formation and union, the fusion of bone with HA cement containing Sr and indicated biocompatibility in vivo. Tetracycline staining showed that the mineralization area was in the order: 3 months -1 month -6 months. In the 1st month, the increase in mineralization was due to the bone healing process. An additional increase in the mineralization area in 3 months indicated that HA containing Sr has a stimulating effect on bone formation. The mineralization area decreased in 6 months because the healing process was concluded with bone remodeling 20 . Among all the studies analyzed, it was observed that HA containing Sr is biocompatible depending on the concentration of Sr and the period of the experiment.