The continuous structure of the joint capsule and meniscus in the pig knee

The knee joint capsules composed of a fibrous layer and a synovial layer. The knee meniscus consists of the superficial network, lamellar layer, tie fibre and circumferential bundles. However, the continuous structure of the knee joint capsule and meniscus has not been reported. Fetal and adult pigs were used to investigate the structural relationship between the stifle joint capsule and meniscus based on the gross anatomy and histological findings. In the gross anatomical examination, the joint capsule appeared to have separated attachments to the meniscus, except for the lower aspect of the popliteal hiatus. Histologically, the lower half of the popliteal hiatus was found to have separated attachments, with vessels running between the attachments of the joint capsules. The synovial layer of the joint capsule continued to the superficial network, and the fibrous layer of the joint capsule continued to the lamellar layer and tie fibres. There were two routes of arterial entry into the meniscus: intracapsular and intercapsular. It appeared that the presence of separated attachments of the joint capsule was necessary to allow the intercapsular route. This study clarified for the first time the routes of feeding vessels entering the meniscus and proposed to call this entry point the meniscus hilum. We consider that this detailed anatomical information is important for understanding the continuation between the joint capsule and the meniscus.


| INTRODUC TI ON
It is well known that the joint capsule (JC) is vital to the normal function and long-term health of the knee joint. It seals the joint space, provides passive stability by limiting movements, and active stability through its proprioceptive nerve endings, and forms articular surfaces for the joint. The knee JC consists of a fibrous layer and a synovium layer (Ralphs & Benjamin, 1994). It varies in thickness according to the stresses to which it is subjected, is locally thickened to form capsular ligaments (Ralphs & Benjamin, 1994) and may also incorporate tendons. The JC is often injured, leading to laxity, constriction and/or adhesion to surrounding structures. It is attached to the bones via the meniscus and forms a sleeve around the joint. The meniscus has been divided into peared to have separated attachments to the meniscus, except for the lower aspect of the popliteal hiatus. Histologically, the lower half of the popliteal hiatus was found to have separated attachments, with vessels running between the attachments of the joint capsules. The synovial layer of the joint capsule continued to the superficial network, and the fibrous layer of the joint capsule continued to the lamellar layer and tie fibres. There were two routes of arterial entry into the meniscus: intracapsular and intercapsular. It appeared that the presence of separated attachments of the joint capsule was necessary to allow the intercapsular route. This study clarified for the first time the routes of feeding vessels entering the meniscus and proposed to call this entry point the meniscus hilum. We consider that this detailed anatomical information is important for understanding the continuation between the joint capsule and the meniscus.

K E Y W O R D S
arterial supply, meniscus hilum, popliteal hiatus, synovial layer, stifle, tie fibre three major layers: (i) the main body, composed of circumferential bundles and tie-fibre sheets; (ii) the superficial network; and (iii) the lamellar layer. The main body occupies the majority of the volume of the tissue (Petersen & Tillmann, 1998).The medial meniscus (MM) can also be divided into five anteroposterior zones ( Figure 1) (Hathila et al., 2019;Śmigielski et al., 2015;Zdanowicz et al., 2016). This includes the anterior root attachment (zone 1), the anteromedial zone between the posterior border of the anterior root and the anterior border of the superficial medial collateral ligament (zone 2a and 2b), the portion of the meniscus that is adjacent to the superficial medial collateral ligament (zone 3), the posterior horn (zone 4), and the posterior root (zone 5). While the lateral meniscus (LM) can be classified into six zones based on the anteroposterior location ( Figure 1) (Zdanowicz et al., 2016). These include the anterior root (zone1), the anterolateral zone between the anterior root and the anterior border of the popliteal hiatus (zone 2), the popliteal hiatus (zone 3), the posteroinferior popliteomeniscal fascicle (zone 4), the ligamentous zone (zone 5), and the posterior root (zone 6). We focused on zones 2-4 of the MM and the zones 2-5 of the LM (Figure 1). Although the structure of each tissue has been clarified, and there have been various reports on the continuation between the JC and the meniscus, a consensus has not been reached. Some researchers have described the attachment of the JC to the meniscus as defective at different sites (Rath & Richmond, 2000;Śmigielski et al., 2015), a common continuation of the tibial side of the knee (stifle) JC (TJC) and the femoral side of the knee (stifle) JC (FJC) on the posterior surface of the meniscus (Dephillipo et al., 2019;Greif et al., 2020), and separate continuation of the TJC and FJC on the posterior surface of the meniscus (Cavaignac et al., 2021;Śmigielski et al., 2015).
Thus, reports vary and only specific sites, such as the posterior horn of the MM, have been examined, which does not speak to the generality of the joint continuation between the JC and the meniscus.
To our knowledge, no anatomical studies on the tissue structure of the meniscus and the continuation of the JC to it have been reported in the relevant literature. A better understanding of the anatomical continuations between the meniscus and the JC may help surgeons understand their importance, as well as improve their anatomical approach to repair. The aim of the present study was to investigate, in detail, the attachment of JC to the meniscus in order to provide a comprehensive anatomic basis that is relevant to JCmeniscus complex.

| Preparation of specimens and gross anatomy
A total of 27 stifles were used and 11 adult pig stifles that had been slaughtered but whose lower limbs had not been dissected were purchased from Nansyu Natural Pork (Kagoshima, Japan) and 16 fetal pig stifles that had been fixed with formalin perfusion were imported from the Bio Corporation Company (Minnesota, USA). Seven of the 11 sides of the adult stifles were used for gross dissection and four were used for histological observation. In the dissection of the adult pigs, to clearly show the peripheral microarteries, 50 mL of a mixture of red ink (Xstamper; Shachihata Inc., Japan) and latex was injected into the femoral artery with pressurization. Then, all the stifles were immersion fixed in mixture fixing solution that contained 5% formalin and 50% alcohol. Prior to immersion fixation, to ensure sufficient fixation, a generous amount of similar fixative was F I G U R E 1 A schematic view from the horizontal plane showing the MM and LM zones. AIL, anterior intermeniscal ligament; MCL, medial collateral ligament; PMT, popliteus muscle tendon.
injected into the deep tissue with a 20-mL syringe. At 3 weeks after immersion fixation, detailed dissection around the stifles joint was performed.
Ten of the 16 sides of fetal stifle were used for gross dissection and six were used for histological observation. Red latex had already been injected into the arterial system of the fetal pigs and blue latex had been injected into the venous system, followed by preservation in 10% formalin solution (Dai et al., 2020). The dissection procedure of the fetal pigs followed the protocol (described below) that was used for the adult pigs.
In dissection, after removing the skin and subcutaneous soft tissues, the patellar tendon was cut from the tibial tuberosity. The JC-menisci were harvested as a block from the femur and tibia. The

| Preparation of tissue specimens and histological evaluation
The remaining stifles were used to prepare serial coronal sections for histological examination. The skin of the specimen was peeled, and the subcutaneous fat and excess muscle tissues were also carefully removed.
A MM-JC complex and a LM-JC complex were harvested as a tissue block. Before tissue embedding, it was re-fixed by immersing it in 5% formalin and 50% alcohol for 24 h, then washed thoroughly for 4-5 h under running tap water and dehydrated and routinely embedded in paraffin. Ten-micrometre-thick sections were cut at 100-300μm intervals. The slides were stained with Haematoxylin and Eosin (HE), Masson's trichrome, Fast green and Safranin-o. Sections were then imaged and photographed using an optical microscope. Images were merged and measured using Adobe Photoshop Elements 9.0 (Adobe Inc., USA).

| Gross observation of the LM and JC in adult pigs
In adult pigs, the total length of the LM was 60 mm (Figure 3a

| Gross observation of the meniscus and JC in fetal pigs
In fetal pigs, separated attachments of the JC to MM were observed, which were similar to those in adult pigs as mentioned above (Figure 4a,b). The total length of the LM was15mm (Figure 4c Petersen and Tillmann (1998). 1, fibrous layer; 2, synovial layer; 3, superficial network; 4, lamellar layer; 5, circumferential bundles; 6, tie fibre; 7, M-shape of the tie fibre; *, peri-meniscal fat pad. CB, circumferential bundles; FJC, femoral side of the stifle JC; FL, fibrous layer of the JC; LL, lamellar layer; MCL, medial collateral ligament; SL, synovial layer of the JC; SN, superficial network; TF, tie fibre; TJC, tibial side of the knee JC; ▲, blood vessel; Scale bars: 1 cm.

(a) (b) (c)
6 mm (Figure 4c). On the lower aspect of the popliteal hiatus, the TJC continued to the lower half of the LM (Figure 4e). The part between the upper and lower aspect of the popliteal hiatus was similar to that in adult pigs, as mentioned above (Figure 4d).

| Histological findings of the MM and JC in adult pigs
As shown in the coronal section of the MM in Figure 5, in adult pigs, the FJC and TJC were attached to the upper and lower halves of the outer surface of the MM, respectively, with a distance of approximately 6.6 mm between the attachments. Between the JCs on both halves, the vascularized peri-meniscal fat pad was present at the outer surface of the MM (Figure 5a,b). The synovial layer of the JC continued to the superficial network of the MM (Figure 5b). The fibrous layer of the JC continued to the lamellar layer and tie fibre of the MM (Figure 5b,c).The outer surface of the meniscus between the JCs attachment was covered with tie fibres (Figure 5c). Tie fibre surrounded circumferential bundles and continued to the peri-meniscal fat pad on the outer surface of the MM (Figure 5b,c). The outer surface of the continuing fibre was M-shaped, with a groove at the entrance of the blood vessel (Figure 5c).

| Histological findings of the LM and JC in adult pigs
As shown in the coronal section of the LM in Figure

| Histological findings of the meniscus and JC in fetal pigs
In fetal pigs, the FJC and TJC were attached to the upper and lower halves of the MM, respectively. Similar to the adult pigs described above, there was a vascularized peri-meniscal fat pad located outside between the JCs on both attachments, (Figure 7a). We could find small amounts of tie fibre, circumferential bundles, the lamellar layer, and the superficial network in fetal pigs (Figure 7a), however, the tie fibres were not as clearly organized as those of adult pigs (Figure 7b).
With  of the meniscus, respectively. We also found that the collagen fibres were underdeveloped in the fetal pig meniscus. We considered the continuity of the pig meniscus and JC to be equivalent to that of the human meniscus. This was based on the fact that the pig model appears to better represent the human meniscus, particularly in size, shape and structure (Joshi et al., 1995), and is considered comparable to the young adult human meniscus (Barber &Herbert, 2000).
First, previous studies have reported two forms of the JC attachment points of the meniscus a common continuation of the meniscotibial ligament (called the TJC in this study) and the meniscocapsular ligament (called the FJC in this study) on the posterior surface of the meniscus (MM zone 4) (Dephillipo et al.,2019;Greif et al., 2020) and that different continuation of the meniscotibial ligament and meniscocapsular ligament on the posterior surface of the meniscus (Cavaignac et al., 2021;Śmigielski et al., 2015). Our results support the latter, and this fact may apply not only to MM Zone 4, but also to MM Zones 2-3 and LM Zones 2-5.
Next, arteries to the menisci pass through the JC (Mameri et al. 2022), but in the present study, in addition to this route, arterial access from the peri-meniscal fat pad to the M-shaped valley of tie fibres also was observed. That is, there were two route of arterial access into the meniscus: an intracapsular route (the synovial layer and the fibrous layer of the JC to between the superficial layer and lamellar layer) and an intercapsular route (the peri-meniscal fat pad to the M-shaped valley of tie fibre).We propose to call the entrance of the two routes the meniscus hilum, since this route connected the inside and outside of the meniscus. Based on the meniscus hilum, the avascularity of the LM adjacent to the popliteus tendon (LM zone 3) might be explained. The relatively avascular zone of the LM adjacent to the popliteus tendon has been reported in humans (Arnoczky& Warren, 1982;Day et al., 1985;Crawford et al., 2020;Markes et al., 2020), dogs (Arnoczky& Warren, 1983), sheep (Swiontkowski et al., 1988), and we also confirmed it in pigs. This LM adjacent to the popliteus tendon was relatively avascular because of the narrow meniscus hilum (Figure 5b,c).
Histologically, we observed differences in the organization of collagen fibres between fetal and adult pigs. However, no differences were observed grossly, suggesting that the JC attachments were separated in both fetal and adult pigs. We consider this finding to be informative for fetal, paediatric, and adult menisci. Histologically, in fetal pigs, the tie fibres were organized indistinctly, although they were partially visible (e.g., in the vascular route) (Figure 7b).
Moreover, the indistinct organization of collagen fibres may be advantageous for fetal meniscus development, allowing for increased blood vessel route as identifies in the popliteal hiatus of fetal pigs (Figure 7d,e).
In conclusion, based on morphological and histological continuity, the JC attached separately and the fibrous layer of the JC continued to the lamellar layer and tie fibre layer of the meniscus, and the synovial layer of the JC continued to the superficial network of the meniscus. The continuity of these layers suggests that there were two arterial routes for the meniscus (intracapsular route and intercapsular route), and we proposed that these entrances are called meniscus hilum. These findings provide beneficial information for understanding the knee.

ACK N O WLE D G E M ENTS
This study was supported by an institutional fund from Tokyo Metropolitan University.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.