Automated bone healing evaluation: New approach to histomorphometric analysis.

This study aimed to assess different approaches for bone healing evaluation on histological images and to introduce a new automatic evaluation method based on segmentation with distinct thresholds. We evaluated the hyperbaric oxygen therapy (HBO) effects on bone repair in type 1 diabetes mellitus rats. Twelve animals were divided into four groups (n = 3): non-diabetic, non-diabetic + HBO, diabetic, and diabetic + HBO. Diabetes was induced by intravenous administration of streptozotocin (50 mg/kg). Bone defects were created in femurs and HBO was immediately started at one session/day. After 7 days, the animals were euthanized, femurs were removed, demineralized, and embedded in paraffin. Histological sections were stained with hematoxylin and eosin (HE) and Mallory's trichrome (MT), and evaluated using three approaches: (1) conventional histomorphometric analysis (HE images) using a 144-point grid to quantify the bone matrix; (2) a semi-automatic method based on bone matrix segmentation to assess the bone matrix percentage (MT images); and (3) automatic approach, with the creation of a plug-in for ImageJ software. The time required to perform the analysis in each method was measured and subjected to Bland-Altman statistical analysis. All three methods were satisfactory for measuring bone formation and were not statistically different. The automatic approach reduced the working time compared to visual grid and semi-automated method (p < .01). Although histological evaluation of bone healing was performed successfully using all three methods, the novel automatic approach significantly shortened the time required for analysis and had high accuracy.

Effectiveness of Photobiomodulation Therapy on Human Bone Healing in Dentistry: A Systematic Review.

Background: Despite the association, possible causality, and contradictory results, numerous studies evaluate photobiomodulation (PBM) therapy on the process of human bone healing. It is of paramount importance to review the available literature to elucidate the effect of laser on the bone healing process in dentistry. Objective: This systematic review analyzes the effectiveness of PBM therapy to improve bone healing in dentistry. Methods: A systematic search of studies published up to September 2021 and listed in PubMed, Cochrane Library, and Embase databases and registered on PROSPERO (CRD42020212790). Twenty-five studies were selected. Results: The most used device was diode laser. PBM therapy parameters varied greatly. From the 25 selected studies, 17 had the primary outcome bone healing. Of these, 11 studies revealed improvement in bone healing with PBM therapy and six studies suggested no effect. The other eight studies evaluated secondary parameters. In seven studies, some of the clinical parameters were improved with the PBM therapy. Conclusions: Within the limitations of this systematic review, bone healing in dentistry was improved with the use of PBM. PBM therapy can promote anti-inflammatory and analgesic effects, improve healing, as well as enhance quality of life related to oral health. Within the areas analyzed in dentistry, laser parameters varied greatly, becoming difficult to consider a definite protocol as a proper one.

Stephalagine, an aporphinic alkaloid with therapeutic effects in acute gout arthritis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Gout is an inflammatory disease characterized by the accumulation of monosodium urate crystals (MSU) in the joints, leading to severe pain and inflammation. Stephalagine is a Brazilian Savanna aporphine alkaloid isolated from Annona crassiflora Mart. Fruit peel, that has been popularly used to treat rheumatism and have been described with antinociceptive properties. However, no studies evaluated the possible therapeutic properties of stephalagine in arthritic pain. AIM OF THE STUDY: To evaluate the possible antinociceptive and anti-inflammatory effects of stephalagine in an acute gout attack in mice. MATERIALS AND METHODS: Adult male wild type C57BL/6/J/UFU mice (20-25 g) were used (process number 018/17). The treated group received stephalagine (1 mg/kg, by gavage) and the vehicle group received saline (10 mL/kg, by gavage), both 1 h before the MSU crystals (100 µg/ankle joint) administration. All groups were analyzed for mechanical allodynia, thermal hyperalgesia, overt pain-like behaviors, and edema development at 2, 4, 6 and 24 h after injections. Synovial fluid and the ankle articulation from the injected joint were collected 4 h after administrations for myeloperoxidase enzyme activity, IL-1ß measurement, and histological analysis. RESULTS: Stephalagine had a significant antinociceptive effect on mechanical allodynia, when compared to vehicle group at 2-24 h after intra-articular injection of MSU and 2 h for spontaneous and cold thermal sensitivity. Stephalagine was also able to significantly reduce the articular edema (45 ± 1%), the activity of the myeloperoxidase enzyme (37 ± 6%), and IL-1ß levels (43 ± 3%). The histological analysis confirms that stephalagine dramatically reduced the number of infiltrating inflammatory cells (75 ± 6%) in MSU injected animals. Also, stephalagine treatment did not alter the uric acid levels, xanthine oxidase activity, AST and ALT activities, urea and creatinine levels, neither cause any macroscopic changes in the mice's weight, deformations, changes in the coat, or feces. CONCLUSION: Stephalagine may be an alternative for the management of gout, once it was able to induce antinociceptive and anti-inflammatory effects without causing adverse effects on the evaluated parameters.

The effect of hyperbaric oxygen therapy on bone macroscopy, composition and biomechanical properties after ionizing radiation injury.

BACKGROUND: Radiotherapy used in tumor treatment compromises vascularization of bone tissue. Hyperbaric oxygenation (HBO) increases oxygen availability and improves vascularization, minimizing the deleterious effects of ionizing radiation (IR). Therefore, the aim of this study was to evaluate HBO therapy effect on bone macroscopy, composition and biomechanical properties after IR damage. METHODS: Twenty male Wistar rats weighing 300 ± 20 g (10 weeks of age) were submitted to IR (30 Gy) to the left leg, where the right leg was not irradiated. After 30 days, ten animals were submitted to HBO therapy, which was performed daily for 1 week at 250 kPa for 90-min sessions. All animals were euthanized 37 days after irradiation and the tibia were separated into four groups (n = 10): from animals without HBO - right tibia Non-irradiated (noIRnoHBO) and left tibia Irradiated (IRnoHBO); and from animals with HBO - right tibiae Non-irradiated (noIRHBO) and left tibia Irradiated (IRHBO). The length (proximal-distal) and thickness (anteroposterior and mediolateral) of the tibiae were measured. Biomechanical analysis evaluated flexural strength and stiffness. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used to calculate the amide I ratio, crystallinity index, and matrix to mineral ratios. RESULTS: In the macroscopic and ATR-FTIR analysis, the IRnoHBO showed lower values of length, thickness and amide I ratio, crystallinity index and matrix to mineral ratios compared to noIRnoHBO (p < 0.03). IRnoHBO showed no statistical difference compared to IRHBO for these analyses (p > 0.05). Biomechanics analysis showed that the IRnoHBO group had lower values of flexural strength and stiffness compared to noIRnoHBO and IRHBO groups (p < 0.04). In addition, the noIRHBO group showed higher value of flexural strength when compared to noIRnoHBO and IRHBO groups (p < 0.02). CONCLUSIONS: The present study concluded that IR arrests bone development, decreases the collagen maturation and mineral deposition process, thus reducing the flexural strength and stiffness bone mechanical parameters. Moreover, HBO therapy minimizes deleterious effects of irradiation on flexural strength and the bone stiffness analysis.

Hyberbaric oxygen and bone reconstruction.

INTRODUCTION: The aim of this literature review was to determine the benefits of hyperbaric oxygen therapy after bone reconstruction procedures in humans and identify information that may be useful for the development of optimal protocols for hyperbaric oxygen therapy to stimulate bone healing. EVIDENCE ACQUISITION: We searched the electronic database PubMed/Medline for studies published between January 1999 and December 2018, using the key words: "bone" or "bone graft" and "mandible reconstruction" or "jaw reconstruction" and "hyperbaric oxygen" or "HBO." First, the titles and abstracts of the studies found were evaluated and those that corresponded to the aims of this review were pre-selected for analysis of the full text. Subsequently, the full texts were analyzed, and those that met the eligibility criteria were pre-selected for the review. The full texts of studies whose abstracts did not provide enough data for decision were also evaluated. Two examiners independently assessed eligibility, risk of bias and extracted data. EVIDENCE SYNTHESIS: A total of 2237 studies were found according to pre-established criteria for data collection, of which only 5 studies were included in this systematic review. Although we observed positive results in the included studies, there are still few standardized clinical studies in the literature, assessing hyperbaric oxygen therapy after extensive bone reconstructive procedures. CONCLUSIONS: It is difficult to compare results found in different studies due to the variety of methodological and clinical conditions assessed.

Protective effects of a polyphenol-enriched fraction of the fruit peel of Annona crassiflora Mart. on acute and persistent inflammatory pain.

Different parts of Annona crassiflora Mart., a native species from Brazilian savanna, were traditionally used for the treatment of a wide variety of ailments including arthritis. Thus, this study aimed to investigate the possible antinociceptive and anti-inflammatory properties of a polyphenol-enriched fraction of the fruit peel of A. crassiflora, named here as EtOAc, in mice. Pro-inflammatory cytokines and nitric oxide (NO) production were evaluated in LPS-activated macrophages. Then, EtOAc fraction was administered by oral route in male C57BL/6/J mice, and the animals were submitted to glutamate-induced nociception and complete Freund's adjuvant (CFA)-induced monoarthritis tests to assess nociception (mechanical, spontaneous and cold pain) and inflammation (edema and neutrophil infiltration), and to the open-field and rotarod tests for motor performance analysis. EtOAc fraction inhibited the production of IL-6 and NO in the LPS-induced macrophages, and reduced spontaneous nociception induced by glutamate, without altering the animals' locomotor activity. In addition, the polyphenol-enriched fraction was able to revert the early and late hyperalgesia induced by CFA, as well as edema at the acute phase. Reduction of myeloperoxidase activity and inflammatory cell infiltration was observed in the paw tissue of mice injected with CFA and treated with EtOAc fraction. Together, our results support the antinociceptive and anti-inflammatory effects of the polyphenol-enriched fraction of A. crassiflora fruit peel and suggest that these effects are triggered, at least in part, by suppressing pro-inflammatory cytokines and neutrophils infiltration.

Influence of hyperbaric oxygen on biomechanics and structural bone matrix in type 1 diabetes mellitus rats.

BACKGROUND: The aim of this study was to evaluate the biomechanics and structural bone matrix in diabetic rats subjected to hyperbaric oxygen therapy (HBO). METHODS: Twenty-four male rats were divided into the following groups: Control; Control + HBO; Diabetic, and Diabetic + HBO. Diabetes was induced with streptozotocin (STZ) in the diabetic Groups. After 30 days, HBO was performed every 48h in HBO groups and all animals were euthanized 60 days after diabetic induction. The femur was submitted to a biomechanical (maximum strength, energy-to-failure and stiffness) and Attenuated Total Reflectance Fourier transform infrared (ATR-FTIR) analyses (crosslink ratio, crystallinity index, matrix-to-mineral ratio: Amide I + II/Hydroxyapatite (M:MI) and Amide III + Collagen/HA (M:MIII)). RESULTS: In biomechanical analysis, diabetic animals showed lower values of maximum strength, energy and stiffness than non-diabetic animals. However, structural strength and stiffness were increased in groups with HBO compared with non-HBO. ATR-FTIR analysis showed decreased collagen maturity in the ratio of crosslink peaks in diabetic compared with the other groups. The bone from the diabetic groups showed decreased crystallinity compared with non-diabetic groups. M:MI showed no statistical difference between groups. However, M:MIII showed an increased matrix mineral ratio in diabetic+HBO and control+HBO compared with control and diabetic groups. Correlations between mechanical and ATR-FTIR analyses showed significant positive correlation between collagen maturity and stiffness. CONCLUSIONS: Diabetes decreased collagen maturation and the mineral deposition process, thus reducing biomechanical properties. Moreover, the study showed that HBO improved crosslink maturation and increased maximum strength and stiffness in the femur of T1DM animals.

Hyperbaric Oxygen therapy effects on bone regeneration in Type 1 diabetes mellitus in rats.

PURPOSE: The aim of this study was evaluate the effect of HBO on diabetic rats. MATERIALS AND METHODS: Twenty rats were distributed into four groups (n = 5): Control (C); Control + HBO (CH); Diabetes (D) and Diabetes + HBO (DH). Diabetes was induced by streptozotocin, and bone defects were created in both femurs in all animals. HBO therapy began immediately after surgery and was performed daily in the CH and DH groups. After 7 days, the animals were euthanized. The femurs were removed, demineralized, embedded in paraffin, and histologic images were analyzed. RESULTS: Qualitative histologic analyses showed more advanced stage bone regeneration in control groups (C and CH) compared with diabetic groups (D and DH). Histomorphometric analysis showed significantly increased bone neoformation in CH compared with the other groups (p < 0.001). Diabetic Group (D) showed decreased bone neoformation compared with non-diabetic groups (C and CH) (p < 0.001); however DH did not differ from C Group (p > 0.05). The mast cell population increased in CH compared with the other groups (C, D, and DH) (p < 0.05). The mast cell population did not differ between D and DH Groups. CONCLUSIONS: This study showed that HBO therapy improved early bone regeneration in diabetic rats and increased the mast cell population only in non-diabetic animals. HBO was shown to be important treatment for minimizing deleterious effects of diabetes on bone regeneration.

Biological Effects of a Root Conditioning Treatment on Periodontally Affected Teeth--An In Vitro Analysis.

The aim of this study was to evaluated the root surfaces modifications resulted by application of different chemicals agents, and their influence on the fibrin network and fibroblasts attachment. From 96 anterior mandibular human extracted incisor teeth, 192 dentin blocks of buccal and lingual surface were obtained and randomly divided into 6 groups: Cont- control group, which received no treatment; Root surface scaling and root planing (Srp); Citric acid-Srp; EDTA-Srp; Tetracycline capsule-Srp; Tetracycline gel-Srp. After dentin treatments the specimens were analyzed as follows: 1) demineralization level and residues of the product by scanning electron microscopy (SEM); 2) adhesion of blood components after 20 min of surface treatment by SEM; 3) fibroblast attachment after 24 h by SEM; 4) cell metabolism after 24 h by MTT assay. Data were analyzed using Fisher Exact, One-way ANOVA test followed by Dunn's test, Tukey test and Dunnett test (α=0.05). Citric acid, EDTA and Tetracycline gel resulted in adequate demineralization with no completely smear layer and smear plug removal on root dentin surface. Tetracycline capsule produced great tetracycline residues with several demineralization areas. Tetracycline gel and EDTA groups presented more fibroblast fixation than other experimental groups. The highest mean blood clot adhesion score was observed in roots treated with tetracycline gel. EDTA and Tetracycline gel surface treatment removed the smear layer over dentin surface and promoted adhesion of fibrin network and fibroblast cells attachment.

Biological Effects of a Root Conditioning Treatment on Periodontally Affected Teeth - An In Vitro Analysis

Abstract The aim of this study was to evaluated the root surfaces modifications resulted by application of different chemicals agents, and their influence on the fibrin network and fibroblasts attachment. From 96 anterior mandibular human extracted incisor teeth, 192 dentin blocks of buccal and lingual surface were obtained and randomly divided into 6 groups: Cont- control group, which received no treatment; Root surface scaling and root planing (Srp); Citric acid-Srp; EDTA-Srp; Tetracycline capsule-Srp; Tetracycline gel-Srp. After dentin treatments the specimens were analyzed as follows: 1) demineralization level and residues of the product by scanning electron microscopy (SEM); 2) adhesion of blood components after 20 min of surface treatment by SEM; 3) fibroblast attachment after 24 h by SEM; 4) cell metabolism after 24 h by MTT assay. Data were analyzed using Fisher Exact, One-way ANOVA test followed by Dunn's test, Tukey test and Dunnett test (α=0.05). Citric acid, EDTA and Tetracycline gel resulted in adequate demineralization with no completely smear layer and smear plug removal on root dentin surface. Tetracycline capsule produced great tetracycline residues with several demineralization areas. Tetracycline gel and EDTA groups presented more fibroblast fixation than other experimental groups. The highest mean blood clot adhesion score was observed in roots treated with tetracycline gel. EDTA and Tetracycline gel surface treatment removed the smear layer over dentin surface and promoted adhesion of fibrin network and fibroblast cells attachment.

Resumo O objetivo deste estudo foi avaliar modificações nas superfícies radiculares sofridas pela aplicação de diferentes agentes químicos, e sua influência sobre a rede de fibrina e adesão de fibroblastos. A partir de 96 incisivos inferiores humanos, 192 blocos de dentina das superfícies vestibular e lingual foram obtidos e divididos aleatoriamente em 6 grupos: Cont-grupo controle, não recebeu nenhum tratamento; Raspagem e alisamento radicular (RAR); Ácido cítrico-SRP; EDTA-SRP; Tetraciclina em cápsula-SRP; Tetraciclina gel-SRP. Após o tratamento da dentina as espécimes foram analisadas: 1, nível de desmineralização e resíduos do produto por microscopia eletrônica de varredura (MEV); 2, adesão dos componentes sanguineos após 20 min na tratamento de superfície por SEM; 3, adesão de fibroblastos após 24h por SEM; 4, o metabolismo celular após 24 h por ensaio MTT. Os dados foram analisados por Fisher Exact, teste one-way ANOVA, seguido pelo teste de Dunn, teste de Tukey e teste de Dunnett (α = 0,05). O ácido cítrico, EDTA e gel tetraciclina resultaram na adequada desmineralização sem remoção completa de camada de smear layer e smear plug sobre a superfície da dentina radicular. Cápsula de tetraciclina produziu grandes resíduos de tetraciclina com várias áreas de desmineralização. Os grupos Gel de tetraciclina e EDTA apresentaram maior adesão de fibroblastos do que os demais grupos experimentais. O maior score de adesão de coágulo sanguineo foi observado nas superfícies tratadas com gel de tetraciclina. EDTA e Gel de tetraciclina removeram a camada de smear layer sobre a superfície da dentina e promoveu adesão da rede de fibrina e de fibroblastos.

Influence of hyperbaric oxygen on the initial stages of bone healing.

OBJECTIVE: The objective of this study was to evaluate, in a rat model, the effect of hyperbaric oxygen (HBO) on the healing of normal bone on day 7. STUDY DESIGN: Forty male rats were used, equally divided into two groups based on treatment and time of sacrifice: the control group had bone defects created; and the HBO group had bone defects and received HBO. HBO sessions were conducted daily, at 2.5 atmosphere absolute for 90 minutes, and the animals were euthanized after 1, 3, 5, or 7 days. Bone density, bone neoformation, and expression of Runt-related transcription factor 2 (Runx2) and tartrate-resistant acid phosphatase were evaluated. RESULTS: Computed tomography analysis revealed significant differences only at 3 days (P=.01) between the control and HBO groups. HBO treatment accelerated the initial events of bone repair, resulting in improved bone neoformation. Increased expression of Runx2 was observed, especially on days 5 and 7 in the HBO group, although not significantly. There was no significant difference (P=.74) in the number of tartrate-resistant acid phosphatase-positive osteoclasts between the control and HBO groups on day 7. CONCLUSIONS: These results suggest that exposure to HBO enhances bone anabolism, reduces inflammation, and accelerates bone healing, with positive results in bone neoformation. Therefore, the aim of this study was to evaluate the effect of HBO on the healing of experimental defects created in normal bone, on the first 7 days, in a rat model.

Low-level laser therapy on bone repair: is there any effect outside the irradiated field?

The biological effects of local therapy with laser on bone repair have been well demonstrated; however, this possible effect on bone repair outside the irradiated field has not been evaluated. The aim of this study was to investigate the effect of low-level laser therapy (LLLT) (λ = 830 nm) on repair of surgical bone defects outside the irradiated field, in rats. Sixty Wistar rats were submitted to osteotomy on the left femur and randomly separated into four groups (n = 15): group I, control, bone defect only; group II, laser applied on the right femur (distant dose); group III, laser applied locally on the bone defect and also on the right femur (local and distant doses); and group IV, laser applied locally on the left femur (local dose). Laser groups received applications within a 48-h interval in one point per session of density energy (DE) = 210 J/cm(2), P = 50 mW, t = 120 s, and beam diameter of 0.028 cm. Five animals of each group were euthanized 7, 15, and 21 days after surgery. Histologic analysis in all groups showed new bone formation in the region of interest (ROI) at 7 days. After 15 days, bone remodeling with a decrease of bone neoformation in the marrow area was observed in all groups. After 21 days, advanced bone remodeling with new bone mostly located in the cortical area was observed. The histomorphometric analysis showed at 7 days a significant increase of bone formation in groups III and IV compared to groups I and II. At days 15 and 21, histomorphometric analysis showed no significant differences between them. Laser therapy presented a positive local biostimulative effect in the early stage of bone healing, but the LLLT effect was not observed a long distance from the evaluated area.

Effect of low-level laser therapy on repair of the bone compromised by radiotherapy.

Radiotherapy (RDT) is commonly used for cancer treatment, but high doses of ionizing radiation can directly affect healthy tissues. Positive biological effects of low-level laser therapy (LLLT) on bone repair have been demonstrated; however, this effect on surgical defects of bone previously compromised by radiotherapy has not been evaluated. The aim of this study was to investigate the influence of LLLT (λ = 830 nm) in femur repair after ionizing radiation. Twenty Wistar rats were divided into four groups: control group (GC, n = 5) creation of bone defects (BDs) only; laser group (GL), with BD and LLLT (n = 5); radiotherapy group (GR), submitted to RDT and BD (n = 5); and radiotherapy and laser group (GRL), submitted to RDT, BD, and LLLT (n = 5). GL and GRL received punctual laser application (DE = 210 J/cm(2), P = 50 mW, t = 120 s, and beam diameter of 0.04 cm(2)) immediately after surgery, with 48-h interval during 7 days. Animals were euthanized at 7 days after surgery, and bone sections were evaluated morphometrically with conventional microscopy. Bone repair was only observed in nonirradiated bone, with significant improvement in GL in comparison to GC. GR and GRL did not present any bone neoformation. The result demonstrated a positive local biostimulative effect of LLLT in normal bone. However, LLLT was not able to revert the bone metabolic damage due to ionizing radiation.