Birefringence analysis of collagen supraorganization in cat corneas with tropical keratopathy.

OBJECTIVE: To evaluate the birefringent properties of the cornea and examine the supraorganizational aspects of collagen fibers in cats with tropical keratopathy. PROCEDURE: In this study, 10-micrometer-thick sections of corneal tissue from cats with tropical keratopathy were examined, both in the opaque and transparent areas of the anterior stroma. Control samples were obtained from healthy cat corneas. Polarized light microscopy was employed to evaluate the birefringent properties using two distinct methods. The first method involved measuring the optical retardation associated with corneal birefringence, while the second method assessed the alignment/waviness of the birefringent collagen fibers. Differences were significant when p < .05. RESULTS: Tropical keratopathy resulted in a significant rise (p < .05) in optical retardation in both opaque and transparent regions of the cat cornea. In the anterior stroma, both the opaque zones and transparent tissue exhibited a higher degree of collagen fiber packing than the control corneas. However, no significant differences (p > .05) in alignment were observed between the transparent tissue of the diseased cornea and the healthy corneas. CONCLUSION: Supraorganizational changes in collagen fiber packing are not restricted to lesion zones in cat corneas affected by tropical keratopathy. Such alterations also occur in the corneal tissue of the anterior stroma adjoining the lesions. Therefore, it is plausible that the transparent tissue of the anterior stroma in corneas affected by the disease may have functional abnormalities, despite its macroscopic healthy appearance. Additional investigations are required to clarify the implications of these potential defects and their conceivable contribution to tropical keratopathy.

Sperm selection by birefringence: a promising non-invasive tool to improve ICSI outcomes.

OBJECTIVE: Despite higher sperm DNA fragmentation may affect intracytoplasmic sperm injection (ICSI) outcomes, sperm selection protocols do not evaluate this parameter. Therefore, sperm's head birefringence has been suggested as an adjuvant of seminal processing to select viable sperm for couples with severe male factor. Considering men with normal seminal parameters may also curse with DNA fragmentation, the aim of this study was to evaluate the impact of sperm selection by birefringence on ICSI outcomes in couples with different infertility factors compared to those submitted to conventional sperm selection. METHODS: In this case-control study, medical records from 181 couples who underwent ICSI from January 2018 to August 2020 (107 from the Conventional and 74 from the Birefringence group) were included in the study. Clinical characteristics and ICSI outcomes were compared between the groups using Student's t test or Chi-square test (p<0.05) and a multivariate logistic regression model was applied regarding clinical pregnancy. RESULTS: Despite the Birefringence group showed higher female age (p=0.01), lower seminal sperm concentration (p<0.01) and higher sperm DNA fragmentation (p<0.01), those patients cursed with both higher cleavage rate (p=0.04), clinical pregnancy rate per transfer (p=0.03) and clinical pregnancy rate per initiated cycle (p=0.02). The logistic regression showed a positive group effect on clinical pregnancy. CONCLUSIONS: The findings suggest a positive clinical impact of this cheap and easily reproducible adjuvant technique on ICSI outcomes in couples with different infertility factors. If confirmed by further methodologically appropriate studies, the sperm's head birefringence could be considered to improve the reproductive chances of those patients.

Variability in the Epithelial Expression of Ki67 and Polarization Characteristics of Collagen in Odontogenic Cysts and Tumours

ABSTRACT Objective: To assess the proliferation of epithelium (using the Ki67 index) and the polarization pattern of collagen in selected odontogenic cysts and tumours. In addition, an exploratory analysis of the effect of inflammation on the proliferation rate was done. Material and Methods: Following immunohistochemical staining, the labelling/proliferation index of Ki67 was calculated. The thickness and corresponding polarization colour of 100 juxta-epithelial picrosirius red-stained collagen fibers were assessed using linear micrometry with an eyepiece reticule under × 1000 magnification. Inflammation was graded subjectively as mild, moderate, and severe. Results: Overall Ki-67 expression was higher in the radicular cyst, Odontogenic Keratocyst, Ameloblastoma, while suprabasal Ki-67 positivity was maximum in Odontogenic Keratocyst. The stromal collagen fibers in Ameloblastoma showed predominantly green birefringence, whereas Odontogenic Keratocyst had orange birefringence. There was no significant association of inflammation with Ki-67 expression or birefringence patterns. Conclusion: The highest Ki67 expression in the radicular cyst, followed by Odontogenic Keratocyst and Ameloblastoma. Differences in the collagen maturation pattern were noted innately in five lesions studied and were further influenced by inflammatory changes. Epithelial proliferation and concomitant expression of thickness and maturity of the stromal collagen are innate features of the lesion further influenced by inflammation in various odontogenic cysts and tumours and may, in turn, guide the clinical behavior.

Birefringence Analyses Reveal Differences in Supramolecular Characteristics of Corneal Stromal Collagen Fibrils Between Falconiformes and Strigiformes.

This study aimed to assess the birefringent properties of corneal stromal collagen fibrils in birds of the orders Falconiformes (diurnal) and Strigiformes (predominantly nocturnal) to compare their supramolecular organizations. In total, 22 corneas of Falconiformes (Caracara plancus, n = 8; Rupornis magnirostris, n = 10; and Falco sparverius, n = 4) and 28 of Strigiformes (Tyto furcata, n = 16; Pseudoscops clamator, n = 6; and Athene cunicularia, n = 6) were processed histotechnically into 8-µm thick sections. Corneal optical retardation (OR) values related to the form and intrinsic fractions of the total birefringence of collagen fibrils were measured using a polarized light microscope equipped with phase compensators. In addition, the coherence coefficients that inform the local orientation of the fibrils were calculated through video image analysis. All assessments were conducted both in the anterior and posterior stroma of the cornea. Differences were significant when P < 0.05. The results showed supraorganizational differences between fibrils in the anterior stroma of Falconiformes and Strigiformes. The OR values were greater (P < 0.0001) for Falconiformes, indicating that the corneas of these birds contain more collagen fibrils or more aggregated collagen fibrils. In contrast, the coherence coefficients were higher (P = 0.016) for Strigiformes, indicating that the corneal collagen fibers in these birds are highly aligned and have few undulations. A multivariate data matrix constructed for Euclidean distance calculations showed that the dissimilarity between Falconiformes and Strigiformes corneas, in terms of the supraorganization of stromal collagen fibrils, was 4.56%. In conclusion, it is possible that the supraorganizational differences reported in this study may be sources of variation in the visual quality of Falconiformes and Strigiformes. This study provides the necessary evidence to encourage further research associating corneal optical performance to supramolecular characteristics of corneal stromal collagen.

In Vivo Biological Behavior of Polymer Scaffolds of Natural Origin in the Bone Repair Process.

Autologous bone grafts, used mainly in extensive bone loss, are considered the gold standard treatment in regenerative medicine, but still have limitations mainly in relation to the amount of bone available, donor area, morbidity and creation of additional surgical area. This fact encourages tissue engineering in relation to the need to develop new biomaterials, from sources other than the individual himself. Therefore, the present study aimed to investigate the effects of an elastin and collagen matrix on the bone repair process in critical size defects in rat calvaria. The animals (Wistar rats, n = 30) were submitted to a surgical procedure to create the bone defect and were divided into three groups: Control Group (CG, n = 10), defects filled with blood clot; E24/37 Group (E24/37, n = 10), defects filled with bovine elastin matrix hydrolyzed for 24 h at 37 °C and C24/25 Group (C24/25, n = 10), defects filled with porcine collagen matrix hydrolyzed for 24 h at 25 °C. Macroscopic and radiographic analyses demonstrated the absence of inflammatory signs and infection. Microtomographical 2D and 3D images showed centripetal bone growth and restricted margins of the bone defect. Histologically, the images confirmed the pattern of bone deposition at the margins of the remaining bone and without complete closure by bone tissue. In the morphometric analysis, the groups E24/37 and C24/25 (13.68 ± 1.44; 53.20 ± 4.47, respectively) showed statistically significant differences in relation to the CG (5.86 ± 2.87). It was concluded that the matrices used as scaffolds are biocompatible and increase the formation of new bone in a critical size defect, with greater formation in the polymer derived from the intestinal serous layer of porcine origin (C24/25).

Análise do processo de reparo alveolar em camundongos fêmeas senescentes 129/Sv-WT e 129/Sv 5-LOKO sob terapia com bifosfonato de alta potência / Alveolar bone repair of 129/Sv WT and 129/Sv 5LOKO female senescent mice under therapy with high potency bisphosphonate

O processo de senescência acarreta uma série de modificações fisiológicas com declínio das funções das atividades celulares e sistêmicas que se manifestam de maneira mais importante na população feminina pelo evento da menopausa, como a osteoporose. A fim de se minimizar tais efeitos, há a possibilidade de se utilizar medicamentos que diminuem o processo de remodelação óssea como os bifosfonatos nitrogenados (BF). Entretanto, o uso dessas drogas está intimamente relacionado ao desenvolvimento de osteonecrose dos maxilares (OM), principalmente quando associado a outros fatores de risco como as cirurgias bucais. Sabe-se que fisiologicamente a dinâmica do tecido ósseo depende também de eicosanóides derivados do metabolismo do ácido araquidônico (AA), como as enzimas cicloxigenase (COX) e 5 lipoxigenase (5LO). Deste modo, o objetivo do presente trabalho foi analisar o efeito BF ácido zoledrônico (ZL) e sua relação com o desenvolvimento da OM em camundongos fêmeas senescentes 129/Sv com e sem modificação genética para a enzima 5LO. Para tanto, foram utilizados 40 camundongos fêmeas senescentes 129/Sv, sendo 20 WT e 20 com alteração no gene 5LO (129 Alox5tm1Fun/J) (5LOKO), divididas em grupos: WT, tratadas com 0,01 ml de solução salina 0,9% estéril (SS) via intraperitoneal (IP) e ZL, tratadas com 250µg/Kg de ácido zoledrônico (ZL) IP diluído em solução salina estéril, ambas administradas 1 vez/semana por 7 semanas. Os grupos foram compostos por 5 animais cada (WT Controle ­ 7 e 21dias, WT ZL ­ 7 e 21 dias, 5LOKO Controle ­ 7 e 21 dias, 5LOKO ZL ­ 7 e 21dias), sendo as maxilas coletadas para análises em microCT, histopatológica, birrefringência, técnica imunohistoquímica e histomorfométricas. De modo geral, a microCT revelou deficiência significativa na microarquitetura óssea nos animais WT ZL em comparação com os demais. Do mesmo modo, a partir da análise histopatológica e de birrefringência da matriz colagenosa, observou-se padrão compatível com o desenvolvimento de OM no grupo WT ZL, com presença de infiltrado inflamatório intenso, atraso na neoformação óssea, presença de fraturas patológicas, e deficiência da matriz colagenosa e de células Runx-2+, TRAP+ e F4/80+. Os animais 5LOKO ZL apresentaram alterações compatíveis com atraso no processo de reparo especialmente no período de 7 dias, com menor quantidade de células Runx-2+ em comparação com o grupo 5LOKO Controle e pela qualidade da matriz óssea colagenosa com menor quantidade de fibras do espectro vermelho neste período, se igualando, porém, aos 21 dias. Deste modo, concluiu-se que o processo de reparo em camundongos fêmeas senescentes da linhagem 129/Sv WT e 5LOKO associados ao uso do BF ZL ocorreu de modo distinto, levando a quadro de OM nos animais WT e atraso nos animais 5LOKO, sem sinais histopatológicos que caracterizassem a doença. Deste modo, a inibição da enzima 5LO parece influenciar de maneira positiva o processo de reparo ósseo intramembranoso alveolar, mesmo na presença de fenótipo esqueletal osteopetrótico, sugerindo outros fatores relacionados à droga que favoreçam o desenvolvimento da OM no presente modelo animal(AU)

Senescence brings a number of physiological modifications with the decrease of cell and systemic activities and function that manifest in an important way in female population due to the event of menopause, as osteoporosis. In order to diminish these effects, there is the possibility of taking medication that decrease bone remodeling process, as the bisphosphonates containing nitrogen (BF). However, the use of these drugs is intimate related with the development of the osteonecrosis of the jaws (ON), especially when associated to other risk factors as oral surgery. It is known that physiologically, the dynamics of bone tissue also depends on the eicosanoids derivate from the arachidonic acid metabolism (AA), such as cyclooxygenase (COX) and 5 lipoxygenase (5LO) enzymes. In this way, the aim of the present study was to analyze the effects of the BF zoledrônico acid (ZL) and its relation with de development of ON in 129/SV old female mice with or without genetic modification for 5LO. Forty animals, 20 WT and 20 with 5LO gene alteration (129 Alox5tm1Fun/J) (5LOKO) were divided in groups: WT, treated with 0.01 ml of sterile 0.9% saline solution (SS) intraperitoneal (IP), and ZL, treated with 250µg/Kg of ZL IP diluted in SS, both administered once a week for 7 weeks. Groups contained 5 animals each (WT Control ­ 7 and 21 days, WT ZL ,7 and 21 days, 5LOKO Control, 7 and 21 days, and 5LOKO ZL, 7 and 21 days), and the maxillae removed for microCT, histopathology, birefringence, immunohistochemistry, and histomorphometric analysis. In general, microCT revealed significant deficiency in bone microarchitecture in WT ZL group in comparison to the other groups. In the same way, histopathological and birefringence analysis revealed histological pattern compatible with ON development in WT ZL group, presenting intense inflammatory infiltrate, late new bone formation, presence of pathological fractures, and deficiency in collagenous matrix, and also in Runx-2+, TRAP+, and F4/80. 5LOKO ZL animals presented alterations compatible with a late bone repair, especially at day 7, with decreased number of Runx-2+ cells in comparison to 5LOKO Control, and by the quality of collagenous bone matrix with decreased number of red spectra fibers in this period, however, being similar at day 21. From this, it could be concluded that alveolar bone repair of 129/SV WT and 5LOKO old female mice associated with the administration of ZL occurred in different ways, leading to a picture of ON in the WT animals, and late bone repair in the 5LOKO animals, without histopathological signs that could characterize the disease. In this way, inhibition of 5LO seems to influence intramembranous alveolar bone repair in a positive way, even in the presence of osteopetrotic skeletal phenotype, suggesting other factors related to the drug that favors the development of the ON in the present animal model(AU)

Transected Tendon Treated with a New Fibrin Sealant Alone or Associated with Adipose-Derived Stem Cells.

Tissue engineering and cell-based therapy combine techniques that create biocompatible materials for cell survival, which can improve tendon repair. This study seeks to use a new fibrin sealant (FS) derived from the venom of Crotalus durissus terrificus, a biodegradable three-dimensional scaffolding produced from animal components only, associated with adipose-derived stem cells (ASC) for application in tendons injuries, considered a common and serious orthopedic problem. Lewis rats had tendons distributed in five groups: normal (N), transected (T), transected and FS (FS) or ASC (ASC) or with FS and ASC (FS + ASC). The in vivo imaging showed higher quantification of transplanted PKH26-labeled ASC in tendons of FS + ASC compared to ASC on the 14th day after transection. A small number of Iba1 labeled macrophages carrying PKH26 signal, probably due to phagocytosis of dead ASC, were observed in tendons of transected groups. ASC up-regulated the Tenomodulin gene expression in the transection region when compared to N, T and FS groups and the expression of TIMP-2 and Scleraxis genes in relation to the N group. FS group presented a greater organization of collagen fibers, followed by FS + ASC and ASC in comparison to N. Tendons from ASC group presented higher hydroxyproline concentration in relation to N and the transected tendons of T, FS and FS + ASC had a higher amount of collagen I and tenomodulin in comparison to N group. Although no marked differences were observed in the other biomechanical parameters, T group had higher value of maximum load compared to the groups ASC and FS + ASC. In conclusion, the FS kept constant the number of transplanted ASC in the transected region until the 14th day after injury. Our data suggest this FS to be a good scaffold for treatment during tendon repair because it was the most effective one regarding tendon organization recovering, followed by the FS treatment associated with ASC and finally by the transplanted ASC on the 21st day. Further investigations in long-term time points of the tendon repair are needed to analyze if the higher tissue organization found with the FS scaffold will improve the biomechanics of the tendons.

Bioactive glass-ceramic bone repair associated or not with autogenous bone: a study of organic bone matrix organization in a rabbit critical-sized calvarial model.

OBJECTIVE: The aim of the study was to analyze bone matrix (BMX) organization after bone grafting and repair using a new bioactive glass-ceramic (Biosilicate®) associated or not with particulate autogenous bone graft. MATERIAL AND METHODS: Thirty rabbits underwent surgical bilateral parietal defects and divided into groups according to the materials used: (C) control-blood clot, (BG) particulate autogenous bone, (BS) bioactive glass-ceramic, and BG + BS. After 7, 14, and 30 days post-surgery, a fragment of each specimen was fixed in - 80 °C liquid nitrogen for zymographic evaluation, while the remaining was fixed in 10% formalin for histological birefringence analysis. RESULTS: The results of this study demonstrated that matrix organization in experimental groups was significantly improved compared to C considering collagenous organization. Zymographic analysis revealed pro-MMP-2, pro-MMP-9, and active (a)-MMP-2 in all groups, showing gradual decrease of total gelatinolytic activity during the periods. At day 7, BG presented more prominent gelatinolytic activity for pro-MMP-2 and 9 and a-MMP-2, when compared to the other groups. In addition, at day 7, a 53% activation ratio (active form/[active form + latent form]) was evident in C group, 33% in BS group, and 31% in BG group. CONCLUSION: In general, BS allowed the production of a BMX similar to BG, with organized collagen deposition and MMP-2 and MMP-9 disponibility, permitting satisfactory bone remodeling at the late period. CLINICAL RELEVANCE: The evaluation of new bone substitute, with favorable biological properties, opens the possibility for its use as a viable and efficient alternative to autologous bone graft.

Alveolar bone healing in mice genetically selected in the maximum (AIRmax) or minimum (AIRmin) inflammatory reaction.

The exact role of inflammatory immune response in bone healing process is still unclear, but the success of the alveolar bone healing process seems to be associated with a moderate and transitory inflammatory response, while insufficient or exacerbated responses seems to have a detrimental influence in the healing outcome. In this context, we performed a comparative analysis of mice strains genetically selected for maximum (AIRmax) or minimum (AIRmin) acute inflammatory response to address the influence of inflammation genes in alveolar bone healing outcome. Experimental groups comprised 8-week-old male or female AIRmax and AIRmin submitted to extraction of upper right incisor, and evaluated at 0, 3, 7, 14 and 21 days after upper incision extraction by micro-computed tomography (µCT), histomorphometry, birefringence, immunohistochemistry and molecular (PCRArray) analysis. Overall, the results demonstrate a similar successful bone healing outcome at the endpoint was evidenced in both AIRmin and AIRmax strains. The histormophometric analysis reveal a slight but significant decrease in blood clot and inflammatory cells density, as well a delay in the bone formation in AIRmax strain in the early times, associated with a decreased expression of BMP2, BMP4, BMP7, TGFb1, RUNX2, and ALP. The evaluation of inflammatory cells nature reveals increased GR1+ cells counts in AIRmax strain at 3d, associated with increased levels of neutrophil chemoattractants such as CXCL1 and CXCL2, and its receptor CXCR1, while F4/80+ cell prevails in AIRmin strain at 7d. Also, our results demonstrate a relative predominance of M2 macrophages in AIRmin strain, associated with an increased expression of ARG1, IL10, TGFb, while M1 macrophages prevail in AIRmax, which parallel with increased IL-1B, IL-6 and TNF expression. At late repair stage, AIRmax presents evidences of increased bone remodeling, characterized by increased density of blood vessels and osteoclasts in parallel with decreased bone matrix density, as well increased levels of MMPs, osteoclastogenic and osteocyte markers. In the view of contrasting inflammatory and healing phenotypes of AIRmin and AIRmax strains in other models, the unpredicted phenotype observed suggests the existence of specific QTLs (Quantitative trait loci) responsible for the regulation 'sterile' inflammation and bone healing events. Despite the similar endpoint healing, AIRmax strain delayed repair was associated with increased presence of neutrophils and M1 macrophages, supporting the association of M2 cells with faster bone healing. Further studies are required to clarify the elements responsible for the regulation of inflammatory events at bone healing sites, as well the determinants of bone healing outcome.

Evaluation of dermal collagen stained with picrosirius red and examined under polarized light microscopy.

The special picrosirius red staining highlights the natural birefringence of collagen fibers when exposed to polarized light. The results from birefringence allow to evaluate the organization of the collagen fibers in the tissues. The authors intend to elucidate all steps to obtain and capture images of histological sections stained with picrosirius red and evaluated under polarized light microscopy, as well as possible artefacts that may occur.

Evaluation of dermal collagen stained with picrosirius red and examined under polarized light microscopy

Abstract: The special picrosirius red staining highlights the natural birefringence of collagen fibers when exposed to polarized light. The results from birefringence allow to evaluate the organization of the collagen fibers in the tissues. The authors intend to elucidate all steps to obtain and capture images of histological sections stained with picrosirius red and evaluated under polarized light microscopy, as well as possible artefacts that may occur.

[Polarized light microscopy for evaluation of oocytes as a prognostic factor in the evolution of a cycle in assisted reproduction]. / Microscopia de luz polarizada para evaluación de ovocitos como factor pronóstico en la evolución de un ciclo de reproducción asistida.

BACKGROUND: Identification of the best embryos to transfer is a key element for success in assisted reproduction. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems has allowed the visualization of the meiotic spindle and the different layers of the zona pellucida in human oocytes on the basis of birefringence in a non-destructive way. Conflicting results have been reported regarding the predictive value in ICSI cycles. OBJECTIVE: To assess the predictive ability of meiotic spindle and zona pellucida of human oocytes to implant by polarized microscopy in ICSI cycles. MATERIAL AND METHODS: Prospective and observational clinical study. 903 oocytes from 94 ICSI cycles were analyzed with polarized microscopy. Meiotic spindle visualization and zona pellucida birefringence values by polarized microscopy were correlated with ICSI cycles results. RESULTS: Meiotic spindle visualization and birefringence values of zona pellucida decreased in a direct basis with increasing age. In patients aged over the 35 years, the percentage of a visible spindle and mean zona pellucida birefringence was lower than in younger patients. Fertilization rate were higher in oocytes with visible meiotic spindle (81.3% vs. 64%; p < 0.0001), as well as embryo quality (47.4% vs. 39%; p=0.01). Fertilization rate was higher in oocytes with positive values of birefringence (77.5 % vs. 68.5% p=0.005) with similar embryo quality. Conception cycles showed oocytes with higher mean value of zona birefringence and visible spindle vs. no-conception cycles (p<0.05). CONCLUSIONS: Polarized light microscopy improves oocyte selection, which significantly impacts in the development of embryos with greater implantation potential. The use of polarized light microscopy with sperm selection methods, blastocyst culture and deferred embryo transfers will contribute to transfer fewer embryos without diminishing rates of live birth and single embryo transfer will be more feasible.

Host inflammatory response to polypropylene implants: insights from a quantitative immunohistochemical and birefringence analysis in a rat subcutaneous model.

OBJECTIVES: To describe acute and sub acute aspects of histological and immunohistochemical response to PP implant in a rat subcutaneous model based on objective methods. MATERIALS AND METHODS: Thirty rats had a PP mesh subcutaneously implanted and the same dissection on the other side of abdomen but without mesh (sham). The animals were euthanized after 4 and 30 days. Six slides were prepared using the tissue removed: one stained with hematoxylin-eosin (inflammation assessment); one unstained (birefringence evaluation) and four slides for immunohistochemical processing: IL-1 and TNF-α (pro-inflammatory cytokines), MMP-2 (collagen metabolism) and CD-31 (angiogenesis). The area of inflammation, the birefringence index, the area of immunoreactivity and the number of vessels were objectively measured. RESULTS: A larger area of inflammatory reaction was observed in PP compared to sham on the 4th and on the 30th day (p=0.0002). After 4 days, PP presented higher TNF (p=0.0001) immunoreactivity than sham and no differences were observed in MMP-2 (p=0.06) and IL-1 (p=0.08). After 30 days, a reduction of IL-1 (p=0.010) and TNF (p=0.016) for PP and of IL-1 (p=0.010) for sham were observed. Moreover, area of MMP-2 immunoreactivity decreased over time for PP group (p=0.018). Birefringence index and vessel counting showed no differences between PP and sham (p=0.27 and p=0.58, respectively). CONCLUSIONS: The implantation of monofilament and macroporous polypropylene in the subcutaneous of rats resulted in increased inflammatory activity and higher TNF production in the early post implant phase. After 30 days, PP has similar cytokines immunoreactivity, vessel density and extracellular matrix organization.

Host inflammatory response to polypropylene implants: insights from a quantitative immunohistochemical and birefringence analysis in a rat subcutaneous model

ABSTRACT Objectives To describe acute and sub acute aspects of histological and immunohistochemical response to PP implant in a rat subcutaneous model based on objective methods. Materials and Methods Thirty rats had a PP mesh subcutaneously implanted and the same dissection on the other side of abdomen but without mesh (sham). The animals were euthanized after 4 and 30 days. Six slides were prepared using the tissue removed: one stained with hematoxylin-eosin (inflammation assessment); one unstained (birefringence evaluation) and four slides for immunohistochemical processing: IL-1 and TNF-α (pro-inflammatory cytokines), MMP-2 (collagen metabolism) and CD-31 (angiogenesis). The area of inflammation, the birefringence index, the area of immunoreactivity and the number of vessels were objectively measured. Results A larger area of inflammatory reaction was observed in PP compared to sham on the 4th and on the 30th day (p=0.0002). After 4 days, PP presented higher TNF (p=0.0001) immunoreactivity than sham and no differences were observed in MMP-2 (p=0.06) and IL-1 (p=0.08). After 30 days, a reduction of IL-1 (p=0.010) and TNF (p=0.016) for PP and of IL-1 (p=0.010) for sham were observed. Moreover, area of MMP-2 immunoreactivity decreased over time for PP group (p=0.018). Birefringence index and vessel counting showed no differences between PP and sham (p=0.27 and p=0.58, respectively). Conclusions The implantation of monofilament and macroporous polypropylene in the subcutaneous of rats resulted in increased inflammatory activity and higher TNF production in the early post implant phase. After 30 days, PP has similar cytokines immunoreactivity, vessel density and extracellular matrix organization.

Birefringence of collagen fibres in rat calcaneal tendons treated with acupuncture during three phases of healing.

BACKGROUND: Birefringence is an optical anisotropy that is investigated by polarisation microscopy, and has been valuable for the study of the oriented organisation of collagen fibres in tendons. However, the application of this technology to evaluate the effect of different acupuncture points during tendon healing has not yet been described. OBJECTIVES: To evaluate the concentration of non-collagenous proteins (NCP) and birefringence in rat calcaneal tendons following injury during the three different phases of healing: inflammatory (7th day), proliferative (14th day), and remodelling (21st day). METHODS: Tendons of 120 Wistar rats were tenotomised and left untreated (teno group, n=24), treated with manual acupuncture at ST36 (ST36 group, n=24), BL57 (BL57 group, n=24) or ST36+BL57 (SB group, n=24), or treated with electroacupuncture at ST36+BL57 (EA group, n=24). Tendon samples were collected at 7, 14 and 21 days after injury (n=8 per group). NCP concentrations were measured using the Bradford method (n=4 each) and birefringence was examined using polarisation microscopy and image analysis (n=4 each). Comparison was also made with healthy (non-tenotomised) tendons in a subgroup of rats (n=4 each). RESULTS: Manual acupuncture at ST36 and BL57 increased molecular organisation of collagen fibres on day 14 and 21 after injury. Isolated use of BL57 and ST36 also increased collagen fibre organisation when examined on day 14 and 21, respectively. No significant increase in NCP concentration was observed in any of the treated tenotomised groups. CONCLUSIONS: Acupuncture, through putative anti-inflammatory and mechanotransductor effects, may have a role in strengthening tendons and increasing resistance to re-rupture.

Efeito da aplicação do ultrassom terapêutico durante 4 e 5 minutos por área do transdutor no processo de reparação de tendão de ratos / Effect of application times 3, 4 and 5 minutes ERA of therapeutic ultrasound in tendon injury of calcaneal rats

O objetivo deste estudo foi avaliar o efeito dos tempos de aplicação 3, 4 e 5 minutos por ERA do ultrassom terapêutico (UST) na organização das fibras de colágeno em lesão do tendão do calcâneo de ratos. Foram utilizados quarenta ratos machos Wistar, dos quais 32 sofreram tenotomia total do tendão do calcâneo e foram divididos em 5 grupos: GC, sem tenotomia e tratamento; GT, com tenotomia e sem tratamento; UST3, UST4 e UST5 submetidos à tenotomia e tratados com UST nos tempos de 3, 4 e 5 minutos por área de radiação efetiva respectivamente. Os animais foram submetidos à primeira aplicação do UST foi 24 horas após a cirurgia de tenotomia. A irradiação ultrassônica foi aplicada com os seguintes parâmetros: 1 MHz, modo pulsado com 20% do ciclo de trabalho (2 ms de emissão / 8 ms de intervalo), frequência de 100 Hz, 0,5 W / cm² de intensidade e ERA de 0,5 cm². A aplicação foi realizada 1x/dia. Os animais foram sacrificados após a 10ª sessão de tratamento, no 12º dia pós-operatório. Os tendões foram retirados cirurgicamente para análise da organização das fibras colágenas através do método de birrefringência (retardo óptico - OR). As fibras colágenas mostraram melhor agregação e organização no grupo UST3, UST4 e UST5 quando comparado ao GT (p<0.05) e o UST5 apresentou melhor resposta na comparação intergrupos. Conclui-se que o UST, aplicado no tempo de 5 minutos por área de radiação efetiva, apresentou a melhor dose-resposta quanto à organização das fibras colágenas no reparo tecidual de tendões de ratos...

The aim of this study was to evaluate the effect of application times 3, 4 and 5 minutes ERA of therapeutic ultrasound in the organization of the collagen fibers in rat calcaneal tendon injury. Forty male Wistar rats were used, of which 32 underwent complete tenotomy of the calcaneal tendon and were divided into 5 groups: GC without tenotomy and treatment; GT tenotomy with and without treatment; UST3, UST4 UST5 and submitted to tenotomy treated with therapeutic ultrasound at times 3, 4 and 5 minutes per effective radiating area respectively. The animals were submitted to the first application of therapy US tenotomy 24 hours after surgery. Ultrasonic irradiation was applied with the following parameters: 1 MHz, pulsed mode at 20% duty cycle (2ms transmission / 8 ms interval), frequency 100 Hz, 0.5 W / cm² intensity and ERA 0.5 cm². The application was performed 1x / day. The animals were sacrificed after the 10th treatment session, on the 12th postoperative day. The tendons were surgically removed for analysis of the organization of the collagen fibers through birefringence method (optical delay - OR). The collagen fibers showed better aggregation and organization in group UST3, UST4 and UST5 when compared to the GT (p <0.05) and UST5 showed better response in the intergroup comparison. We conclude that the UST, applied in time of 5 minutes for effective radiation area, presented the best dose-response as the organization of the collagen fibers in tissue repair of rat tendons...

Birefringence-induced wavelength mismatch between the polarization rotation and resonant modes in magnetophotonic structures containing nematic layers.

The present work is devoted to the study of spectral characteristics of normal incident light transmitted by a multilayered structure composed of an alternated sequence of nematic and magnetic layers presenting a central magneto-optical defect. Using the Berreman 4 × 4 matrix formalism, we numerically obtain the transmission spectrum and the polarization rotation angle of the system as a function of the nematic optical axis direction. Our results reveal the emergence of a shift between the wavelengths of the resonant mode and polarization rotation angle, which strongly depends on the birefringence of the nematic layers. In particular, we show the existence of distinct regimes for the wavelength mismatch between the transmission of resonant modes and the maximum polarization rotation angle, which are governed by the ordinary and extraordinary refractive indices of nematic layers. The mechanism behind such shift is discussed under the light of propagation eigenmodes for a medium presenting circular and linear birefringence. The effects associated with the defect thickness are also analyzed.

Films from resistant starch-pectin dispersions intended for colonic drug delivery.

Free films were obtained by the solvent casting method from retrograded starch-pectin dispersions at different polymer proportions and concentrations with and without plasticizer. Film forming dispersions were characterized according to their hardness, birefringence and rheological properties. The polymer dispersions showed a predominantly viscous behavior (G″>G') and the absence of plasticizers lead to building of stronger structures, while the occurrence of Maltese crosses in the retrograded dispersions indicates the occurrence of a crystalline organization. Analyses of the films included mechanical properties, thickness, superficial and cross sectional morphology, water vapor permeability, liquid uptake ability, X-ray diffractometry, in vitro dissolution and enzymatic digestion. The high resistant starch content (65.8-96.8%) assured the resistance of materials against enzymatic digestion by pancreatin. Changes in the X-ray diffraction patterns indicated a more organized and crystalline structure of free films in relation to isolated polymers. Increasing of pectin proportion and pH values favored the dissolution and liquid uptake of films. Films prepared with lower polymer concentration presented better barrier function (WVP and mechanical properties).

Photoelastic analysis of stress distribution with different implant systems.

The aim of this study was to evaluate stress distribution with different implant systems through photoelasticity. Five models were fabricated with photoelastic resin PL-2. Each model was composed of a block of photoelastic resin (10 × 40 × 45 mm) with an implant and a healing abutment: model 1, internal hexagon implant (4.0 × 10 mm; Conect AR, Conexão, São Paulo, Brazil); model 2, Morse taper/internal octagon implant (4.1 × 10 mm; Standard, Straumann ITI, Andover, Mass); model 3, Morse taper implant (4.0 × 10 mm; AR Morse, Conexão); model 4, locking taper implant (4.0 × 11 mm; Bicon, Boston, Mass); model 5, external hexagon implant (4.0 × 10 mm; Master Screw, Conexão). Axial and oblique load (45°) of 150 N were applied by a universal testing machine (EMIC-DL 3000), and a circular polariscope was used to visualize the stress. The results were photographed and analyzed qualitatively using Adobe Photoshop software. For the axial load, the greatest stress concentration was exhibited in the cervical and apical thirds. However, the highest number of isochromatic fringes was observed in the implant apex and in the cervical adjacent to the load direction in all models for the oblique load. Model 2 (Morse taper, internal octagon, Straumann ITI) presented the lowest stress concentration, while model 5 (external hexagon, Master Screw, Conexão) exhibited the greatest stress. It was concluded that Morse taper implants presented a more favorable stress distribution among the test groups. The external hexagon implant showed the highest stress concentration. Oblique load generated the highest stress in all models analyzed.

Polarization microscopy of extended chromatin fibers.

Analysis of the formation of extended chromatin fibers (ECFs) in response to the action of gravity following lysis by hypertonic and detergent solutions is a useful technical procedure relevant for studies of the positioning of particular DNA signals on chromatin filaments. Additionally, if toluidine blue molecules are allowed to bind electrostatically to available DNA phosphates on ECFs, the birefringence brightness generated in these filaments, as observed by polarization microscopy, facilitates the description of the frequency of ECF formation and extension of the chromatin filaments generated. Thus, different patterns of DNA-nuclear matrix protein associations related to varying transcriptional activities and chromatin organization in isolated cells can be assessed. A technique for producing ECFs in different isolated cell types under variable physiological and/or pathological conditions is detailed in this chapter.