Advertisement

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

Open AccessPublished:September 27, 2022DOI:https://doi.org/10.1016/j.knee.2022.07.013

      Abstract

      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.

      Keywords

      1. Introduction

      Injuries to the medial collateral ligament (MCL) and the anterior cruciate ligament (ACL) are frequent. MCL has a relatively efficient healing capacity; however, the healing process leads to a structure with mechanical properties that are inferior to those of the native ligament. Unlike MCL, an injured ACL shows a very limited healing capacity, with a response defined as “functionally insufficient” [
      • Frank C.B.
      Ligament healing: Current knowledge and clinical applications.
      ,
      • Woo S.L.
      • Abramowitch S.D.
      • Kilger R.
      • Liang R.
      Biomechanics of knee ligaments: Injury, healing, and repair.
      ,
      • Woo S.L.
      • Vogrin T.M.
      • Abramowitch S.D.
      Healing and repair of ligament injuries in the knee.
      ]. There are many possible explanations for the differences in healing between the MCL and ACL. Different factors, including matrix biochemical composition, biomechanical forces, intra- versus extrasynovial environment, blood supply, and/or growth factors, should be considered in relation to these explanations [
      • Amiel D.
      • Nagineni C.N.
      • Choi S.H.
      • Lee J.
      Intrinsic properties of ACL and MCL cells and their responses to growth factors.
      ,
      • Bray R.C.
      • Leonard C.A.
      • Salo P.T.
      Correlation of healing capacity with vascular response in the anterior cruciate and medial collateral ligaments of the rabbit.
      ]. Recently, a novel epiligament (EL) theory was proposed based on studies that presented the EL as a donor of fibroblasts and other progenitor cells, blood vessels, and connective tissue cells that migrate towards the body of the ligament via the endoligament and are vital for ligament repair [
      • Georgiev G.P.
      Epiligament or paratenon is more appropriate for describing the enveloping tissue of the cruciate ligaments of the human knee?.
      ,
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Kinov P.
      • Slavchev S.
      • Landzhov B.
      Light and electron microscopic study of the medial collateral ligament epiligament tissue in human knees.
      ,
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Nedialkova V.K.
      • Kirkov V.
      • Landzhov B.
      Epiligament Tissue of the medial collateral ligament in rat knee joint: Ultrastructural study.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Kinov P.
      • Angelova J.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Dimitrova I.N.
      • Malinova L.
      • Ovtscharoff W.
      Expression of fi-bronectin during early healing of the medial collateral ligament epiligament in rat knee model.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Kotov G.
      • Slavchev S.A.
      • Iliev A.
      Matrix metalloproteinase-2 and -9 expression in the epiligament of the medial collateral and anterior cruciate ligament in human knees: A comparative study.
      ,
      • Georgiev G.P.
      • Vidinov N.K.
      • Kinov P.S.
      Histological and ultrastructural evaluation of the early healing of the lateral collateral ligament epiligament tissue in a rat knee model.
      ]. Fibroblasts in particular are responsible for synthesizing several types of collagen, matrix metalloproteinases (MMPs), fibromodulin, decorin, and fibronectin. These proteins are involved in both the degradation and proliferation and remodeling of the ligament after injury [
      • Georgiev G.P.
      Epiligament or paratenon is more appropriate for describing the enveloping tissue of the cruciate ligaments of the human knee?.
      ,
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Kinov P.
      • Slavchev S.
      • Landzhov B.
      Light and electron microscopic study of the medial collateral ligament epiligament tissue in human knees.
      ,
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Nedialkova V.K.
      • Kirkov V.
      • Landzhov B.
      Epiligament Tissue of the medial collateral ligament in rat knee joint: Ultrastructural study.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Kinov P.
      • Angelova J.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Dimitrova I.N.
      • Malinova L.
      • Ovtscharoff W.
      Expression of fi-bronectin during early healing of the medial collateral ligament epiligament in rat knee model.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Kotov G.
      • Slavchev S.A.
      • Iliev A.
      Matrix metalloproteinase-2 and -9 expression in the epiligament of the medial collateral and anterior cruciate ligament in human knees: A comparative study.
      ,
      • Georgiev G.P.
      • Vidinov N.K.
      • Kinov P.S.
      Histological and ultrastructural evaluation of the early healing of the lateral collateral ligament epiligament tissue in a rat knee model.
      ,
      • Chamberlain C.S.
      • Crowley E.
      • Vanderby R.
      The spatio-temporal dynamics of ligament healing.
      ,
      • Iliev A.
      • Kotov G.
      • Stamenov N.
      • Landzhov B.
      • Kirkov V.
      • Georgiev G.P.
      A comparative im-munohistochemical and quantitative study of the epiligament of the medial collateral and anterior cruciate ligament in rat knee.
      ]. Georgiev et al. [
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ] considered that the EL theory could explain the differences between the healing potential of MCL and ACL; according to these authors, the smaller number of cells in the EL of the ACL compared with MCL, as well as the lower expression of collagen type I (responsible for the ligament’s tensile strength), collagen type V (organizing collagen type I fibrils and regulating their diameters) and procollagen type III (integral to proper ligament healing in fibroblasts) in the EL of the ACL than in the MCL cannot provide adequate healing in ACL.
      To enrich the existing literature data concerning EL theory, we aimed to analyze the expression of different molecules known to be important for ligament healing and we analyzed the expression of vascular endothelial growth factor (VEGF), CD34, and α-smooth muscle actin (α-SMA) immunohistochemically in the ELs of the MCL and ACL. We also explored the significance of the difference in their expression between the two ligaments in relation to EL theory.

      2. Material and methods

      2.1 Tissue preparation

      For histology and immunohistochemistry, we used samples from the mid-substance of the MCL and the ACL from 12 fresh knee joints. The knee joints were obtained from five fresh male and seven female European cadavers in the Department of Anatomy, Histology, and Embryology at the Medical University of Sofia. The mean age of the cadavers was 55 years (minimum 49, maximun 62). There were no clinical data indicating knee osteoarthritis and no scars around the knee joint from previous surgery. The study was approved by the Medical-Legal Office, the Local Ethics Committee, and the Institutional Review Board (No. 4866). There was no medical or surgical history of previous trauma of the knees in the cadavers investigated.
      After the skin incision, the underlying subcutaneous tissue was dissected to expose the MCL of the knee. The MCL and the external surface of the surrounding EL were dissected with precision, and samples from the middle third of the ligament were immediately fixed for 24 h in 10% neutral phosphate-buffered formalin, prepared under laboratory conditions from 37% formaldehyde solution (Merck Catalog No. 1040031000, Merck KGga, Darmstadt, Germany). They were then dehydrated in increasing concentrations of ethanol (70%, 80%, 95%, 100%) (Merck Catalog No. 1009835000). After the knee joint was opened, samples were also acquired from the mid-substance of the ACL and fixed in the same way. The ethanol was then removed using cedar oil until the samples became translucent. The samples were rinsed in xylene (Merck Catalog No. 1082984000) and embedded in paraffin (Merck Catalog No. 1071511000).

      2.2 Light microscopy

      For routine light microscopy, sections were cut on a microtome (Leica, Wetzlar, Germany) at a thickness of 5 μm. The paraffin sections obtained were then mounted on microscope slides. They were stained routinely with Mallory’s trichrome stain, hematoxylin and eosin stain and Van Gieson’s stain, all according to the standard methods.

      2.3 Immunohistochemistry

      The specimens were fixed in 10% buffered formalin, embedded in paraffin, and cut to 4 μm thick. They were deparaffinated, and endogenous peroxidase was blocked for 5 min with a blocking reagent according to the manufacturer’s protocol. Then, they were washed three times with phosphate-buffered saline and incubated with primary antibody for 1 h, followed by incubation with marked polymer and another wash. The tissue samples were incubated with 3,3′-diaminobenzidine (DAB) substrate-chromogen and counterstained with Mayer’s hematoxylin after washing. The following antibodies were used for immunohistochemistry: monoclonal mouse anti-human VEGF antibody (M7273, DAKO, Agilent), monoclonal mouse anti-human α-SMA antibody (M0851, DAKO, Agilent) and monoclonal mouse anti-human CD34 antibody (M7165, DAKO, Agilent), all diluted 1:100; the detection system was EnVision™ FLEX+, Mouse, High pH (Link) (K8002, DAKO Cytomation, Denmark, Agilent). The analysis followed the manufacturer’s protocols. Eighteen sections were used as controls.
      Photomicrographs of representative fields of immunohistochemical staining were obtained using an Olympus CX 21 microscope fitted with an Olympus C5050Z digital camera (Olympus Optical Co., Ltd, Tokyo, Japan).

      2.4 Semiquantitative analysis

      For semiquantitative analysis of VEGF, CD 34, and α-SMA expression, we used ImageJ 1.53f51 software, which was free to download from the website of the National Institute of Health (NIH) (https://imagej.nih.gov/ij/) [
      • Schneider C.A.
      • Rasband W.S.
      • Eliceiri K.W.
      NIH Image to ImageJ: 25 years of image analysis.
      ]. The intensity of staining was assessed through the IHC Profiler plugin, another free download from the Sourceforge website (https://sourceforge.net/projects/ihcprofiler/), according to a well-established protocol [
      • Varghese F.
      • Bukhari A.B.
      • Malhotra R.
      • De A.
      IHC Profiler: An open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples.
      ]. The IHC profiler assigned a score to each visual field in a four-tier system: high positive (3+), positive (2+), low positive (1+), and negative (0). Five slides were used from each ligament. On each slide, we analyzed at least 10 random visual fields. The final score was the average of the scores of all visual fields as calculated by the IHC Profiler.
      For the comparative DAB stain localization analysis, we further subjected the IHC Profiler-generated deconvoluted images to expert-trained supervised machine learning-based pixel classification by ilastik software [
      • Berg S.
      • Kutra D.
      • Kroeger T.
      • Straehle C.N.
      • Kausler B.X.
      • Haubold C.
      • et al.
      ilastik: Interactive machine learning for (bio)image analysis.
      ]. To distinguish pixel intensities in connective tissue and vascular visual fields, we first generated masks and applied them to each region of interest of the deconvoluted DAB channel images. Then, we separately measured the mean pixel intensities for connective and vascular tissue visual fields in ImageJ and determined their ratio for each image. We used this ratio as a comparative index of stain localization; lower ratios indicated predominantly blood vessel-localized DAB and vice versa.
      To eliminate inter- and intraobserver variation, we used the IHC Profiler plugin for ImageJ software. A thorough validation by Varghese et al. [
      • Varghese F.
      • Bukhari A.B.
      • Malhotra R.
      • De A.
      IHC Profiler: An open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples.
      ] shows that the accuracy between IHC profiler and blinded manual scoring by pathologists is high, with 88.6% of the scores being in agreement (P < 0.0001).
      Graphic data representations were created by the open-source data visualization package ggplot2 v3.3.5 [
      • Wickham H.
      Elegant graphics for data analysis.
      ], running on the integrated development environment R Studio v1.4.1106 [

      RStudio Team. RStudio: Integrated Development Environment for R [Internet] (2021). Boston, MA: RStudio, PBC. Available at: http://www.rstudio.com/.

      ] for the statistical programming language R v4.04 [

      Core Team RR. A language and environment for statistical computing. R Foundation for Statistical Computing (2021). Vienna Austria. Available at: https://www R-Proj OrgGoogle Sch.

      ].

      3. Results

      3.1 Light microscopic observations

      The normal structure of the EL compared to the ligament proper of the MCL and ACL in humans is quite varied; the morphology of the EL substance was well described in our previous studies [
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Kinov P.
      • Slavchev S.
      • Landzhov B.
      Light and electron microscopic study of the medial collateral ligament epiligament tissue in human knees.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Kotov G.
      • Slavchev S.A.
      • Iliev A.
      Matrix metalloproteinase-2 and -9 expression in the epiligament of the medial collateral and anterior cruciate ligament in human knees: A comparative study.
      ]. The external surface of the EL of the MCL (Figure 1(a), (b)) and ACL (Figure 1(c), (d)) are morphologically similar and comprised of various types of connective tissue cells, including active fibroblasts and nonactive fibroblasts (fibrocytes), fat tissue cells; extracellular collagen fibers, which had uniformly small diameters single or in groups were also observed in the EL tissue of both ligaments; the main parts of neurovascular bundles of the EL–ligament complex were predominantly localized in the EL. The number of fibroblasts in the EL of the MCL was higher than that in the EL of the ACL, which was assessed quantitatively in our previous studies [
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Kotov G.
      • Slavchev S.A.
      • Iliev A.
      Matrix metalloproteinase-2 and -9 expression in the epiligament of the medial collateral and anterior cruciate ligament in human knees: A comparative study.
      ]. The internal surface of the EL of the MCL was closely related and connected to the medial meniscus. The EL morphology was notably quite similar to that of the synovium.
      Figure thumbnail gr1
      Figure 1Normal morphology of the epiligament (EL) of the medial collateral ligament (a, b) and anterior cruciate ligament (c, d) in the human knee. L, ligament tissue; arrowheads, blood vessels in the EL; asterisks, adipocytes. (a) Van Gieson’s stain, (c) hematoxylin and eosin stain; (b), (d) Mallory’s trichrome stain. Scale bar, 200 µm.
      Figure thumbnail gr2
      Figure 2Immunohistochemical staining for CD34, α-smooth muscle actin (α-SMA), and vascular endothelial growth factor (VEGF) in the epiligament of the medial collateral ligament in the human knee. (a), (b) Immunohistochemical staining for CD34. (a) Scale bar, 100 µm; (b) scale bar, 50 µm. (c), (d) Immunohistochemical staining for α-SMA. (c) Scale bar, 100 µm; (d) scale bar, 50 µm. (e), (f) Immunohistochemical staining for VEGF. (e) Scale bar,100 µm; (f) scale bar, 50 µm.

      3.2 Expression of CD34, α-SMA, and VEGF in MCL and ACL EL

      In the EL of the MCL and ACL, immunostaining for CD34 was observed predominantly in the endothelial layer of the blood vessels and was uniformly expressed in the EL tissue (Figures 2(a), (b) and 3(a), (b)). The immunohistochemical reaction for α-SMA was detected predominantly in the smooth muscle cells of the tunica media of blood vessels and the superficial layer of the EL of the MCL (Figure 2(c), (d)) and ACL (Figure 3(c), (d)). Positive VEGF immunohistochemical expression was localized mostly in the endothelial layer of the blood vessels and the superficial layer of the EL in both ligaments (Figures 2(e), (f) and 3(e), (f)).
      Figure thumbnail gr3
      Figure 3Immunohistochemical staining for CD34, α-smooth muscle actin (α-SMA), and vascular endothelial growth factor (VEGF) in the epiligament of the anterior cruciate ligament in the human knee. (a), (b) Immunohistochemical staining for CD34. (a) Scale bar, 100 µm; (b) scale bar, 50 µm. (c), (d) Immunohistochemical staining for α-SMA. (c) Scale bar, 100 µm; (d) scale bar, 50 µm. (e), (f) Immunohistochemical staining for VEGF. (e) Scale bar, 100 µm; (f) Scale bar, 50 µm.
      Because the intensity of the immunohistochemical reactions for CD34, α-SMA, and VEGF varied between the EL and ligament tissue of the MCL and ACL, it was calculated using the IHC Profiler Plugin for ImageJ (Table 1). We took into account two parameters: the number of images with a specific overall score (Figure 4) and the distribution of visual fields with specific scores across images of the same type of EL stained with the same marker (Table 1). Analysis of CD34, α-SMA, and VEGF DAB-stained images showed that among images of ACL EL, there was the highest fraction of positive scores (1+ and 2+) compared with the MCL EL images (Figure 4). Most of the images with high positive and positive overall scores were of ACL EL, stained for CD34, followed by the fraction of images of ACL EL, stained for VEGF. However, the percentage of visual fields that scored highly positive was highest for α-SMA images, followed by VEGF and CD34 (Table 1). The most visual fields with positive scores were observed in ACL EL images stained for CD34, followed by VEGF and α-SMA (Table 1). There were considerable fractions of images of ACL EL which scored low positive for α-SMA and high positive for VEGF, but the number of negative scores was higher for α-SMA than for VEGF (Figure 4). There were only small differences in the expression of CD34, α-SMA, and VEGF in ACL EL, which was overall of considerable intensity. Among images of MCL EL, the highest overall scores were low positive for the majority of images stained for CD34 and a smaller fraction of images stained for α-SMA. All MCL EL images stained for VEGF had negative overall scores (Figure 4). In MCL EL images, there was a noteworthy fraction of visual fields with low positive scores for α-SMA and CD34, and a smaller fraction of visual fields scored low positive for VEGF (Table 1). Overall, among MCL EL images, the results consistently showed that CD34 expression was highest among the tested markers, followed by α-SMA and VEGF.
      Table 1Semiquantitative analysis of the immunohistochemical expression of vascular endothelial growth factor (VEGF), CD34, and α-smooth muscle actin (α-SMA) in the epiligament of the medial collateral and anterior cruciate ligament.
      IHC markerACL ELMCL EL
      VEGFHigh positive (3+) (7.7%)High positive (3+) (3.1%)
      Positive (2+) (9.8%)Positive (2+) (4.7%)
      Low positive (1+) (28.9%)Low positive (1+) (13.1%)
      Negative (0) (53.6%)Negative (0) (79.1%)
      CD34High positive (3+) (4.5%)High positive (3+) (3.9%)
      Positive (2+) (10.0%)Positive (2+) (8.2%)
      Low positive (1+) (21.1%)Low positive (1+) (21.1%)
      Negative (0) (64.4%)

      Negative (0) (44.8%)
      α-SMAHigh positive (3+) (10.6%)High positive (3+) (3.0%)
      Positive (2+) (9.3%)Positive (2+) (5.5%)
      Low positive (1+) (20.8%)Low positive (1+) (21.2%)
      Negative (0) (59.3%)Negative (0) (70.3%)
      The percentage for each score represents the percentage of visual fields to which the IHC Profiler assigned this score. ACL, anterior cruciate ligament; EL, epiligament; MCL, medial collateral ligament.
      Figure thumbnail gr4
      Figure 4Percentages of overall image grades, according to IHC Profiler-generated scores for CD34, α-smooth muscle actin (α-SMA), and vascular endothelial growth factor (VEGF) in the epiligament of the medial collateral ligament (MCL) in the human knee. Bars are color-coded according to the immunohistochemical staining: CD34, orange; α-SMA, green; VEGF, blue. Top row, anterior cruciate ligament (ACL) epiligament images; bottom row, MCL epiligament images.
      The segmentation of images in vascular and connective tissue parts allowed a semiquantitative comparison of the distribution of CD34, α-SMA, and VEGF in the ELs of ACL and MCL tissue slices [
      • Yordanov Y.I.
      Hep G2 cell culture confluence measurement in phase-contrast micrographs - a user-friendly, open-source software-based approach.
      ]. The results were plotted as a bar graph, with higher values representing a comparatively higher distribution in connective tissue and lower values representing a comparatively higher distribution in vascular tissue (Figure 5). All of the observed ratio values were lower than one, meaning that the mean pixel intensities of all markers were higher in vascular zones. However, there were some differences in this distribution among the analyzed markers and ELs. Higher fractions of CD34 and α-SMA seemed to be found in the connective tissue of the MCL EL than in the EL of the ACL, where the fraction of those markers localized in the vasculature was higher. Notably, higher scores of α-SMA-stained images of ACL EL were related to increased relative vascular localization. In contrast, higher scores of CD34 in images of both ACL EL and MCL EL appeared to show a trend towards increasing connective tissue localization. It was difficult to find such a trend in images of the ELs in ACL and MCL, stained for VEGF, and in MCL images, stained for α-SMA.
      Figure thumbnail gr5
      Figure 5Spatial distribution of immunohistochemical staining across connective tissue and blood vessels. Higher mean pixel intensity ratios (connective tissue/vasculature) represent predominantly connective tissue-localized staining in comparison to the intensity in blood vessels and vice versa. Box plots are color-coded according to the immunohistochemical staining: CD34, orange; α-smooth muscle actin (α-SMA), green; vascular endothelial growth factor (VEGF), blue. Top row, anterior cruciate ligament (ACL) epiligament images; bottom row, medial collateral ligament (MCL) epiligament images. DAB, 3,3′-diaminobenzidine.

      4. Discussion

      To elaborate the existing literature data concerning the EL theory and to further develop knowledge of the EL of the MCL and ACL, these ligaments were investigated immunohistochemically for CD34, α-SMA, and VEGF, which have roles in ligament healing.
      Firstly, we would like to explain how the EL theory appears and to present it briefly. It is well known that the ligament is a hypocellular and hypovascular structure. In contrast, the EL is hypercellular and contains numerous cells such as fibroblasts, fibrocytes, adipocytes, and mast cells. These cells are not static and produce numerous molecules, e.g., collagens, matrix metalloproteinases, and fibronectin, that are important for the ligament homeostasis [
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Kinov P.
      • Slavchev S.
      • Landzhov B.
      Light and electron microscopic study of the medial collateral ligament epiligament tissue in human knees.
      ,
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Nedialkova V.K.
      • Kirkov V.
      • Landzhov B.
      Epiligament Tissue of the medial collateral ligament in rat knee joint: Ultrastructural study.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Kinov P.
      • Angelova J.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Dimitrova I.N.
      • Malinova L.
      • Ovtscharoff W.
      Expression of fi-bronectin during early healing of the medial collateral ligament epiligament in rat knee model.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Kotov G.
      • Slavchev S.A.
      • Iliev A.
      Matrix metalloproteinase-2 and -9 expression in the epiligament of the medial collateral and anterior cruciate ligament in human knees: A comparative study.
      ,
      • Georgiev G.P.
      • Vidinov N.K.
      • Kinov P.S.
      Histological and ultrastructural evaluation of the early healing of the lateral collateral ligament epiligament tissue in a rat knee model.
      ,
      • Iliev A.
      • Kotov G.
      • Stamenov N.
      • Landzhov B.
      • Kirkov V.
      • Georgiev G.P.
      A comparative im-munohistochemical and quantitative study of the epiligament of the medial collateral and anterior cruciate ligament in rat knee.
      ]. All these molecules are also implicated in the degradation, proliferation, and remodeling of the ligament after trauma. The fibroblast cells due to their ultrastructural characteristics have been accepted as being involved in the differentiation, phagocytosis, and collagen synthesis [
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Kinov P.
      • Slavchev S.
      • Landzhov B.
      Light and electron microscopic study of the medial collateral ligament epiligament tissue in human knees.
      ,
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Nedialkova V.K.
      • Kirkov V.
      • Landzhov B.
      Epiligament Tissue of the medial collateral ligament in rat knee joint: Ultrastructural study.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Kinov P.
      • Angelova J.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Vidinov N.K.
      • Kinov P.S.
      Histological and ultrastructural evaluation of the early healing of the lateral collateral ligament epiligament tissue in a rat knee model.
      ]. In the EL, numerous collagen fibers in different directions were also observed. It was hypothesized that the single collagen fibers or those grouped in bundles may respond to ligament tension in different directions [
      • Georgiev G.P.
      • Vidinov N.K.
      • Kinov P.S.
      Histological and ultrastructural evaluation of the early healing of the lateral collateral ligament epiligament tissue in a rat knee model.
      ]. The adipocytes localized in the EL serve as excellent packing material [
      • Georgiev G.P.
      • Vidinov N.K.
      • Kinov P.S.
      Histological and ultrastructural evaluation of the early healing of the lateral collateral ligament epiligament tissue in a rat knee model.
      ]. It should be pointed out that the main neurovascular bundles are predominantly localized it the EL tissue of MCL and ACL [
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Kinov P.
      • Slavchev S.
      • Landzhov B.
      Light and electron microscopic study of the medial collateral ligament epiligament tissue in human knees.
      ,
      • Georgiev G.P.
      • Iliev A.
      • Kotov G.
      • Nedialkova V.K.
      • Kirkov V.
      • Landzhov B.
      Epiligament Tissue of the medial collateral ligament in rat knee joint: Ultrastructural study.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Kinov P.
      • Angelova J.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Dimitrova I.N.
      • Malinova L.
      • Ovtscharoff W.
      Expression of fi-bronectin during early healing of the medial collateral ligament epiligament in rat knee model.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Kotov G.
      • Slavchev S.A.
      • Iliev A.
      Matrix metalloproteinase-2 and -9 expression in the epiligament of the medial collateral and anterior cruciate ligament in human knees: A comparative study.
      ,
      • Georgiev G.P.
      • Vidinov N.K.
      • Kinov P.S.
      Histological and ultrastructural evaluation of the early healing of the lateral collateral ligament epiligament tissue in a rat knee model.
      ,
      • Iliev A.
      • Kotov G.
      • Stamenov N.
      • Landzhov B.
      • Kirkov V.
      • Georgiev G.P.
      A comparative im-munohistochemical and quantitative study of the epiligament of the medial collateral and anterior cruciate ligament in rat knee.
      ]. Due to the aforementioned facts, it could be accepted that this enveloping tissue is essential for normal ligament homeostasis, function, and growth and may control water and metabolite influx into the ligament. Thereafter, based on this, Georgiev et al. [
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Kinov P.
      • Angelova J.
      • Landzhov B.
      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.
      ] studied the role of EL in a rat model of MCL injury. The authors clearly presented in detail on histological and ultrastructural micrographs that the EL cells migrate to the gap within the torn ligaments and fill the cavity between the injured ends and thus ensure ligament healing. Moreover, they also pointed out that the main collagens for ligament healing are localized predominantly the EL of MCL [
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ,
      • Iliev A.
      • Kotov G.
      • Stamenov N.
      • Landzhov B.
      • Kirkov V.
      • Georgiev G.P.
      A comparative im-munohistochemical and quantitative study of the epiligament of the medial collateral and anterior cruciate ligament in rat knee.
      ]. Thus, the EL theory was accepted. To enlarge the theory, Georgiev et al. [
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ] compared the EL tissues of MCL and ACL. They found similar morphological characteristics of EL in both ligaments, but a significant difference in the number of cells, which was higher in MCL. Based on these findings and the established differences in main collagens for healing and different matrix metalloproteinases in the ELs and ligament proper, the EL theory was developed and explained the failure in ACL healing. Briefly, based on the presented data, we described the main idea of the novel EL theory. Below, we present previous reports of the presence of CD34, α-SMA, and VEGF which are known to have a role in ligament healing and direct these reports toward EL theory.
      The walls of blood vessels have been described as a rich supply of stem/progenitor cells with characteristic expression of CD34 surface cell markers [
      • Howson K.M.
      • Aplin A.C.
      • Gelati M.
      • Alessandri G.
      • Parati E.A.
      • Nicosia R.F.
      The postnatal rat aorta contains pericyte progenitor cells that form spheroidal colonies in suspension culture.
      ,
      • Tavian M.
      • Zheng B.
      • Oberlin E.
      • Crisan M.
      • Sun B.
      • Huard J.
      • et al.
      The vascular wall as a source of stem cells.
      ]. Circulating human CD34 cells exert potent vasculogenesis in an MCL injury-induced environment, enabling them to make a remarkable contribution to morphological ligament healing. The technical feasibility in a clinical situation and the present preclinical findings demonstrate morphological changes during healing through concurrent vasculogenesis. The results presented by Tei et al. [
      • Tei K.
      • Matsumoto T.
      • Mifune Y.
      • Ishida K.
      • Sasaki K.
      • Shoji T.
      • et al.
      Administrations of peripheral blood CD34-positive cells contribute to medial collateral ligament healing via vasculogenesis.
      ] are promising for future clinical applications of circulating CD34 cells to ligament healing and remodeling. Lee et al. [
      • Lee J.K.
      • Jo S.
      • Lee Y.L.
      • Park H.
      • Song J.S.
      • Sung I.H.
      • et al.
      Anterior cruciate ligament remnant cells have different potentials for cell differentiation based on their location.
      ] showed that a ruptured human ACL could contain vascular-derived stem cells and that ACL-derived CD34 + cells can promote healing. Different studies have indicated that blood vessels have a rich supply of stem/progenitor cells expressing the surface marker CD34 [
      • Howson K.M.
      • Aplin A.C.
      • Gelati M.
      • Alessandri G.
      • Parati E.A.
      • Nicosia R.F.
      The postnatal rat aorta contains pericyte progenitor cells that form spheroidal colonies in suspension culture.
      ,
      • Crisan M.
      • Yap S.
      • Casteilla L.
      • Chen C.-W.
      • Corselli M.
      • Park T.S.
      • et al.
      A perivascular origin for mesenchymal stem cells in multiple human organs.
      ,
      • Zengin E.
      • Chalajour F.
      • Gehling U.M.
      • Ito W.D.
      • Treede H.
      • Lauke H.
      • et al.
      Vascular wall resident progenitor cells: A source for postnatal vasculogenesis.
      ]. These CD34-expressing vascular cells are present in ACL tissue and demonstrate potential for multilineage differentiation and the ability to migrate to a site of ACL rupture to contribute to ligament healing [
      • Matsumoto T.
      • Ingham S.M.
      • Mifune Y.
      • Osawa A.
      • Logar A.
      • Usas A.
      • et al.
      Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells.
      ]. Furthermore, Matsumoto et al. [
      • Matsumoto T.
      • Kubo S.
      • Sasaki K.
      • Kawakami Y.
      • Oka S.
      • Sasaki H.
      • et al.
      Acceleration of tendon-bone healing of anterior cruciate ligament graft using autologous ruptured tissue.
      ] tested the maturation of bone–tendon integration in a dog model of ACL reconstruction and found endochondral ossification-like integration that had enhanced angiogenesis in grafts of tissue treated with CD34 + cells. Ruptured ACL tissue contains higher percentages of CD34 + cells (46%) than peripheral blood (1%) [
      • Matsumoto T.
      • Kubo S.
      • Sasaki K.
      • Kawakami Y.
      • Oka S.
      • Sasaki H.
      • et al.
      Acceleration of tendon-bone healing of anterior cruciate ligament graft using autologous ruptured tissue.
      ,
      • Matsumoto T.
      • Kawamoto A.
      • Kuroda R.
      • Ishikawa M.
      • Mifune Y.
      • Iwasaki H.
      • et al.
      Therapeutic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34-positive cells for functional bone healing.
      ]. Using immunohistochemistry and flow cytometry, Matsumoto et al. [
      • Matsumoto T.
      • Kubo S.
      • Sasaki K.
      • Kawakami Y.
      • Oka S.
      • Sasaki H.
      • et al.
      Acceleration of tendon-bone healing of anterior cruciate ligament graft using autologous ruptured tissue.
      ] confirmed that CD34 +  and CD146 + cells are recruited to the site of rupture and that there are significantly more of these cell types in this area than in the mid-substance region. Mifune et al. [
      • Mifune Y.
      • Matsumoto T.
      • Ota S.
      • Nishimori M.
      • Usas A.
      • Kopf S.
      • et al.
      Therapeutic potential of anterior cruciate ligament-derived stem cells for anterior cruciate ligament reconstruction.
      ] used CD34 + endothelial progenitor cells sorted by a fluorescence-activated cell sorter from the ACL remnants of human patients undergoing surgery to improve tendon–bone healing in a rat model of ACL rupture. Later, Mifune et al. [
      • Mifune Y.
      • Matsumoto T.
      • Takayama K.
      • Terada S.
      • Sekiya N.
      • Kuroda R.
      • et al.
      Tendon graft revitalization using adult anterior cruciate ligament (ACL)-derived CD34+ cell sheets for ACL reconstruction.
      ] demonstrated that ACL-derived CD34 + cells contributed to tendon–bone healing via angiogenesis and enhanced osteogenesis. These authors reported that the remnants of ruptured ACL tissue included more abundant vascular-derived CD34 + stem cells than the uninjured ACL mid-substance, further emphasizing that CD34 + cells had a high potential for proliferation and multilineage differentiation. Nakano et al. [
      • Nakano N.
      • Matsumoto T.
      • Takayama K.
      • Matsushita T.
      • Araki D.
      • Uefuji A.
      • et al.
      Age-dependent healing potential of anterior cruciate ligament remnant-derived cells.
      ] found that ACL-derived cells from a younger group enhanced bone–tendon healing in an immunodeficient ACL reconstruction rat model. It has also been reported that ACL remnants in young patients exhibit higher proliferation and multilineage differentiation potentials. This potential showed a decrease with age, as CD34 + cells were more prevalent in ACL remnants from younger patients. Kirizuki et al. [
      • Kirizuki S.
      • Matsumoto T.
      • Ueha T.
      • Uefuji A.
      • Inokuchi T.
      • Takayama K.
      • et al.
      The influence of ruptured scar pattern on the healing potential of anterior cruciate ligament remnant cells.
      ] studied the potential for ACL healing by morphological pattern (attachment of the remnants to surrounding tissues) and found significantly more CD34 + cells in the non-reattachment group than in the reattachment group. The distal third region of the ACL remnant demonstrated a stronger tendency toward chondrogenic differentiation with more CD34 + cells. Furthermore, the more proximal portion of the remnants had a stronger tendency towards ligamentous and osteogenic differentiation. The characteristics of the ACL remnant tissue should be studied during remnant-preserving ACL reconstruction or harvesting of ACL remnants for tissue engineering [
      • Lee J.K.
      • Jo S.
      • Lee Y.L.
      • Park H.
      • Song J.S.
      • Sung I.H.
      • et al.
      Anterior cruciate ligament remnant cells have different potentials for cell differentiation based on their location.
      ].
      Our results revealed that in the EL of the MCL, CD34 was expressed in the endothelium, the superficial layer of the EL, and the EL tissue. In the EL of the ACL, the reaction for CD34 was also positive in the endothelium and uniformly expressed in the EL tissue. In ACL EL, there was the highest fraction of positive scores (1+ and 2+) for CD34 compared with the MCL EL images. If we accept the view of Kirizuki et al. [
      • Kirizuki S.
      • Matsumoto T.
      • Ueha T.
      • Uefuji A.
      • Inokuchi T.
      • Takayama K.
      • et al.
      The influence of ruptured scar pattern on the healing potential of anterior cruciate ligament remnant cells.
      ] that remnant-preserving ACL reconstruction should be performed when the ligament ends do not reattach, it should be remembered that the EL tissue is the main donor of CD34 + cells and will therefore ensure better healing.
      Myofibroblasts have been identified in intact and injured human ACLs [
      • Murray M.M.
      • Martin S.D.
      • Martin T.L.
      • Spector M.
      Histological changes in the human anterior cruciate ligament after rupture.
      ,
      • Murray M.M.
      • Spector M.
      Fibroblast distribution in the anteromedial bundle of the human anterior cruciate ligament: The presence of alpha-smooth muscle actin-positive cells.
      ]. They contribute significantly to the contractile phase of MCL healing, allowing its original length to be recovered [
      • Faryniarz D.A.
      • Chaponnier C.
      • Gabbiani G.
      • Yannas I.V.
      • Spector M.
      Myofibroblasts in the healing lapine medial collateral ligament: Possible mechanisms of contraction.
      ]. In the healing MCL, blood clots form and provide a provisional scaffold as a basis for repair. This scaffold is invaded progressively by surrounding cells that proliferate and can differentiate into myofibroblasts to form a functional scar. Myofibroblasts in the MCL granulation tissue are subjected to mechanical factors that favor their development and maintain their contractile phenotype [
      • Frank C.
      • Amiel D.
      • Akeson W.H.
      Healing of the medial collateral ligament of the knee. A morphological and biochemical assessment in rabbits.
      ].
      Myofibroblasts can have widely heterogeneous origins; however, their development follows a consistent sequence [
      • Hinz B.
      • Celetta G.
      • Tomasek J.J.
      • Gabbiani G.
      • Chaponnier C.
      • Matsudaira P.T.
      Alpha-smooth muscle actin expression upregulates fibroblast contractile activity.
      ]. After injury, fibroblasts subjected to inflammatory stimuli (cytokines, mechanical microenvironment) have been reported to acquire the ‘protomyofibroblast’ phenotype and then differentiate into typical myofibroblasts characterized by de novo expression of α-SMA, the actin isoform predominant in vascular smooth muscle cells [
      • Hinz B.
      Formation and function of the myofibroblast during tissue repair.
      ]. The expression of α-SMA allows myofibroblast contractile activity to be significantly increased [
      • Hinz B.
      • Celetta G.
      • Tomasek J.J.
      • Gabbiani G.
      • Chaponnier C.
      • Matsudaira P.T.
      Alpha-smooth muscle actin expression upregulates fibroblast contractile activity.
      ]. Menetrey et al. [
      • Menetrey J.
      • Laumonier T.
      • Garavaglia G.
      • Hoffmeyer P.
      • Fritschy D.
      • Gabbiani G.
      • et al.
      α-Smooth muscle actin and TGF-β receptor I expression in the healing rabbit medial collateral and anterior cruciate ligaments.
      ] observed that α-SMA-positive cells appeared in the MCL as early as the third day postlesion and migrated towards the center of the lesion, while only low levels of α-SMA expression were observed in the healing ACL. Interestingly, this difference in α-SMA expression persisted up to day 21 postinjury, when MCL healing was quite advanced. This discrepancy could at least partly explain the difference in healing efficiency between the two ligaments, particularly given the previous observation that myofibroblasts contribute to the restoration of the original length and in situ strain of the MCL [
      • Faryniarz D.A.
      • Chaponnier C.
      • Gabbiani G.
      • Yannas I.V.
      • Spector M.
      Myofibroblasts in the healing lapine medial collateral ligament: Possible mechanisms of contraction.
      ]. The low myofibroblast density in the healing ACL could correspond to the relatively limited number of precursor cells available at the injured site, as suggested by Kanaya et al. [
      • Kanaya A.
      • Deie M.
      • Adachi N.
      • Nishimori M.
      • Yanada S.
      • Ochi M.
      Intra-articular injection of mesenchymal stromal cells in partially torn anterior cruciate ligaments in a rat model.
      ], who showed that injection of mesenchymal stromal cells into a partially torn rat ACL accelerates and improves healing.
      Murray and Spector [
      • Murray M.M.
      • Spector M.
      Fibroblast distribution in the anteromedial bundle of the human anterior cruciate ligament: The presence of alpha-smooth muscle actin-positive cells.
      ] demonstrated that myofibroblast-like cells that contain α-SMA are present in the mid-substance of the intact human ACL. Later, Murray et al. [
      • Murray M.M.
      • Martin S.D.
      • Martin T.L.
      • Spector M.
      Histological changes in the human anterior cruciate ligament after rupture.
      ] suggested that a contiguous layer of α-SMA-containing cells around the tissue, and not individual myofibroblasts distributed through it, could be responsible for contraction (that is, retraction of the remnants of the ACL). Moreover, the formation of a synovial layer comprising cells with a contractile actin isoform over the epiligamentous tissue could partly explain the retraction of the remnants, which disfavors reparative bridging tissue [
      • Murray M.M.
      • Martin S.D.
      • Martin T.L.
      • Spector M.
      Histological changes in the human anterior cruciate ligament after rupture.
      ].
      Our results showed a positive reaction for α-SMA predominantly in the smooth muscle cells of the tunica media of blood vessels and the superficial layer of the EL of both the MCL and ACL. The expression of α-SMA was most pronounced in ACL EL images. Our results corroborated the finding by Murray et al. [
      • Murray M.M.
      • Martin S.D.
      • Martin T.L.
      • Spector M.
      Histological changes in the human anterior cruciate ligament after rupture.
      ] of a positive reaction for α-SMA in the superficial layer of the EL of the ACL. However, Murray et al. [
      • Murray M.M.
      • Martin S.D.
      • Martin T.L.
      • Spector M.
      Histological changes in the human anterior cruciate ligament after rupture.
      ] recognized EL tissue and described it together with synovial tissue. The EL of the ACL was clearly defined in our previous reports [
      • Georgiev G.P.
      Epiligament or paratenon is more appropriate for describing the enveloping tissue of the cruciate ligaments of the human knee?.
      ,
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ,
      • Georgiev G.P.
      • Landzhov B.
      • Kotov G.
      • Slavchev S.A.
      • Iliev A.
      Matrix metalloproteinase-2 and -9 expression in the epiligament of the medial collateral and anterior cruciate ligament in human knees: A comparative study.
      ] as tissue enveloping the ACL ligament. It is similar in morphology to synovial tissue and is considered a specialized form of it [
      • Georgiev G.P.
      • Kotov G.
      • Iliev A.
      • Slavchev S.
      • Ovtscharoff W.
      • Landzhov B.
      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.
      ,
      • Key J.A.
      The reformation of synovial membrane in the knees of rabbits after synovectomy.
      ].
      VEGF is a potent mediator of angiogenesis, which involves the activation, migration, and proliferation of endothelial cells in various pathological conditions [
      • Ferrara N.
      • Davis-Smyth T.
      The biology of vascular endothelial growth factor.
      ]. VEGF is integral to the early proliferative and remodeling phases, in which it is a powerful stimulator of angiogenesis [
      • Molloy T.
      • Wang Y.
      • Murrell G.
      The roles of growth factors in tendon and ligament healing.
      ]. It is expressed in developing blood vessels, and its receptors are found exclusively on endothelial cells [
      • Nicosia R.F.
      • Lin Y.J.
      • Hazelton D.
      • Qian X.
      Endogenous regulation of angiogenesis in the rat aorta model. Role of vascular endothelial growth factor.
      ]. VEGF is considered the most powerful vascular growth promoter and is directly involved in the control and regulation of endothelial cell behaviors such as migration, proliferation, and differentiation [
      • Jackson J.R.
      • Minton J.A.
      • Ho M.L.
      • Wei N.
      • Winkler J.D.
      Expression of vascular endothelial growth factor in synovial fibroblasts is induced by hypoxia and interleukin 1beta.
      ]. Stimulation of angiogenesis by VEGF facilitates access to the healing site. Whether increased neovascularization is advantageous for the clinical outcome is unclear [
      • Zumstein M.A.
      • Rumian A.
      • Lesbats V.
      • Schaer M.
      • Boileau P.
      Increased vascularization during early healing after biologic augmentation in repair of chronic rotator cuff tears using autologous leukocyte- and platelet-rich fibrin (L-PRF): A prospective randomized controlled pilot trial.
      ]. VEGF is an endogenous stimulator of both angiogenesis and increased vascular permeability [
      • Flamme I.
      • von Reutern M.
      • Drexler H.C.
      • Syed-Ali S.
      • Risau W.
      Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation.
      ]. Different studies have shown that it is essential for tissue healing through the induction of angiogenesis [
      • Corral C.J.
      • Siddiqui A.
      • Wu L.
      • Farrell C.L.
      • Lyons D.
      • Mustoe T.A.
      Vascular endothelial growth factor is more important than basic fibroblastic growth factor during ischemic wound healing.
      ,
      • Nishimori M.
      • Matsumoto T.
      • Ota S.
      • Kopf S.
      • Mifune Y.
      • Harner C.
      • et al.
      Role of angiogenesis after muscle derived stem cell transplantation in injured medial collateral ligament.
      ,
      • Phillips G.D.
      • Stone A.M.
      • Jones B.D.
      • Schultz J.C.
      • Whitehead R.A.
      • Knighton D.R.
      Vascular endothelial growth factor (rhVEGF165) stimulates direct angiogenesis in the rabbit cornea.
      ]. Some studies have reported that VEGF production peaks only after the inflammatory phase during natural healing of the injured ligament and tendon and that VEGF and blood vessel formation peak between five and nine days postinjury [
      • Chamberlain C.S.
      • Crowley E.
      • Vanderby R.
      The spatio-temporal dynamics of ligament healing.
      ,
      • Molloy T.
      • Wang Y.
      • Murrell G.
      The roles of growth factors in tendon and ligament healing.
      ]. Increased levels of angiogenic growth factors such as VEGF within an injury site are correlated with a well-defined pattern of vascular ingrowth from the epi- and intratendinous blood supply toward the site of repair. This neovascularization proceeds along the surface of the epitenon through a normally avascular area and provides extrinsic cells, nutrients, and growth factors to the injured site [
      • Molloy T.
      • Wang Y.
      • Murrell G.
      The roles of growth factors in tendon and ligament healing.
      ]. VEGF is an endothelial mitogen that promotes angiogenesis, increases capillary permeability, and contributes to fibrous integration between tendon and bone during the early postoperative stage [
      • Kanazawa T.
      • Soejima T.
      • Murakami H.
      • Inoue T.
      • Katouda M.
      • Nagata K.
      An immunohistological study of the integration at the bone-tendon interface after reconstruction of the anterior cruciate ligament in rabbits.
      ].
      The results presented here revealed that positive immunohistochemical expression of VEGF was located predominantly in the blood vessel walls and the superficial layer of the EL in both ligaments, re-emphasizing the validity of the EL theory and confirming that the EL is the main donor of blood vessels and that VEGF is involved in ligament healing.
      Of course, we would like to point out that the presented data that enlarge the knowledge about the EL tissue, which is on the basis of the EL theory, is only one of the existing explanations for differences in healing of ACL and MCL. Various theories that explain the different healing potentials of these ligaments have been proposed. It should be pointed out that ACL healing failure is ‘multifactorial’, and no single theory can fully explain the reasons for an inadequate healing process. Below, we briefly summarize the existing theories that explain differences in the healing process of MCL and ACL. When culturing fibroblasts from human ACL and MCL, Andrish and Holmes [
      • Andrish J.
      • Holmes R.
      Effects of synovial fluid on fibroblasts in tissue culture.
      ] established weaker fibroblast growth from ACL after exposure to synovial fluid. Moreover, the growth rates of fibroblasts from MCL were higher than those of fibroblasts from ACL. In conclusion, the authors accept that the combination of the inhibitory role of the synovial fluid and different growth rates of fibroblasts from these ligaments may explain ACL healing failure. Amiel et al. [
      • Amiel D.
      • Nagineni C.N.
      • Choi S.H.
      • Lee J.
      Intrinsic properties of ACL and MCL cells and their responses to growth factors.
      ], based on the evaluation of explants from rabbit ACL and MCL, described a higher outgrowth of cells in ACL than in MCL. Moreover, morphological differences between the cells of these ligaments were also established. Based on this, the authors considered that different intrinsic properties of both ligament cells could explain one of the reasons for ACL healing failure. Yoshida and Fujii [
      • Yoshida M.
      • Fujii K.
      Differences in cellular properties and responses to growth factors between human ACL and MCL cells.
      ], after culturing cells from human ACL and MCL, established that ACL cells have poorer distinctive cellular features and weaker responses to growth factors than MCL cells. Therefore, these authors considered that these differences could explain the lower capacity of ACL cells for healing compared with MCL. Cao et al. [
      • Cao M.
      • Stefanovic-Racic M.
      • Georgescu H.I.
      • Fu F.H.
      • Evans C.H.
      Does nitric oxide help explain the differential healing capacity of the anterior cruciate, posterior cruciate, and medial collateral ligaments?.
      ] established that ACL produced significant levels of nitric oxide in response to the inflammatory cytokine interleukin-1. In contrast, MCL synthesized only modest levels of nitric oxide. The authors determined that when applied to these ligaments, nitric oxide inhibited the synthesis of collagen and proteoglycans in ACL, in contrast to MCL. Based on this, Cao et al. [
      • Cao M.
      • Stefanovic-Racic M.
      • Georgescu H.I.
      • Fu F.H.
      • Evans C.H.
      Does nitric oxide help explain the differential healing capacity of the anterior cruciate, posterior cruciate, and medial collateral ligaments?.
      ] proposed that high levels of nitric oxide in ACL could explain its healing failure. Lyon et al. [
      • Lyon R.M.
      • Akeson W.H.
      • Amiel D.
      • Kitabayashi L.R.
      • Woo S.L.
      Ultrastructural differences between the cells of the medical collateral and the anterior cruciate ligaments.
      ], by light microscopic and ultrastructural studies, found significant differences in the ACL and MCL cells in rabbits. These authors considered cells from the MCL as typical fibroblasts and those from the ACL as fibrocartilage cells. In accordance with the reported morphologic differences, the authors considered that this could explain differences in healing of both ligaments. Bray et al. [
      • Bray R.C.
      • Leonard C.A.
      • Salo P.T.
      Correlation of healing capacity with vascular response in the anterior cruciate and medial collateral ligaments of the rabbit.
      ], based on a study in which a hemisection of rabbit ACL and MCL was performed, established a higher capacity of the MCL for angiogenesis than ACL, which is essential for healing of torn ligaments; thus, these authors explain the difference in healing between the ACL and MCL. Murray et al. [
      • Murray M.M.
      • Spindler K.P.
      • Ballard P.
      • Welch T.P.
      • Zurakowski D.
      • Nanney L.B.
      Enhanced histologic repair in a central wound in the anterior cruciate ligament with a collagen-platelet-rich plasma scaffold.
      ] considered that ACL healing failure is due to the inability to form a fibrin-platelet clot between the ruptured ends, as in MCL. Because of its lack and the gap between the ends of the ruptured ACL, the healing process is disrupted at its earliest stage. According to Brommer et al. [
      • Brommer E.J.
      • Dooijewaard G.
      • Dijkmans B.A.
      • Breedveld F.C.
      Depression of tissue-type plasminogen activator and enhancement of urokinase-type plasminogen activator as an expression of local inflammation.
      ], plasmin in the intraarticular synovial fluid demolishes the fibrin clot. Nishikawa et al. [
      • Nishikawa Y.
      • Kokubun T.
      • Kanemura N.
      • Takahashi T.
      • Matsumoto M.
      • Maruyama H.
      • et al.
      Effects of controlled abnormal joint movement on the molecular biological response in intra-articular tissues during the acute phase of anterior cruciate ligament injury in a rat model.
      ], based on the expression of matrix metalloproteinase-13 and the ratio of this matrix metalloproteinase and tissue inhibitors of metalloproteinases, showed that the control of abnormal movement inhibits the inflammatory reaction in the joint after ACL injury, which is important for the healing process.
      Limitations of this study should be pointed out. (1) The age of the cadavers could compromise the results owing to age-related alterations [
      • Fishkin Z.
      • Miller D.
      • Ritter C.
      • Ziv I.
      Changes in human knee ligament stiffness secondary to osteoarthritis.
      ,
      • Hill C.L.
      • Seo G.S.
      • Gale D.
      • Totterman S.
      • Gale M.E.
      • Felson D.T.
      Cruciate ligament integrity in osteoarthritis of the knee.
      ,
      • Nur H.
      • Aytekin A.
      • Gilgil E.
      Medial collateral ligament bursitis in a patient with knee osteoarthritis.
      ]. Therefore, we used fresh cadavers with a mean age of 55 years with no previous history of osteoarthritis or trauma. (2) Visual quantification of immunohistochemical images is susceptible to significant inter- and intraobserver variation. To eliminate this, we used the IHC Profiler plugin for ImageJ software. (3) The EL was investigated only in the middle part of the ligaments.

      5. Conclusion

      Our results show for the first time that CD34, α-SMA, and VEGF are expressed in the EL of human MCL and ACL. All these molecules are involved in the healing of ligaments and are expressed mainly in the blood vessel walls. It should also be pointed out that the EL is the major source of blood vessels to the ligament–EL complex, which is crucial for ligament nutrition and healing. In addition, it should be noted that both ELs had more pronounced α-SMA expression than VEGF and CD34 expression, and the ACL showed slightly higher CD34 expression than the MCL. The reported results supplement the existing EL theory of MCL healing and the failure of ACL healing and show that future investigations regarding optimal ligament healing should not overlook the EL.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      Author contributions

      All the authors read and approved the final version submitted, met the criteria for authorship as established by the International Committee of Medical Journals Editors, believe that the paper represents honest work, and can verify the validity of the results reported.

      Ethics Information

      The study was approved by the Medical-Legal Office, the Local Ethics Committee, and the Institutional Review Board (No. 4866).

      Declaration of Competing Interest

      The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Georgi P. Georgiev reports a relationship with Medical Iniversity of Sofia that includes: employment.]

      Acknowledgements

      The authors sincerely thank those who donated their bodies to science such that anatomical research could be performed. The results from such research can potentially increase mankind’s overall knowledge which can then improve patient care. Therefore, these donors and their families deserve our highest gratitude [
      • Iwanaga J.
      • Singh V.
      • Ohtsuka A.
      • Hwang Y.
      • Kim H.-J.
      • Moryś J.
      • et al.
      Acknowledging the use of human cadaveric tissues in research papers: Recommendations from anatomical journal editors.
      ].

      References

        • Frank C.B.
        Ligament healing: Current knowledge and clinical applications.
        J Am Acad Orthop Surg. 1996; 4: 74-83https://doi.org/10.5435/00124635-199603000-00002
        • Woo S.L.
        • Abramowitch S.D.
        • Kilger R.
        • Liang R.
        Biomechanics of knee ligaments: Injury, healing, and repair.
        J Biomech. 2006; 39: 1-20https://doi.org/10.1016/j.jbiomech.2004.10.025
        • Woo S.L.
        • Vogrin T.M.
        • Abramowitch S.D.
        Healing and repair of ligament injuries in the knee.
        J Am Acad Orthop Surg. 2000; 8: 364-372https://doi.org/10.5435/00124635-200011000-00004
        • Amiel D.
        • Nagineni C.N.
        • Choi S.H.
        • Lee J.
        Intrinsic properties of ACL and MCL cells and their responses to growth factors.
        Med Sci Sports Exerc. 1995; 27: 844-851
        • Bray R.C.
        • Leonard C.A.
        • Salo P.T.
        Correlation of healing capacity with vascular response in the anterior cruciate and medial collateral ligaments of the rabbit.
        J Orthop Res. 2003; 21: 1118-1123https://doi.org/10.1016/S0736-0266(03)00078-0
        • Georgiev G.P.
        Epiligament or paratenon is more appropriate for describing the enveloping tissue of the cruciate ligaments of the human knee?.
        Folia Morphol (Warsz). 2021 Feb 26; 81: 258-259
        • Georgiev G.P.
        • Iliev A.
        • Kotov G.
        • Kinov P.
        • Slavchev S.
        • Landzhov B.
        Light and electron microscopic study of the medial collateral ligament epiligament tissue in human knees.
        World J Orthop. 2017; 8: 372-378https://doi.org/10.5312/wjo.v8.i5.372
        • Georgiev G.P.
        • Iliev A.
        • Kotov G.
        • Nedialkova V.K.
        • Kirkov V.
        • Landzhov B.
        Epiligament Tissue of the medial collateral ligament in rat knee joint: Ultrastructural study.
        Cureus. 2019; 11: e3812
        • Georgiev G.P.
        • Kotov G.
        • Iliev A.
        • Kinov P.
        • Angelova J.
        • Landzhov B.
        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.
        Cells Tissues Organs. 2018; 206: 165-182
        • Georgiev G.P.
        • Kotov G.
        • Iliev A.
        • Slavchev S.
        • Ovtscharoff W.
        • Landzhov B.
        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.
        Ann Anat. 2019; 224: 88-96https://doi.org/10.1016/j.aanat.2019.04.002
        • Georgiev G.P.
        • Landzhov B.
        • Dimitrova I.N.
        • Malinova L.
        • Ovtscharoff W.
        Expression of fi-bronectin during early healing of the medial collateral ligament epiligament in rat knee model.
        Compt Rend Acad Bulg Sci. 2016; 69: 639-644
        • Georgiev G.P.
        • Landzhov B.
        • Kotov G.
        • Slavchev S.A.
        • Iliev A.
        Matrix metalloproteinase-2 and -9 expression in the epiligament of the medial collateral and anterior cruciate ligament in human knees: A comparative study.
        Cureus. 2018; 10: e3550
        • Georgiev G.P.
        • Vidinov N.K.
        • Kinov P.S.
        Histological and ultrastructural evaluation of the early healing of the lateral collateral ligament epiligament tissue in a rat knee model.
        BMC Musculoskelet Disord. 2010; 11: 117https://doi.org/10.1186/1471-2474-11-117
        • Chamberlain C.S.
        • Crowley E.
        • Vanderby R.
        The spatio-temporal dynamics of ligament healing.
        Wound Repair Regen. 2009; 17: 206-215https://doi.org/10.1111/j.1524-475X.2009.00465.x
        • Iliev A.
        • Kotov G.
        • Stamenov N.
        • Landzhov B.
        • Kirkov V.
        • Georgiev G.P.
        A comparative im-munohistochemical and quantitative study of the epiligament of the medial collateral and anterior cruciate ligament in rat knee.
        Int J Morphol. 2021; 39: 151-159
        • Schneider C.A.
        • Rasband W.S.
        • Eliceiri K.W.
        NIH Image to ImageJ: 25 years of image analysis.
        Nat Methods. 2012; 9: 671-675https://doi.org/10.1038/nmeth.2089
        • Varghese F.
        • Bukhari A.B.
        • Malhotra R.
        • De A.
        IHC Profiler: An open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples.
        PLoS ONE. 2014; 9: e96801
        • Berg S.
        • Kutra D.
        • Kroeger T.
        • Straehle C.N.
        • Kausler B.X.
        • Haubold C.
        • et al.
        ilastik: Interactive machine learning for (bio)image analysis.
        Nat Methods. 2019; 16: 1226-1232
        • Wickham H.
        Elegant graphics for data analysis.
        Media. 2009; 35: 211
      1. RStudio Team. RStudio: Integrated Development Environment for R [Internet] (2021). Boston, MA: RStudio, PBC. Available at: http://www.rstudio.com/.

      2. Core Team RR. A language and environment for statistical computing. R Foundation for Statistical Computing (2021). Vienna Austria. Available at: https://www R-Proj OrgGoogle Sch.

        • Yordanov Y.I.
        Hep G2 cell culture confluence measurement in phase-contrast micrographs - a user-friendly, open-source software-based approach.
        Toxicol Mech Methods. 2020; 30: 146-152https://doi.org/10.1080/15376516.2019.1695303
        • Howson K.M.
        • Aplin A.C.
        • Gelati M.
        • Alessandri G.
        • Parati E.A.
        • Nicosia R.F.
        The postnatal rat aorta contains pericyte progenitor cells that form spheroidal colonies in suspension culture.
        Am J Physiol Cell Physiol. 2005; 289: C1396-C1407https://doi.org/10.1152/ajpcell.00168.2005
        • Tavian M.
        • Zheng B.
        • Oberlin E.
        • Crisan M.
        • Sun B.
        • Huard J.
        • et al.
        The vascular wall as a source of stem cells.
        Ann N Y Acad Sci. 2005; 1044: 41-50https://doi.org/10.1196/annals.1349.006
        • Tei K.
        • Matsumoto T.
        • Mifune Y.
        • Ishida K.
        • Sasaki K.
        • Shoji T.
        • et al.
        Administrations of peripheral blood CD34-positive cells contribute to medial collateral ligament healing via vasculogenesis.
        Stem Cells. 2008; 26: 819-830https://doi.org/10.1634/stemcells.2007-0671
        • Lee J.K.
        • Jo S.
        • Lee Y.L.
        • Park H.
        • Song J.S.
        • Sung I.H.
        • et al.
        Anterior cruciate ligament remnant cells have different potentials for cell differentiation based on their location.
        Sci Rep. 2020; 10: 3097https://doi.org/10.1038/s41598-020-60047-w
        • Crisan M.
        • Yap S.
        • Casteilla L.
        • Chen C.-W.
        • Corselli M.
        • Park T.S.
        • et al.
        A perivascular origin for mesenchymal stem cells in multiple human organs.
        Cell Stem Cell. 2008; 3: 301-313
        • Zengin E.
        • Chalajour F.
        • Gehling U.M.
        • Ito W.D.
        • Treede H.
        • Lauke H.
        • et al.
        Vascular wall resident progenitor cells: A source for postnatal vasculogenesis.
        Development. 2006; 133: 1543-1551
        • Matsumoto T.
        • Ingham S.M.
        • Mifune Y.
        • Osawa A.
        • Logar A.
        • Usas A.
        • et al.
        Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells.
        Stem Cells Dev. 2012; 21: 859-872
        • Matsumoto T.
        • Kubo S.
        • Sasaki K.
        • Kawakami Y.
        • Oka S.
        • Sasaki H.
        • et al.
        Acceleration of tendon-bone healing of anterior cruciate ligament graft using autologous ruptured tissue.
        Am J Sports Med. 2012; 40: 1296-1302
        • Matsumoto T.
        • Kawamoto A.
        • Kuroda R.
        • Ishikawa M.
        • Mifune Y.
        • Iwasaki H.
        • et al.
        Therapeutic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34-positive cells for functional bone healing.
        Am J Pathol. 2006; 169: 1440-1457
        • Mifune Y.
        • Matsumoto T.
        • Ota S.
        • Nishimori M.
        • Usas A.
        • Kopf S.
        • et al.
        Therapeutic potential of anterior cruciate ligament-derived stem cells for anterior cruciate ligament reconstruction.
        Cell Transplant. 2012; 21: 1651-1665
        • Mifune Y.
        • Matsumoto T.
        • Takayama K.
        • Terada S.
        • Sekiya N.
        • Kuroda R.
        • et al.
        Tendon graft revitalization using adult anterior cruciate ligament (ACL)-derived CD34+ cell sheets for ACL reconstruction.
        Biomaterials. 2013; 34: 5476-5487
        • Nakano N.
        • Matsumoto T.
        • Takayama K.
        • Matsushita T.
        • Araki D.
        • Uefuji A.
        • et al.
        Age-dependent healing potential of anterior cruciate ligament remnant-derived cells.
        Am J Sports Med. 2015; 43: 700-708
        • Kirizuki S.
        • Matsumoto T.
        • Ueha T.
        • Uefuji A.
        • Inokuchi T.
        • Takayama K.
        • et al.
        The influence of ruptured scar pattern on the healing potential of anterior cruciate ligament remnant cells.
        Am J Sports Med. 2018; 46: 1382-1388
        • Murray M.M.
        • Martin S.D.
        • Martin T.L.
        • Spector M.
        Histological changes in the human anterior cruciate ligament after rupture.
        J Bone Joint Surg Am. 2000; 82: 1387-1397https://doi.org/10.2106/00004623-200010000-00004
        • Murray M.M.
        • Spector M.
        Fibroblast distribution in the anteromedial bundle of the human anterior cruciate ligament: The presence of alpha-smooth muscle actin-positive cells.
        J Orthop Res. 1999; 17: 18-27https://doi.org/10.1002/jor.1100170105
        • Faryniarz D.A.
        • Chaponnier C.
        • Gabbiani G.
        • Yannas I.V.
        • Spector M.
        Myofibroblasts in the healing lapine medial collateral ligament: Possible mechanisms of contraction.
        J Orthop Res. 1996; 14: 228-237https://doi.org/10.1002/jor.1100140210
        • Frank C.
        • Amiel D.
        • Akeson W.H.
        Healing of the medial collateral ligament of the knee. A morphological and biochemical assessment in rabbits.
        Acta Orthop Scand. 1983; 54: 917-923https://doi.org/10.3109/17453678308992934
        • Hinz B.
        • Celetta G.
        • Tomasek J.J.
        • Gabbiani G.
        • Chaponnier C.
        • Matsudaira P.T.
        Alpha-smooth muscle actin expression upregulates fibroblast contractile activity.
        Mol Biol Cell. 2001; 12: 2730-2741
        • Hinz B.
        Formation and function of the myofibroblast during tissue repair.
        J Invest Dermatol. 2007; 127: 526-537https://doi.org/10.1038/sj.jid.5700613
        • Menetrey J.
        • Laumonier T.
        • Garavaglia G.
        • Hoffmeyer P.
        • Fritschy D.
        • Gabbiani G.
        • et al.
        α-Smooth muscle actin and TGF-β receptor I expression in the healing rabbit medial collateral and anterior cruciate ligaments.
        Injury. 2011; 42: 735-741
        • Kanaya A.
        • Deie M.
        • Adachi N.
        • Nishimori M.
        • Yanada S.
        • Ochi M.
        Intra-articular injection of mesenchymal stromal cells in partially torn anterior cruciate ligaments in a rat model.
        Arthroscopy. 2007; 23: 610-617https://doi.org/10.1016/j.arthro.2007.01.013
        • Key J.A.
        The reformation of synovial membrane in the knees of rabbits after synovectomy.
        J Bone Joint Surg. 1925; 7: 793-813
        • Ferrara N.
        • Davis-Smyth T.
        The biology of vascular endothelial growth factor.
        Endocr Rev. 1997; 18: 4-25https://doi.org/10.1210/edrv.18.1.0287
        • Molloy T.
        • Wang Y.
        • Murrell G.
        The roles of growth factors in tendon and ligament healing.
        Sports Med. 2003; 33: 381-394https://doi.org/10.2165/00007256-200333050-00004
        • Nicosia R.F.
        • Lin Y.J.
        • Hazelton D.
        • Qian X.
        Endogenous regulation of angiogenesis in the rat aorta model. Role of vascular endothelial growth factor.
        Am J Pathol. 1997; 151: 1379-1386
        • Jackson J.R.
        • Minton J.A.
        • Ho M.L.
        • Wei N.
        • Winkler J.D.
        Expression of vascular endothelial growth factor in synovial fibroblasts is induced by hypoxia and interleukin 1beta.
        J Rheumatol. 1997; 24: 1253-1259
        • Zumstein M.A.
        • Rumian A.
        • Lesbats V.
        • Schaer M.
        • Boileau P.
        Increased vascularization during early healing after biologic augmentation in repair of chronic rotator cuff tears using autologous leukocyte- and platelet-rich fibrin (L-PRF): A prospective randomized controlled pilot trial.
        J Shoulder Elbow Surg. 2014; 23: 3-12https://doi.org/10.1016/j.jse.2013.08.017
        • Flamme I.
        • von Reutern M.
        • Drexler H.C.
        • Syed-Ali S.
        • Risau W.
        Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation.
        Dev Biol. 1995; 171: 399-414https://doi.org/10.1006/dbio.1995.1291
        • Corral C.J.
        • Siddiqui A.
        • Wu L.
        • Farrell C.L.
        • Lyons D.
        • Mustoe T.A.
        Vascular endothelial growth factor is more important than basic fibroblastic growth factor during ischemic wound healing.
        Arch Surg. 1999; 134: 200-205https://doi.org/10.1001/archsurg.134.2.200
        • Nishimori M.
        • Matsumoto T.
        • Ota S.
        • Kopf S.
        • Mifune Y.
        • Harner C.
        • et al.
        Role of angiogenesis after muscle derived stem cell transplantation in injured medial collateral ligament.
        J Orthop Res. 2012; 30: 627-633
        • Phillips G.D.
        • Stone A.M.
        • Jones B.D.
        • Schultz J.C.
        • Whitehead R.A.
        • Knighton D.R.
        Vascular endothelial growth factor (rhVEGF165) stimulates direct angiogenesis in the rabbit cornea.
        In Vivo. 1994; 8: 961-965
        • Kanazawa T.
        • Soejima T.
        • Murakami H.
        • Inoue T.
        • Katouda M.
        • Nagata K.
        An immunohistological study of the integration at the bone-tendon interface after reconstruction of the anterior cruciate ligament in rabbits.
        J Bone Joint Surg Br. 2006; 88: 682-687https://doi.org/10.1302/0301-620X.88B5.17198
        • Andrish J.
        • Holmes R.
        Effects of synovial fluid on fibroblasts in tissue culture.
        Clin Orthop Relat Res. 1979; 138: 279-283
        • Yoshida M.
        • Fujii K.
        Differences in cellular properties and responses to growth factors between human ACL and MCL cells.
        J Orthop Sci. 1999; 4: 293-298https://doi.org/10.1007/s007760050106
        • Cao M.
        • Stefanovic-Racic M.
        • Georgescu H.I.
        • Fu F.H.
        • Evans C.H.
        Does nitric oxide help explain the differential healing capacity of the anterior cruciate, posterior cruciate, and medial collateral ligaments?.
        Am J Sports Med. 2000; 28: 176-182https://doi.org/10.1177/03635465000280020701
        • Lyon R.M.
        • Akeson W.H.
        • Amiel D.
        • Kitabayashi L.R.
        • Woo S.L.
        Ultrastructural differences between the cells of the medical collateral and the anterior cruciate ligaments.
        Clin Orthop Relat Res. 1991; 272: 279-286
        • Murray M.M.
        • Spindler K.P.
        • Ballard P.
        • Welch T.P.
        • Zurakowski D.
        • Nanney L.B.
        Enhanced histologic repair in a central wound in the anterior cruciate ligament with a collagen-platelet-rich plasma scaffold.
        J Orthop Res. 2007; 25: 1007-1017https://doi.org/10.1002/jor.20367
        • Brommer E.J.
        • Dooijewaard G.
        • Dijkmans B.A.
        • Breedveld F.C.
        Depression of tissue-type plasminogen activator and enhancement of urokinase-type plasminogen activator as an expression of local inflammation.
        Thromb Haemost. 1992; 68: 180-184
        • Nishikawa Y.
        • Kokubun T.
        • Kanemura N.
        • Takahashi T.
        • Matsumoto M.
        • Maruyama H.
        • et al.
        Effects of controlled abnormal joint movement on the molecular biological response in intra-articular tissues during the acute phase of anterior cruciate ligament injury in a rat model.
        BMC Musculoskelet Disord. 2018; 19: 175https://doi.org/10.1186/s12891-018-2107-6
        • Fishkin Z.
        • Miller D.
        • Ritter C.
        • Ziv I.
        Changes in human knee ligament stiffness secondary to osteoarthritis.
        J Orthop Res. 2002; 20: 204-207https://doi.org/10.1016/S0736-0266(01)00087-0
        • Hill C.L.
        • Seo G.S.
        • Gale D.
        • Totterman S.
        • Gale M.E.
        • Felson D.T.
        Cruciate ligament integrity in osteoarthritis of the knee.
        Arthritis Rheum. 2005; 52: 794-799https://doi.org/10.1002/art.20943
        • Nur H.
        • Aytekin A.
        • Gilgil E.
        Medial collateral ligament bursitis in a patient with knee osteoarthritis.
        J Back Musculoskelet Rehabil. 2018; 31: 589-591https://doi.org/10.3233/BMR-169741
        • Iwanaga J.
        • Singh V.
        • Ohtsuka A.
        • Hwang Y.
        • Kim H.-J.
        • Moryś J.
        • et al.
        Acknowledging the use of human cadaveric tissues in research papers: Recommendations from anatomical journal editors.
        Clin Anat. 2021; 34: 2-4