Swine Meniscus: Are Femoral-Tibial Surfaces Properly Tuned to Bear the Forces Exerted on the Tissue?

Abstract

Menisci are subjected to different pathologies that affect the proper functions and biology of the knee. Over the years, different techniques were tried to repair meniscus injury and, when the reparation was not possible, to replace or regenerate it. These techniques still present a lack of controlled and independent clinical studies that do not allow identifying their effective failure rate. This rate could be due to a still incomplete knowledge about meniscal biology, its composition, and biomechanical properties. The purpose of this study was to analyze the relationship between the contact forces and the meniscal structures at the level of the femoral and tibial surfaces of the meniscus to improve the knowledge about this tissue, in view of the possible application in tissue engineering, for the production of meniscal scaffolds. Swine meniscal samples were studied for morphological (Safranin-O, Sirius Red, and collagen type I and II), biochemical (DNA, glycosaminoglycans [GAGs] and GAGs/DNA ratio), computed tomography scanning and biomechanical analyses (compression and traction tests) of femoral and tibial meniscal surfaces. Results revealed a biomechanical-dependent characterization of the meniscus. The femoral surface is characterized by a higher quantity of GAGs and a greater amount of cells (p < 0.01 for each analysis), with the interposition of radial and oblique fibers. These features are responsible of a higher resistance (p < 0.05) to compressive forces like that acted on by the femoral condyles. On the contrary, the tibial surface shows a circumferential arrangement of the fibers and a poorer GAGs presence and cellular spread (p < 0.01); these characteristics seem to allow a higher resistance (p < 0.05) of the tibial surface to traction forces. Results from this work provide useful information for the design and creation of meniscal substitute and suggest that the features of the meniscus are biomechanical-dependent, and that its composition and structure are dependent to the different forces that femur and tibia generate upon its surfaces.

Impact Statement

The importance of the present study is linked to how the contact forces act on the knee meniscus in particular, considering the femoral condyles and tibial plateau: this can be useful as a base for the ultimate creation of tissue-engineered biphasic scaffolds, which can mimic the native tissue complex, for meniscal repair or regeneration.

Introduction

The knee joint comprises the meniscus, which is a structure composed of a medial and a lateral component and both located between the corresponding femoral condyle and the tibial plateau.¹ It is a white and glossy complex tissue composed of cells specialized to produce extracellular matrix (ECM). Menisci are characterized by specific regional innervation and vascularization.² Both are critical components of a healthy knee joint.³

Moreover, meniscal tissue comprises a large network of collagen fibrils, which embed the ECM as well as water. Collagen fibrils networks are indirectly involved in the joint biomechanics by strengthening the tissue fluid pressure under load and directly by resisting tensile forces.⁴ The collagen fibrils network (considering composition, structure, and arrangement) spatially vary within the meniscus along the depth, because collagen is the meniscal element that can contribute to the main function of this tissue itself, that is, loading transmission.⁵

The meniscus bears many different forces such as traction and compression. It also plays a crucial role in the transmission of load, shock absorption, lubrication, and nutrition of articular cartilage.^6,7 All these functions are very complex and require a specialization of the meniscus itself. Since the meniscus structure is wedge-shaped, it is very efficient in stabilizing the round-shaped femoral condyle as well as the flat tibial plate.⁸

The contact forces acting on the meniscus inside the knee joint have been extensively studied. It was calculated that the whole and undamaged meniscus occupies ∼60% of the contact area between the articular cartilage of the femoral condyles and the tibial plate, while it transmits >50% of the total axial load applied to the joint.⁹

Menisci are subjected to different pathologies (principally tears and degeneration) that can affect their fundamental functions for the knee joints. It is demonstrated that pathologies of meniscus lead to gonarthrosis, with higher impact on human welfare.^10–12 For these reasons, different techniques were developed during this time to try to treat meniscal injuries.¹³

The first technique adopted was the meniscectomy,¹¹ this technique contemplated the removal, initially, of the whole meniscus, then partially renovated, trying to diminish, only the place damaged, the entity of meniscal tissue removed.

However, a lack of meniscal tissue was associated with the development of arthrosis and even a partial meniscectomy (i.e., currently the principal method to treat this pathologies) lead, with different grades, to the same result.¹¹ Then, removal was abandoned, when not necessary, in favor of reparation: different techniques of reparation were developed to treat meniscal injuries,¹⁴ but these kinds of techniques were seen to be functional only in the cases when the lesions affected the peripheral region of meniscus (i.e., the most vascularized^2,15 and inclined to regeneration¹⁴).

For these reasons, in the past years, the request of new techniques, which lead to the ultimate replacement or regeneration of the meniscus, increased. The focus of these techniques is to restore the knee biomechanics, distribute the load across a larger contact area (compared to meniscectomy), and potentially delay the onset of osteoarthritis.¹⁰

In the last years, different techniques were developed in this direction, with scaffolds replacements and allograft transplantations,^10–12,14 but due to their relatively new creations, these techniques still present a lack of controlled and independent long-term clinical studies that do not allow identifying their effective outcomes.^10,11,13

Nevertheless, a certain failure rate is present in any case.¹⁰ Probably, this can be ascribed to a still incomplete knowledge about meniscal biology, its composition, and biomechanical properties.

The aim of the present study was to deepen the relationship between the contact forces and the knee structures, in particular to search for the differences between the femoral condyles and tibial plateau surfaces of menisci, as a base for the ultimate creation of tissue-engineered biphasic scaffolds, which can mimic the native tissue complex, for meniscal repair or regeneration.

Materials and Methods

Study design

Seen the similarity with human meniscus^16,17 and the huge utilization as model for meniscal tissue engineering¹⁷ and repair,^17,18 pig's menisci were chosen as samples for this study. The knee joints of 26 adult (∼9 months old) female pigs (Landrace × Large white, average weight 75–90 kg; n = 104 meniscal samples) were obtained from a local slaughterhouse and dissected to isolate the tibia and remove the menisci. Capsular tissue and ligaments were removed, and the menisci were sectioned according to the purpose of the study as described below (Fig. 1). Only joints presenting as healthy at dissection were included in the study. Furthermore, giving the similarities between medial and lateral menisci observed by previous microanatomical analysis,^18,19 medial and lateral meniscal samples were pooled for the biochemical and biomechanical analyses. Moreover, limbs from six pigs were used for computed tomography (CT) scanning as described below. The Ethics Committee of the University of Milan (OPBA, 58/2016) approved the use of cadavers for research purpose; furthermore, all the animals were dead for causes not related with the present study.

FIG. 1.

Study design. The preparation of the meniscal samples in base of the type of technique performed is shown on the top. The table, on the bottom, resumes the number of each sample utilized for each technique and the type of cuts performed. AH, anterior horn; BO, body; PH, posterior horn. Original picture by U.P.

Morphological analyses: histology, and immunohistochemistry

Both the medial and lateral menisci (n = 28) were transversally sectioned into the three parts, corresponding to the anterior horn, central body, and posterior horn (Fig. 1). Menisci were sampled and fixed in buffered 10% formalin (Bio-Optica, Milan, Italy) for 24 h, dehydrated, and embedded in paraffin (total number of specimen = 84). Microtome sections (4-μm thickness) were analyzed with Safranin-O staining for describing both the menisci structure and the glycosaminoglycans (GAGs) in the matrix as follows.^20,21 Other sections were used for Sirius Red staining²⁰ and upon the stained samples; the polarized light microscopy was performed to highlight the birefringence of the collagen fibers to assess the spatial orientation of the fibers networks.

Finally, sections (Fig. 1) were used to detect Collagen type I and II as described elsewhere.^15,19,21 In brief, dewaxed and rehydrated sections were treated with 5% hydrogen peroxide in phosphate-buffered saline (PBS; pH 7.4) for 10 min, to inhibit nonspecific reactivity. Subsequently, sections were incubated with 1:50 rabbit polyclonal anti-collagen type I (cod. NB600-408; Novus Biologicals, Littleton, CO) or 1:100 mouse monoclonal anti-collagen type II (cod. 7005; Chondrex, Redmond, WA) for 24 h at 18–20°C in humid chamber, then washed in PBS, and subsequently treated with the rabbit or mouse EnVision system, respectively, for 120 min at room temperature (DakoCytomation, Milan, Italy). Peroxidase activity was detected with diaminobenzidine (DakoCytomation) as the substrate. Appropriate washing with PBS was performed between each step, and all incubations were carried out in a moist chamber. All sections were finally weakly counterstained with Mayer's hematoxylin, dehydrated, and permanently mounted.

The specificity tests for the antibodies were verified by incubating sections with (1) PBS instead of the specific primary antibody and (2) PBS instead of the secondary antibodies. The results of these controls were negative (i.e., staining was abolished). The samples were analyzed with an Olympus BX51 light microscope (Olympus; OperaZerbo, Milan, Italy) equipped with a digital camera, and final magnifications were calculated.

Biochemical analyses

Sectioned menisci (Fig. 1, left) of all animals (n = 20) were treated as described elsewhere.^15,21,22 In brief, the samples were digested in papain (Sigma-Aldrich, Milan, Italy) for 16–24 h at 60°C: 125 mg/mL of papain in 100 mM sodium phosphate, 10 mM sodium ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich), 10 mM cysteine hydrochloride (Sigma-Aldrich), and 5 mM EDTA adjusted to pH 6.5 and brought to 100 mL of solution with distilled water. Later, the digested samples were assayed separately for proteoglycan and DNA contents. Proteoglycan content was estimated by quantifying the amount of sulfated GAGs using the 1,9-dimethylmethylene blue dye-binding assay (Polysciences, Inc., Warrington, PA) and a microplate reader (wavelength: 540 nm). The standard curve for the analysis was generated by using bovine trachea chondroitin sulfate A (Sigma-Aldrich). DNA content was evaluated with the Quant-iT PicoGreen dsDNA Assay Kit (Molecular Probes; Invitrogen, Carlsbad, CA) and a fluorescence microplate reader and standard fluorescein wavelengths (excitation 485 nm, emission 538 nm, cutoff 530 nm). The standard curve for the analysis was generated using the bacteriophage lambda DNA supplied with the kit.

CT examination

Six hind limbs harvested from pig cadaver were evaluated. Medio-lateral and caudo-cranial radiographs of each stifle were performed to exclude abnormal findings. Each limb was positioned foot-first toward the gantry mimicking dorsal recumbence, with the caudal surface of the limb apposed to the CT couch. Images were acquired with a 16-slices CT scanner (Ge Brightspeed^®; GE Healthcare, Italy), using a bone algorithm. Scanning parameters were set as follows: kV = 120, mA = 230, slice thickness = 0.625 mm, and pitch = 0.5625. Each limb was initially positioned in a neutral position. Transverse prearthrography CT images were acquired from 2 cm proximal to the patella to 2 cm distal to the tibial tuberosity. Twenty-five milliliters of iomeprol 150 mg/mL (Iomeron 150; Bracco, Italy) was injected through a 20-gauge hypodermic needle inserted into the stifle joint medial to the midpoint of the patellar tendon (adaptation of the paraligamentous technique described in the ovine stifles²³). The joint was repeatedly flexed and extended to ensure adequate distribution of contrast medium. The limb was then repositioned on the CT couch, and the CT acquisition protocol was repeated. After the acquisition in neutral position, further arthrographic images were acquired with the limb in flexion and in extension. Each limb was flexed and extended as much as manually achievable and secured to the CT couch with large Velcro bands (extended: 140 ± 2°; neutral: 116 ± 2°; and flexed: 54 ± 4°). The same imaging protocol was then repeated. Images were reviewed by a single radiologist to verify meniscal movements, the contact between femoral/tibial condyles and meniscal surfaces, and meniscal compression with different positioning, focusing on the lateral meniscus. Meniscal thickness was measured at the level of the anterior and posterior horn on a multiplanar reformation sagittal image passing through the midpoint of the femoral condyle, at a distance of 1 mm from its cranial and caudal surface, respectively (Supplementary Fig. S1A, red and pink lines; Supplementary Data are available online at www.liebertpub.com/tea). Body portion thickness was evaluated in the same point as the measure of the midpoint of the meniscus (Supplementary Fig. S1A, yellow lines). On the same image, the lengths of the contact surface between the femoral surface of the meniscus and the femoral condyle and between the tibial surface of the meniscus and the tibial condyle were also measured (Supplementary Fig. S1B, dark and light blue lines). Finally, the total length of the meniscus was evaluated (Supplementary Fig. S1C, green line). All measures were repeated three times for each positioning (extension, neutral, and flexion).

Biomechanical testing

Menisci from adult swine (n = 50) were stored in saline solution NaCl 0.9% and frozen at −80°C until the time of testing. At least 24 h before test execution, samples were taken to a temperature of −24°C and then completely thawed at room temperature (23°C). Each meniscus was prepared and cut along the longitudinal axis, in its half height, to obtain a meniscal femoral surface and a meniscal tibial surface, too (Fig. 1). Compression and circumferential traction tests were performed using EnduraTEC ELF^® 3200 machine, equipped with a 220 or 22N load cell depending on the test and sample.

For compression tests, 25 menisci were used. Specimens were obtained dividing each meniscal surface in the three portions after the longitudinal cut (Fig. 1): anterior horn, body, and posterior horn (n = 75). Subsequently, for each zone, a cylindrical sample perpendicular to the femoral and tibial surfaces was cut using a die cutter (Fig. 1, in the middle). Before testing, dimensional measurements on the specimens were made with a digital caliper (Mitutoyo Corp, Kanogawa, Japan; number of series 06,081,911, accuracy class 1). The samples were inserted into a Plexiglas cell, and PBS solution was added into the cell to avoid dehydration of the specimen during the test. The samples thickness was measured from the position of the testing machine actuator, after imposing a preload of 0.1N. The sample was then subjected to a multiramp stress relaxation test, made of five increasing 4% strains at a velocity of 0.1%/s, followed by stress relaxation to equilibrium for 600 s. The compressive modulus, Ec, was obtained for each ramp from the equilibrium data as the ratio between values of relaxation stress and the corresponding values of strain.

For circumferential tension test (n = 25 menisci), we obtained representative fragments of the meniscus anterior horn, body, and posterior horn on both femoral and tibial surfaces (n = 75). These fragments were cut out using a scalpel, trying to obtain samples with a shape as similar as possible to a parallelepiped (Fig. 1, on the right) and following the circumferential tensile force direction. Before testing, dimensional measurements on the specimens were made with the same digital calipers previously indicated. Width and thickness of each sample were obtained using the calipers, length instead was measured on the mounted specimen, considering length as the distance between the two grips after a preload of 0.1N application. The specimens were subjected to a multiramp stress-relaxation test, made of four increasing 4% strains at a velocity of 0.1%/s, followed by stress relaxation to equilibrium for 1200 s. A custom made chamber filled with PBS was used to keep the samples hydrated during the test.

Statistical analyses

Statistical analyses of the biochemical (GAGs, DNA, and GAGs/DNA ratio), biomechanical results (compression and traction tests), and CT measures (horns' thickness, body thickness, meniscal length, and femoral and tibial contact surfaces) were analyzed with two-way analysis of variance with surfaces (femoral and tibial surfaces) and meniscal portions (anterior horn, body, and posterior horn) as main factors. The statistical analysis was performed using the general linear model of the SAS (version 8.1; Cary Inc., NC). The individual meniscal samples were considered to be the experimental unit of all response variables. The data are presented as least squared means ± standard errors of the mean. Differences between means were considered significant at p < 0.05.

Results and Discussion

Morphological analyses: histochemistry and immunohistochemistry

Considering the similarities of the medial and lateral meniscus already described in a previous study of our research group,¹⁹ only data regarding morphology of the lateral meniscus are presented. Safranin-O histochemistry assessed that the amount of matrix (proteoglycans) was more evident in the inner part both in anterior and posterior horns (Fig. 2A, B, respectively, asterisks), similarly to the results of previous works, that showed the same aspect in the inner part of ovine meniscus.²⁴

FIG. 2.

Histochemical findings of the lateral meniscus (Safranin-O staining). (A) Anterior horn, (A1) femoral surface, (A3) tibial surface. Body (B): (B1) femoral surface, (B3) tibial surface. Posterior horn (C): (C1) femoral surface, (C3) tibial surface. Histochemical findings of the meniscus (Sirius red staining—polarized light microscopy). Anterior horn: (A2) femoral surface, (A4) tibial surface. Body: (B2) femoral surface, (B4) tibial surface. Posterior horn: (C2) femoral surface, (C4) tibial surface. (A, B), Scale bar 5000 μm; A1–A4; B1–B4, Scale bar 200 μm. Black arrows: circumferential fibers, white arrowheads: radial fibers, black arrowheads: oblique fibers, and asterisk: matrix deposition.

Proteoglycans were also predominant within the anterior horn (Fig. 2A 1, A3, asterisks) when compared with the posterior one (Fig. 2B 1, B3, asterisks) and in the femoral meniscal surfaces (Fig. 2A 1, B1, asterisks) when compared with the corresponding tibial surfaces (Fig. 2A 3, B3, asterisks). Body portion seemed to have poorer in GAGs quantity, with a very pale staining in the middle of the tissue (Fig. 2C, asterisk).

FIG. 3.

Immunohistochemistry for collagen type I of the lateral meniscus: Anterior horn: (A1) femoral surface; (A2) tibial surface; (A3) inner part. Body: (C1) femoral surface; (C2) tibial surface; (C3) inner part. Posterior horn: (E1) femoral surface; (E2) tibial surface; (E3) inner part. Immunohistochemistry for collagen type II. Anterior horn: (B1) femoral surface; (B2) tibial surface; (B3) inner part. Body: (D1) femoral surface; (D2) tibial surface; (D3) inner part. Posterior horn: (F1) femoral surface; (F2) tibial surface; (F3) inner part. Arrows: fibers; asterisk: matrix deposition. All the figures have the same scale bar: 100 μm.

These results may suggest a role in the development of the ECM of dynamic compression forces, typically acting on the femoral surface of the meniscus. Moreover, these results suggest, accordingly with a previous work,¹⁹ an earlier development of the meniscal anterior horn with respect to the posterior one.

Sirius red-stained samples were evaluated to enhance the arrangement of collagen fibers in the central point of the meniscus.

The spatial disposition of the fibers varied, considering the different meniscal portions and regions. The femoral and tibial surfaces of anterior horn presented principally a radial pattern of the fibers in the superficial region, the one closer to the femoral condyle or to the tibial plateau (Fig. 2A2, A4, white arrowhead), as seen in Refs.^24–26 in ovine, human, and bovine, respectively.

The main differences observed in this study between femoral and tibial surfaces of the anterior horn were related to a higher incidence of oblique (Fig. 2A2, black arrowheads) and circumferential (Fig. 2A2, black arrows) fibers in the femoral surfaces respect to the tibial ones, while the opposite trend was observed for the radial fibers (Fig. 2A 4, white arrowheads). These kinds of fibers recall those observed in the study of Andrews et al.²⁷ and denominated braided organized type, present in the superficial layer and the woven organized type, present in the deeper layer.

FIG. 4.

Biochemical analysis results. DNA, GAGs, and GAGs/DNA ratios (A–C) are shown. DNA values (i.e., cellularity, A) and GAGs quantification (B) are higher among the femoral meniscal contact surface when compared with the tibial one. Values with different subscripts (a, b) for p < 0.05 and (A, B) for p < 0.01. GAGs, glycosaminoglycans.

In contrast, the same study (performed upon the anterior horn of bovine medial meniscus) showed no differences between three-dimensional fibers arrangement in the femoral and the tibial surfaces.²⁷

The posterior horn showed much different patterns between the femoral and tibial surfaces and between the deep and superficial regions of both surfaces. In the superficial femoral surface (the one closer to the femoral condyle), it was possible to recognize two layers of fibers disposed in a radial arrangement (the most superficial one; Fig. 2B2, white arrowhead) and a circumferential arrangement (deeper with respect to the previous; Fig. 2B2, black arrows), while in the deepest region of the femoral surface, there was a wider occurrence of radial (Fig. 2B2, white arrowhead), oblique (Fig. 2B2, black arrowheads), and circumferential fibers (Fig. 2B2, black arrow).

Moreover, the tibial surface of the posterior horn presented a wider radial disposition of the fibers in the superficial region (the ones closer to the tibial plateau; Fig. 2B 4, white arrowhead), while in the deepest region, there are fibers mostly arranged in a circumferential pattern (Fig. 2B 4, black arrow).

Generally, in the posterior horn, femoral and tibial surfaces showed a conventional fiber arrangement, as already described by other works, the differences seen in this article between the two surfaces differed from those described in Refs.^24,26 for ovine and bovine menisci, respectively, but could be grossly assimilate to those reported by AufderHeide and Athanasiou²⁵ as well as Petersen and Tillmann,²⁸ in human.

Body portion seemed to have an intermediate pattern of fibers, with a higher prevalence of circumferential fibers (Fig. 2B2, black arrows) in the femoral surface and a higher disposition in radial (Fig. 2B 4, white arrowhead) direction in the tibial one.

Immunohistochemistry showed a regional dependent matrix deposition: collagen I was present principally in the posterior horn (Fig. 3E1, E2, E3), in the femoral (Fig. 3E 1) and tibial (Fig. 3E 2) surfaces of the meniscus, and in the inner region (Fig. 3E3). In the anterior horn, collagen I was shown only by a nuclear staining (Fig. 3A 1, A2, A3, brown in the pictures).

Collagen II was present in the anterior horn (for the inner, femoral, and tibial parts; Fig. 3B 1, B2, B3) and in the posterior horn (almost only in the inner part; Fig. 3F3). These results agreed with previous work that showed an age-dependent localization of the collagen disposition in ovine and swine.^19,24 Body shows once again an intermediate pattern, with a poor, almost only nuclear, expression of collagen I (as seen in anterior horn; Fig. 3C 1, C2, C3) and a poor expression of collagen II (as seen in posterior horn; Fig. 3D 1, D2, D3), but a higher presence of collagen II in the inner part with respect to the latter (Fig. 3D3, asterisk).

Collagen I, much more abundant in immature meniscal tissue¹⁹ and tendons,²⁵ was mainly expressed by the posterior horn (Fig. 3E 1, E2, E3), as seen previously,¹⁹ developed later with respect to the anterior one. On the contrary, collagen II, much more abundant in mature meniscal tissue¹⁹ and cartilage,²⁵ was expressed in the anterior horn (Fig. 3: B1, B2, B3), which developed earlier in the meniscal tissue. Note that, only the inner and much compressed part of the posterior horn expressed collagen II in its matrix (Fig. 3F3). Once again, these results showed how biomechanical forces are essential for the right development of the meniscal structure.

Biochemical analysis

Biochemical analysis quantified the cellular spread and the GAGs amount through the meniscal tissue. Both DNA and GAGs contents were higher in the femoral surface (p < 0.05) when compared with the tibial one (Fig. 4A, B), while the GAGs/DNA ratio showed no significant differences, but a higher value for the femoral surface (Fig. 4C).

In particular, DNA content was higher in both anterior and posterior horns of the femoral surface when compared with the corresponding tibial surface portions (Fig. 4A; p < 0.01 and p < 0.05, respectively). Body portions of both femoral and tibial surfaces presented no statistical significant differences (Fig. 4A).

A similar trend was present in GAGs quantification that is characterized by a higher value in both anterior and posterior horns of the femoral side when compared with the corresponding tibial portions (Fig. 4B; p < 0.01). GAGs quantification showed also a poorer (p < 0.05) statistical significant difference between the femoral and tibial body portion, with a higher value present in the femoral surface (Fig. 4B).

GAGs were evaluated to analyze matrix content in relationship with the response to compressive forces^29,30 as observed by Bursac et al.²⁹ in human and by Abdelgaied et al. in pigs.³⁰

The GAGs/DNA ratio showed no significant differences among all portions (Fig. 4F). This may be due to the fact that adult meniscus had reached a complete development, and so, cells, matrix, and collagen network achieved an equilibrate connection throughout the entire tissue, differently from what could be seen in growing menisci.³⁰

Biochemical analysis reiterated, once again, the presence of two well-differentiated portions: a femoral (rich of cells and GAGs) and a tibial one (characterized by the greater relative amount of fibers). The obtained data were in agreement with previous description of the meniscus as a tissue having an inner proteoglycan rich matrix^25,31 that resembles hyaline cartilage and an external fibrous region, more similar to ligament and tendons.^25,32,33,34

CT scanning

The results of CT analysis (Fig. 5) provided a hint of meniscal behavior during knee's movement. During standing and movement, neutral positioning corresponded to full loading, while flexion corresponded to general stifle unloading.³⁵ The thickness of the meniscus in different positioning seemed to suggest that compressive forces on the anterior horn, body, and posterior horns (Fig. 5D) have no effects upon their macroscopic structure. The same happened for the total length of the meniscus (Fig. 5E), as their complex structure allows them to hold their shape under different stimuli. Flexion seemed to induce a noticeable reduction in the length of the contact surface among the meniscus and both the bone surfaces (for both, p < 0.01; Fig. 5A–C, F, G). However, the femoral contact surfaces seemed to be less affected by the extension (Fig. 5F; p < 0,05), and the tibial contact surface seemed to be not affected at all (not statistically significant respect to the neutral position; Fig. 5G). A large amount of contrast medium seemed to fill the space between the anterior horn of the meniscus and the femoral and tibial condyles (femur >tibia), suggesting a fan-like separation between the horn and the bone surfaces during flexion (Fig. 5C, red arrows). Moreover, caudal displacement of the meniscus was visible, which seemed to slip caudally along the tibial condyle (Fig. 5C, yellow arrow), as it was described in Refs.^7,36

FIG. 5.

CT images analysis. Representative images of meniscal changes with different positioning (lateral meniscus): (A) in extension, (B) neutral, (C) in flexion. CT images analysis shows no statistically significant differences both for body and horns' thickness (D) and meniscal length (E), while a reduction in bone/meniscus contact surfaces is evident in flexion (F, G). Light blue lines: femoral contact surface; pink lines: tibial contact surface. Contrast mean is noticed between the meniscus and the femoral and tibial surfaces (red arrows), suggesting a fan-like separation of the cranial part of the meniscus from the bones. Caudal displacement of the meniscus along the tibial condyle is also evident (emphasized by the yellow arrow). Values with different subscripts (a, b) for p < 0.05 and (A, B) for p < 0.01. Scale bar = 1 cm. CT, computed tomography.

The observed differences were related to the decreasing contact between the cranial horn of the meniscus and the two joint heads, while the contact of the caudal horn seemed to be less influenced by the joint movement (Fig. 5A–C).

Biomechanical tests

Results of biomechanical tests showed a higher compression elastic modulus (Ec) in the femoral surface with respect to the tibial one (Fig. 6A). In particular, anterior and posterior horns presented higher Ec moduli in the femoral surfaces respect to tibial one (Fig. 6A; p < 0.05), while no differences were recorded for the bodies.

FIG. 6.

Biomechanical results. Compression test results (A) and circumferential traction test results (B). Femoral meniscal contact surface shows a higher elastic modulus in compression (Ec; A), while tibial meniscal contact surface shows a higher elastic modulus in traction (Et; B). Values with different superscripts (a, b) for p < 0.05.

The influence of GAGs on tissue compressive force response has been previously demonstrated in Refs.^29,30 both in human and pigs.

In contrast with our results, Proctor et al.⁶ did not find any differences in unconfined elastic modulus in compression, moving from femoral superficial to deeper regions in bovine medial meniscus.³⁷ A higher Ec modulus in the anterior horn (at 12% strain), with respect to the posterior one, was reported by Chia and Hull,³⁸ in human meniscus. Unfortunately, both these studies did not consider the differences between femoral and tibial surfaces.

On the contrary, it was possible to observe opposite results for what concerned the traction elastic modulus (Et) with a higher value for the tibial surface respect to the femoral one (Fig. 6B). It is well known that the Et property is dependent on collagen.³⁹ In particular, all the three regions (anterior horns, posterior horns, and bodies) presented higher values of Et in the tibial surfaces with respect to the femoral ones (Fig. 6B; p < 0.05 for all comparisons). These results showed a different biomechanical behavior in the different regions and surfaces of the meniscus related to the type of forces that act upon them.

Tensile properties of the meniscus are related with the collagen fibers presence and disposition.^30,39 A higher tensile modulus value of the anterior horn was also reported by other studies⁴⁰ in porcine meniscus. However, in a study, performed on bovine meniscus, the highest value of tensile modulus was measured in the posterior horn.³⁹ Therefore, these differences may be due simply to interspecific features, but their importance has to be better evaluated in the future.

Biomechanical results draw a picture that characterized the femoral surfaces like more subjected to compressive forces (as previously suspected, based on the GAGs presence enhanced by the Safranin-O staining and GAGs biochemical quantification), while the tibial surfaces, in particular that of the anterior horn, like more subjected to traction forces. In fact, during physiological loading, tensile, compressive, and shear forces are generated. Consequently, vertical and horizontal forces result from the femur compressive action on the curved superior surface of the meniscus. From the horizontal force originated a radial reaction force: when the femur compresses the meniscus, it tends to deform radially, but because of its anchorages to the tibial plateau, it generates a tensile hoop stress resulting from the radial deformation.^42,43

The role of biomechanical forces is essential in the morphofunctional development of the synovial joints components, and in particular, for the meniscus, in fact, it was demonstrated that the immobilization of the embryo's hind limb causes the degeneration and the disappearance of this structure.⁴⁴

Conclusion

Morphological, biochemical, CT imaging, and biomechanical analysis highlight the presence of two well-differentiated meniscal components: a femoral (rich of cells and GAGs) and a tibial one (characterized by a greater amount of circumferential fibers). Their different compositions reflect their natural functions: femoral surface act as the springs of a car in response to the transmission of the compressive weight-bearing loads, consequently, the tibial surface acts to dissipate the compressive forces through the collagen fibers network with a hoop-stress mechanism, comparable to car dampers.

These functions are biomechanical-dependent and linked to the fibers organization: compressive forces, due to the femoral condyles compression, sliding, and rolling induce a higher deposition of ECM, as well as a circumferential arrangement of the fibers associated with the interposition of some radial and oblique fibers in the corresponding surface of the meniscus. Moreover, traction forces are highly active on the tibial surfaces of the meniscus, inducing a circumferential deposition of fibers, which are highly resistant to traction.

Results from this work, even if preliminary, provide useful information for the design and creation of meniscal substitute and suggest that the features of the meniscus are biomechanical-dependent and that its composition and structure are dependent on the different forces that femur and tibia generate upon its surfaces.

Footnotes

Acknowledgments

This paper was supported by the Italian Ministry of Health.

Disclosure Statement

No competing financial interests exist.

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