Objective The aim of this study was to investigate the in vitro and in vivo biological responses to nanostructured carbonated hydroxyapatite/calcium alginate (CHA) microspheres used for alveolar bone repair, compared to sintered hydroxyapatite (HA).Material and Methods The maxillary central incisors of 45 Wistar rats were extracted, and the dental sockets were filled with HA, CHA, and blood clot (control group) (n=5/period/group). After 7, 21 and 42 days, the samples of bone with the biomaterials were obtained for histological and histomorphometric analysis, and the plasma levels of RANKL and OPG were determined via immunoassay. Statistical analysis was performed by Two-Way ANOVA with post-hoc Tukey test at 95% level of significance.Results The CHA and HA microspheres were cytocompatible with both human and murine cells on an in vitro assay. Histological analysis showed the time-dependent increase of newly formed bone in control group characterized by an intense osteoblast activity. In HA and CHA groups, the presence of a slight granulation reaction around the spheres was observed after seven days, which was reduced by the 42nd day. A considerable amount of newly formed bone was observed surrounding the CHA spheres and the biomaterials particles at 42-day time point compared with HA. Histomorphometric analysis showed a significant increase of newly formed bone in CHA group compared with HA after 21 and 42 days from surgery, moreover, CHA showed almost 2-fold greater biosorption than HA at 42 days (two-way ANOVA, p<0.05) indicating greater biosorption. An increase in the RANKL/OPG ratio was observed in the CHA group on the 7th day.Conclusion CHA spheres were osteoconductive and presented earlier biosorption, inducing early increases in the levels of proteins involved in resorption.

Background and Objective Tissue regeneration remains a challenge for orthopedic and craniomaxillofacial surgery to treat bone loss. The use of low‐level laser therapy suggests a promise on this road with positive results for narrow defects. However, temporal and quantitative evaluations are required to understand the healing process of large injuries. The aim of this study was to investigate the repair of critical‐size bone defects in rat calvaria using a GaAlAs laser. Study Design/Materials and Methods Bone defects (9 mm in diameter) were created on the skull of 30 Wistar rats separated in control or irradiated group. GaAlAs laser (λ = 830 nm, energy density = 2.5 J/cm2 and output power = 50 mW) was applied after surgery and six times more at 48 hours intervals. The animals were euthanized after 2, 4, and 8 weeks. Digital radiographs, descriptive histological and histomorphometric analyses were carried out. Results Radiographic analysis showed greater bone formation in the irradiated group than control at 8 weeks, covering 45% and 28% of the defect, respectively (P < 0.05). Histological analysis showed in the irradiated groups a higher amount of bone neoformation and greater maturity at 4 and 8 weeks. Histomorphometric analysis showed that the volume density of bone tissue at 4 weeks in the irradiated group was two times higher than the control (P < 0.01). Conclusion The biomodulation of low‐level laser therapy using 830 nm wavelength light was effective in promoting bone healing in critical defects despite the unfavorable prognosis as well as it accelerated the maturation of bone tissue. Lasers Surg. Med. 46:61–67, 2014. © 2013 Wiley Periodicals, Inc.

The objective of the present study was to evaluate the quality and intensity of the tissue response to two synthetic hydroxyapatites implanted in critical defects in the skulls of rats. Sixty animals were divided into three experimental groups: I (control), II (HA-1 = HA with 28% crystallinity) and III (HA-2 = HA with 70% crystallinity). They were sacrificed 1, 3, 6, and 9 months after implantation (n = 5 individuals per group/period). Histomorphometric analysis included i) counting of polymorphonuclear leucocytes, mast cells, macrophages and foreign body multinucleated giant cells stained with anti-lysozyme; ii) microvascular density stained with anti-Factor VIII and iii) degree of cell proliferation stained with anti-PCNA. There were no significant differences between the experimental groups in either the quality or quantity of cells in the inflammatory infiltrate, or the degree of angiogenesis and cell proliferation. We conclude that HA-1 and HA-2 are biocompatible and that the physico-chemical differences of these biomaterials did not affect cellular response

Hydroxyapatite (HA) is one of the most employed materials for bone therapy due to its structural similarity with bone, its biocompatibility and physicochemical properties. Additionally, HA performance may be improved by ionic substitution of calcium with divalent bioactive metallic cations such as zinc. In this context, zinc incorporation into HA have been well studied, in spite of conflicting results regarding its biocompatibility: while previous reports on in vitro cytocompatibility have described 5% zinc containing HA (ZnHA) as slightly cytotoxic, this material presented an excellent response on in vivo studies. In order to bring more information on ZnHA biocompatibility, we performed a multiparametric assay evaluating sequentially on the same cells three different viability parameters: mitochondrial activity (XTT), membrane integrity (Neutral Red) and cell density (Crystal Violet Dye Exclusion test). Additionally, we intended to complement the existing data on ZnHA in vivo performance, by assessing its ability to affect the arrangement of collagen fibers on the grafted area, an important indicative of bone maturation. MC3T3-E1 cells were exposed to 24-hours extracts of ZnHA or stoichiometric HA on culture medium (DMEM) and cell viability was assayed. ZnHA was very cytocompatible, since the levels of viable cells on all 3 tests were similar to the HA and polystyrene (negative control) extracts, but significantly higher than cells treated with 4% phenol (positive control). For the in vivo studies, critical size defects in rats calvaria were filled with HA or ZnHA granules. The histological evaluation after 30 and 180 days revealed an increase along time. Event tough ZnHA is cytocompatible the presence of Zn was unable to alter the interaction between collagen fibers and the mineral bone phase, as compared to stoichiometric HA.

The aim of this paper was to evaluate the usefulness of coupling digital image analysis with immunohistochemistry and histomorphometry data to the study of tissue response to hydroxyapatite in a model of critical size bone defect in calvaria of rats. A transosseous defect measuring 8 mm in diameter was performed with a surgical trephine in the parietal bone of 40 rats and divided into two experimental groups according to the treatment: group I (blood clot, control), group II (HA) and killed 1, 3, 6 and 9 months after implantation (n=5/group/period). The skullcaps with overlaying skin were collected and processed for paraffin embedding. The specimens were cut in the laterolateral direction into 5-µm thick semi-serial sections and stained with hematoxylin-eosin for identification and counting of polymorphonuclears cells, mastocytes, and multinucleated giant cells, MNG, or immunolabeled with anti- lysozyme, -factor VIII and –PCNA. Digital images were obtained and analyzed with the ImagePro-Plus® software for cell couting (polymorphonuclears cells, mastocytes, macrophages and MNG) and microvessel density. Image segmentation of anti-PCNA immunostaining was used for cell proliferation analysis. The digital images obtained allowed clear identification of cells of interest by through morphological aspects or immunostaining. Data recording and analysis was facilitated by the use of specific software for image processing and graphical and statistical analysis. It can be concluded that the techniques applied were usefull to identify and count cells, structures and process of interest making easier the effectiveness of hydroxyapatite in the critical size defect in rat calvaria model.