Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds.

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds.

O diabetes Mellitus é uma doença na qual o organismo perde a habilidade de regular adequadamente o metabolismo dos carboidratos e é caracterizada entre outros sintomas, por hiperglicemia permanente. Especificamente, o diabetes Tipo I, está associado com a diminuição da integridade do tecido ósseo e, por isso, mais propenso à fraturas. Esta pesquisa refere-se ao estudo aplicação do ultra-som e do exercício físico (natação) como formas de consolidação de fraturas em fíbulas de ratos diabéticos e apresenta resultados das avaliações glicêmicas, radiológicas, histológica e de microscopia de luz polarizada. A avaliação do grau de birrefringência nos permitiu observar que os animais diabéticos, tanto os exercitados quanto os tratados apresentaram melhores resultados que os controles. O tratamento com ultra-som de baixa intensidade e o exercício físico foram eficazes na consolidação de osteotomias experimentais / Diabetes Mellitus is a desease characterized by lost of ability in the regulating the carbohydrate metabolism and it is represented by constant hiperglycaemia and others sintoms. Among the types of diabetes, the type I, is associated with the decrease of the bone tissue integrity and therefore more susceptible to fractures. This research is related to the aplication of ultrasound and physical exercise(swimming) to help healing of osteotomized fibulae in diabetics rats. The results were evaluated by glycaemics, radiological, hystological and polarized light microscopy analysis. Birefringence analysis showed that diabetic animals, exercited and treated with ultrasound presented better results as compared to the controls. The treatment with low intensity ultrasound and physical exercise demonstrated efficacy in experimental healing fractures

Bone is refractory to most conventional biochemical Procedures. However because it is now possible to cut sections (e. g. lopm) of fresh, undemineralized adult bone, this tissue can be analyzed by suitably modified methods of quantitative cytochemistry. A new substrate for assaying hydroxyacyl dehydrogenase activity demonstrated that bone cells may use fatty acids as a major source of energy: detailed analysis of the activities of key enzymes indicated that the paradox of ‘aerobic glycolysis’ of bone could be explained by fatty acid oxidation satisfying the requirements of the Krebs' cycle and directing the conversion of pyruvate to lactate The influence of glucose 6-phosphate dehydrogenase (G6PD) activity in aerobic glycolysis has been considered. The inverse relationships between this activity and that of Na-K-ATPase led to the development of a new method for the latter, based on a new concept in cytochemistry ('hidden-capture' procedure). A major feature of fracture-healing is increased periosteal G6PD activity. The association with the vitamin K cycle has been investigated by feeding rats with dicoumarol which not only inhibited bone-formation but also G6PD activity. The stimulation of this activity in fracture-healing has been linked with ornithine decarboxylase (ODC) activity, for which a new method has been developed. Rats deficient in pyridoxal phosphate (cofactor for ODC) had decreased G6PD responses and also appeared to become osteoporotic. Studies on osteoporotic fractures in the human showed the presence of relatively large apatite crystals close to the fracture-site, and disorganized glycosaminoglycans (demonstrated by the new method of ‘induced birefringence’).

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

O diabetes Mellitus é uma doença na qual o organismo perde a habilidade de regular adequadamente o metabolismo dos carboidratos e é caracterizada entre outros sintomas, por hiperglicemia permanente. Especificamente, o diabetes Tipo I, está associado com a diminuição da integridade do tecido ósseo e, por isso, mais propenso à fraturas. Esta pesquisa refere-se ao estudo aplicação do ultra-som e do exercício físico (natação) como formas de consolidação de fraturas em fíbulas de ratos diabéticos e apresenta resultados das avaliações glicêmicas, radiológicas, histológica e de microscopia de luz polarizada. A avaliação do grau de birrefringência nos permitiu observar que os animais diabéticos, tanto os exercitados quanto os tratados apresentaram melhores resultados que os controles. O tratamento com ultra-som de baixa intensidade e o exercício físico foram eficazes na consolidação de osteotomias experimentais / Diabetes Mellitus is a desease characterized by lost of ability in the regulating the carbohydrate metabolism and it is represented by constant hiperglycaemia and others sintoms. Among the types of diabetes, the type I, is associated with the decrease of the bone tissue integrity and therefore more susceptible to fractures. This research is related to the aplication of ultrasound and physical exercise(swimming) to help healing of osteotomized fibulae in diabetics rats. The results were evaluated by glycaemics, radiological, hystological and polarized light microscopy analysis. Birefringence analysis showed that diabetic animals, exercited and treated with ultrasound presented better results as compared to the controls. The treatment with low intensity ultrasound and physical exercise demonstrated efficacy in experimental healing fractures

Alguns trabalhos sobre a resposta do tecido ósseo ao Cianocrilato podem ser encontrados na literatura, embora nenhum deles avalie a resposta histológica, micro-tomográfica e biomolecular na fixação de enxertos ósseos onlay com o N-Butil-2-Cianocrilato (IndermilTM). O objetivo do estudo proposto foi comparar o processo de incorporação, remodelação, revascularização e manutenção do volume de enxertos fixados com parafuso ou adesivo, além de mapear os eventos biomoleculares nos quais os enxertos ósseos onlay possam estar envolvidos. Oitenta e oito coelhos adultos foram envolvidos nesse estudo. Dois blocos ósseos provenientes da calvária dos coelhos foram transplantados para a mandíbula, em que de cada lado do leito receptor o osso autógeno foi fixado com parafuso de osteossíntese de forma aposicional, lag screw, cianoacrilato ou parafuso e cianoacrilato. O sacrifício dos animais ocorreu após 3, 7, 20 e 40 dias do procedimento cirúrgico inicial, quando então os animais foram submetidos às análises micro-tomográficas, histológicas e biomoleculares. Cortes histológicos das áreas enxertadas foram preparados para se avaliar o reparo dos enxertos ósseos no sítio receptor. Os resultados biomoleculares mostraram que o método de fixação utilizando o composto N-Butil-2-Cianocrilato (NB-Cn) apresentou um maior potencial anti-inflamatório, revascularizador e de formação óssea, bem como, uma menor reabsorção óssea. Na avaliação histológica, observou-se que embora o NB-Cn tenha impedido a formação de novo osso na área onde foi aplicado, a estabilidade promovida pela cola permitiu que a revascularização e incorporação do enxerto ocorresse de forma semelhante aos demais grupos. Esses resultados indicam que o NB-Cn se comportou de forma semelhante ao parafuso como material de osteossínte. / Some experimental studies on the bone tissue responses to cyanoacrylate can be found in the literature, none of them evaluate the histological response, micro-tomographic and biomolecular on fixation of onlay bone grafts with N-butyl-2-cyanoacrylate (IndermilTM). The aim of the proposed study was to compare the process of incorporation, remodeling, and volume maintenance of grafts set with screw or adhesive and map biomolecular events in which onlay bone grafts may be involved. Eighty-eight adult rabbits were submitted to calvaria onlay bone graft on both sides of the mandible. The grafts were fixated on each side of mandible by a fixation screw appositionally, lag screw, cyanoacrilate or both. The animals were sacrificed on 3rd, 7th, 20th and 40th day after the initial surgical procedure, then they were subjected to micro-computed tomography, histological and biomolecular analysis. Histological sections of the grafted areas were prepared to evaluate the healing of bone grafts in the receptor site. The biomolecular analysis showed that fixation method using N-butyl-2-cyanoacrylate (NB-Cn) presented a greater potential on anti-inflammatory, revascularization and bone formation properties, as well as reduced bone resorption. Histological evaluation observed that although the NB-Cn has prevented the formation of new bone in the area where it was applied, the stability achieved by Nb-Cn allowed revascularization and graft incorporation just like other groups. These results indicate that NB- Cn behaved similarly to the screw as osteosynthesys material.

Alguns trabalhos sobre a resposta do tecido ósseo ao Cianocrilato podem ser encontrados na literatura, embora nenhum deles avalie a resposta histológica e a fixação de enxertos ósseos onlay com o N-Butil-2-Cianocrilato (Indermil™). O objetivo do estudo proposto foi (1) analisar a manutenção do volume de enxertos ósseos onlay fixados à mandíbula de coelhos usando N-Butil-2-Cianocrilato (NB-Cn) ou parafuso de titânio, assim como avaliar (2) a remodelação e incorporação desses enxertos ao leito receptor e também (3) observar a diferença do nível de expressão da proteína tartarato-resistente ácido fosfatase (Trap) envolvida na absorção dos mesmos na presença do NB-Cn e do parafuso de titânio. Dezoito coelhos adultos foram envolvidos nesse estudo. Dois blocos ósseos provenientes da calvária dos coelhos foram transplantados para a mandíbula, em que de um lado do leito receptor o osso autógeno foi fixado com parafuso de osteossíntese (Grupo I - controle), e do outro lado com NB-Cn (Grupo II). Após o procedimento cirúrgico, os animais foram submetidos a exame tomográfico. O sacrifício dos animais ocorreu após 1 (n=9) e 6 (n=9) semanas do procedimento cirúrgico inicial, quando então os animais foram submetidos à nova tomografia. As imagens de tomografia foram usadas para estimativa da manutenção do volume dos enxertos. Cortes histológicos das áreas enxertadas foram preparados para se avaliar o reparo dos enxertos ósseos no sítio receptor e o nível de expressão da proteína Trap. Os resultados tomográficos mostraram melhor manutenção do volume dos enxertos fixados com NB-Cn (p≤0,05) em comparação àqueles fixados com parafuso, em ambos os tempos experimentais. Na avaliação imunohistoquímica, observou-se que a marcação da proteína Trap no período de 6 semanas foi significativamente maior em comparação ao tempo de 1 semana, sem apresentar diferença significante entre os grupos. A análise histológica revelou que embora o NB-Cn tenha provocado a destruição do periósteo, a estabilidade promovida pela cola permitiu que a revascularização e incorporação do enxerto ocorresse de forma semelhante ao grupo controle. Esses resultados indicam que o NB-Cn se comportou de forma superior ao parafuso como material de osteossínte. No entanto, estudos adicionais são necessários para se investigar a toxicidade deste composto quando utilizado como meio de fixação óssea. / Some experimental studies on the bone tissue responses to cyanoacrylate can be found in the literature, although neither evaluating the histological response and the fixation of onlay bone grafts with Indermil™. The aim of the proposed study is (1) to analyze the bone grafts volume maintenance fixed to the mandible of rabbits using Indermil™ or titanium screw, as well as (2) assess the healing of onlay grafts to the recipient bed and (3) the differences of level expression of Trap protein involved in reabsortion of these bone grafts using Indermil™ or an osteosynthesis screw. Eighteen adult New Zealand White rabbits were submitted to calvaria onlay bone grafts on both sides of the mandible. On one side the onlay bone graft were fixated with Indermil™, and on the other side bone graft with an osteosynthesis screw. The wounds were then closed, and a computed tomography was realized immediately after this procedure. The animals were killed after 1 (n=9) and 6 weeks (n=9), then the animals were submitted to another computed tomography (CT). The CT images were used to estimate the bone grafts volume maintenance. Histological sections of the grafted areas were prepared to evaluate the healing of bone grafts in the receptor site and the expression level of Trap protein. The CT scan showed better volume maintenance of the bone grafts fixed with Indermil™ (p≤0.05) compared to those fixed with screws, in both the experimental times. The immunohistochemical evaluation showed that the protein Trap expression in 6-week period was significantly higher compared to the 1-week period, without showing significant difference between the groups. Histological analysis revealed that although the Indermil™ has caused the destruction of the periosteum, the stability provided by the glue allowed that the bone graft revascularization and incorporation has occurred in a similar manner to the control group. These results indicate that the Indermi™ behaved than the screw as osteosynthesys material. However, further studies are needed to investigate the toxicity of this compound when used as a means of bone fixation.

Alguns trabalhos sobre a resposta do tecido ósseo ao Cianocrilato podem ser encontrados na literatura, embora nenhum deles avalie a resposta histológica e a fixação de enxertos ósseos onlay com o N-Butil-2-Cianocrilato (Indermil™). O objetivo do estudo proposto foi (1) analisar a manutenção do volume de enxertos ósseos onlay fixados à mandíbula de coelhos usando N-Butil-2-Cianocrilato (NB-Cn) ou parafuso de titânio, assim como avaliar (2) a remodelação e incorporação desses enxertos ao leito receptor e também (3) observar a diferença do nível de expressão da proteína tartarato-resistente ácido fosfatase (Trap) envolvida na absorção dos mesmos na presença do NB-Cn e do parafuso de titânio. Dezoito coelhos adultos foram envolvidos nesse estudo. Dois blocos ósseos provenientes da calvária dos coelhos foram transplantados para a mandíbula, em que de um lado do leito receptor o osso autógeno foi fixado com parafuso de osteossíntese (Grupo I - controle), e do outro lado com NB-Cn (Grupo II). Após o procedimento cirúrgico, os animais foram submetidos a exame tomográfico. O sacrifício dos animais ocorreu após 1 (n=9) e 6 (n=9) semanas do procedimento cirúrgico inicial, quando então os animais foram submetidos à nova tomografia. As imagens de tomografia foram usadas para estimativa da manutenção do volume dos enxertos. Cortes histológicos das áreas enxertadas foram preparados para se avaliar o reparo dos enxertos ósseos no sítio receptor e o nível de expressão da proteína Trap. Os resultados tomográficos mostraram melhor manutenção do volume dos enxertos fixados com NB-Cn (p≤0,05) em comparação àqueles fixados com parafuso, em ambos os tempos experimentais. Na avaliação imunohistoquímica, observou-se que a marcação da proteína Trap no período de 6 semanas foi significativamente maior em comparação ao tempo de 1 semana, sem apresentar diferença significante entre os grupos. A análise histológica revelou que embora o NB-Cn tenha provocado a destruição do periósteo, a estabilidade promovida pela cola permitiu que a revascularização e incorporação do enxerto ocorresse de forma semelhante ao grupo controle. Esses resultados indicam que o NB-Cn se comportou de forma superior ao parafuso como material de osteossínte. No entanto, estudos adicionais são necessários para se investigar a toxicidade deste composto quando utilizado como meio de fixação óssea. / Some experimental studies on the bone tissue responses to cyanoacrylate can be found in the literature, although neither evaluating the histological response and the fixation of onlay bone grafts with Indermil™. The aim of the proposed study is (1) to analyze the bone grafts volume maintenance fixed to the mandible of rabbits using Indermil™ or titanium screw, as well as (2) assess the healing of onlay grafts to the recipient bed and (3) the differences of level expression of Trap protein involved in reabsortion of these bone grafts using Indermil™ or an osteosynthesis screw. Eighteen adult New Zealand White rabbits were submitted to calvaria onlay bone grafts on both sides of the mandible. On one side the onlay bone graft were fixated with Indermil™, and on the other side bone graft with an osteosynthesis screw. The wounds were then closed, and a computed tomography was realized immediately after this procedure. The animals were killed after 1 (n=9) and 6 weeks (n=9), then the animals were submitted to another computed tomography (CT). The CT images were used to estimate the bone grafts volume maintenance. Histological sections of the grafted areas were prepared to evaluate the healing of bone grafts in the receptor site and the expression level of Trap protein. The CT scan showed better volume maintenance of the bone grafts fixed with Indermil™ (p≤0.05) compared to those fixed with screws, in both the experimental times. The immunohistochemical evaluation showed that the protein Trap expression in 6-week period was significantly higher compared to the 1-week period, without showing significant difference between the groups. Histological analysis revealed that although the Indermil™ has caused the destruction of the periosteum, the stability provided by the glue allowed that the bone graft revascularization and incorporation has occurred in a similar manner to the control group. These results indicate that the Indermi™ behaved than the screw as osteosynthesys material. However, further studies are needed to investigate the toxicity of this compound when used as a means of bone fixation.

Alguns trabalhos sobre a resposta do tecido ósseo ao Cianocrilato podem ser encontrados na literatura, embora nenhum deles avalie a resposta histológica, micro-tomográfica e biomolecular na fixação de enxertos ósseos onlay com o N-Butil-2-Cianocrilato (IndermilTM). O objetivo do estudo proposto foi comparar o processo de incorporação, remodelação, revascularização e manutenção do volume de enxertos fixados com parafuso ou adesivo, além de mapear os eventos biomoleculares nos quais os enxertos ósseos onlay possam estar envolvidos. Oitenta e oito coelhos adultos foram envolvidos nesse estudo. Dois blocos ósseos provenientes da calvária dos coelhos foram transplantados para a mandíbula, em que de cada lado do leito receptor o osso autógeno foi fixado com parafuso de osteossíntese de forma aposicional, lag screw, cianoacrilato ou parafuso e cianoacrilato. O sacrifício dos animais ocorreu após 3, 7, 20 e 40 dias do procedimento cirúrgico inicial, quando então os animais foram submetidos às análises micro-tomográficas, histológicas e biomoleculares. Cortes histológicos das áreas enxertadas foram preparados para se avaliar o reparo dos enxertos ósseos no sítio receptor. Os resultados biomoleculares mostraram que o método de fixação utilizando o composto N-Butil-2-Cianocrilato (NB-Cn) apresentou um maior potencial anti-inflamatório, revascularizador e de formação óssea, bem como, uma menor reabsorção óssea. Na avaliação histológica, observou-se que embora o NB-Cn tenha impedido a formação de novo osso na área onde foi aplicado, a estabilidade promovida pela cola permitiu que a revascularização e incorporação do enxerto ocorresse de forma semelhante aos demais grupos. Esses resultados indicam que o NB-Cn se comportou de forma semelhante ao parafuso como material de osteossínte. / Some experimental studies on the bone tissue responses to cyanoacrylate can be found in the literature, none of them evaluate the histological response, micro-tomographic and biomolecular on fixation of onlay bone grafts with N-butyl-2-cyanoacrylate (IndermilTM). The aim of the proposed study was to compare the process of incorporation, remodeling, and volume maintenance of grafts set with screw or adhesive and map biomolecular events in which onlay bone grafts may be involved. Eighty-eight adult rabbits were submitted to calvaria onlay bone graft on both sides of the mandible. The grafts were fixated on each side of mandible by a fixation screw appositionally, lag screw, cyanoacrilate or both. The animals were sacrificed on 3rd, 7th, 20th and 40th day after the initial surgical procedure, then they were subjected to micro-computed tomography, histological and biomolecular analysis. Histological sections of the grafted areas were prepared to evaluate the healing of bone grafts in the receptor site. The biomolecular analysis showed that fixation method using N-butyl-2-cyanoacrylate (NB-Cn) presented a greater potential on anti-inflammatory, revascularization and bone formation properties, as well as reduced bone resorption. Histological evaluation observed that although the NB-Cn has prevented the formation of new bone in the area where it was applied, the stability achieved by Nb-Cn allowed revascularization and graft incorporation just like other groups. These results indicate that NB- Cn behaved similarly to the screw as osteosynthesys material.