Fibrin Network and Platelets Densities in Platelet-Rich Fibrin (PRF) Membranes Produced from Plastic Tubes Without Additives: A New Approach to PRF Clinical Use.

Background/Purpose: The present study aimed to investigate plastic tubes without additives as alternatives to glass and silica-coated plastic tubes, in the production of PRF membranes. Materials and Methods: Nine blood samples were collected from eight volunteers (n = 8) separated into three groups, according to tube material: glass, silica-coated plastic, and plastic without additives. In each group, the samples were centrifuged using different relative centrifugation forces: L-PRF (700 g/12 min), A-PRF (200 g/14 min), and A-PRF + (200 g/8 min). The generated membranes were evaluated by histomorphometry, considering the fibrin network, platelet aggregates, and cellular morphology, by light microscopy. The ultrastructural cellular morphology integrity was evaluated by transmission electron microscopy. Results: The L-PRF (p < 0.019) and A-PRF (p < 0.001) membranes showed a significantly lower fibrin network density in plastic tubes without additives compared to glass and silica-coated plastic tubes. Plastic tubes without additives revealed a significantly higher platelet percentage, regardless of the protocol (p < 0.005). In all groups, TEM analysis showed preserved normal morphological ultrastructure, maintaining the integrity of cellular components. Conclusion: Plastic tubes without additives offer a viable alternative for producing PRF membranes. They exhibited a higher platelet density and demonstrated fibrin network and cellular morphology similar to those of glass and silica-coated plastic tubes, irrespective of the centrifugation protocol.

Insulin Therapy on Bone Macroscopic, Microarchitecture, and Mechanical Properties of Tibia in Diabetic Rats.

BACKGROUND: This study evaluated tibia's macroscopic structure, mechanical properties, and bone microarchitecture in rats with type 1 diabetes mellitus (T1DM). METHODS: Eighteen animals were divided into three groups (n=6): non-diabetic (ND), diabetic (D), and diabetic+insulin (DI). T1DM was induced by streptozotocin; insulin was administered daily (4IU). The animals were euthanized 35 days after induction. The tibiae were removed and analyzed using macroscopic, micro-computed tomography (micro-CT) and three-point bending. The macroscopic analysis measured proximal-distal length (PD), antero-posterior thickness (AP) of proximal (AP-P) and distal (AP-D) epiphysis, and lateral-medial thickness (LM) of proximal (LM-P) and distal (LM-D) epiphysis. Micro-CT analysis closed porosity, tissue mineral density, and cortical thickness. The three-point bending test measured maximum strength, energy, and stiffness. RESULTS: The macroscopic analysis showed that D presented smaller measures of length and thickness (AP and AP-P) than ND and DI. More extensive measurements were observed of LM and AP-D thickness in DI than in D. In micro-CT, DI showed larger cortical thickness than D. Mechanical analysis showed lower strength in D than in other groups. CONCLUSIONS: T1DM reduces bone growth and mechanical strength. Insulin therapy in diabetic rats improved bone growth and fracture resistance, making diabetic bone similar to normoglycemic animals.

Platelet-Rich Fibrin Progressive Protocol: Third Generation of Blood Concentrates.

PURPOSE: Platelet-rich fibrin (PRF) has been used in several fields of dentistry to improve tissue healing. However, PRF from glass tubes results in a limited number of small membranes, increasing clinical difficulty and work time. The aim of this study was to evaluate cell and platelet amounts and biomechanical strength of PRF-giant membranes produced from plastic tubes without additives. MATERIAL AND METHODS: The investigators designed an ex vivo study, to compare 3 different centrifugation protocols for obtaining PRF: 700 × g/12 minutes (leukocyte and PRF [L-PRF]), 350 × g/14 minutes (GM350), and 60-700 × g more than 15 minutes total (progressive PRF [PRO-PRF]). We collected blood samples from 5 volunteers aged 25-54 years, over 3 different time periods (triplicate and paired study). From each venipuncture, 4 mL of blood was collected in vacutainers with ethylenediamine tetraacetic acid (EDTA) and approximately 104 mL in 12 plastic tubes without additives, which were separated into 3 groups, as per the centrifugation protocols (n = 5): L-PRF, GM350, and PRO-PRF. The PRF from the tubes of the same protocol was aspirated and 9 mL were placed in polylactic acid (PLA) forms and 3 mL were placed in a glass receptacle. The membranes from PLA forms were tested for tensile strength and the membranes from glass receptacles were evaluated by histomorphometry, while platelets and leukocytes were counted for those in tubes with EDTA. Statistical analyses were performed using Shapiro-Wilk normality test and then a one-way repeated measures analysis followed by Tukey multiple comparisons test (α < 0.05). RESULTS: In tensile analyses, PRO-PRF (0.85 ± 0.23 N) showed a significantly higher maximum breaking strength than L-PRF (0.61 ± 0.26 N, P = .01) and GM350 (0.58 ± 0.23 N, P < .01). The histomorphometry revealed no significant statistical difference in cell counts between the groups (P = .52). Furthermore, there was no significant difference between the leukocyte (P = .25) and platelet counts (P = .59) in whole blood between the groups. CONCLUSION: The progressive protocol (PRO-PRF) enabled the production of PRF giant membranes with greater tensile strength and adequate cell distribution. Moreover, it allows biomaterial incorporation during production and enables clinical control of membrane thickness and size as per the surgical procedure.

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.

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.

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.

Ketal Sugar Conversion Into Green Hydrocarbons by Faujasite Zeolite in a Typical Catalytic Cracking Process.

Fluidized catalytic cracking (FCC) converts hydrocarbons in the presence of a catalyst based on faujasite zeolite (USY and REY). While hydrocarbon is poorly reactive, biomass and its derived compounds are highly functionalized and not suitable to a typical FCC process. To overcome this limitation biomass was first converted into a dense and stable bio-crude composed mainly of ketal-sugar derivatives by using acetone in diluted acid. Here, a representative compound of this bio-crude, 1,2:3,5-di-O-isopropylidene-α-D-xylofuranose (DX) in n-hexane, was converted by USY and a commercial FCC catalyst containing USY, at 500°C, in a fixed bed and fluidized bed reactors, respectively. Faujasite Y is very efficient in converting DX. More than 95% conversion was observed in all tests. Over 60 wt.% was liquid products, followed by gas products and only around 10% or less in coke. The higher the catalyst activity the greater the aromatics in the liquid products and yet higher coke yields were observed. In particular, simulating more practical application conditions: using deactivated catalyst in a fluidized bed reactor, improved green hydrocarbons production (mono-aromatic up to 10 carbons and light hydrocarbon up to eight carbons) and unprecedented lower coke yield (≈5 wt.%) for bio-feeds. The present results further suggest that catalyst will play a primary role to convert the bio-crude into target hydrocarbons and overcome the transition of a non-renewable to a renewable refinery feed.

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.