Direct inhibitory effect of caffeine on viability, synthesis activity and gene expression in cultures of chondrocytes extracted from the articular cartilage of rats / Efeito inibidor direto da cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos da cartilagem articular de ratos

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)

O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)

The chondrotoxic and apoptotic effects of levobupivacaine and bupivacaine on the rabbit knee joint / Os efeitos condrotóxicos e apoptóticos de levobupivacaína e bupivacaína na articulação do joelho de coelhos

Abstract Background: A single dose injection or continuous infusion of local anesthetics into the joint space is considered to be a well-defined analgesia technique. The aim of this study was to investigate the chondrotoxic and apoptotic effects of single-dose intra-articular injection of levobupivacaine and bupivacaine on rabbit knee joint tissues. Materials and methods: The animals were allocated into two groups each containing 20 rabbits. 0.5% levobupivacaine (Group L) and 0.5% bupivacaine (Group B) were applied intra-articularly to the left posterior joints of rabbits. At the same time, normal saline was applied to the right posterior leg knee joints of rabbits in both groups and used as a control (Group S). At the end of the 7th and 28th days after the intraarticular injections, ten randomly chosen rabbits in each group were killed by applying intraperitoneal thiopental. Sections of cartilage tissue samples were stained for light microscopic examinations and the TUNEL method was used to investigate apoptotic cells. Results: As a result of immunofluorescence microscopic examination, the number of apoptotic cells in Group B at day 7 and day 28 were both significantly higher than Group L and S (p < 0.05). Also, the number of apoptotic cells in Group L at day 7 and day 28 were both significantly higher than Group S (p < 0.05). Conclusions: We found that bupivacaine is more chondrotoxic than other anesthetic agent and increases the number of apoptotic cells. These results indicated that bupivacaine caused high chondrotoxic damage and it led to more apoptotic activation than levobupivacaine.

Resumo Justificativa: Uma injeção em dose única ou infusão contínua de anestésicos locais no espaço articular é considerada uma técnica de analgesia bem definida. O objetivo deste estudo foi investigar os efeitos condrotóxicos e apoptóticos da injeção intra-articular com dose única de levobupivacaína e bupivacaína em tecidos articulares do joelho de coelho. Material e métodos: Os animais foram alocados em dois grupos, cada um contendo 20 coelhos. Levobupivacaína a 0,5% (Grupo L) e bupivacaína a 0,5% (Grupo B) foram aplicadas intra-articularmente nas articulações posteriores esquerdas de coelhos. Ao mesmo tempo, solução salina normal foi aplicada nas articulações do joelho da perna posterior direita de coelhos em ambos os grupos e usada como controle (Grupo S). Ao fim do 7° e 28° dias após as injeções intra-articulares, 10 coelhos escolhidos aleatoriamente em cada grupo foram mortos por aplicação de tiopental intraperitoneal. Seções de amostras de tecido cartilaginoso foram coradas para exames de microscopia de luz, e o método TUNEL foi usado para investigar células apoptóticas. Resultados: Como resultado do exame microscópico de imunofluorescência nos dias 7 e 28, o número de células apoptóticas no Grupo B foi significativamente maior que nos grupos L e S (p < 0,05). Além disso, o número de células apoptóticas nos dias 7 e 28 foi significativamente maior no Grupo L do que no Grupo S (p < 0,05). Conclusões: Demonstramos que a bupivacaína é mais condrotóxica do que o outro agente anestésico e aumenta o número de células apoptóticas. Esses resultados indicaram que a bupivacaína causou intensa lesão condrotóxica e levou a uma ativação apoptótica maior do que a levobupivacaína.

Strontium ranelate as a possible disease-modifying osteoarthritis drug: a systematic review

Considering that osteoarthritis (OA) is the most prevalent joint disease worldwide, multiple pharmacological treatments have been proposed to alter the articular structure with potential benefit in the progression of the disease. The so-called disease-modifying OA drugs have been frequently investigated but conclusive findings are rare. Strontium ranelate (SrRan) is a drug usually prescribed to treat osteoporosis, with proven effects in decreasing the risk of fractures and possible effect in reducing the progression of OA. The objective of this review was to demonstrate the current panorama of knowledge on the use of SrRan in clinical and experimental models, clarifying its mechanisms of action and describing possible anti-nociceptive and anti-inflammatory effects. The systematic review was based on the PRISMA statement and included articles that are indexed in scientific databases. Fifteen studies were included: seven pre-clinical and eight clinical studies. Despite the limited number of studies, the results suggest a positive effect of SrRan in patients with OA, through changes in functional capacity and reduction of progression of morphological parameters and joint degradation, with moderate quality of evidence for those clinical outcomes. Novel studies are necessary to elucidate the molecular targets of SrRan, focusing on anti-inflammatory effects and histological changes promoted by SrRan, which seemed to reduce the progression of OA in the experimental and clinical studies.

Development of a rat model of knee osteoarthritis by a combination of monoiodoacetate and streptozotocin / Desarrollo de un modelo de rata de osteoartritis de rodilla por una combinación de monoiodoacetato y estreptozotocina

SUMMARY: Osteoarthritis (OA) caused by ageing joints or as a secondary complication of diabetes is a common health problem. We sought to develop an animal model of OA induced by a combination of the chondrocyte glycolytic inhibitor mono-iodoacetate (MIA) and streptozotocin (STZ), the agent that induces diabetes mellitus. We then hypothesized that the extent of damages to the knee joint induced by this model can be greater than OA induced by either MIA or STZ. Rats were either injected with MIA (model 1) or STZ (model 2) or both agents (model 3). After 8 weeks, harvested tissues from the knee joint of these groups were examined using scanning and transmission electron microscopy. In addition, blood samples were assayed for tumor necrosis factor alpha (TNF-α) and interleukin -6 (IL-6) that are known to be modulated in OA and diabetes. Compared to control group, substantial damages to the articular cartilage of the knee joint were observed in the three models with the severest in model 3. In addition, rats in model 3 showed significant (P<0.0001) increase in TNF-α and IL-6 compared to model 1 and 2. Thus, we have developed a new model of knee OA in rats that mimics a type of OA that is common among elderly people who have both, "ageing" joints and diabetes.

RESUMEN: La osteoartritis (OA) es un problema generalizado de salud a causa de un envejecimiento de las articulaciones, o bien de una complicación secundaria de la diabetes. El objetivo de este estudio fue desarrollar un modelo animal de OA inducido por una combinación dos drogas, un inhibidor de los condrocitos glucolíticos, el mono-iodoacetato (MIA), y la estreptozotocina (STZ), agente que induce la diabetes mellitus. Se consideró como hipótesis que el alcance de los daños a la articulación de la rodilla inducida por este modelo puede ser mayor que la OA inducida por MIA o STZ. Las ratas fueron inyectadas con MIA (grupo 1) o STZ (grupo 2) o ambos agentes (grupo 3). Se extrajeron muestras de la articulación de la rodilla de estos grupos al término de 8 semanas, y se examinaron mediante microscopía electrónica de barrido y de transmisión. Además, se analizaron muestras de sangre para el factor de necrosis tumoral alfa (TNF-α) e interleucina-6 (IL-6), que están moduladas en OA y en la diabetes. En comparación con el grupo control, se observaron daños sustanciales en el cartílago articular de la articulación de la rodilla en los tres modelos, encontrándose los daños más severos en el grupo 3. Además, las ratas del grupo 3 mostraron un aumento significativo (P <0,0001) de los niveles de TNF-α e IL- 6, en comparación con los grupos 1 y 2. Hemos desarrollado un nuevo modelo de OA de rodilla en ratas que imita un tipo de OA el cual, además de la diabetes, es común entre las personas mayores con un nivel importante de daño en las articulaciones.

The effects of chitosan oligosaccharides on OPG and RANKL expression in a rat osteoarthritis model

Abstract Purpose: To investigate the effect of chitosan oligosaccharides (COS) against osteoarthritis (OA) and preliminarily discuss the osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL) and RANK expression in a rat OA model. Methods: Thirty-six 6-week-old Male SD rats were randomly divided into three groups: sham-operated group(CON), OA-induction group(OA), COS intervention group(n=12/group). At 4 weeks after the operation, COS (50 ul) intervention weekily for consecutive 5 weeks. The OA and CON groups received an injection of 50 ul physiological saline. At death, 11 weeks following surgery, cartilage was harvested and total RNA and protein were extracted. Both the morphological changes of the cartilage were observed and harvested the total RNA and protein. Meanwhile, the expression of OPG, RANKL and RANK in cartilage were determined. Results: The expression of OPG and RANKL were both enhanced in the cartilage of the OA model. Compared with the OA group, COS treatment improved the cartilage damage (both extent and grade). Furthermore, the COS group showed highly OPG and lower RANKL. Simultaneously, COS treatment upregulated the ratio of OPG/RANKL and downregulated the RANKL/RANK. Conclusion: Chitosan oligosaccharides may be used as a unique biological agent to prevent and treat osteoarthritis, and this effect is associated with modulation of the expression of osteoprotegerin and receptor activator of NF-κB ligand.

The effect of intra-articular levobupivacaine on shoulder cartilage at different doses - experimental study / O efeito de levobupivacaína intra-articular na cartilagem do ombro em doses diferentes-estudo experimental

Abstract Background and objectives: In this study it was aimed to examine the histological and morphometric effects on cartilage structure of intra-articular application of levobupivacaine to the shoulder joint. Methods: In twenty New Zealand adult male rabbits, 35 shoulders were used for the study and prepared in 5 groups of 7. These groups were defined as Groups L1, L2, L3 and L4 which were right shoulders administered with 0.25% and 0.5% levobupivacaine, Group C which were left shoulders as the control group and Groups S1 and S2 which were left shoulders administered with 0.9% saline. On the 2nd and 15th days the animals were killed, the glenohumeral joints were evaluated macroscopically then cartilage samples were taken. These samples were evaluated with Mankin score, and histomorphometrically by measuring the thickness of the cartilage between the superficial cartilage layer and the tidemark and the thickness of calcified cartilage between the tidemark and the subchondral bone. Results: Macroscopically, on the 15th day the joint fluid was seen to have reduced in all the groups. After microscopic evaluation, the highest Mankin score (mean: 3.14 ± 2.1/14) was in the L4 group (15th day 0.5% levobupivacaine) and was found to be statistically significant (p < 0.05). No statistically significant difference was determined between the other groups. Conclusions: Histologically, as the highest Mankin score was in the L4 group, this indicates that in a single intra-articular injection of levobupivacaine a low concentration should be selected. Level of evidence: Level 5, animal study.

Resumo Justificativa e objetivo: Neste estudo o objetivo foi examinar os efeitos histológicos e morfométricos sobre a estrutura da cartilagem da aplicação intra-articular de levobupivacaína em articulação do ombro. Métodos: Trinta e cinco ombros de 20 coelhos New Zealand, machos e adultos, foram usados para o estudo e divididos em cinco grupos de sete. Os grupos foram definidos como L1, L2, L3 e L4, consistiram em ombros direitos nos quais levobupivacaína a 0,25% e 0,5% foi administrada; o Grupo C, que consistiu em ombros esquerdos, foi o grupo controle; os grupos S1 e S2, que consistiram em ombros esquerdos, receberam solução salina a 0,9%. Os animais foram sacrificados no segundo e no 15º dia; as articulações glenoumerais foram avaliadas macroscopicamente e, em seguida, amostras de cartilagem foram coletadas. As amostras foram avaliadas com o escore de Mankin e histomorfometricamente. Mediu-se a espessura da cartilagem entre a camada superficial e a "linha de maré" (tidemark) e a espessura da cartilagem calcificada entre a tidemark e o osso subcondral. Resultados: Macroscopicamente, observou-se no 15º dia que o líquido articular havia reduzido em todos os grupos. Após a avaliação microscópica, o maior escore de Mankin (média: 3,14 ± 2,1/14) foi observado no grupo L4 (15º dia levobupivacaína a 0,5%), considerado estatisticamente significativo (p < 0,05). Nenhuma diferença estatisticamente significativa foi determinada entre os outros grupos. Conclusões: Histologicamente, como o maior escore de Mankin foi observado no Grupo L4, isso indica que em uma única injeção intra-articular de levobupivacaína uma concentração baixa deve ser selecionada. Nível de evidência: Nível 5, estudo em animais.

Comparison between two different experimental models of osteoarthritis in rabbits. Intra-articular collagenase injection and anterior cruciate ligament transection

ABSTRACT PURPOSE: To compare two different experimental models of osteoarthritis in rabbits: intra-articular collagenase injection and anterior cruciate ligament transection. METHODS: Ten adult rabbits were randomly divided in two groups: COLL (collagenase group) and ACLT (anterior cruciate ligament transection). The COLL group was treated with 0.5 ml collagenase solution (2mg collagenase/0.5 ml sterile PBS), and the ACTL group was subjected to anterior cruciate ligament. After six and twelve weeks, respectively, the animals in the COLL and ACTL groups were euthanized. The gross appearance and histological examinations conducted in the cartilage articular surface was blindly scored according to the criteria developed by Yoshimi et al. (1994) and Mankin et al. (1971), respectively. RESULTS: The gross morphologic observation, macroscopic score and histological examinations have demonstrated that the ACTL group presented the highest scores, and lesions more severe than those in the COLL group. CONCLUSIONS: Both methods, anterior cruciate ligament transection and collagenase, applied to the stifle joint of the rabbits have effectively induced degenerative changes in the cartilage tissue, through statistically significant analysis (p≤0.05). The ACTL method has presented more severe lesions.

Protective effects of tumor necrosis factor-α blockade by adalimumab on articular cartilage and subchondral bone in a rat model of osteoarthritis

We aimed to investigate the effects of an anti-tumor necrosis factor-α antibody (ATNF) on cartilage and subchondral bone in a rat model of osteoarthritis. Twenty-four rats were randomly divided into three groups: sham-operated group (n=8); anterior cruciate ligament transection (ACLT)+normal saline (NS) group (n=8); and ACLT+ATNF group (n=8). The rats in the ACLT+ATNF group received subcutaneous injections of ATNF (20 μg/kg) for 12 weeks, while those in the ACLT+NS group received NS at the same dose for 12 weeks. All rats were euthanized at 12 weeks after surgery and specimens from the affected knees were harvested. Hematoxylin and eosin staining, Masson's trichrome staining, and Mankin score assessment were carried out to evaluate the cartilage status and cartilage matrix degradation. Matrix metalloproteinase (MMP)-13 immunohistochemistry was performed to assess the cartilage molecular metabolism. Bone histomorphometry was used to observe the subchondral trabecular microstructure. Compared with the rats in the ACLT+NS group, histological and Mankin score analyses showed that ATNF treatment reduced the severity of the cartilage lesions and led to a lower Mankin score. Immunohistochemical and histomorphometric analyses revealed that ATNF treatment reduced the ACLT-induced destruction of the subchondral trabecular microstructure, and decreased MMP-13 expression. ATNF treatment may delay degradation of the extracellular matrix via a decrease in MMP-13 expression. ATNF treatment probably protects articular cartilage by improving the structure of the subchondral bone and reducing the degradation of the cartilage matrix.

Influence of different body positions in vital capacity in patients on postoperative upper abdominal / Influência de diferentes posições corporais na capacidade vital em pacientes no pós-operatório abdominal superior / Influencia de diferentes posiciones corporales en la capacidad vital en pacientes en el postoperatorio abdominal superior

RATIONALE: The changes in body position can cause changes in lung function, and it is necessary to understand them, especially in the postoperative upper abdominal surgery, since these patients are susceptible to postoperative pulmonary complications. OBJECTIVE: To assess the vital capacity in the supine position (head at 0° and 45°), sitting and standing positions in patients in the postoperative upper abdominal surgery. METHODS: A cross-sectional study conducted between August 2008 and January 2009 in a hospital in Salvador/BA. The instrument used to measure vital capacity was analogic spirometer, the choice of the sequence of positions followed a random order obtained from the draw of the four positions. Secondary data were collected from the medical records of each patient. RESULTS: The sample consisted of 30 subjects with a mean age of 45.2 ± 11.2 years, BMI 20.2 ± 1.0 kg/m2. The position on orthostasis showed higher values of vital capacity regarding standing (mean change: 0.15 ± 0.03 L; p = 0.001), the supine to 45 (average difference: 0.32 ± 0.04 L; p = 0.001) and 0° (0.50 ± 0.05 L; p = 0.001). There was a positive trend between the values of forced vital capacity supine to upright posture (1.68 ± 0.47; 1.86 ± 0.48; 2.02 ± 0.48 and 2.18 ± 0.52 L; respectively). CONCLUSION: Body position affects the values of vital capacity in patients in the postoperative upper abdominal surgery, increasing in postures where the chest is vertical. .

JUSTIFICATIVA: As alterações no posicionamento corporal podem ocasionar mudanças na função respiratória e é necessário compreendê-las, principalmente no pós-operatório abdominal superior, já que os pacientes estão suscetíveis a complicações pulmonares pós-operatórias. OBJETIVO: Verificar a capacidade vital nas posições de decúbito dorsal (cabeceira a 0° e 45°), sentado e em ortostase em pacientes no pós-operatório de cirurgia abdominal superior. MÉTODOS: Estudo transversal, feito entre agosto de 2008 e janeiro de 2009, em um hospital na cidade de Salvador (BA). O instrumento usado para mensuração da capacidade vital (CV) foi o ventilômetro analógico e a escolha da sequência das posições seguiu uma ordem aleatória obtida a partir de sorteio das quatro posições. Os dados secundários foram colhidos nos prontuários de cada paciente. RESULTADOS: A amostra foi composta por 30 indivíduos com idade média de 45,2 ± 11,2 anos e IMC 20,2 ± 1,0 kg/m2. A posição em ortostase apresentou valores maiores da CV em relação à sedestração (média das diferenças: 0,15 ± 0,03 litros; p = 0,001), ao decúbito dorsal a 45° (média das diferenças: 0,32 ± 0,04 litros; p = 0,001) e 0° (0,50 ± 0,05 litros; p = 0,001). Houve um aumento positivo entre os valores de CVF do decúbito dorsal para a postura ortostática (1,68 ± 0,47; 1,86 ± 0,48; 2,02 ± 0,48 e 2,18 ± 0,52 litros; respectivamente). CONCLUSÃO: A posição do corpo afeta os valores da CV em pacientes no pós-operatório de cirurgia abdominal superior, com aumento nas posturas em que o tórax encontra-se verticalizado. .

JUSTIFICACIÓN: Las alteraciones en el posicionamiento corporal pueden ocasionar cambios en la función respiratoria y es necesario comprenderlas, principalmente en el postoperatorio abdominal superior, ya que los pacientes son susceptibles a complicaciones pulmonares postoperatorias. OBJETIVO: Verificar la capacidad vital en las posiciones de decúbito dorsal (cabeza a 0° y 45°), sentado y en ortostasis en pacientes en el postoperatorio de cirugía abdominal superior. MÉTODOS: Estudio transversal realizado entre agosto de 2008 y enero de 2009, en un hospital en la ciudad de Salvador (BA). El instrumento usado para la medición de la capacidad vital (CV) fue el espirómetro analógico y la elección de la secuencia de las posiciones siguió un orden aleatorio que se obtuvo a partir de un sorteo de las 4 posiciones. Los datos secundarios fueron extraídos de las historias clínicas de cada paciente. RESULTADOS: La muestra se compuso de 30 individuos con edades medias de 45,2 ± 11,2 años e IMC de 20,2 ± 1 kg/m2. La posición en ortostasis presentó valores mayores de CV con relación a la posición sedente (media de las diferencias: 0,15 ± 0,03 L; p = 0,001), al decúbito dorsal a 45° (media de las diferencias: 0,32 ± 0,04 L; p = 0,001) y a 0° (0,50 ± 0,05 L; p = 0,001). Hubo un aumento positivo entre los valores de CV forzada del decúbito dorsal para la postura ortostática (1,68 ± 0,47; 1,86 ± 0,48; 2,02 ± 0,48 y 2,18 ± 0,52 L, respectivamente). CONCLUSIÓN: La posición del cuerpo afecta los valores de la CV en pacientes durante el postoperatorio de cirugía abdominal superior, con aumento en las posturas en las que el tórax está verticalizado. .

Análisis histopatológico del cartílago articular expuesto a metilprednisolona, ropivacaína y ácido hialurónico en un modelo animal de osteoartrosis / Histopathological analysis of the articular cartilage exposed to methylprednisolone, ropivacaine and hyaluronic acid in an animal model of osteoarthrosis

La osteoartrosis es un padecimiento del aparato locomotor con una prevalencia elevada y en crecimiento, paralela al envejecimiento de la población. La infiltración intraarticular de sustancias para aliviar la sintomatología de la osteoartrosis es una práctica común en el consultorio médico de los especialistas que atienden esta enfermedad. Aunque la sintomatología mejora con la infiltración de anestésicos locales, corticoesteroides y suplementos viscosantes, es aún incierto el efecto que estas sustancias tienen sobre la integridad del cartílago articular. Este estudio explora a nivel macroscópico e histológico el efecto de la infiltración de ropivacaína, metilprednisolona y ácido hialurónico sobre el cartílago articular en un modelo de osteoartrosis química en conejos (n=24). Nuestros resultados indican que en los grupos infiltrados con metilprednisolona (n=8) y ropivacaína (n=8) la estructura del cartílago articular presento alteraciones más severas con respecto a su grupo control, además de una disminución importante en la síntesis de matriz extracelular. En el grupo infiltrado con ácido hialurónico (n=8), las alteraciones macroscópicas e histológicas del cartílago articular mejoraron con respecto a su grupo control, presentando una estructura integra y síntesis de matriz extracelular normal.

Osteoarthritis is a musculoskeletal condition with a high prevalence, increasing with the aging of population. The intraarticular infiltration of substances to relieve the symptoms of osteoarthritis is a common practice in medical practice. Although symptoms improved with the infiltration of local anesthetics, corticosteroids and supplements, it is still uncertain what effect these substances have on the integrity of articular cartilage. This study explores the macroscopic and histological effects of infiltration of Ropivacaine, Methylprednisolone and Hyaluronic Acid on articular cartilage in a model of chemical osteoarthritis in rabbits (n=24). Our results indicate that in the infiltrated groups with Methylprednisolone (n=8) and Ropivacaine (n=8) the structure of articular cartilage present more severe alterations with respect to its control group and an important decrease in the synthesis of extracellular matrix. In-group infiltrated with hyaluronic acid (n=8), macroscopic and histological changes of articular cartilage improved with respect to its control group, presenting a normal structure and normal extracellular matrix synthesis.

Glucosamine and chondroitin sulfate association increases tibial epiphyseal growth plate proliferation and bone formation in ovariectomized rats

OBJECTIVE: The growth plate consists of organized hyaline cartilage and serves as a scaffold for endochondral ossification, a process that mediates longitudinal bone growth. Based on evidence showing that the oral administration of glucosamine sulfate (GS) and/or chondroitin sulfate (CS) is clinically valuable for the treatment of compromised articular cartilage, the current study evaluated the effects of these molecules on the tibial epiphyseal growth plate in female rats. METHOD: The animals were divided into two control groups, including vehicle treatment for 45 days (GC45) and 60 days (GC60) and six ovariectomized (OVX) groups, including vehicle treatment for 45 days (GV45), GS for 45 days (GE45GS), GS+CS for 45 days (GE45GS+CS), vehicle for 60 days (GV60), GS for 60 days (GE60GS) and GS+CS for 60 days (GE60GS+CS). At the end of treatment, the tibias were dissected, decalcified and processed for paraffin embedding. Morphological and morphometric methods were employed for analyzing the distal tibial growth plates using picrosirius red staining and the samples were processed for histochemical hyaluronan detection. Morphometric analyses were performed using the 6.0ProPlus¯ Image system. RESULTS: Notably, after 60 days of treatment, the number of proliferative chondrocytes increased two-fold, the percentage of remaining cartilage increased ...

SKI306X inhibition of glycosaminoglycan degradation in human cartilage involves down-regulation of cytokine-induced catabolic genes

BACKGROUND/AIMS: SKI306X, a mixed extract of three herbs, Clematis mandshurica (CM), Prunella vulgaris (PV), and Trichosanthes kirilowii (TK), is chondroprotective in animal models of osteoarthritis (OA). The objectives of this study were to investigate its effect on interleukin (IL)-1beta-induced degradation of glycosaminoglycan (GAG) and the basis of its action in human OA cartilage, as well as to screen for the presence of inhibitors of matrix metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 in SKI306X and its component herbs, as well as in fractions from SKI306X. METHODS: Human OA chondrocytes and cartilage explants were obtained during total knee replacements and incubated with IL-1beta +/- oncostatin M with or without SKI306X or its component herb extracts. GAG degradation was assayed in cartilage explants using a commercial kit. Expression of genes involved in cartilage destruction was measured by real-time polymerase chain reaction using chondrocyte RNA. SKI306X was fractionated by preparative liquid chromatography to test for the presence of inhibitors of MMP-13 and ADAMTS-4. RESULTS: SKI306X and PV inhibited IL-1beta-induced GAG release from cartilage explants, and SKI306X, CM, PV, and TK inhibited IL-1beta-induced MMP gene expression. Unexpectedly, SKI306X greatly stimulated IL-1beta + oncostatin M-induced ADAMTS-4 gene expression, probably due to its TK component. Some fractions of SKI306X also inhibited ADAMTS-4 activity. CONCLUSIONS: SKI306X and its herbal components inhibit GAG degradation and catabolic gene expression in human OA chondrocytes and cartilage explants. SKI306X likely also contains one or more ADAMTS-4 inhibitor.

The metabolic dynamics of cartilage explants over a long-term culture period

INTRODUCTION: Although previous studies have been performed on cartilage explant cultures, the generalized dynamics of cartilage metabolism after extraction from the host are still poorly understood due to differences in the experimental setups across studies, which in turn prevent building a complete picture. METHODS: In this study, we investigated the response of cartilage to the trauma sustained during extraction and determined the time needed for the cartilage to stabilize. Explants were extracted aseptically from bovine metacarpal-phalangeal joints and cultured for up to 17 days. RESULTS: The cell viability, cell number, proteoglycan content, and collagen content of the harvested explants were analyzed at 0, 2, 10, and 17 days after explantation. A high percentage of the cartilage explants were found to be viable. The cell density initially increased significantly but stabilized after two days. The proteoglycan content decreased gradually over time, but it did not decrease to a significant level due to leakage through the distorted peripheral collagen network and into the bathing medium. The collagen content remained stable for most of the culture period until it dropped abruptly on day 17. CONCLUSION: Overall, the tested cartilage explants were sustainable over long-term culture. They were most stable from day 2 to day 10. The degradation of the collagen on day 17 did not reach diseased levels, but it indicated the potential of the cultures to develop into degenerated cartilage. These findings have implications for the application of cartilage explants in pathophysiological fields.

healing effect of bioglue in articular cartilage defect of femoral condyle in experimental rabbit model

The full-thickness articular cartilage defects of knee have a poor healing capacity that may progress to osteoarthritis and need a knee replacement. This study determines the healing effect of bioglue in full-thickness articular cartilage defect of femoral condyle in rabbit. Forty-eight male rabbits were randomly divided into four equal groups. In group A, 4 mm articular cartilage defects were created in the right and left medial femoral condyles. Then a graft from xiphoid cartilage was transferred into the defect together with a designed bioglue and the knees were closed. In group B, an articular cartilage defect was created identical to group A, but the defect size was 6 mm. In group C, 4 and 6 mm articular cartilage defects were created in the right and left medial femoral condyles respectively. The graft was transferred into the defect and the knees were stitched. In group D, articular cartilage defects were created similar to group C, just filled with bioglue and closed. The rabbits were euthanized and subgroups were defined as A1, B1, C1 and D1 after 30 days and A2, B2, C2 and D2 after 60 days. The cartilages were macroscopically and histologically investigated for any changes. Microscopic and macroscopic investigations showed that bioglue had a significant healing effect in the femoral condyle. Addition of bioglue can effectively promote the healing of articular cartilage defects

Análise fotocolorimétrica computadorizada dos efeitos da betametasona intra-articular sobre a concentração de proteoglicanos da matriz cartilaginosa de joelhos leporinos: influência do número de injeções intra-articulares / Computerized photocolorimetric analysis of the effects of intraarticular betamethasone on the proteoglycan concentration of leporine knee cartilage matrix: influence of the number of intraarticular injections

OBJETIVO: Analisar os efeitos da injeção repetida de betametasona na concentração de proteoglicanos da cartilagem articular do joelhos normais de coelhos californianos de ambos os sexos. MÉTODOS: Os animais foram randomizados em oito grupos de dez animais cada. Três grupos controle (injeção ou não de solução salina isotônica) e cinco grupos de estudo - doses terapêuticas, repetidas ou não, de betametasona injetadas no joelho direito de cada coelho, com intervalos semanais. Após oito dias da última injeção prevista, cortes histológicos da cartilagem das áreas de apoio dos platôs tibiais foram corados com hematoxilina e eosina para análise por microscopia óptica, e com safranina O para a pesquisa da quantidade de proteoglicanos. A intensidade da coloração da safranina O foi quantificada em aparelho de histomorfometria, composto por microscópio Olympus BX 50 e microcomputador com software Image Pro-plus 4.5Ò. RESULTADOS: Não houve diferenças nos animais que tiveram seus joelhos injetados com betametasona uma, duas e quatro vezes quando comparados com os grupos controle. Nos animais que receberam seis e oito aplicações a intensidade da coloração com safranina O reduziu-se significativamente (p < 0,05) quando comparada tanto com grupos controle quanto com os outros de estudo. CONCLUSÃO: Foi possível demonstrar redução da concentração de proteoglicanos na matriz cartilaginosa articular dependente do efeito deletério cumulativo das repetidas injeções intra-articulares de betametasona.

OBJECTIVE: To study the effects of repeated injections of betamethasone on proteoglycan concentration in the articular cartilage of normal knees of Californian rabbits of both sexes. METHODS: Eighty animals were randomly divided into eight groups of ten animals each. Three control groups (saline solution injected or not) and five study groups - therapeutical doses, repeated or not, of betamethasone injected into the right knee of each animal at weekly intervals. After eight days from the last injection, sections of articular cartilage from tibial plateaus collected from weight-bearing surfaces were stained with hematoxylin and eosin for light microscopy analysis and with safranin O for the proteoglycan content assay. The staining intensity of safranin O was quantified by histomorphometry using an Olympus BX 50 microscope and a microcomputer with the Image Pro-plus 4.5Ò software. RESULTS: Animals receiving one, two and four betamethasone injections showed no differences when compared to normal controls. Animals receiving six and eight injections had a significant decrease in safranin O staining intensity (p < 0.05) as compared to the control groups and the other study groups. CONCLUSION: A decrease in the concentration of articular cartilage proteoglycans dependent on repeated betamethasone injection was effectively demonstrated.

Effect of intraarticular injection of lornoxicam on the articular cartilage & synovium in rat.

BACKGROUND & OBJECTIVE: Intraarticular (i.a) drug application is consider to be a new therapeutic approach for the treatment of postoperative pain after arthroscopic knee surgery without any systemic adverse effects. Lornoxicam, a nonsteroid anti-inflammatory drug is a short acting agent, and its anti-inflammatory and analgesic activity may be effective in the postoperative pain management in minor surgery. In this study, the effects of intraarticular administration of lornoxicam on the synovium and articular cartilage in the rat knee joint were investigated. METHODS: Lornoxicam (0.25 ml) was given as an injection into the right knee joint and 0.25 ml of 0.9 per cent saline solution by injection into the left knee joint as a control in 25 rats. Groups of five rats were sacrificed by a lethal injection of ketamine 1st, 2nd, 7th, 14th and 21st days after lornoxicam administration. Knee joints were detached, fixed in 10 per cent buffered formalin and decalcified. Serial sections of 5 microm were stained with haematoxylin-eosin and evaluated for the presence of inflammation in the articular, periarticular regions and synovium. Inflammatory changes in the joints were graded according to a five-point scale, histologically. RESULTS: There were no significant differences in inflammation and cartilage degeneration, between control and lornoxicam applied knees. Grade 3 inflammatory changes occurred only in one knee in lornoxicam group, at 24 h after injection. No pathological changes were observed in both groups at any time point. INTERPRETATION & CONCLUSION: Lornoxicam did not show significant effect on inflammation on rat synovia in knee joint. Further studies including in human need to be done before any recommendations are made for i.a. administration of lornoxicam.

Efectos de Diclofenac, Celecoxib y sulfato de glucosamina sobre marcadores inflamatorios en cultivo de condrocitos de pacientes con osteoartritis / Articular cartilage in osteoarthritic patients: effects of diclofenac, celecoxib and glucosamine sulfate on inflammatory markers

La osteoartritis (OA) es una enfermedad articular crónica, progresiva que se instala como consecuencia de un proceso complejo que involcra alteraciones mecánicas y biológicas del sistema músculo-esquelético, siendo resultante de múltiples interacciones entre factores genéticos e injurias extrínsecas. La patogenia de esta enfermedad se ralaciona con alta y desviada producción de citokinas flogógenas y de enzimas proteolíticas, que degradan y destruyen la matriz extracelular en tejidos articulares y peri-articulares. Se estudiaron 20 casos con OA, de los cuales se obtuvo cartílago durante intervenciones quirúrgicas programadas. El cartílago se cultivó en medio Dulbecco-Eagle, con o sin agregado de AINEs o condromodulares. En los sobrenadantes se determinaron óxido nítrico por reacción de Giess y la medición espectrofotométrica; y colagenasa por ELISA doble sándwich en presencia de anticuerpos monoclonales. En ausencia de AINEs, los cultivos de condrocitos produjeron 1950 ± 665ng/ml de MMP-1, La adición de Diclofenac redujo esa cifra a 1140 ± 155 ng/ml, aunque esta diferencia no fue estadisticamente significativa, (p<0.60). Por el contrario, Celecoxib redujo el nivel de la enzima a 760 ± 75ng/ml (p<0,01) y la Glucosamina también provocó un descenso (950 ± 89 ng/ml) significativo (p<0.05). Los niveles de ON en ausencia de AINEs llegaron a 47,3 ± 4,9 µM. Su producción no varió significativamente con la adición de Diclofenac, Ceecoxib o Glucosamina (p=ns). Los resultados indicarían la incapacidad de Diclofenac para modificar la generación de enzimas proteolíticas, mientras que Celecoxib y Glucosamina disminuyen su producción significativamente. Ninguno de los fármacos utilizados en nuestro trabajo ha logrado alterar la concentración de ON. Muchos integrantes quedan aún sin resolver y todavía se carece de fármacos de eficacia comprobada para alterar el curso natural de la enfermedad.

Osteoarthritis is a chronic and progressive joint disease. It is established by a complex process involving mechanical and biological alterations of the musculoskeletal system, which are generated by a great variety of interactions between genetic factors and extrinsic injuries. The pathogenesis of this disease is related to an increased and divergent production of inflammatory markers and proteolytic enzymes that promote the degradation and destruction of the extracellular matrix of articular and periarticular tissues. Cartilage samples were taken from 20 osteoarthritic patients during programmed surgical interventions. The cartilage samples were cultured in Dulbecco-Eagle medium, with or without the addition of NSAIDs or modulators of chondrocyte metabolism. The content of nitric oxide in the supernatant was quantified using the Griess reaction; the concentration of MMP-1 was quantified via double-sandwich ELISA. Untreated chondrocyte cultures produced 1950 +/- 665 ng/ml MMP-1. With the addition of Diclofenac this value decreased to 1140 +/- 155 ng/ml, although this difference was not statistically significant (p < 0.06). However, in the presence of Celecoxib the level significantly dropped to 760 +/- 75 ng/ml (p < 0.01). Although the addition ofglucosamine did not produce such a noticeable reduction in the level of MMP-1 (950 +/- 89 ng/ml), it was statistically significant (p < 0.05). On the contrary, none of the drugs (Diclofenac, Celecoxib, Glucosamine) modified the level of nitric oxide which had a mean value of 47.3 +/- 4.9 microM in the control samples. This investigation evidenced the inability of Diclofenac to significantly modify the production of proteolytic enzymes in osteoarthritic chondrocyte cultures. However, both Celecoxib and Glucosamine significantly reduced the production of MMP-1. On the contrary, none of the drugs used in this study managed to modify the concentration of nitric oxide. To the present day, no drugs have been found to be...

Effects of intraarticular contrast media on synovial membrane and cartilage. An electron microscopic evaluation in rabbit knees

To evaluate the histological and ultrastructural alterations in rabbit knee joint cartilage and synovia induced by intraarticular injections of 2 water soluble contrast agents. The study was conducted at the Department of Orthopedics and Traumatology, Medical Faculty, Osmangazi University, Eskisehir, Turkey in January 2002. To examine the effect of contrast agents on articular cartilage and synovial membrane, rabbit model was used. Specimens from 62 knee joints were examined by light microscopy and transmission electron microscopy one hour, one day, one week and 2 weeks after intraarticular administration of gadolinium-diethylenetriamine pentaacetic acid, iopromide or saline. In the knees injected with saline, light microscopic changes of the synovium consisted of edema only. Edema and hyperemia were seen in contrast agent injected knees. Ultrastructurally, numerous and large pinocytotic vesicles in A cells of the synovial membrane were seen in contrast agent injected groups. In the knees injected with saline the cartilage were ultrastructurally normal but contrast agent injected knees showed increased activation of chondrocytes with increase of dense glycogen accumulation, large lipid vacuoles and matrix material. There were very rare pycnotic cells in these samples. The rating scale has been used and the means of the total scores were determined for the groups. The effects of contrast agents reduced gradually on the cartilage and synovium in general but did not become completely normal in the observation period

The use of resorcine adhesive in repairing osteochondral's defect in knees of rabbits

PURPOSE: To study the morphology of the articulation of the knee of rabbits after the repairing of the defect osteochondral standardized with resorcina adhesive or metallic synthesis. METHODS: The procedure was to the creation of the defect osteochondral in femoral medial condylus of the knee of 80 rabbits, The animals were distributed in two groups with continuations of 7 and 42 days and submitted to the technique G (resection and retreat of the fragment osteochondral of the femoral medial condylus and relocation with resorcina adhesive), technique S (resection and retreat of the fragment osteochondral of the femoral medial condylus and relocation and metallic synthesis) or technique C (resection and retreat of the fragment osteochondral of the femoral medial condylus, leaving the empty standard defect the control). It was Made clinical study, radiographic, macroscopic and histological in two groups. RESULTS: the resorcina adhesive provokes: necrosis of the fragment osteochondral in 100 percent and 95 percent, degeneration 90 percent and 100 percent, free body in 80 percent and 65 percent respectively in the group I and II; compared with the metallic synthesis that it presented: necrosis in 25 percent and 35 percent, degeneration 25 percent and 35 percent, free body in 35 percent and 10 percent respectively in the group I and II. CONCLUSION: the resorcinol adhesive, related with the necrosis, cartilaginous degeneration and detachment of the fragment osteochondral lives frequently that the metallic synthesis.

OBJETIVO: Estudar a morfologia da articulação do joelho de coelhos após a reparação de um defeito osteocondral padronizado com adesivo de resorcina ou síntese metálica. MÉTODOS: Procedeu-se à criação de um defeito osteocondral em côndilo femoral medial do joelho de 80 coelhos. Os animais foram distribuídos em dois grupos com seguimentos de 7 e 42 dias e submetidos à técnica G (ressecção e retirada do fragmento osteocondral do côndilo femoral medial e recolocação com adesivo de resorcina), técnica S (ressecção e retirada do fragmento osteocondral do côndilo femoral medial e recolocação e síntese metálica) ou técnica C (ressecção e retirada do fragmento osteocondral do côndilo femoral medial, deixando o defeito padrão vazio como controle). Fez-se estudo clínico, radiográfico, macroscópico e histológico nos dois grupos. RESULTADOS: o adesivo de resorcina provoca: necrose do fragmento osteocondral em 100 por cento e 95 por cento, degeneração 90 por cento e 100 por cento, corpo livre em 80 por cento e 65 por cento respectivamente no grupo I e II; comparado com a síntese metálica que apresentou: necrose em 25 por cento e 35 por cento, degeneração 25 por cento e 35 por cento, corpo livre em 35 por cento e 10 por cento respectivamente no grupo I e II. CONCLUSÃO: o adesivo de resorcina, está relacionado com a necrose, degeneração cartilaginosa e despreendimento do fragmento osteocondral com maior freqüência que a síntese metálica.

Comparative efficacy and tolerability of nimesulide and piroxicam in osteoarthritis with specific reference to chondroprotection: a double blind randomised study.

The aim of the study was to assess the efficacy, tolerability and chondroprotection afforded by nimesulide, a selective cyclooxygenase-2 inhibitor and piroxicam in a randomised, double blind, controlled clinical trial in 90 patients suffering from osteoarthritis of the knee joint. A significant improvement in the osteoarthritis severity index at 2 weeks (p < 0.01) and an improvement in physicians assessment of global arthritic condition at 4 weeks (p < 0.01) was seen with both the treatments. A significant decrease in articular index of joint tenderness (p < 0.05) at 8 weeks and in self assessment of handicap at 4 weeks (p < 0.05), in comparison to baseline, was observed only in patients receiving nimesulide. Rescue therapy was required by a greater percentage of patients being administered piroxicam. Functional capacity improved in 64% of the patients on nimesulide and 74.5% of the patients receiving piroxicam. Adverse effects were observed in 6 patients on nimesulide and 9 patients receiving piroxicam. No significant difference was found in any of the efficacy and tolerability parameters between the two treatment groups. Magnetic resonance imaging evaluation of the knee joint of 10 patients showed no significant change in the articular cartilage and associated joint structures after 6 months of therapy with both the treatments. The results show that nimesulide and piroxicam are comparable in efficacy and tolerability in patients suffering from osteoarthritis.