Characterization and Biological Performance of Marine Sponge Collagen

Abstract This study characterized the morphological aspects of marine collagen - spongin (SPG) extract from marine sponges, as well as, evaluating its in vitro and in vivo biological performance. Aplysina fulva marine sponge was used for the SPG extraction. It was investigated the physicochemical and morphological properties of SPG by using scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction and compared to PMMA and bovine collagen. Additionally, the SPG cytotoxicity and its influence on cell proliferation, through in vitro tests. Moreover, the in vivo biological response was investigated using an experimental model of tibial bone defect. The results demonstrated that SPG presented an irregular granular aspect, with a composition of OH, C=O, NH, CN and an amorphous profile. Also, in vitro viability results for the L929 and MC3T3 cell lines cultured with SPG extracts demonstrated normal growth in comparison to controls, except for MC3T3 viability at day 3. For in vivo analysis, using tibial bone defects in rats, SPG treated animals presented an increased rate of material resorption and higher granulation and bone formation deposition in the region of the defect, mainly after 45 days. As a conclusion, SPG was successfully extracted. The in vitro and in vivo studies pointed out that SPG samples produced an increase in L929 and MC3T3 viability and improved the performance in tibial bone defects. It can be concluded that SPG can be used as a bone graft for bone regeneration.

In vitro and in vivo genotoxicity and cytotoxicity analysis of protein extract from Aplysina fulva sponges

This study evaluated the physicochemical and morphological properties of a marine sponge protein extract (PE) using scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDS), analysis of mass loss and pH and in vitro and in vivo. Scanning electron microscopy showed that PE fibers present a granular aspect and irregular structure and the element carbon followed by oxygen was detected in the EDS analysis. Moreover, a 29% of mass loss was observed after 14 days and the pH slightly modified after 14 days. Cell viability of fibroblast cells (L929) of control and PE at a concentration of 25% demonstrated higher values compared to the groups. Osteoblast cell viability of PE at 25 and 50% was significantly higher. Comet assay on day 1 showed higher values for PE at 25%. In addition, in vivo experiments demonstrated that in the treated animals, the bone defects were filled with biomaterial particles, granulation tissue and some areas of newly formed bone. Furthermore, similar immunoexpression of Runx-2 and Cox-2 was observed. Taken together, all results suggest that PE is biocompatible, present non-citotoxicity in the in vitro studies (at the lower concentration) and in the in vivo studies and it can be considered as an alternative source of collagen for tissue engineering proposals.

The photobiomodulation (658, 830 and 904nm) on wound healing in histomorphometric analysis / A fotobiomodulação (658, 830 e 904nm) na cicatrização de feridas na análise histomorfométrica / La fotobiomodulación (658, 830 y 904nm) en la cicatrización de heridas en el análisis histomorfométrico

Abstract Introduction: Photobiomodulation (PBM) assists in the processes of angiogenesis and cellular mitosis after skin lesion, contributing to tissue repair. Objective: to investigate the effects of photobiomodulation (during the proliferative phase) of 658 nm, 830 nm and 904 nm in the repair of skin lesions in an animal model. Method: 658 nm (G658), 830 nm (G830), 904 nm (G904) PBM, and control group (CG) integrated the research. We submitted the animals to an excisional wound and treatment at different wavelengths for 14 days. On the seventh and 14-1485004059th postoperative days, we calculated the area and percentage of lesion contraction. The animals were sacrificed on the 14-1485004056th postoperative day and cutaneous section of the injured region was collected for histomorphometric evaluation of the cellularity, neovascularization, thickness of the epidermis and volume density of collagen fibers colored with H&E and Picross Sirius respectively. For the statistical analysis, we applied the ANOVA test. Results: the G658 presented higher cellularity than GC (p = 0.03). The animals in the G658 group showed a significant increase in the neovascularization in relation to the CG (p = 0.01). Type III collagen significantly increased in G904 compared to G830 (p < 0.0001) and CG (p < 0.0001). The G658 had a significant increase in type III collagen fibers compared to G830 (p < 0.0001) and GC (p < 0.0001). We found no significant difference in the thickness of the epidermis, wound area, and in the percentage wound of contraction between the analyzed groups. Conclusion: PBM was effective to stimulate the tissue repair process, with better results for the 658 nm wavelength.

Resumo Introdução: A Fotobiomodulação (FBM) auxilia nos processos de angiogênese e mitose celular após lesão cutânea, contribuindo para reparo do tecido. Objetivo: investigar os efeitos da fotobiomodulação (durante a fase proliferativa) com comprimento de onda de 658 nm, 830 nm e 904 nm no reparo de lesões cutâneas em modelo animal. Método: FBM 658 nm (G658), 830 nm (G830), 904 nm (G904) e controle (GC) integraram a pesquisa. Os animais foram submetidos a uma ferida excisional e receberam tratamento em diferentes comprimentos de por 14 dias. No 7º e 14º dia pós-operatório, calculou-se a área e a porcentagem de contração da lesão. Os animais foram sacrificados no 14º dia pós-operatório e a secção cutânea da região lesada foi coletada para avaliação histomorfométrica da celularidade, neovascularização, espessura da epiderme e densidade volumétrica das fibras colágenas, corados com H&E e Picross Sirius respectivamente. Para a análise estatística, foi aplicado o teste ANOVA. Resultados: o G658 apresentou maior celularidade que GC (p = 0,03). Os animais do grupo G658 apresentaram aumento significativo da neovascularização em relação ao GC (p = 0,01). Houve aumento significativo do colágeno tipo III no G904 em relação ao G830 (p < 0,0001) e GC (p < 0,0001). O G658 teve um aumento significativo nas fibras colágenas tipo III em comparação ao G830 (p < 0,0001) e GC (p < 0,0001). Nenhuma diferença significativa foi encontrada na espessura da epiderme, área da ferida e na porcentagem de contração da ferida entre os grupos analisados. Conclusão: a PBM foi efetiva para estimular o processo de reparo tecidual, com melhores resultados para o comprimento de onda de 658 nm.

Resumen Introducción: La fotobiomodulación (FBM) auxilia en los procesos de angiogénesis y mitosa celular después de lesión cutánea, contribuyendo para la reparación. Objetivo: investigar los efectos de la fotobiomodulación (durante la fase proliferativa) con longitud de onda de 658 nm, 830 nm y 904 nm en la reparación de lesiones cutáneas en modelo animal. Método: grupos de FBM 658 nm (G658), 830 nm (G830), 904 nm (G904) y control (GC) integraron la investigación. Los animales fueron sometidos a una herida excisional y recibieron tratamiento 14 días. En el 7º y 14º día postoperatorio, se calculó el área y el porcentaje de contracción de la lesión. Los animales fueron sacrificados en el 14º día postoperatorio y la sección cutánea de la región lesada fue recolectada para evaluación histomorfométrica de la celularidad, neovascularización, espesor de la epidermis y densidad volumétrica de las fibras colágenas, colorados con H & E y Picross Sirius respectivamente. Para el análisis estadístico, se aplicó la prueba ANOVA. Resultados: G658 presentó mayor celularidad que GC (p = 0,03). G658 presentaron un aumento significativo de la neovascularización en relación al GC (p = 0,01). Se observó un aumento significativo del colágeno tipo III en el G904 con respecto al G830 (p < 0,0001) y GC (p < 0,0001). El G658 tuvo un aumento significativo en las fibras colágenas tipo III en comparación con el G830 (p < 0,0001) y GC (p < 0,0001). Ninguna diferencia significativa se encontró en el espesor de la epidermis, área de la herida entre los grupos. Conclusión: la PBM fue efectiva para estimular el proceso de reparación del tejido, con mejores resultados para grupo 658 nm.

In Vivo Biological Effects of Marine Biosilica on a Tibial Bone Defect in Rats

Abstract Research on biomaterials of natural origin has gained prominence in the literature. Above all, marine sponges, due to their architecture and structural components, present a promising potential for the engineering of bone tissue. In vitro studies demonstrate that a biosilica of marine sponges has osteogenic potential. However, in vivo works are needed to elucidate the interaction of biosilica (BS) and bone tissue. The objective of the study was to evaluate the morphological and chemical characteristics of BS compared to Bioglass (BG) by scanning electron microscopy (SEM) and X-ray dispersive energy (EDX) spectroscopy. In addition, to evaluate the biological effects of BS, through an experimental model of tibial bone defect using histopathological, histomorphometric, immunohistochemical (IHC) and mechanical tests. SEM and EDX demonstrated the successful extraction of BS. Histopathological analysis demonstrated that Control Group (GC) had greater formation of newly formed bone tissue compared to BG and BS, yet BG bone neoformation was greater than BS. However, BS showed material degradation and granulation tissue formation, with absence of inflammatory process and formation of fibrotic capsule. The results of histomorphometry corroborate with those of histopathology, where it is worth emphasizing the positive influence of BS in osteoblastic activity. IHQ demonstrated positive VEGF and TGF-β immunoexpression for GC, BS and BG. In the mechanical test no significant differences were found. The present results demonstrate the potential of BS in bone repair, further studies are needed other forms of presentation of BS are needed.

Alternative animal model for studies of total skin thickness burns

Abstract Purpose: To present an alternative experimental model of third degree burn of easy reproducibility. Methods: Eighteen male Wister rats were randomly divided into three groups, 6 of which were allocated to each group. A soldering iron coupled to an aluminum plate was used to produce burn, at a temperature of 150ºC, with different exposure times per group. Group 5 (G5) animals were burned at 150°C with exposure time of 5 seconds; Group 10 (G10) the animals were burned at 150°C with exposure time of 10 seconds and group 15 (G15) the animals were burned at 150°C with exposure time of 15 seconds. Results: Histopathological analyzes showed that all three groups had similar morphological characteristics, with total thickness involvement. Conclusion: The technique is effective to reproduce a third degree burn and suggests the temperature of 150ºC with 5 seconds of exposure in order to minimize the risks to the animals.

Opioidergic effects of transcutaneous electrical nerve stimulation on pain and inflammatory edema in a rat model of ankle sprain / Efeitos de opioidérgico da estimulação elétrica nervosa transcutânea sobre a dor e edema inflamatório em um modelo de entorse de tornozelo em ratos / Efectos opioidergicos de la estimulación nerviosa eléctrica transcutánea sobre el dolor y el edema inflamatorio en un modelo de rata con esguince de tobillo

ABSTRACT Although transcutaneous electrical nerve stimulation (TENS) has been proposed to modulate pain and the mechanisms underlying analgesia remain poorly understood, evidence of anti-inflammatory effect is more limited. The purpose of this study was to examine the opioidergic mechanisms of TENS effects in two different frequencies on pain and inflammatory edema in the ankle sprain model in rats. Threshold to mechanical stimulation was utilized to examine the changes produced by intraperitoneal injection of non-selective opioid antagonist naloxone on the antihyperalgesic effect induced by a 20-min period of 2Hz or 100Hz TENS in the ankle sprain model, produced by manually overextending the lateral ligaments. Ankle sprain induced a long-lasting reduction in paw withdrawn latency (PWL) after 30 minutes for up to 24 hours in sham TENS (SH-TENS) treated rats. The reduced PWL after the induction of ankle sprain was restored partially at 0,1,2,3 and 6, but not 24 hours, after the termination of 2 Hz-TENS (LF-TENS). In 100Hz (HF-TENS) the reduction in PWL was shorter than LF-TENS and both LF and HF effects were fully blocked in naloxone-treated rats. LF- and HF-TENS treated rats did not reach the elevation of edema and presented a progressive edema reduction for over 24 hours when compared to SH-TENS group. Both effects were reduced by naloxone. TENS-induced antihyperalgesic and anti-edematous effects observed in ankle sprain model were mediated by the endogenous opioid system.

RESUMO Embora estimulação elétrica nervosa transcutânea (TENS) tem sido proposta para modular a dor e os mecanismos subjacentes a analgesia permanecem mal compreendidos, evidências do efeito anti-inflamatório são mais limitadas. O objetivo deste estudo foi examinar os mecanismos opioidérgicos de efeitos de TENS em duas frequências diferentes sobre dor e edema inflamatório no modelo de entorse de tornozelo em ratos. Limiar de estimulação mecânica foi utilizado para examinar as alterações produzidas pela injeção intraperitoneal de naloxona, um antagonista opioide não-seletivo, sobre o efeito anti-hiperalgésico induzido por um período de 20 min de 2Hz ou 100Hz de TENS no modelo de entorse de tornozelo, produzido ultrapassando manualmente os ligamentos laterais. Entorse de tornozelo induziu uma redução duradoura na latência de retirada da pata (PWL) depois de 30 minutos até 24 horas em ratos tratados para TENS "simulada" (SH-TENS). A PWL reduzida após a indução de entorse de tornozelo foi restaurada parcialmente em 0,1,2,3 e 6, mas não em 24 horas, após o término do 2 Hz-TENS (LF-TENS). Em 100Hz (HF-TENS) a redução de PWL foi menor do que LF-TENS e tanto os efeitos HF e LF foram totalmente bloqueados em ratos tratados com naloxona. Ratos tratados com LF- e HF-TENS não alcançou a elevação do edema e apresentaram uma redução progressiva do edema por mais de 24 horas, quando comparado ao grupo SH-TENS. Ambos os efeitos foram reduzidos pela naloxona. Efeitos anti-hiperalgésicos induzidos por TENS e efeitos antiedematosos observados no modelo de entorse de tornozelo foram mediados pelo sistema de opioides endógenos.

RESUMEN Aunque la estimulación nerviosa eléctrica transcutánea (TENS) ha sido propuesta para modular el dolor y los mecanismos subyacentes a la analgesia sigue siendo mal entendida, la evidencia del efecto antiinflamatorio es limitada. El propósito de este estudio fue examinar los mecanismos opioidérgicos de los efectos de la TENS en dos frecuencias diferentes sobre el dolor y el edema inflamatorio en un modelo de ratas con esguince de tobillo. Se utilizó el umbral a la estimulación mecánica para examinar los cambios producidos por inyección intraperitoneal del antagonista opiáceo no selectivo naloxona sobre el efecto antihiperalgésico inducido por un período de 20 minutos de 2Hz o 100Hz TENS en el modelo con esguince de tobillo, producido por sobrecarga manual de los ligamentos laterales. El esguince de tobillo indujo una reducción de larga duración en latencia de la pata retraída (PWL) después de 30 minutos por hasta 24 horas en simulación de la TENS (SH-TENS) para las ratas tratadas. El PWL reducido después de la inducción del esguince de tobillo fue restaurado parcialmente en 0,1,2,3 y 6, pero no por 24 horas, después de la terminación de 2 Hz-TENS (LF-TENS). La reducción en PWL fue menor que LF-TENS en 100Hz (HF-TENS) y tanto los efectos de LF como de HF fueron completamente bloqueados en ratas tratadas con naloxona. Las ratas tratadas con LF- y HF-TENS no alcanzaron la elevación del edema y presentaron una reducción progresiva del edema durante más de 24 horas en comparación con el grupo SH-TENS. Ambos efectos fueron reducidos por la naloxona. Efectos antihiperalgésicos y antiedematosos TENS-inducidos observados en el modelo con esguince de tobillo fueron mediados por el sistema opioide endógeno.