Are There Any Differences in the Healing Capacity between the Medial Collateral Ligament's (MCL) Proximal and Distal Parts in the Human Knee? Quantitative and Immunohistochemical Analysis of CD34, α-Smooth Muscle Actin (α-SMA), and Vascular Endothelial Growth Factor (VEGF) Expression Regarding the Epiligament (EL) Theory.

The human knee is a complex joint that comprises several ligaments, including the medial collateral ligament (MCL). The MCL provides stability to the knee and helps prevent its excessive inward movement. The MCL also has a thin layer of connective tissue known as the epiligament (EL), which adheres to the ligament. This unique feature has drawn attention in the field of ligament healing research, as it may have implications for the recovery process of MCL injuries. According to the EL theory, ligament regeneration relies heavily on the provision of cells, blood vessels, and molecules. The present study sought to compare the expression of vascular endothelial growth factor (VEGF), CD34, and α-smooth muscle actin (α-SMA) in healthy knees' proximal and distal MCL segments to better understand how these proteins affect ligament healing. By improving the EL theory, the current results could lead to more effective treatments for ligament injury. To conduct the present analysis, monoclonal antibodies were used against CD34, α-SMA, and VEGF to examine samples from 12 fresh knee joints' midsubstance MCLs. We identified a higher cell density in the EL than in the ligament connective tissue, with higher cell counts in the distal than in the proximal EL part. CD34 immunostaining was weak or absent in blood vessels and the EL, while α-SMA immunostaining was strongest in smooth muscle cells and the EL superficial layer. VEGF expression was mainly in the blood vessels' tunica media. The distal part showed more SMA-positive microscopy fields and higher cell density than the proximal part (4735 vs. 2680 cells/mm2). Our study identified CD34, α-SMA, and VEGF expression in the MCL EL, highlighting their critical role in ligament healing. Differences in α-SMA expression and cell numbers between the ligament's proximal and distal parts may explain different healing capacities, supporting the validity of the EL theory in ligament recovery.

Do the Differences in the Epiligament of the Proximal and Distal Parts of the Anterior Cruciate Ligament Explain Their Different Healing Capacities? Quantitative and Immunohistochemical Analysis of CD34 and α-SMA Expression in Relation to the Epiligament Theory.

The aim of this study was to assess the epiligament theory by determining the normal epiligament morphology of the proximal and distal parts of the anterior cruciate ligament in humans and analyzing the differences between them and the midportion of the ligament in terms of cell numbers and expression of CD34 and α-SMA. Samples were obtained from the anterior cruciate ligaments of 12 fresh knee joints. Monoclonal antibodies against CD34 and α-SMA were used for immunohistochemistry. Photomicrographs were analyzed using ImageJ software, version 1.53f. The cell density was higher in the epiligament than in the ligament connective tissue. Cell counts were higher in the proximal and distal thirds than in the midsubstance of the epiligament. CD34 was expressed similarly in the proximal and distal thirds, although it seemed slightly more pronounced in the distal third. α-SMA expression was more robust in the proximal than the distal part. The results revealed that CD34 and α-SMA are expressed in the human epiligament. The differences between the numbers of cells in the proximal and distal parts of the epiligament and the expression of CD34 and α-SMA enhance epiligament theory. Future investigations into improving the quality of ligament healing should not overlook the epiligament theory.

A comparative study of the epiligament of the medial collateral and anterior cruciate ligaments in the human knee: Immunohistochemical analysis of CD 34, α-smooth muscle actin and vascular endothelial growth factor in relation to epiligament theory.

BACKGROUND: This study evaluated and compared the expression of VEGF, CD34, and α-SMA in the anterior cruciate ligaments and medial collateral ligaments in healthy human knees in order to enrich the epiligament theory regarding ligament healing after injury. METHODS: Samples from the mid-substance of the anterior cruciate ligament and the medial collateral ligament of 12 fresh knee joints were used. Monoclonal antibodies against CD34, α-SMA, and VEGF were used for immunohistochemical analysis. Photomicrographs were analyzed using the ImageJ software. RESULTS: The epiligament of the anterior cruciate ligament showed slightly higher expression of CD34, α-SMA, and VEGF than the epiligament of the medial collateral ligament. Overall, among the tested markers, α-SMA expression was most pronounced in anterior cruciate ligament epiligament images and CD34 dominated in medial collateral ligament epiligament images. The intensity of DAB staining for CD34, α-SMA, and VEGF was higher in vascular areas of the epiligament than in epiligament connective tissue. CONCLUSIONS: The results illustrate that CD34, α-SMA, and VEGF are expressed in the human epiligament. The differences between the epiligament of the investigated ligaments and the fact that CD34, α-SMA, and VEGF, which are known to have a definite role in ligament healing, are predominantly expressed in the main vascular part of the ligament-epiligament complex enlarge the existing epiligament theory. Future investigations regarding better ligament healing should not overlook the epiligament tissue.

The novel epiligament theory: differences in healing failure between the medial collateral and anterior cruciate ligaments.

According to current literature, 90% of knee ligament injuries involve the medial collateral ligament or the anterior cruciate ligament. In contrast to the medial collateral ligament, which regenerates relatively well, the anterior cruciate ligament demonstrates compromised healing. In the past, there were numerous studies in animal models that examined the healing process of these ligaments, and different explanations were established. Although the healing of these ligaments has been largely investigated and different theories exist, unanswered questions persist.Therefore, the aim of this article is 1) to review the different historical aspects of healing of the medial collateral ligament and present the theories for healing failure of the anterior cruciate ligament; 2) to examine the novel epiligament theory explaining the medial collateral ligament healing process and failure of anterior cruciate ligament healing; and 3) to discuss why the enveloping tissue microstructure of the aforementioned ligaments needs to be examined in future studies.We believe that knowledge of the novel epiligament theory will lead to a better understanding of the normal healing process for implementing optimal treatments, as well as a more holistic explanation for anterior cruciate ligament healing failure.

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.

The Epiligament: Structure, Postnatal Development and Role in Ligament Healing.

While much is known about the ligament, the precise morphology and function of the thin layer of connective tissue lining its surface, termed the epiligament, have not been fully studied yet. Herein, we aimed at reviewing the recent findings on the structural and functional significance of the epiligament in both animal models and human tissue. The epiligament is made up of various connective tissue cells such as fibroblasts, fibrocytes, mast cells, and adipocytes and contains a number of neurovascular bundles. Arrangement of collagen fibers in the epiligament is rather chaotic, in multiple directions, which allows for greater mobility and resistance to stress. Differences in the collagen content and types of enzymes of the group of matrix metalloproteinases between the epiligament and the underlying ligament tissue have been reported and are reviewed herein. While the ligament tissue mainly contains collagen type I, the epiligament is also rich in collagen types III and V. As suggested by a number of studies, the epiligament plays a key role in ligament repair as a donor of cells and matrix metalloproteinases, particularly matrix metalloproteinase-2 and 9, which are essential for scar tissue remodeling. In conclusion, future studies will likely reveal additional functional aspects of the epiligament, which may allow scientists to devise more suitable treatment strategies for damaged ligaments in a world where injuries resulting from sports activities or daily routine have long merited their due attention.

A comparative study of the epiligament of the medial collateral and the anterior cruciate ligament in the human knee. Immunohistochemical analysis of collagen type I and V and procollagen type III.

BACKGROUND: Recent reports in rat models have shown that fibroblasts in the epiligament, an enveloping tissue of the ligament, are not static cells and play an important role during the early ligament healing of isolated grade III injury of the collateral ligaments of the knee. Fibroblasts produce collagen types I, III and V and infiltrate within the ligament body via the endoligament. In addition, similarities have been reported between the structure of the epiligament of the medial collateral ligament and anterior cruciate ligament of the knee in rat and in human. In line with the ascribed role of the epiligament tissue and the synthesis of these collagens and their role in ligament healing, the aim of this study was to determine their presence in the normal epiligament of the aforementioned ligaments in humans, to compare their differential expression and to present a novel hypothesis about the failure of healing of the anterior cruciate ligament in contrast to the medial collateral ligament. MATERIALS AND METHODS: We used samples from the mid-substance of the medial collateral and the anterior cruciate ligament of the knee joint, acquired from 12 fresh knee joints. Routine histological analysis was performed through hematoxylin and eosin stain, Mallory's trichrome stain and Van Gieson's stain. The immunohistochemical analysis was conducted using monoclonal antibodies against collagen type I and V and procollagen type III. The number of cells in the epiligament, endoligament and the ligament tissue was assessed quantitatively through a computerized system for image analysis NIS-Elements Advanced Research and Statistica software. RESULTS: Our observations revealed certain differences in the morphology of the epiligament, as well as variations in the expression of the investigated molecules. Expression of collagen type I was mostly low-positive (1+) in the epiligament and positive (2+) in the ligament tissue of both ligaments. Expression of procollagen type III was mostly positive (2+) in the epiligament and ligament tissue of the medial collateral ligament, low-positive (1+) in the epiligament and negative (0) in ligament tissue of the anterior cruciate ligament. Expression of collagen type V was predominantly low-positive (1+) in the epiligament and negative (0) in the ligament tissue of both ligaments. The immunoreactivity for all three molecules was always higher in the epiligament of the medial collateral ligament than that of the anterior cruciate ligament. CONCLUSIONS: The results of our study illustrate for the first time that fibroblasts in the human epiligament are indeed responsible for the synthesis of the main types of collagen participating in the early ligament healing, thus corresponding to previous data of the medial collateral ligament healing in animal models. The differences between the epiligament of the investigated ligaments could add a novel explanation for the failed anterior cruciate ligament healing.

Epiligament Tissue of the Medial Collateral Ligament in Rat Knee Joint: Ultrastructural Study.

Introduction Recent studies stressed the importance of the epiligament in ligament nutrition and healing. While ligaments of the knee joint have been the subject of extensive research, the epiligament of the medial collateral ligament has received only limited attention. The aim of our study was to present the ultrastructural morphological features of the epiligament of the medial collateral ligament in a rat knee joint. Materials and methods For the present study, we used eight eight-month-old male Wistar rats. A transmission electron microscopic study of the epiligament was conducted according to standard protocol. Results In the epiligament, we described the presence of fibroblasts with the typical features of protein-synthesizing cells, as well as fibrocytes and adipocytes. We noted an abundance of blood vessels and nerve elements. Collagen fibers were organized in multidirectional bundles. Conclusions Our findings confirm that the cells and structures of the epiligament play an important role in the nutrition and healing of the medial collateral ligament.

Role of Epiligament in Ligamentum Flavum Hypertrophy in Patients with Lumbar Spinal Canal Stenosis:a Pilot Study.

Ligamentum flavum (LF) hypertrophy is one of the main factors of lumbar spinal canal stenosis (LSCS). The primary object of this study is to clarify the existence of epiligament in the LF and its role in hypertrophy, and to develop an LF hypertrophy animal model. A cadaveric spine from a 30-year-old man was used to investigate the existence of epiligament in LF. Five LF samples from LSCS patients were obtained to evaluate hypertrophied LF. To create a rat model, we destabilized the lumbar spine. Each LF was sagittally cut for histological evaluation. The epiligament was clearly evident in normal LF specimens, which stained pink on Elastica van Gieson and green on Masson Trichrome. One layer was observed on the dural side and another on the dorsal side of the LF. LSCS patients had an enlarged dorsal epiligament, at around 30 times that of the regular thin epiligament on the dural side. The destabilized rat model showed an enlarged dorsal epiligament, with a mean thickness 8-fold that of the control. LF hypertrophy may be due to enlargement of the dorsal epiligament. Mechanical loading of the LF is an important factor for inducing hypertrophy in the rat model. J. Med. Invest. 65:85-89, February, 2018.

Comparison between Operative and Non-Operative Treatment of the Medial Collateral Ligament: Histological and Ultrastructural Findings during Early Healing in the Epiligament Tissue in a Rat Knee Model.

The medial collateral ligament of the knee joint is one of the most commonly injured ligaments of the knee. Recent data have shown that the thin layer of connective tissue covering the ligament, known as the epiligament, is essential for its nutrition and normal function, as well as its healing after injury. The aim of the present study was to investigate and compare the changes in the epiligament of the medial collateral ligament which occurred during operative and non-operative treatment throughout the first month after injury. We used 27 male Wistar rats randomly allocated to three groups. In the 9 rats belonging to the first group, the medial collateral ligament was fully transected and left to heal spontaneously without suture. In the 9 rats belonging to the second group, the transected ends were marked with a 9-0 nylon monofilament suture. The 9 rats in the third group were used as normal controls. Three animals from each group were sacrificed on days 8, 16, and 30 after injury. Light microscopic analysis was performed on semi-thin sections stained with 1% methylene blue, azure II, and basic fuchsin. Transmission electron microscopy was used to study and compare the ultrastructural changes in the epiligament. The statistical analysis of the obtained data was performed using the Kruskal-Wallis H test and Mood's median test. The normal structure of the epiligament of the medial collateral ligament was presented by fibroblasts, fibrocytes, adipose cells, mast cells, collagen fibers, and neuro-vascular bundles. On days 8 and 16 postinjury, the epiligament appeared hypercellular and returned to its normal appearance on the thirtieth day postinjury. The electron microscopic study revealed the presence of different types of fibroblasts with the typical ultrastructural features of collagen-synthetizing cells. The comparative statistical analysis on the respective day showed that there was no statistically significant difference in the number of cells between spontaneously healing animals and animals recovering with suture application. These data further prove that spontaneous healing of the medial collateral ligament yields similar results to surgical treatment and may be used as a basis for the development of treatment regimens with improved patient outcome.

Matrix Metalloproteinase-2 and -9 Expression in the Epiligament of the Medial Collateral and Anterior Cruciate Ligament in Human Knees: A Comparative Study.

Aim Ninety percent of knee ligament injuries involve the medial collateral ligament (MCL) and the anterior cruciate ligament (ACL) of the knee joint. Matrix metalloproteinases (MMPs) are a large group of calcium- and zinc-dependent endopeptidases responsible for cleaving and rebuilding various connective tissue components. Previous studies showed that MMP-2 and 9 have a significant effect on the healing process of injured ligaments. Therefore, the aim of this study was to evaluate for the first time in literature the expression and localization of MMP-2 and 9 in the epiligament (EL) and the ligament tissue of the MCL and the ACL of the human knee joint in order to assess their role in ligament healing. Materials and methods For the present study, we used histological material from the mid-portion of the MCL and the ACL of 14 knee joints from fresh cadavers. For the purpose of the immunohistochemical analysis, we used primary polyclonal antibodies against MMP-2 and 9. The obtained results were evaluated semi-quantitatively through ImageJ. Results Immunoreactivity for MMP-2 was predominantly positive (2+) in the EL of the MCL and remained mostly negative (0) in the ligament tissue. The expression of MMP-9 was mostly low-positive (1+) in the EL of the MCL and almost entirely negative (0) in the ligament tissue. In the EL of the ACL, the immunohistochemical expression of MMP-2 was predominantly low-positive (1+) and that of the MMP-9 was read as mostly low-positive (1+). Expression of the two enzymes in the ligament tissue was similar to the MCL. Conclusion The present study is the first comparison of the expression of the aforementioned MMPs in the EL tissue of the MCL and the ACL in human knees, which may play a key role in physiological and pathophysiological processes such as tissue healing and repair and basement membrane degradation.

Light and electron microscopic study of the medial collateral ligament epiligament tissue in human knees.

AIM: To examine the normal morphology of the epiligament tissue of the knee medial collateral ligament (MCL) in humans. METHODS: Several samples of the mid-substance of the MCL of the knee joint from 7 fresh human cadavers (3 females and 4 males) were taken. Examination of the epiligament tissue was conducted by light microscopy and photomicrography on semi-thin sections of formalin fixed paraffin-embedded blocks that were routinely stained with haematoxylin and eosin, Mallory stain and Van Gieson's stain. Electron microscopy of the epiligament tissue was performed on ultra-thin sections incubated in 1% osmium tetroxide and contrasted with 2.5% uranyl acetate, lead nitrate, and sodium citrate. RESULTS: The current light microscopic study demonstrated that the epiligament of the MCL consisted of fibroblasts, fibrocytes, adipocytes, neuro-vascular bundles and numerous multidirectional collagen fibers. In contrast, the ligament body was poorly vascularised, composed of hypo-cellular fascicles which were formed of longitudinal groups of collagen fibers. Moreover, most of the vessels of the epiligament-ligament complex were situated in the epiligament tissue. The electron microscopic study revealed fibroblasts with various shapes in the epiligament substance. All of them had the ultrastructural characteristics of active cells with large nuclei, well developed rough endoplasmic reticulum, multiple ribosomes, poorly developed Golgi apparatus, elliptical mitochondria and oval lysosomes. The electron microscopy also confirmed the presence of adipocytes, mast cells, myelinated and unmyelinated nerve fibers and chaotically oriented collagen fibers. CONCLUSION: Significant differences exist between the normal structure of the ligament and the epiligament whose morphology and function is to be studied further.