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.

Ligaments of the knee joint have been the subject of extensive research (by transmission electron microscopy) while the EL of the medial collateral ligament (MCL) received only limited attention. The latter was examined in current literature by several studies describing the ultrastructural morphology of the EL in mature animals or comparing mature and immature animals [3][4][5].
The aim of our study was to present the ultrastructural morphological features of the EL of the MCL in a rat knee joint.

Materials And Methods
For the present study, we used eight eight-month-old male Wistar rats.

Results
The EL tissue of the MCL consists of fibroblasts, fibrocytes, fat cells, and neurovascular bundles, as well as single blood vessels. Their ultrastructural characteristics are presented below.
The fibroblasts were large and well formed. They displayed a very delicate chromatin structure and a prominent nucleolus. Their cytoplasm contained a considerable number of free ribosomes, polysomes, well-presented rough endoplasmic reticulum, poorly developed Golgi apparatus, spherical mitochondria, and single lysosomes. Spindle-shaped fibrocytes with similar nuclei and rough endoplasmic reticulum were reported too. The third cell type was presented by unilocular spherical and polyhedral adipose cells, which were found closely packed with one another. The adipocytes had large vacuoles and eccentric, flat nuclei, surrounded by a basal lamina (Figures 1a-1d). In the intercellular space, we discovered collagen fibers with multiple orientations (Figure 1e). In the same layer, myelinated and unmyelinated nerve fibers formed nerve trunks. The external fibrous coat of the trunk (epineurium) filled the space between the bundles of nerve fibers, each surrounded by perineurium. Schwann cells were also detected. The blood vessels had thin walls and their intima consisted of layers of endothelial cells, with a number of pericytes around them (Figures 1f-1h). h) Epiligament with fibroblast with triangular shape and spindle-shaped fibrocytes in the intercellular matrix with large nucleus (N), blood vessel with erythrocytes (Ery) and pericytes (arrowheads) around the blood vessel wall. n -nucleolus; Scale bar -1.5 µm.

Discussion
According to Chowdhury et al., who described the external surface of the MCL in rabbits, there were two types of cells -spinous-shaped fibroblasts and fat cells, which differed from ligament cells [1]. These authors did not present the ultrastructural findings in detailed description. On the contrary, fibroblasts in the EL of MCL were of various types: spinous-shaped, spindleshaped, elongated, and with irregular form [3,5,7]. The data from the present study gave a detailed examination of the morphological changes in the midsubstance of the EL of MCL, fibroblasts, fibrocytes, fat cells, and neurovascular bundles in particular. Similar ultrastructural characteristics were discussed by Georgiev et al. [12]. Their study described the special characteristics of fibroblasts, supporting the hypothesis of their importance in the nutrition and healing of the MCL, along with the abundant blood vessels.
Chowdhury et al. speculated that the spinous-shaped cells are primarily responsible for the synthesis of collagen fibers [1]. Our observations supported those earlier reports. Fat cells in the EL appear to be typical adipocytes that form white adipose tissue [16]. These cells metabolize and store lipids and may function as a packing material that could confer the distinct properties of the EL [1]. It is well-known that both small and large collagen fibers in the ligament are aligned parallel to its long axis and form fascicles separated by the transverse sections of septae while the whole structure is enveloped in thin connective tissue called endoligament [16]. In contrast, our research showed uniform small collagen fibers in the EL organized in bundles with multiple orientations and that the fibers of the EL of MCL continued as the endoligament. The EL layer appeared to contain a relative abundance of blood vessels and nerve elements compared to the ligament tissue [5]. The blood vessels in the EL were randomly dispersed in a loose connective tissue matrix while the nerve bundles often accompanied them, but apparently not all blood vessels formed part of a neurovascular bundle [2,5]. We found that the EL of the MCL had both myelinated and unmyelinated nerve fibers, which occurred frequently in contrast to the ligament [17]. The axonal density of nerve fiber distribution is the greatest in the fibular collateral ligament in the knee joint of rats as compared to the other ligaments [18]. On the contrary, nerve types and their endings found in the MCL during an examination by McDougall et al. appeared to have a similar innervation pattern as the medial collateral ligament [19].

Conclusions
Different types of cells and cell complexes (fibroblasts, fibrocytes, and fat cells) can be found in the EL of the MCL. Fibroblasts in the EL may be involved in processes such as differentiation, phagocytosis, and collagen synthesis due to their ultrastructural characteristics. Collagen fibers in the midsubstance of the external surface of EL in the MCL have uniformly small diameters and are organized in bundles with varying orientations, opposed to collagen fibers in the ligament. These differences in orientation suggest that the EL may respond to tension differently in different directions having better mobility than the ligament. The EL contains a multitude of blood vessels and nerve fibers (the latter can be both myelinated and unmyelinated), as opposed to the ligament substance. In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.