A comparative immunohistochemical and quantitative study of the epiligament of the medial collateral and anterior cruciate ligament in rat knee / Estudio inmunohistoquímico y cuantitativo del epiligamento del ligamento colateral medial y cruzado anterior en rodilla de rata

SUMMARY: The aim of the present study was to evaluate the importance of the epiligament for the difference in the healing potential of the knee anterior cruciate and medial collateral ligament. To do so, we compared the structure of the anterior cruciate and the medial collateral ligament and evaluated the differences in the expression of collagen types I, III and V in a rat knee. We have also conducted a comparative quantitative analysis of the number of cells per mm2 in the two ligaments. Tissue samples were obtained from the anterior cruciate and medial collateral ligament of 10 knee joints taken from five 8-month-old Wistar rats. We used standard hematoxylin and eosin staining, in addition to immunohistochemical staining with monoclonal antibodies against collagen types I, III and V. A semi-quantitative analysis of the expression was made through ImageJ, while Student's T-test was used for the statistical analysis. Our results showed higher expression of all collagen types in the epiligament, compared to the ligament proper and difference in the expression between the medial collateral and the anterior cruciate ligament in favor of the first. We also reported a statistically significant difference in the number of cells per mm2 between the two ligaments and their epiligaments. Our findings show a higher number of cells and a stronger expression of certain collagen types in the epiligament of the medial collateral compared to the anterior cruciate ligament, which may be related to the difference in their healing potential.

RESUMEN: El objetivo del presente estudio fue evaluar la importancia del epiligamento para la diferencia en el potencial de curación del ligamento cruzado anterior y colateral medial de la rodilla. Comparamos la estructura del ligamento cruzado anterior y el ligamento colateral medial y evaluamos las diferencias en la expresión de los tipos de colágeno I, III y V en una rodilla de rata. También se realizó un análisis cuantitativo comparativo del número de células por mm2 en los dos ligamentos. Se obtuvieron muestras de tejido del ligamento cruzado anterior y colateral medial de 10 articulaciones de rodilla tomadas de cinco ratas Wistar de 8 meses de edad. Utilizamos tinción estándar con hematoxilina y eosina, además de tinción inmunohistoquímica con anticuerpos monoclonales contra colágeno tipo I, III y V. Se realizó un análisis semicuantitativo de la expresión mediante ImageJ, mientras que para el análisis estadístico se utilizó la prueba T de Student. Nuestros resultados mostraron una mayor expresión de todos los tipos de colágeno en el epiligamento, en comparación con el ligamento y una diferencia en la expresión entre el ligamento colateral medial y el ligamento cruzado anterior. También informamos una diferencia estadísticamente significativa en el número de células por mm2 entre los dos ligamentos y sus epiligamentos. Nuestros hallazgos muestran un mayor número de células y una expresión mayor de ciertos tipos de colágeno en el epiligamento colateral medial en comparación con el ligamento cruzado anterior, lo que puede estar relacionado con la diferencia en su potencial de curación.

Immunohistochemical Study of Matrix Metalloproteinase-9 in Medial Collateral Ligament Epiligament in Rat Knee after Grade III Injury.

Background: In the present study, we conduct an immunohistochemical analysis to evaluate the expression of matrix metalloproteinase-9 (MMP-9) in the epiligament (EL) after grade III injury of the medial collateral ligament (MCL) in an experimental model of a rat knee. Methods: Twelve 8-month old male Wistar rats were used in this study. Three animals were used as controls, while the remaining nine underwent grade III injury of the MCL. The MMP-9 immunoreactivity was evaluated on the 8th, 16th and 30th day after injury. Results: We observed an intensive expression of the enzyme in all periods after injury in contrast with the control group. We also discovered that the main source of matrix metalloproteinase-9 was localized in the epiligament tissue. Immunoreactivity was highest and homogeneously distributed on the 8th day and gradually diminished, concentrating on the EL-ligament border and the perivascular zones on the 30th day. Conclusion: We present the first immunohistochemical study of the expression and distribution of the enzyme MMP-9 in the EL of the MCL and track the changes in enzyme activity on the 8th, 16th and 30th day after damage.

Expression of Matrix Metalloproteinase-2 and 9 in the Medial Collateral Ligament Epiligament in Rat Knee.

Background: In this study, we describe and compare the different expression of matrix metalloproteinase-2 and 9 (MMP-2 and 9) in the epiligament (EL) of the medial collateral ligament (MCL) of the rat knee. Methods: Twelve 8-month old male Wistar rats were used in this study. The MCL and the external surface of the surrounded EL were precisely dissected. The description of the structure of EL and the expression of MMP-2 and 9 were implemented by light microscopic and imunohistochemical analysis. Results: In the ligament, a classic morphological structure was found. The expression of the MMP-2 and MMP-9 was fSound in the adventitia of the blood vessels and in the fibroblast in the EL. The reaction of MMP-2 was more intensive in comparison to MMP-9. Conclusion: We present one of the first study of immunohistochemical localization and distribution of the enzyme MMP-2 and 9 in EL tissue and furthermore we describe and compare the enzyme activity of MMP-2 and 9 in the EL.