Localization of matrix metalloproteinase-2 in injured medial collateral ligament epiligament in rat knee

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Abstract
Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases, which are responsible for the tissue remodelling and degradation of the extra-cellular matrix. They are secreted by fibroblasts, osteoblasts, endothelial cells, macrophages, neutrophils and lymphocytes. These enzymes have a devastating effect on the healing process of injured ligaments. The aim of this study was to investigate the presence of matrix metalloproteinase-2 during early healing of the medial collateral ligament epiligament in rat knee model. The medial collateral ligament of the knee joint of twelve male Wistar rats was surgically transected and was left to heal spontaneously in nine of them. On the eighth, sixteenth, and thirtieth day after injury, the animals were sacrificed and the ligaments were examined. The matrix metalloproteinase-2 distribution was investigated in these periods. We observed a well defined immunopositive reaction in all periods after surgical trauma, in contrast with normal tissue. We also discovered that the main source of matrix metalloproteinase-2 is localized in the epiligament tissue. The current study offers a more complete description of the epiligament healing process and illustrates for the first time the presence of matrix metalloproteinase-2 during epiligament healing.
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Доклади на Българската академия на науките
Comptes rendus de l’Acad´emie bulgare des Sciences
Tome 70, No 2, 2017
MEDECINE
Morphologie
LOCALIZATION OF MATRIX METALLOPROTEINASE-2
IN INJURED MEDIAL COLLATERAL LIGAMENT
EPILIGAMENT IN RAT KNEE
Georgi P. Georgiev, Alexandar Iliev, Boycho Landzhov,
Iva N. Dimitrova∗∗, Georgi Kotov, Lina Malinova,
Wladimir Ovtscharoff
(Submitted on July 11, 2016)
Abstract
Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases,
which are responsible for the tissue remodelling and degradation of the extra-
cellular matrix. They are secreted by fibroblasts, osteoblasts, endothelial cells,
macrophages, neutrophils and lymphocytes. These enzymes have a devastating
effect on the healing process of injured ligaments. The aim of this study was
to investigate the presence of matrix metalloproteinase-2 during early healing
of the medial collateral ligament epiligament in rat knee model. The medial
collateral ligament of the knee joint of twelve male Wistar rats was surgically
transected and was left to heal spontaneously in nine of them. On the eighth,
sixteenth, and thirtieth day after injury, the animals were sacrificed and the
ligaments were examined. The matrix metalloproteinase-2 distribution was
investigated in these periods. We observed a well defined immunopositive re-
action in all periods after surgical trauma, in contrast with normal tissue. We
also discovered that the main source of matrix metalloproteinase-2 is localized
in the epiligament tissue. The current study offers a more complete description
of the epiligament healing process and illustrates for the first time the presence
of matrix metalloproteinase-2 during epiligament healing.
Key words: epiligament, immunohistochemistry, matrix metalloproteinase-
2, medial collateral ligament, rat knee model
Introduction. Despite the fact that the ligaments of the knee joint have
been studied extensively, very little is known about the thin layer of connective
tissue adherent to these ligaments, termed the epiligament (EL) (epi-[Greek-on
or upon]; ligament [Latin-ligare, to bind]. In 1990 Bray et al. [1] described the
epiligament as a “surrounding adherent connective tissue removed simultaneously
with the ligament but which was grossly distinguishable from ligament tissue
This research was supported by Grant No. 19/2016 of the Medical University of Sofia,
Bulgaria.
273
proper”. The medial collateral ligament (MCL) is the most commonly injured
ligament structure of the knee joint used to compare normal healing and non-
healing in ligaments. The incidence of this injury has increased in recent years
and represents a commonly encountered problem in modern sports medicine [2–4 ].
After injury, ligaments do not heal by regeneration, but by a formation of scar
tissue similar to other wound healing models [2, 5]. Numerous studies investigated
an insufficient MCL repair process, as well as in different treatment regimens,
including tissue engineering approaches, non-steroidal anti-inflammatory drugs,
local corticosteroids, etc [6–10 ].
Matrix metalloproteinases (MMPs) are a major group of enzymes that reg-
ulate cell-matrix composition. The MMPs are zinc-dependent endopeptidases
known for their ability to cleave one or several extracellular matrix (ECM) con-
stituents, as well as non-matrix proteins [11]. MMPs family members have been
classified into different but closely related subgroups with fairly close charac-
teristic features. This classification recognises collagenases (MMP-1, 8, 13, 18),
gelatinases (MMP-2 and 9), stromelysins (MMP-3, 10, 11), elastases (MMP-7,
12), and membrane type MMPs (MT-MMPs, MMP-14, 15, 16, 17) and a group
of unnamed members [12]. These enzymes have a devastating effect on the healing
process of the injured ligaments [13]. MMP-2 has been found to be involved in
many cellular processes such as tissue remodelling, repair and basement mem-
brane degradation and it has been proved that it is involved in the healing of
acute tears, tumour invasion, neovascularization and metastases [14]. There is no
literature data concerning the localization of MMP-2 in the EL in the process of
healing in the knee after injury. Therefore, the aim of this study was to investigate
for the first time in literature the presence, expression and localization of MMP-2
in EL and the role of this enzyme in tissue repair processes of the knee joint.
Materials and methods. Twelve 8-month-old male Wistar rats, ranging
in weight from 350 g to 400 g, were used for this study with the approval of
the University Committee on Animal Resources. All animals received humane
care in compliance with the “Principles of laboratory animal care” formulated
by the National Society for Medical Research and the “Guide for the care and
use of laboratory animals” prepared by the National Institute of Health (NIH
publication No. 86–23, revised 1996). The experimental animals were divided
in four groups, each group containing three subjects. The last group of animals
underwent no transection and served as intact controls.
Nine rats were anesthetized by intraperitoneal injection using a mixture of
5 mg/kg b.w. Xylasine and 45 mg/kg b.w. Calypsol. Under sterile conditions
a small incision (10 mm) was made in their skin on the knee joint of the left
hind limb over the site of MCL. After skin incision, the overlying connective tis-
sue was dissected to expose the MCL. Then, a 1-mm gap in the mid-substance
was surgically created and the gap was left without suturing. The right knees
of the animals were preserved intact. After surgery, the rats were allowed free
274 G. P. Georgiev, A. Iliev, B. Landzhov et al.
Fig. 1. (a) Immunohistochemical localization of MMP-2 in con-
trols. Magnification ×200; (b) Immunohistochemical localiza-
tion of MMP-2 on the 8th day after injury. Magnification ×200
Fig. 1. (c) Immunohistochemical localization of MMP-2 on the
16th day after injury. Magnification ×200; (d) Immunohisto-
chemical localization of MMP-2 on the 30th day after injury.
Magnification ×200
cage activities. No infections or complications were observed. On the 8th, 16th,
and 30th day after surgery, the animals were sacrificed with intracardiac injection
of Thiopental. The injured ligaments were carefully removed without disturbing
the scar region and were immediately fixed in 10% buffered formalin for three
days. After fixation, the tissue samples were washed in water for 24 h and then
dehydrated in increasing concentrations of ethanol. Alcohol was removed using
cedar oil until the pieces became translucent. Samples were rinsed in xylene and
embedded in paraffin. Paraffin sections 7 µm in thickness were mounted on ad-
hesive slides. Sections were deparaffinised, dehydrated and washed in phosphate
buffered saline. Endogenous peroxidase activity was blocked with 3% H2O2for
15 min at room temperature. The sections were rinsed in PBS and nonspecific
binding sites were blocked with 2.5% horse serum in PBS for 20 min. Primary
polyclonal antibody for MMP-2 at a dilution 1:1000 were added and sections
were incubated overnight at 4 C, rinsed in PBS and incubated with biotinylated
horse anti-rabbit IgG, diluted 1:400 in 1.5% horse serum for 60 min at room
temperature. Sections were rinsed as before and incubated with streptavidin-
HRP for 45 min at room temperature. Antibody binding was visualized using
3,3’-diaminobenzidine (DAB) as chromogen for 10 min. Sections were counter-
stained with haematoxylin, dehydrated, cleared in xylene and cover-slipped with
Canada balsam. The antibody binding was visualized for 10 min using DAB as
chromogen. The sections were counterstained with haematoxylin, washed, dehy-
drated and cover-slipped in Canada balsam. As a negative control, the primary
antibody was replaced with isotype-matched control antibody.
Results. In control animals, the normal morphology of the EL of the MCL
is quite different. The external surface of the EL of the MCL was comprised of
fibroblasts, fibrocytes, adipocytes, and neuro-vascular bundles, numerous multi-
directional collagen fibres, as we have described previously in details. The most
interesting finding was the distribution of MMP-2 in EL and the ligament sub-
stance. It should be pointed out that the positive reaction of this enzyme was
predominantly localized in the EL compared to the ligament substance. The ad-
ventitia of the blood vessels in the EL also showed a distinct positive staining
for MMP-2. In the ligament, a classic morphological structure was found. The
fibroblasts in the ligament also stained positive for MMP-2 (Fig. 1a).
The histological study on the eighth day after injury showed a significant
amount of granulation tissue between the damaged edges of the ligament. The
EL tissue was hyper-cellular and intensive angiogenesis was presented, as we
have previously reported. The most interesting finding was the results from the
immunohistochemical study. It showed an intensive reaction for MMP-2 present
in the EL scar tissue (Fig. 1b). Thus, we could conclude that EL is the main
donor of this enzyme in the EL-ligament complex during this period of tissue
regeneration.
The morphological changes on the sixteenth day after injury also revealed the
Compt. rend. Acad. bulg. Sci., 70, No 2, 2017 275
hyper-cellular granulation margins in the EL. However, the number of cells had
decreased compared to the previous period and they were more organized. The
cells, mainly presented by fibroblasts and progenitor cells migrated into the en-
doligament, as we have presented previously. The most important finding was the
intensive immunohistochemical expression of the MMP-2 in the EL granulation
tissue (Fig. 1c).
On the thirtieth day after MCL injury the healing process advanced and cells
of the EL infiltrated most of the scar tissue. The EL tissue in this period was
similar to the one in the controls. Intensive immunostaining reaction for MMP-2
was detected throughout the EL-ligament complex (Fig. 1d). These data revealed
the key role of MMP-2 during ligament repair.
Discussion. Numerous studies have investigated the healing process of the
collateral ligaments of the knee in animal models. However, very little is known
about the changes, which occur in the EL after ligament rupture [6–8, 15, 16].
Herein, we present the modification of the EL scar throughout the initial 30
days after injury of the ligament and described changes in the activity of the
enzyme MMP-2 within the EL healing.
The primary roles of MMPs are to break down and remove ECM molecules
from the tissue. A number of non-ECM molecules also become potential sub-
strates of MMPs [17]. Gelatinases include MMP-2 and MMP-9 proteins. They
primarily cleave denatured collagen and intact collagen type IV in basal mem-
branes. MMP-2 is known to cleave native collagen type I [18]. Modulation of
cell-matrix interactions occurs through the action of unique proteolytic systems
responsible for hydrolysis of a variety of ECM components. By regulating the
integrity and composition of the ECM structure, these enzyme systems play a
pivotal role in the control of signals elicited by matrix molecules, which regulate
cell proliferation, differentiation and cell death. MMPs are also involved in wound
healing, a tissue-remodelling process which involves the migration and differen-
tiation of connective tissue cells. The role of MMPs in angiogenesis is also wide
and complex. Many MMPs are produced by endothelial cells and have been de-
scribed as important for the formation of new blood vessels in both physiological
and pathological conditions. MMPs are considered to participate in the dissem-
ination of cancer cells by breaking down ECM and they are also important for
creating an environment that supports the initiation and maintenance of growth
of primary and metastatic tumors [11, 19].
The MCL heals much better and faster than the anterior cruciate ligament
(ACL) of the knee joint. This is most likely due to the specific characteristics
of the EL, located above the MCL. In this study we found a significant increase
in the activity of MMP-2 in the 8th and 16th day after the injury. In a study
of Zhou et al. [20 ] the expression and localization of different types of MMPs,
including MMP-2 in MCL and ACL were compared. They reported a significantly
higher expression of MMP-2 in the MCL than in the ACL. They concluded that
276 G. P. Georgiev, A. Iliev, B. Landzhov et al.
numerous MMP family members that are expressed in various quantities in the
MCL might be involved in the different matrix remodelling process as well as the
different healing ability of MCL. In our study, we observed similar but relatively
detailed results. High levels of active MMP-2 might disrupt the delicate balance
between the removal of damaged matrix components and the deposition of newly
synthesized materials. Overall, our results show that MCL fibroblasts normally
generate low levels of MMP-2. After injury and especially during the early periods
of healing, however, high levels of MMP-2 are expressed. This study reveals the
necessity for further investigation of the role of MMP-2, as well as its regulation,
in the MCL injury and repair processes.
In conclusion, herein we present the first study of immunohistochemical lo-
calization and distribution of the enzyme MMP-2 in EL tissue. In addition we
describe the enzyme activity of MMP-2 in the early recovery period after a lig-
ament injury to the MCL and track the changes in enzyme activity on the 8th,
16th and 30th day after damage. Changes in the expression of the enzyme MMP-
2 in EL fibroblasts plays a major role in the reconstruction of the MCL knee after
mechanical damage.
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Department of Orthopaedics and Traumatology
University Hospital Tsaritsa Yoanna – ISUL
Medical University of Sofia
8, Bialo more St
1527 Sofia, Bulgaria
e-mail:georgievgp@yahoo.com
Department of Anatomy, Histology
and Embryology
Medical University of Sofia
15 Acad. I. Geshov Blvd
1431 Sofia, Bulgaria
∗∗Department of Cardiology
University Hospital “St. Ekaterina”
Medical University of Sofia
52A P. Slaveikov Blvd
1431 Sofia, Bulgaria
278 G. P. Georgiev, A. Iliev, B. Landzhov et al.
  • ... Major differences were reported between the structural composition of the EL and the underlying ligament tissue (LT) [6][7]. In addition, the EL has been found to express various connective tissue components, as well as enzymes responsible for tissue breakdown and remodeling [8][9][10][11]. Herein, we aimed at reviewing the recent findings on the structural and functional significance of the EL both in animal models and in human tissue. ...
    ... It has been shown that as a more cellular structure the EL may play a central role in the formation of scar tissue and the healing process of the ligament [16]. Fibroblasts in the EL are not static cells; instead, they produce varying quantities of collagen types I, III, and V, fibronectin, and enzymes of the group of matrix metalloproteinases (MMPs), especially MMP-2 and 9 [8][9][10][19][20][21]. These substances are all upregulated to promote adequate ligament repair after injury. ...
    ... Thus, MMPs are implicated in a number of physiological and pathological processes throughout the body, including wound healing, ligament repair, myocardial remodeling induced by hypertension, tumor invasion, and metastasis and many others [36][37][38][39][40]. They are secreted by various cells -fibroblasts, osteoblasts, endothelial cells, macrophages, neutrophils, lymphocytes [10]. Karousou et al. were among the first to study the expression of MMPs in the ligament and found higher levels of MMP-2 and 9 in the zone of injury in human Achilles tendon as opposed to a healthy zone [41]. ...
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    Full-text available
    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.
  • ... In 2017, Georgiev et al. [4] hypothesised that the EL of the MCL has specific characteristics and suggested that the fibroblasts in particular, together with the abundant blood vessels are essential for its nutrition and healing. These data were in line with the previous studies on the MCL in rat model [9][10][11][12][13][14][15][16][17]. According to Georgiev and Vidinov [13][14][15] and Georgiev et al. [9][10][11][12] the EL of the MCL is a donor of fibroblasts, progenitor cells, and blood vessels during the process of ligament recovery. ...
    ... These data were in line with the previous studies on the MCL in rat model [9][10][11][12][13][14][15][16][17]. According to Georgiev and Vidinov [13][14][15] and Georgiev et al. [9][10][11][12] the EL of the MCL is a donor of fibroblasts, progenitor cells, and blood vessels during the process of ligament recovery. In contrast, the biological basis for failure of the human ACL to heal after rupture is still unknown. ...
    ... Furthermore, they suggested that the differential expression of MMPs in the MCL and in the ACL may be involved in the differential healing potential of the two ligaments. Previous studies by Georgiev et al. [9] and Iliev et al. [16][17] observed the distribution and expression of MMP-2 and MMP-9 in normal rat tissue and also during the process of healing after acute injury. They showed that fibroblasts in the EL of the MCL normally generate low levels of MMP-2 and 9. ...
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    Full-text available
    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 lowpositive (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.
  • ... [1] The medial collateral ligament (MCL) of the knee joint is a commonly injured ligament used in order to compare normal healing and non-healing in ligaments. [2,3] Type I collagen is the main structural component in both normal and injured ligaments; [4,5] type III and V collagen have also been reported to participate in their structure. [6,7] Type I is primarily responsible for the ligament tensile strength and is apparently key for the long-term properties of the tissue matrix, [4,5] while type III is needed for ligament repair. ...
    ... [12] MMPs family members have been classified into different but closely related subgroups with fairly close characteristic features. [3] This classification recognises collagenases (MMP-1, 8,13,18), gelatinases (MMP-2 and 9), stromelysins (MMP-3, 10, 11), elastases (MMP-7, 12), and membrane type MMPs (MT-MMPs, 15,16,17) and a group of unnamed members. [13] Gelatinases play an important role in many extracellular mechanisms involved in tissue remodelling, repair and basement membrane degeneration and it has been proved that they are involved in the healing of acute tears, tumour invasion, neovascularisation and metastases. ...
    ... However, it has been increasingly evident that the breakdown of ECM or cell surface molecules can influence cell-matrix and cell-cell interactions and lead to the release of growth factors bound to the ECM. [3,11] More specifically, processes such as morphogenesis and tissue regeneration require ECM and basal membranes degradation to allow for the migration of different cells participating in tissue remodeling. [11,17,18] MMPactivity is also implicated in the process of angiogenesis. ...
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    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.
  • ... In 1990, Bray et al [15] described the epiligament as a "surrounding adherent connective tissue removed simultaneously with the ligament but which was grossly distinguishable from ligament tissue proper". Our previous studies on the MCL in rat knee models led to the conclusion that the EL tissue plays a key role in the healing of the ligament tissue after injury [13,16,17] . According to Georgiev and Vidinov [18][19][20] and Georgiev et al [12,13,16,21,22] the EL is a donor of fibroblasts, progenitor cells and blood vessels, which proliferate and migrate towards the body of the ligament through the endoligament during the process of ligament recovery. ...
    ... According to Georgiev and Vidinov [18][19][20] and Georgiev et al [12,13,16,21,22] the EL is a donor of fibroblasts, progenitor cells and blood vessels, which proliferate and migrate towards the body of the ligament through the endoligament during the process of ligament recovery. Fibroblasts in the EL tissue normally produce collagen types Ⅰ, Ⅲ, Ⅴ, fibronectin (FN) and matrix metalloproteinases-2 and -9 (MMP-2, -9) which are essential for the normal functioning of the ligament and their synthesis is increased in order to promote adequate repair after injury [13,16,17,21,23] . Therefore, detailed knowledge of the morphology and function of the EL during physiological conditions and post injury is important in deepening our knowledge with regard to ligament healing and may thus lead to proposal of better treatment options in the future. ...
    ... Collagen fibres in the ligaments are organized in longitudinal groups and form fascicles [11,24,25] . The thin layer of connective tissue separating these fascicles is known as endoligament and is related to another connective tissue structure, containing more blood vessels, which envelops the entire ligament and is known as epiligament [12,13,[16][17][18] . In rabbits, Chowdhury et al [26] (1991) examined the external surface of the MCL EL and described two types of cells -spinousshaped adipocytes and fibroblasts. ...
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    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.
  • ... This structure has been described as a "surrounding adherent connective tissue removed simultaneously with the ligament but which was grossly distinguishable from ligament tissue proper." Recent reports by Georgiev and Vidinov [2009ac], Georgiev et al. [2010aGeorgiev et al. [ , b, 2015aGeorgiev et al. [ , b, 2016Georgiev et al. [ , 2017bGeorgiev et al. [ , 2018, and Iliev et al. [2016] in a rat model have turned the attention to the role of the EL. These studies described in detail the histological appearance, ultrastructural characteristics, and immunohistochemical expression of various substances in the EL under normal conditions and in the setting of experimental ligament injury. ...
    ... According to these authors, the EL is the main source of fibroblasts, progenitor cells, and blood vessels, which proliferate and migrate towards the body of the ligament through the endoligament during ligament healing. Fibroblasts in the EL tissue are not static cells -they normally produce collagen types I, III, V [Georgiev et al., 2010a[Georgiev et al., , 2015a, fibronectin [Georgiev et al., 2016], and matrix metalloproteinases-2 and 9 [Georgiev et al., 2017b[Georgiev et al., , 2018, which are essential for the normal functioning of the ligament and their synthesis is increased in order to promote adequate repair after injury. They also emphasized its important role in understanding the normal recovery of the ligament, thus providing a basis for new treatment strategies. ...
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    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.
  • ... In 1990, Bray et al. defined, for the first time, the term "epiligament" (EL) as a "surrounding adherent connective tissue removed simultaneously with the ligament but which was grossly distinguishable from ligament tissue proper" [2]. Recent studies stressed the importance of the EL in ligament nutrition and healing [3][4][5][6][7][8][9][10][11][12][13][14][15]. ...
    ... 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. ...
  • ... In 1990, Bray et al. defined, for the first time, the term "epiligament" (EL) as a "surrounding adherent connective tissue removed simultaneously with the ligament but which was grossly distinguishable from ligament tissue proper" [2]. Recent studies stressed the importance of the EL in ligament nutrition and healing [3][4][5][6][7][8][9][10][11][12][13][14][15]. ...
    ... 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. ...
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    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 proteinsynthesizing 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.
  • ... Collagen fibers in the ligament are organized in fascicles, enveloped by thin connective tissue sheath, known as the endoligament (Landzhov et al., 2015). The endoligament in turn is connected to a more vascular connective tissue layer, which covers the entire ligament, termed the epiligament (EL) (epi-[Greek -on or upon]; ligament [Latin -ligare, to bind]) (Georgiev et al., 2017b). ...
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    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 lowpositive (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.
  • ... They participate in a variety of processes such as regeneration, migration and proliferation not only in the myocardium, but in other organs too, both under physiological and pathological conditions. [16][17][18][19] The connective tissue, which surrounds the cardiomyocytes includes three layers -epimysium, perimysium and endomysium. The normal extracellular matrix (cardiac gel) is produced by the cardiomyocytes and the fibroblasts in the myocardium. ...
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    Ligaments are composed of dense connective tissue and attach bones in joints. The thin connective tissue sheath, covering these fascicles is called endoligament and is connected to a more vascular connective tissue structure that envelopes the entire ligament and is referred to as epiligament. The tissue of the epiligaments is composed of different cell types such as: fibroblasts, fibrocytes, adipocytes, neurovascular bundles, and a multitude of collagen fibers, disposed in different directions. The main structural protein of epiligament is collagen type I. Collagens types III and V were also found in the structure of epiligament. Type I collagen is the main collagen in normal and healing ligaments. The ligament repair requires presence of collagen type III. Collagen type V is associated with collagen type I and regulates the collagen fibril diameter. Knowledge of variation of cells and collagen types of epiligament in normal and injured ligaments is crucial for understanding of the healing process.
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