Lycopene prevents bone loss in ovariectomized rats and increases the number of osteocytes and osteoblasts.

Osteoporosis is a prevalent disease with a high incidence in women at the onset of menopause mainly because of hormonal changes, genetics, and lifestyle, leading to decreased bone mass and risk of fractures. Maintaining bone mass is a challenge for postmenopausal women, with calcium-rich food intake being essential for bone health. Nevertheless, other nutrients such as carotenoids may influence bone metabolism because of their high antioxidant properties. This study aimed to evaluate the effect of the carotenoid lycopene on bone cells and in the microarchitecture of ovariectomized rats employing in vitro and in vivo assays. After 8 weeks of ovariectomy, femurs were removed to isolate bone marrow mesenchymal cells to be cultured in osteogenic medium (sham and ovariectomized/OVX) or with 1 µmol/L lycopene (OVX/Lyc). There were performed assays for alkaline phosphatase activity and its in situ detection, mineralization nodules, and quantitative expression of genes associated with osteogenesis. Daily ingestion of 10 mg/kg of lycopene by oral gavage for 8 weeks after ovariectomy was conducted for stereological evaluation of the number and volume of osteoblasts, osteoclasts, and osteocytes of femur distal epiphysis and for microtomographic evaluation of the bone microarchitecture of the femoral proximal epiphysis. Data were normalized and analyzed by comparison among the groups using one-way ANOVA followed by post hoc tests with the significance level set out at 5%. Results showed that lycopene promoted an increase in ALP in situ detection as well as a significant increase in mineralized nodules deposition and expression of genes Runx2 and Bglap when compared with the OVX group. The administration by oral gavage of lycopene increased the total number of osteoblasts and osteocytes when compared to sham and ovariectomized groups. Additionally, it decreased the volume and number of osteoclasts and also reduced the volume of osteocytes compared to the sham group. These results suggest that lycopene improves bone cell metabolism and bone remodeling with the onset of osteoporosis. Future studies with different concentrations and periods of administration should be carried out to shed further light on it.

Texturized P(VDF-TrFE)/BT membrane enhances bone neoformation in calvaria defects regardless of the association with photobiomodulation therapy in ovariectomized rats.

OBJECTIVES: The purpose of this investigation was to evaluate in vivo the response of bone tissue to photobiomodulation when associated with texturized P(VDF-TrFE)/BT in calvaria defects of ovariectomized rats. MATERIALS AND METHODS: Wistar Hannover rats were submitted to ovariectomy/control surgery. Calvaria bone defects of 5-mm diameter were performed after 90 days of ovariectomy. The animals were divided into OVX (without laser (L) and membrane), OVX + P(VDF-TrFE)/BT, OVX + P(VDF-TrFE)/BT + L, and OVX + PTFE + L. It was utilized a low-intensity gallium-aluminum-arsenide laser (GaAlAs) with 780-nm wavelength and 30-J/cm2 energy density in 12 sessions (120 s). Thirty days after the bone defect the animals were euthanized for histological, microtomographic, and molecular evaluation. Quantitative analysis was analyzed by statistical software for p < 0.05. RESULTS: Histological parameters showed bone tissue formation at the borders of all group defects. The association of photobiomodulation and texturized P(VDF-TrFE)/BT was not synergistic and did not show significant changes in morphometric analysis and biomarkers gene expression. Nevertheless, texturized P(VDF-TrFE)/BT membrane enhanced bone repair regardless of the association with photobiomodulation therapy, with an increase of connectivity density when compared to the OVX + PTFE + L group. The association of photobiomodulation therapy and PTFE was synergistic, increasing the expression of Runx2, Alp, Bsp, Bglap, Sp7, and Rankl, even though not enough to reflect significance in the morphometric parameters. CONCLUSIONS: The utilization of texturized P (VDF-TrFE)/BT, regardless of the association with photobiomodulation therapy, enhanced bone repair in an experimental model of osteoporosis.

Green tea extract rich in epigallocatechin gallate impairs alveolar bone loss in ovariectomized rats with experimental periodontal disease.

Periodontal disease and osteoporosis are characterized by bone resorption, and researchers have shown an association between these two diseases through increasing loss of systemic bone mass and triggering alveolar bone loss. Green tea is a common and easily accessible beverage, and evidences show that flavonoid epigallocatechin gallate (EGCG) could decrease bone loss in pathologies such as osteoporosis and periodontal disease. In order to verify its possible effects and apply them in the treatment and prevention of these diseases, this investigation aimed to evaluate the influence of green tea extract (GTE) on bone metabolism of ovariectomized rats after experimental periodontal disease (EPD) by histological, morphological and microtomographic parameters. Wistar female rats were divided into Sham, Sham + EPD, Sham + EPD + GTE, OVX, OVX + EPD and OVX + EPD + GTE groups. Immediately after surgery, gavage administration of 50 mg/kg of green tea extract (GTE) was performed for 60 days, with subsequent induction of periodontal disease by ligature 15 days before euthanasia. Mandible and femur samples were collected for histological, morphometric and microtomographic analysis. The results were analysed by means of statistical software with significance set at 5%. Histological and morphometric analysis showed a significant decrease in alveolar and femoral trabecular bone loss in groups that received GTE. Microtomographic results showed that trabecular thickness and bone surface density values in alveolar bone interradicular septum of the OVX + EPD + GTE groups were similar to the Sham group. The results obtained suggest that green tea extract may improve bone metabolism in osteoporotic rats with periodontal disease.

Cell therapy stimulates bone neoformation in calvaria defects in rats subjected to local irradiation.

BACKGROUND: The purpose of the study was to analyze the effect of cell therapy on the repair process in calvaria defects in rats subjected to irradiation. METHODS: Bone marrow mesenchymal cells were characterized for osteoblastic phenotype. Calvariae of male Wistar rats were irradiated (20 Gy) and, after 4 weeks, osteoblastic cells were placed in surgically created defects in irradiated (IRC) and control animals (CC), paired with untreated irradiated (IR) and control (C) animals. After 30 days, histological and microtomographic evaluation was performed to establish significant (P < 0.05) differences among the groups. RESULTS: Higher alkaline phosphatase detection and activity, along with an increase in mineralized nodules, in the IRC, C and CC groups compared to the IR group, confirmed an osteoblastic phenotype. Histology showed impaired bone neoformation following irradiation, affecting bone marrow composition. Cell therapy in the IRC group improved bone neoformation compared to the IR group. Microtomography revealed increased bone volume, bone surface and trabecular number in IRC group compared to the IR group. CONCLUSION: Cell therapy may improve bone neoformation in defects created after irradiation.

Low-level laser therapy enhances the number of osteocytes in calvaria bone defects of ovariectomized rats.

BACKGROUND: Osteoporosis can make bone repair difficult. Low-level laser therapy (LLLT) has been shown to be a promising tool for bone neoformation. This study aimed to analyze the effect of LLLT on calvaria bone defects of ovariectomized rats using stereology. METHODS: Fifty-four Wistar rats were subjected to bilateral ovariectomy, and bone defects were created in calvaria after 150 days. The animals were divided into nine groups (n =  6 per group), and 24 hours after the bone defects were created they received 3, 6 or 12 sessions of LLLT at 0, 20 or 30 J/cm2, using a 780-nm low-intensity GaAlAs laser. One-way ANOVA followed by Tukey's post hoc test was used for data processing. A difference of P < 0.05 was considered statistically significant. The parameters evaluated were osteocyte density (Nv ost), total osteocyte number (Nto ost), trabecular surface density (Sv t), and trabecular surface area (Sa t). RESULTS: Data obtained showed that Nto ost, Sv t, and Sa t in group G2 rats were significantly different from G1 (0 J/cm2) (P < 0.05). Compared to group G4, G5 presented higher values for the parameters Sv t and Sa t, and G6 presented significantly higher values for almost all the analyzed parameters (Nv ost, Nto ost, Sv t, and Sa t) (P < 0.05). Compared to group G7, G8 showed a higher value only for the parameter Sa t, and G9 showed significantly higher values for parameters Nv ost, Nto ost, Sv t, and Sa t. CONCLUSION: We conclude that LLLT stimulated bone neoformation and contributed to an increase in the total number of osteocytes, especially with a laser energy density of 30 J/cm2 given for 6 and 12 sessions.

Lycopene influences osteoblast functional activity and prevents femur bone loss in female rats submitted to an experimental model of osteoporosis.

Antioxidant properties of several nutrients may influence bone metabolism, affording protection against damaging effects caused by oxidative stress. Thus, we hypothesized that lycopene may benefit bone tissue metabolism and functional activity of osteoblastic cells from bone marrow of osteoporotic female rats. Wistar rats were ovariectomized and paired with sham animals. In vitro evaluations were performed after 60 days of surgery, when cells were cultured in osteogenic medium and divided in control (C), ovariectomized (OVX) and ovariectomized + 1 µmol/L lycopene (OVXL) groups. Besides, in vivo studies were carried out to evaluate femur bone remodeling by histological and histomorphometric analyses after daily intake of 10 mg/kg of lycopene for 30 and 60 days after ovariectomy. Cell proliferation was significantly higher in OVX and OVXL groups after 10 days of culture. Alkaline phosphatase activity (ALP) was higher in OVXL group in later periods of cell culture, whereas its in situ detection was higher for this group in all experimental periods; nevertheless, mineralization did not show significant differences among the groups. There was a significant upregulation of genes Sp7, Runx2 and Bsp after 3 days and genes Runx2 and Bglap after 10 days from OVXL when compared to OVX. In vivo results demonstrated that daily intake of 10 mg/kg of lycopene for 60 days decreased bone loss in femur epiphysis in ovariectomized rats by maintaining trabecular bone similar to controls. Data obtained suggest that lycopene might benefit the functional activity of osteoblastic cells from ovariectomized rats, as well as avoid further bone resorption.

Poly(Vinylidene Fluoride-Trifluorethylene)/barium titanate membrane promotes de novo bone formation and may modulate gene expression in osteoporotic rat model.

Osteoporosis is a chronic disease that impairs proper bone remodeling. Guided bone regeneration is a surgical technique that improves bone defect in a particular region through new bone formation, using barrier materials (e.g. membranes) to protect the space adjacent to the bone defect. The polytetrafluorethylene membrane is widely used in guided bone regeneration, however, new membranes are being investigated. The purpose of this study was to evaluate the effect of P(VDFTrFE)/BT [poly(vinylidene fluoride-trifluoroethylene)/barium titanate] membrane on in vivo bone formation. Twenty-three Wistar rats were submitted to bilateral ovariectomy. Five animals were subjected to sham surgery. After 150 days, bone defects were created and filled with P(VDF-TrFE)/BT membrane or PTFE membrane (except for the sham and OVX groups). After 4 weeks, the animals were euthanized and calvaria samples were subjected to histomorphometric and computed microtomography analysis (microCT), besides real time polymerase chain reaction (real time PCR) to evaluate gene expression. The histomorphometric analysis showed that the animals that received the P(VDF-TrFE)/BT membrane presented morphometric parameters similar or even better compared to the animals that received the PTFE membrane. The comparison between groups showed that gene expression of RUNX2, BSP, OPN, OSX and RANKL were lower on P(VDF-TrFE)/BT membrane; the gene expression of ALP, OC, RANK and CTSK were similar and the gene expression of OPG, CALCR and MMP9 were higher when compared to PTFE. The results showed that the P(VDF-TrFE)/BT membrane favors bone formation, and therefore, may be considered a promising biomaterial to support bone repair in a situation of osteoporosis.

Low-level laser therapy improves bone formation: stereology findings for osteoporosis in rat model.

Low-level laser therapy (LLLT) benefits bone metabolism, but its use needs to be standardized. We evaluated the effects of LLLT on bone defects in calvaria of ovariectomized rats. Stereology was used to calculate tissue repair volume (V tr ), density of trabecular bone volume (Vv t ), total volume of newly formed trabecular bone (Vtot), and the area occupied by collagen fibers (A C ). Fifty-four Wistar rats were submitted to bilateral ovariectomy, and bone defects were created in calvaria after 150 days. The animals were divided into nine groups (n = 6), and 24 h after defects, the treatment started with a 780-nm low-intensity GaAlAs laser: G1, G2, and G3 received 3 sessions of 0, 20, and 30 J/cm(2) respectively; G4, G5, and G6 received 6 sessions of 0, 20, and 30 J/cm(2), respectively; and G7, G8, and G9 received 12 sessions of 0, 20, and 30 J/cm(2), respectively. A normal distribution was found for all of the data. The test used to verify the normality was the Kolmogorov-Smirnov (KS, p > 0.05). The one-way ANOVA followed by Tukey's post hoc test was used for data processing. A difference of p < 0.05 was considered statistically significant. Groups G2 and G1 showed significance for V tr , Vv t , Vtot, and (A C ). Results were significant for (Vv t ) and (Vtot) between G3 and G1. There were no significant results between G5 and G4 as well as between G8 and G7. Groups G6 and G4 results showed statistical difference for V tr , Vv t , Vtot, and (A C ). Groups G9 and G7 showed significance for V tr , Vv t , Vtot, and (A C ). In conclusion, there was new bone formation in the groups that received 20 and 30 J/cm(2) when compared to control groups, but over time, the dose of 30 J/cm(2) showed better stereological parameters when compared to 20 J/cm(2).

Morphological and morphometric analyses of crushed sciatic nerves after application of a purified protein from natural latex and hyaluronic acid hydrogel.

Hyaluronic acid hydrogels (HAHs) have been used as a carrier of substances and factors in the repair of nervous tissue. Natural latex protein (Hevea brasiliensis, F1) has shown positive effects in treating various types of tissues, including peripheral nerves. This study evaluated the F1 associated with a HAH in a controlled crush injury (axonotmesis) of the sciatic nerve in Wistar rats. The samples were photomicrographed for morphometric and quantitative analyzes using ImageJ 1.47k software (NIH, Bethesda, MD). Morphological, quantitative (myelin area/nerve area ratio and capillary density) and morphometric (minimum nerve fiber diameter, G-Ratio) data revealed an improvement in the recovery of the sciatic nerve with the application of HAH and the combination of HAH and F1 after 4 and 8 weeks of nerve injury. The most efficacious results were observed with the combination of both substances, F1 and HAH, revealing the regenerative capacity of this new biomaterial, which was hardly tested on nerve tissue.

Effects of the combination of low-level laser irradiation and recombinant human bone morphogenetic protein-2 in bone repair.

Low-level laser irradiation (LLLI) and recombinant human bone morphogenetic protein type 2 (rhBMP-2) have been used to stimulate bone formation. LLLI stimulates proliferation of osteoblast precursor cells and cell differentiation and rhBMP-2 recruits osteoprogenitor cells to the bone healing area. This in vivo study evaluated the effects of LLLI and rhBMP-2 on the bone healing process in rats. Critical bone defects were created in the parietal bone in 42 animals, and the animals were divided into six treatment groups: (1) laser, (2) 7 µg of rhBMP-2, (3) laser and 7 µg of rhBMP-2, (4) 7 µg of rhBMP-2/monoolein gel, (5) laser and 7 µg rhBMP-2/monoolein gel, and (6) critical bone defect controls. A gallium-aluminum-arsenide diode laser was used (wavelength 780 nm, output power 60 mW, beam area 0.04 cm(2), irradiation time 80 s, energy density 120 J/cm(2), irradiance 1.5 W/cm(2)). After 15 days, the calvarial tissues were removed for histomorphometric analysis. Group 3 defects showed higher amounts of newly formed bone (37.89%) than the defects of all the other groups (P < 0.05). The amounts of new bone in defects of groups 1 and 4 were not significantly different from each other (24.00% and 24.75%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). The amounts of new bone in the defects of groups 2 and 5 were not significantly different from each other (31.42% and 31.96%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). Group 6 defects had 14.10% new bone formation, and this was significantly different from the amounts in the other groups (P < 0.05). It can be concluded that LLLI administered during surgery effectively accelerated healing of critical bone defects filled with pure rhBMP-2, achieving a better result than LLLI alone or the use of rhBMP-2 alone.

Application of low-level laser irradiation (LLLI) and rhBMP-2 in critical bone defect of ovariectomized rats: histomorphometric evaluation.

OBJECTIVES: The aim of this study was to evaluate the osteogenic potential of recombinant human bone morphogenetic protein-2 (rhBMP-2) and low-level laser irradiation (LLLI), isolated or combined in critical bone defects (5 mm) in parietal bone using ovariectomized female rats as an experimental animal model. MATERIALS AND METHODS: Forty-nine female Wistar rats, bilaterally ovariectomized (OVX), were divided into seven treatment groups of seven animals each: (I) laser in a single application, (II) 7 µg of pure rhBMP-2, (III) laser and 7 µg of pure rhBMP-2, (IV) 7 µg of rhBMP-2/monoolein gel, (V) laser and 7 µg of rhBMP-2/monoolein gel, (VI) laser and pure monoolein gel, and (VII) critical bone defect controls. The low-level laser source used was a gallium aluminum arsenide semiconductor diode laser device (λ = 780 nm, D = 120 J/cm(2)). RESULTS: Groups II and III presented higher levels of newly formed bone than all other groups with levels of 40.57% and 40.39%, respectively (p < 0.05). The levels of newly formed bone of groups I, IV, V, and VI were similar with levels of 29.67%, 25.75%, 27.75%, and 30.64%, respectively (p > 0.05). The area of new bone formation in group VII was 20.96%, which is significantly lower than groups I, II, III, and VI. CONCLUSIONS: It was concluded that pure rhBMP-2 and a single dose of laser application stimulated new bone formation, but the new bone formation area was significantly increased when only rhBMP-2 was used. Additionally, the laser application in combination with other treatments did not influence the bone formation area.

Macroscopic and microscopic aspects of the temporomandibular joint related to its clinical implication.

In order to obtain a precise diagnosis and treatment for temporomandibular dysfunctions, it is necessary to have morphological and functional knowledge of the temporomandibular joint. Anatomic components are important to the understanding of the complexity of temporomandibular joint. Nonetheless, just as important are the anatomic relationships that this joint has with the neighboring structures. Thus, the aim of this study was to present the anatomic relationships of the temporomandibular joint in its various surfaces: external or lateral, anterior, posterior, medial, superior, and posterosuperior, considering the morphological and histological aspects. Nine human heads, fixed in formalin (10%) underwent sagittal medial section and were subsequently dissected, evidencing the anatomic components of all surfaces to be analyzed. Components of the external surface were: skin, subcutaneous tissue, lymphatic ganglia, parotid gland, superficial temporal artery, transverse facial artery, zygomatic-orbital artery, superficial temporal vein, facial and auriculotemporal nerves, masseter muscle, and pre-auricular lymphonodus. The anterior surface comprised the masseter and lateral pterygoid muscles (upper and inferior heads), pterygoid venous plexus, mandibular notch, posterior deep temporal artery, masseteric nerve, and deep posterior temporal branches. Medial surface components were: internal maxillary artery, of which middle meningeal artery was one of the closest branches to the TMJ, anterior tympanic artery, inferior alveolar, lingual, auriculotemporal, and chorda tympani nerves, which belonged to the surface posterior to the anterior wall of the auditory duct; auricolotemporal nerve, parotid gland; and petrotympanic fissure. The cerebral fossa (meninges and encephalon) belonged to the superior surface and the ear belonged to the posterosuperior surface. Histologically, the temporomandibular joint is composed by different tissues that compound the mandibular head, mandibular fossa, and articular eminence of the temporomandibular joint. It was concluded that the temporomandibular joint is a complex structure and the clinician must have the ability to formulate the diagnosis based on the understanding of morphological aspects of the structures that compose the stomatognatic system.

Educational material of dental anatomy applied to study the morphology of permanent teeth.

The purpose of this report is to present educational material that would allow the dental student to learn to easily identify the morphologic characteristics of permanent teeth, and how they fit together (occlusion). In order to do this, macro models of permanent teeth with no attrition were carved in wax and later molded with alginate. These molds were filled with plaster, dental stone and/or cold-cured acrylic resin. The large individual dental stone tooth models were mounted on a wax base, thus obtaining maxillary and mandibular arches which were occluded. These dental arches were molded with plaster or dental stone. The authors suggest that these types of macro models allow an excellent visualization of the morphologic characteristics of permanent teeth and occlusion. Dental students are able to carve the permanent dentition in wax with great facility when they can observe macro models.