Quantitative three-dimensional collagen orientation analysis of human meniscus posterior horn in health and osteoarthritis using micro-computed tomography.

OBJECTIVE: Knee osteoarthritis (OA) is associated with meniscal degeneration that may involve disorganization of the meniscal collagen fiber network. Our aims were to quantitatively analyze the microstructural organization of human meniscus samples in 3D using micro-computed tomography (µCT), and to compare the local microstructural organization between OA and donor samples. METHOD: We collected posterior horns of both medial and lateral human menisci from 10 end-stage medial compartment knee OA patients undergoing total knee replacement (medial & lateral OA) and 10 deceased donors without knee OA (medial & lateral donor). Posterior horns were dissected and fixed in formalin, dehydrated in ascending ethanol concentrations, treated with hexamethyldisilazane (HMDS), and imaged with µCT. We performed local orientation analysis of collagenous microstructure in 3D by calculating structure tensors from greyscale gradients within selected integration window to determine the polar angle for each voxel. RESULTS: In donor samples, meniscus bundles were aligned circumferentially around the inner border of meniscus. In medial OA menisci, the organized structure of collagen network was lost, and main orientation was shifted away from the circumferential alignment. Quantitatively, medial OA menisci had the lowest mean orientation angle compared to all groups, -24° (95%CI -31 to -18) vs medial donor and -25° (95%CI -34 to -15) vs lateral OA. CONCLUSIONS: HMDS-based µCT imaging enabled quantitative analysis of meniscal collagen fiber bundles and their orientations in 3D. In human medial OA menisci, the collagen disorganization was profound with overall lower orientation angles, suggesting collagenous microstructure disorganization as an important part of meniscus degradation.

Molecular and macromolecular diffusion in human meniscus: relationships with tissue structure and composition.

OBJECTIVE: To date, the pathophysiology of the meniscus has not been fully elucidated. Due to the tissue's limited vascularization, nutrients and other molecular signals spread through the extracellular matrix via diffusion or convection (interstitial fluid flow). Understanding transport mechanisms is crucial to elucidating meniscal pathophysiology, and to designing treatments for repair and restoration of the tissue. Similar to other fibrocartilaginous structures, meniscal morphology and composition may affect its diffusive properties. The objective of this study was to investigate the role of solute size, and tissue structure and composition on molecular diffusion in meniscus tissue. DESIGN: Using a custom FRAP technique developed in our lab, we measured the direction-dependent diffusivity in human meniscus of six different molecular probes of size ranging from ∼300Da to 150,000Da. Diffusivity measurements were related to sample water content. SEM images were used to investigate collagen structure in relation to transport mechanisms. RESULTS: Diffusivity was anisotropic, being significantly faster in the direction parallel to collagen fibers when compared the orthogonal direction. This was likely due to the unique structural organization of the tissue presenting pores aligned with the fibers, as observed in SEM images. Diffusion coefficients decreased as the molecular size increased, following the Ogston model. No significant correlations were found among diffusion coefficients and water content of the tissue. CONCLUSIONS: This study provides new knowledge on the mechanisms of molecular transport in meniscal tissue. The reported results can be leveraged to further investigate tissue pathophysiology and to design treatments for tissue restoration or replacement.

Tie-fibre structure and organization in the knee menisci.

The collagenous structure of the knee menisci is integral to the mechanical integrity of the tissue and the knee joint. The tie-fibre structure of the tissue has largely been neglected, despite previous studies demonstrating its correlation with radial stiffness. This study has evaluated the structure of the tie-fibres of bovine menisci using 2D and 3D microscopy techniques. Standard collagen and proteoglycan (PG) staining and 2D light microscopy techniques were conducted. For the first time, the collagenous structure of the menisci was evaluated using 3D, second harmonic generation (SHG) microscopy. This technique facilitated the imaging of collagen structure in thick sections (50-100 µm). Imaging identified that tie-fibres of the menisci arborize from the outer margin of the meniscus toward the inner tip. This arborization is associated with the structural arrangement of the circumferential fibres. SHG microscopy has definitively demonstrated the 3D organization of tie-fibres in both sheets and bundles. The hierarchy of the structure is related to the organization of circumferential fascicles. Large tie-fibre sheets bifurcate into smaller sheets to surround circumferential fascicles of decreasing size. The tie-fibres emanate from the lamellar layer that appears to surround the entire meniscus. At the tibial and femoral surfaces these tie-fibre sheets branch perpendicularly into the meniscal body. The relationship between tie-fibres and blood vessels in the menisci was also observed in this study. Tie-fibre sheets surround the blood vessels and an associated PG-rich region. This subunit of the menisci has not previously been described. The size of tie-fibre sheets surrounding the vessels appeared to be associated with the size of blood vessel. These structural findings have implications in understanding the mechanics of the menisci. Further, refinement of the complex structure of the tie-fibres is important in understanding the consequences of injury and disease in the menisci. The framework of meniscus architecture also defines benchmarks for the development of tissue-engineered replacements in the future.

An evaluation of meniscal collagenous structure using optical projection tomography.

BACKGROUND: The collagenous structure of menisci is a complex network of circumferentially oriented fascicles and interwoven radially oriented tie-fibres. To date, examination of this micro- architecture has been limited to two-dimensional imaging techniques. The purpose of this study was to evaluate the ability of the three-dimensional imaging technique; optical projection tomography (OPT), to visualize the collagenous structure of the meniscus. If successful, this technique would be the first to visualize the macroscopic orientation of collagen fascicles in 3-D in the meniscus and could further refine load bearing mechanisms in the tissue. OPT is an imaging technique capable of imaging samples on the meso-scale (1-10 mm) at a micro-scale resolution. The technique, similar to computed tomography, takes two-dimensional images of objects from incremental angles around the object and reconstructs them using a back projection algorithm to determine three-dimensional structure. METHODS: Bovine meniscal samples were imaged from four locations (outer main body, femoral surface, tibial surface and inner main body) to determine the variation in collagen orientation throughout the tissue. Bovine stifles (n = 2) were obtained from a local abattoir and the menisci carefully dissected. Menisci were fixed in methanol and subsequently cut using a custom cutting jig (n = 4 samples per meniscus). Samples were then mounted in agarose, dehydrated in methanol and subsequently cleared using benzyl alcohol benzyl benzoate (BABB) and imaged using OPT. RESULTS: Results indicate circumferential, radial and oblique collagenous orientations at the contact surfaces and in the inner third of the main body of the meniscus. Imaging identified fascicles ranging from 80-420 µm in diameter. Transition zones where fascicles were found to have a woven or braided appearance were also identified. The outer-third of the main body was composed of fascicles oriented predominantly in the circumferential direction. Blood vessels were also visualized using this technique, as their elastin content fluoresces more brightly than collagen at the 425 nm wavelength used by the OPT scanner. CONCLUSIONS: OPT was capable of imaging the collagenous structure, as well as blood vessels in the bovine meniscus. Collagenous structure variability, including transition zones between structural regions not previously described in the meniscus, was identified using this novel technique.

Cartilage intermediate layer protein 2 (CILP-2) is expressed in articular and meniscal cartilage and down-regulated in experimental osteoarthritis.

Using transcriptome profiling to determine differential gene expression between the permanent mouse articular cartilage and the transient growth plate cartilage, we identified a highly expressed gene, Cilp2, which is expressed differentially by articular chondrocytes. CILP-2 is highly homologous to CILP-1 (cartilage intermediate layer protein 1), which is expressed in the intermediate zone of articular cartilage and has been linked to cartilage degenerative diseases. We demonstrated that Cilp2 has a restricted mRNA distribution at the surface of the mouse articular cartilage during development, becoming localized to the intermediate zone of articular cartilage and meniscal cartilage with maturity. Although the extracellular CILP-2 protein localization is broadly similar to CILP-1, CILP-2 appears to be more localized in the deeper intermediate zone of the articular cartilage extracellular matrix at maturity. CILP-2 was shown to be proteolytically processed, N-glycosylated, and present in human articular cartilage. In surgically induced osteoarthritis in mice, Cilp1 and Cilp2 gene expression was dysregulated. However, whereas Cilp1 expression was increased, Cilp2 gene expression was down-regulated demonstrating a differential response to mechanically induced joint destabilization. CILP-2 protein was reduced in the mouse osteoarthritic cartilage. Ultrastructural analysis also suggested that CILP-2 may be associated with collagen VI microfibrils and thus may mediate interactions between matrix components in the territorial and inter-territorial articular cartilage matrix. mRNA expression analysis indicated that whereas Cilp1 and Cilp2 are expressed most abundantly in cartilaginous tissues, expression can be detected in muscle and heart.

Cryopreservation increases apoptosis in human menisci.

PURPOSE: Removal of the meniscus leads to progressive degenerative arthritis of the knee on a long-term basis; therefore, meniscal allograft transplantation has been proposed as an alternative to meniscectomy. Preservation methods are required to build up operational stocks and to provide living grafts of a practical size at the right time for patients. Methods for meniscus preservation have been published, and relevant literature confirms that using standard cryopreservation, the chondrocyte survival in situ is inadequate and extremely variable and the cryoinjury mechanisms are not completely established. The aim of the present study is to further investigate possible cellular injury caused by cryopreservation by analysing apoptosis and ultrastructural damage to menisci. METHODS: Seven human menisci that were cryopreserved by standard method were used. All tissue samples were processed simultaneously for routine light microscopy, scanning and transmission electron microscopy as well as apoptosis assessment by the use of ISOL method. RESULTS: With respect to cellularity, significant differences (P < 0.05) between the fresh (14.6 ± 3.5) (mean ± SD) and cryopreserved menisci (9.2 ± 2.8) (mean ± SD) were observed. Apoptosis using ISOL method was observed in fibrochondrocytes of fresh and cryopreserved menisci. The quantitative analysis revealed significant differences (P < 0.05) between fresh meniscus samples, where the apoptotic index was 0.8 ± 2.3% (mean ± SD), and cryopreserved meniscus samples, where this index was 50 ± 18.1% (mean ± SD). CONCLUSION: The results suggest that apoptosis occurs during meniscus cryopreservation. The major findings of this study are cellular damage in meniscus cryopreservation suggesting apoptosis-mediated cell loss. The findings reported herein encourage to further investigations in preservation procedures to enhance maximum long-term clinical survival.

Host serum is not indispensable in collagen performance in viable meniscal transplantation at 4-week incubation.

PURPOSE: Viable meniscal transplantation has been criticized as an expensive and logistically demanding technique. The purpose was to compare the standard culture medium with another culture medium that is more widely available and easier to work with and to assess the collagen net ultrastructure architecture and the capacity of the preserved cells to produce proteins. METHODS: Ten fresh lateral menisci were harvested. Each meniscus was divided into three parts; control group, fetal-bovinum-serum group and Insulin-Transferrin-Selenium group during 4 weeks. Cell metabolism was assessed with the gene expression of type I collagen, type II collagen and aggrecan. Collagen ultrastructure was assessed with transmission electron microscopy. The Collagen Meniscal Architecture scoring system was used to evaluate the degree of meniscal disarray. RESULTS: Type I collagen was expressed more in the fetal-bovinum-serum group than in the ITS group (P = 0.036). No differences were found between cultured samples and control groups. Type II collagen showed decreased expression in both cultured groups compared with the control group. No differences were observed in the gene expression of aggrecan in either group. No differences were observed when the Collagen Meniscal Architecture scoring system was applied. CONCLUSIONS: Insulin-Transferrin-Selenium-supplemented medium is at least as effective as the fetal-bovinum-serum-supplemented medium to preserve the net architecture of the meniscal tissue. Gene expression of the studied proteins was similar in the Insulin-Transferrin-Selenium group to that observed in the control group at 4 weeks. Insulin-Transferrin-Selenium might be a better alternative and might be used instead of fetal-bovinum-serum or an autologous host serum in order to preserve meniscal tissue, which precludes the necessity of obtaining host serum previously. Thus, viable meniscal transplantation would logistically be less complicated to perform.

A new technique for seeding chondrocytes onto solvent-preserved human meniscus using the chemokinetic effect of recombinant human bone morphogenetic protein-2.

Many investigators are currently studying the use of decellularized tissue allografts from human cadavers as scaffolds onto which patients' cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. However, it is difficult to seed cells onto very dense regular connective tissue which has few interstitial spaces. Here, we discuss the development of a chemotactic cell seeding technique using solvent-preserved human meniscus. A chemokinetic response to recombinant human bone morphogenetic protein-2 (rhBMP-2) was observed in a monolayer culture of primary chondrocytes derived from femoral epiphyseal cartilage of 2-day-old rats. The rhBMP-2 significantly increased their migration upto 10 ng/ml in a dose-dependent manner. When tested with solvent-preserved human meniscus as a scaffold, which has few interstitial spaces, rhBMP-2 was able to induce chondrocytes to migrate into the meniscus. After a 3-week incubation, newly-formed cartilaginous extracellular matrix was synthesized by migrated chondrocytes throughout the meniscus, down to a depth of 3 mm. These findings demonstrate that rhBMP-2 may be a natural chemokinetic factor in vivo, which induces migration of proliferative chondrocytes into the narrow interfibrous spaces. Our results suggest a potential application of rhBMP-2 for the designed distribution of chondrocytes into a scaffold to be used for tissue engineering.

Development of rabbit meniscus acellular matrix.

OBJECTIVE: To prepare a rabbit meniscus acellular matrix scaffold and explore the histomorphological and biomechanical properties of the scaffold. METHODS: Rabbit meniscuses were collected and acellularized using a modified eight-step detergent process with hydrogen peroxide, distilled water, Triton X-100, and sodium deoxycholate. Its color and texture were observed. Histomorphological assessment was performed using routine hematoxylin-eosin stain, toluidine blue stain, Saffron stain, Hoechst-33258 stain, and immunohistochemical staining of collagen I. The ultrastructure of the specimens was observed with inverted phase contrast microscopy. Transient recovery rate of deformation, maximal recovery rate of deformation, and maximal compressive strength were tested to determine the biomechanical properties of the scaffold. RESULTS: The processed meniscus was milk-white in color with loose structure. It histologically appeared cell-free, stained positively for collagen I, and had abundant micropores according to phase-contrast microscopy. The transient recovery rate of deformation was (76.65∓4.61)%, the maximal recovery rate of deformation was 100%, and the maximal compressive strength was (4.51∓0.69) N when the specimens were compressed 40%. CONCLUSIONS: The rabbit meniscus acellular matrix scaffold, with numerous micropores, is easy to be recovered from deformation and suitable for the adhesiveness and growth of breeding cells. This scaffold can be used as an ideal implant for future tissue engineering of the meniscus.

Changes of human menisci in osteoarthritic knee joints.

OBJECTIVE: To investigate the changes of knee menisci in osteoarthritis (OA) in human. METHODS: OA and control menisci were obtained from 42 end-stage OA knees with medial involvement and 28 non-arthritic knees of age-matched donors, respectively. The change of menisci in OA was evaluated by histology, and gene expression of major matrix components and anabolic factors was analyzed in the anterior horn segments by quantitative PCR (qPCR). In those regions of menisci, the rate of collagen neo-synthesis was evaluated by [(3)H]proline incorporation, and the change of matrix was investigated by ultrastructural observation and biomechanical measurement. RESULTS: In OA menisci, the change in histology was rather moderate in the anterior horn segments. However, despite the modest change in histology, the expression of type I, II, III procollagens was dramatically increased in those regions. The expression of insulin-like growth factor 1 (IGF-1) was markedly enhanced in OA menisci, which was considered to be responsible, at least partly, for the increase in procollagen gene expression. Interestingly, in spite of marked increase in procollagen gene expression, incorporation of [(3)H]proline increased only modestly in OA menisci, and impaired collagen synthesis was suggested. This finding was consistent with the results of ultrastructural observation and biomechanical measurement, which indicated that the change of meniscal matrix was modest in the macroscopically preserved areas of OA menisci. CONCLUSION: Although the expression of major matrix components was markedly enhanced, matrix synthesis was enhanced only modestly, and the changes of matrix in human OA menisci were rather modest in the non-degenerated areas.

Collagen morphology in human meniscal attachments: a SEM study.

Qualitative analysis of meniscal attachments from five human knees was completed using scanning electron microscopy (SEM). In addition, quantitative analysis to determine the collagen crimping angle and length in each attachment was done. Morphological differences were revealed between the distinct zones of the attachments from the meniscus transition to the bony insertion. Collagen fibers near to the meniscus appeared inhomogeneous in a radial cross-section view. The sheath surrounding the fibers seemed loose compared with the membrane wrapping around the fibers in the menisci. The midsubstance of human meniscal attachments was composed of collagen fibers running parallel to the longitudinal axis, with a few fibers running obliquely, and others transversely. The bony insertion showed that the crimping pattern vanishes as the collagen fibers approach the fibrocartilagenous enthesis. There were no differences between attachments for crimping angle or length. Collagen crimping angles for all attachments were similar with values of approximately 22°. Crimp length values tended to be smaller for the medial attachments (MA: 4.76 ± 1.95 µm; MP: 3.72 ± 2.31 µm) and higher for the lateral (LA: 6.49 ± 2.34 µm, LP: 6.91 ± 2.29 µm). SEM was demonstrated to be an effective method for revealing the morphology of fibrous connective tissue. The data of collagen fiber length and angle found in this study will allow for better development of microstructural models of meniscal attachments. This study will help to better understand the relation between the morphology and the architecture of collagen and the mechanical behavior of meniscal attachments.

An acellular, allograft-derived meniscus scaffold in an ovine model.

PURPOSE: The purpose of this study was to develop a meniscus scaffold that has increased porosity and maintains the native meniscus extracellular matrix in an ovine model. METHODS: The medial menisci of skeletally mature ovine (n = 16) were harvested; half were made into meniscus scaffolds (n = 8), and half remained intact (n = 8). Intact and scaffold meniscus tissues were compared by use of histology, DNA content analysis, in vitro cellular biocompatibility assays, and ultrastructural analysis. An additional 16 knees were used to investigate the biomechanics of the intact meniscus compared with the meniscus scaffold. RESULTS: DNA content and histology showed a significant decrease in cellular and nuclear content in the meniscus scaffold (P < .003). Biocompatibility was supported through in vitro cellular assays. Scanning electron microscopy and micro-computed tomography showed a substantial increase in porosity and pore connectivity in the meniscus scaffold compared with the intact meniscus (P < .01). There was no statistical difference between the ultimate load or elastic modulus of the intact and meniscus scaffolds. CONCLUSIONS: In this study a meniscus scaffold was evaluated for potential clinical application as a meniscus transplant construct in an ovine model. The data showed that a decellularized meniscus scaffold with increased porosity was comparable to the intact meniscus, with an absence of in vitro cellular toxicity. Although some compositional alterations of the extracellular matrix are to be expected during processing, it is evident that many of the essential structural components remained functional with maintenance of biomechanical properties. CLINICAL RELEVANCE: This meniscus scaffold has potential for future clinical application as a meniscus transplant construct.

Cryopreservation does not alter the ultrastructure of the meniscus.

Fresh frozen menisci have recently been shown to have an altered meniscal ultrastructure. The cause might be a deterioration of its permeability due to collagen net disarray. The purpose of this study was to evaluate the cryopreserved meniscus in terms of ultrastructure and cellularity. Ten fresh human lateral menisci were harvested. Collagen architecture was evaluated with transmission electron microscopy. The Collagen Meniscal Architecture scoring system was used to assess the degree of meniscal disarray. Cell population, was also evaluated. The fibril collagen diameters of those menisci which had been previously cryopreserved showed an average size in the longitudinal section of 12.6 +/- 1.3 nm, whereas it was 13.4 +/- 2.2 nm in the menisci used as controls (n.s.). In the transverse section, the cryopreserved menisci averaged 15.5 +/- 2.4 and 16.7 +/- 3.5 nm in the controls (n.s.). The study group scored 4.8 points +/- 1.7, whereas the control group did so at 4.1 +/- 1.3 (n.s.). The percentage of cell survival after the cryopreservation ranged from 4 to 54. The fibril diameters and degree of disarray showed a similar distribution in both groups. The results suggest that meniscal cryopreservation does not alter the meniscal ultrastructure. Therefore, an allograft stored in that way would not alter its biomechanical properties, although its cellular viability is highly unpredictable.

Healing of meniscal tissue by cellular fibrin glue: an in vivo study.

Menisci represent fundamental structures for the maintenance of knee homeostasis, playing a key role in knee biomechanics. However, their intrinsic regenerative potential is poor. As a consequence, when a lesion occurs and the meniscus is partially removed by surgery, knee mechanics is subject to dramatic changes. These have been demonstrated to lead often to the development of early osteoarthritis. Therefore, menisci should be repaired whenever possible. In the last decades, tissue engineering approaches have been advocated to improve the reparative processes of joint tissues. In this study, the bonding capacity of an articular chondrocytes-fibrin glue hydrogel was tested as a biologic glue to improve the bonding between two swine meniscal slices in a nude mouse model. The composites were wrapped with acellular fibrin glue and implanted in subcutaneous pouches of nude mice for 4 weeks. Upon retrieval, a firm gross bonding was observed in the experimental samples while none of the control samples, prepared with acellular fibrin glue at the interface, presented any sign of bonding. This was consistent with the histological and scanning electron microscope findings. In particular, a fibrocartilaginous tissue was found at the interface between the meniscal slices, partially penetrating the native meniscus tissue. In order to overcome the lack of regenerative properties of the meniscus, the rationale of using cellular fibrin glue is that fibrin provides immediate stability while carrying cells in the site of lesion. Moreover, fibrin gel is recognized as an optimal scaffold for cell embedding and for promoting fibrocartilaginous differentiation of the cells which synthesize matrix having healing property. These results demonstrated the potential of this model for improving the meniscal bonding. However, further orthotopic studies in a large animal model are needed to evaluate its potential for clinical application.

Confocal arthroscopic assessment of osteoarthritis in situ.

PURPOSE: This study aimed to assess the ability of the laser scanning confocal arthroscope (LSCA) to evaluate cartilage microstructure, particularly in differentiating stages of human osteoarthritis (OA) as classified by the International Cartilage Repair Society (ICRS) OA grade definitions. METHODS: Ten tibial plateaus from total knee arthroplasty patients were obtained at the time of surgery. Cartilage areas were visually graded based on the ICRS classification, imaged by use of a 7-mm-diameter LSCA (488-nm excitation with 0.5% [wt/vol] fluorescein, 20-minute staining period), and then removed with underlying bone for histologic examination with H&E staining. The 2 imaging techniques were then compared for each ICRS grade to ascertain similarity between the methods and thus gauge the techniques' diagnostic resolution. Cartilage surface degeneration was readily imaged and OA severity accurately gauged by the LSCA and confirmed by histology. RESULTS: LSCA and histologic images of specimens in the late stages of OA were seen to be mutually related even though they were imaged in planes that were orthogonal to each other. Useful and comparable diagnostic resolution was obtained in all imaged specimens from subjects with various stages of OA. CONCLUSIONS: This study showed the LSCA's ability to image detailed cartilage surface morphologic features that identify grade 1 through 4 of the ICRS OA grading system. The LSCA's imaging potential was best shown by its ability to resolve the fine collagen network present under the lamina splendens. The incorporation of high-magnification confocal technology within the confines of an arthroscopic probe has proved to provide the imaging requirements necessary to perform detailed cartilage condition assessment. CLINICAL RELEVANCE: In comparison to video arthroscopy, LSCA provides increased magnification along with improved contrast and resolution.

Effect of 3 Preservation Methods (Freezing, Cryopreservation, and Freezing + Irradiation) on Human Menisci Ultrastructure: An Ex Vivo Comparative Study With Fresh Tissue as a Gold Standard.

BACKGROUND: Three main meniscus preservation methods have been advocated: freezing (-80°C), freezing with gamma irradiation (-80°C + 25 kGy), and cryopreservation (-140°C). HYPOTHESIS: All preservation methods will result in structural and architectural properties similar to those of fresh meniscus, defined as the gold standard. STUDY DESIGN: Controlled laboratory study. METHODS: Five human intact menisci were collected from 5 patients undergoing total knee arthroplasty. The inclusion criteria were patients <70 years old with primary unilateral (medial) femorotibial knee osteoarthritis and without surgical or traumatic history on the operated knee. Four cubes (9 mm3) were cut inside of the white, or avascular, area of each specimen's middle horn and divided into 4 groups: "fresh" control, frozen (-80°C), cryopreserved (-140°C), and frozen + irradiated (-80°C + 25 kGy). Specimens of the control group were evaluated at day 1, and specimens from the frozen, cryopreserved, and frozen + irradiated groups were evaluated after 1 month of storage. Evaluation was performed with electron microscopy according a validated protocol to analyze (1) mean diameters of the collagen fibers in longitudinal and transverse sections in 5 points per section and (2) validated architectural scores. RESULTS: No significant difference was found between the control and cryopreserved groups regarding mean transverse and longitudinal diameters (transverse: 95.39 ± 15.87 nm vs 99.62 ± 19.23 nm, P = .1; longitudinal: 96.31 ± 13.96 nm vs 94.57 ± 16.42 nm, P = .1). Significant differences were found between the control and frozen groups (transverse: 95.39 ± 15.87 nm vs 70.20 ± 13.94 nm, P < .001; longitudinal: 96.31 ± 13.96 nm vs 71.28 ± 10.64 nm, P < .001) and the control and frozen + irradiated groups (transverse: 95.39 ± 15.87 nm vs 63.1 ± 15.57 nm, P < .001; longitudinal: 96.31 ± 13.96 nm vs 60.9 ± 14.8 nm, P < .001). Regarding architectural score calculation, there were significant differences between the control and frozen groups (4.5 ± 1.3 vs 2.3 ± 1.4, P = .02) and the control and frozen + irradiated groups (4.5 ± 1.3 vs 1.4 ± 0.9, P = .02). CONCLUSION: Cryopreservation is the only method that preserves fresh meniscus architectural specificities. Freezing and freezing + irradiation methods modify histologic properties of meniscal allograft. Irradiation deeply alters diameters and the organization of collagen fibers, and this method should be used with caution to preserve and sterilize meniscus tissue. CLINICAL RELEVANCE: The results of our study exhibited detrimental effects of simple freezing and freezing + irradiation on the collagen network of sample meniscus. If those effects occur in menisci prepared for allograft procedures, important differences could appear on the basis of the preservation procedure in terms of the graft's mechanical properties and, thus, the patient's outcomes.

Discoid meniscus: an ultrastructural study with transmission electron microscopy.

BACKGROUND: The ultrastructure of the normal menisci has been thoroughly investigated and found to correlate with meniscal tears. Although discoid menisci are accepted to have the same composition as their normal counterparts, to our knowledge, no study in the literature has investigated this issue. PURPOSE: To investigate the ultrastructure of the discoid menisci and compare it with nondiscoid menisci. STUDY DESIGN: Descriptive laboratory study. METHODS: Meniscal biopsies were taken from 12 patients who were operated for meniscus tear and diagnosed with discoid meniscus and from 6 patients who were operated for meniscal tear and did not have a diagnosis of discoid meniscus. The samples were examined with transmission electron microscopy. RESULTS: The study group demonstrated a decrease in the number of collagen fibers compared with the control group. Also, the homogeneous course of the collagen fibers observed in the control group was replaced by a heterogeneous course in the study group. CONCLUSION: The ultrastructure of the discoid meniscus is different from the normal menisci. CLINICAL RELEVANCE: This difference may contribute to the vulnerability of the discoid meniscus to tears.

Collagenous fibril texture of the discoid lateral meniscus.

PURPOSE: To provide the theoretic basis for treatment and to increase the understanding of the tear patterns of the discoid meniscus, we observed the collagen orientation of the discoid meniscus. METHODS: Ten meniscus specimens were used to observe the collagen fibril orientation of the complete type of the discoid lateral menisci. The samples were observed layer by layer under a polarizing filter microscope by using Sirius red staining, and they were also observed under a scanning electron microscope. RESULTS: The lateral discoid meniscus is classified into 7 layers based on collagen fibril orientation. The femoral surface of the discoid meniscus is covered by dense and well-arranged thick fibrils, which very much resembles a bunched streak. The fibrils show a sagittal isotropic-arranged orientation. However, the tibial surface shows an irregular and anisotropically arranged orientation. In the outer layer, a meshwork of thin fibrils has been observed. The collagen fibrils in the inner layer are radially orientated from the lateral side to the medial side. In the central layer, the peripheral collagen fibrils are displayed as dense bundles running in a circumferential pattern, whereas its medial zone shows as thin, loosely, and irregularly arranged fibrils without a bundle formation. The anterior and posterior zones of the central layer show the collagen fibrils with a straight arrangement in the radial direction. CONCLUSIONS: In the lateral middle zone of discoid meniscus, the collagen fibrils run parallel to the periphery of the meniscus. Therefore, it would be ideally suited for resisting hoop stresses. From this anatomic study, it is apparent that the peripheral portion of the meniscus is constructed to bear a load. CLINICAL RELEVANCE: It is strongly recommended that the peripheral portion of the discoid meniscus should be preserved when a resection of the meniscus is mandatory.

Overlap Between Anterior Cruciate Ligament and Anterolateral Meniscal Root Insertions: A Scanning Electron Microscopy Study.

BACKGROUND: The anterolateral meniscal root (ALMR) has been reported to intricately insert underneath the tibial insertion of the anterior cruciate ligament (ACL). Previous studies have begun to evaluate the relationship between the insertion areas and the risk of iatrogenic injuries; however, the overlap of the insertions has yet to be quantified in the sagittal and coronal planes. PURPOSE: To investigate the insertions of the human tibial ACL and ALMR using scanning electron microscopy (SEM) and to quantify the overlap of the ALMR insertion in the coronal and sagittal planes. STUDY DESIGN: Descriptive laboratory study. METHODS: Ten cadaveric knees were dissected to isolate the tibial ACL and ALMR insertions. Specimens were prepared and imaged in the coronal and sagittal planes. After imaging, fiber directions were examined to identify the insertions and used to calculate the percentage of the ACL that overlaps with the ALMR instead of inserting into bone. RESULTS: Four-phase insertion fibers of the tibial ACL were identified directly medial to the ALMR insertion as they attached onto the tibial plateau. The mean percentage of ACL fibers overlapping the ALMR insertion instead of inserting into subchondral bone in the coronal and sagittal planes was 41.0% ± 8.9% and 53.9% ± 4.3%, respectively. The percentage of insertion overlap in the sagittal plane was significantly higher than in the coronal plane ( P = .02). CONCLUSION: This study is the first to quantify the ACL insertion overlap of the ALMR insertion in the coronal and sagittal planes, which supplements previous literature on the insertion area overlap and iatrogenic injuries of the ALMR insertion. Future studies should determine how much damage to the ALMR insertion is acceptable to properly restore ACL function without increasing the risk for tears of the ALMR. CLINICAL RELEVANCE: Overlap of the insertion areas on the tibial plateau has been previously reported; however, the results of this study demonstrate significant overlap of the insertions superior to the insertion sites on the tibial plateau as well. These findings need to be considered when positioning for tibial tunnel creation in ACL reconstruction to avoid damage to the ALMR insertion.

Collagenous Ultrastructure of the Discoid Meniscus: A Transmission Electron Microscopy Study.

BACKGROUND: The collagen ultrastructure of the discoid lateral meniscus (DLM) has not been precisely defined. PURPOSE: To investigate the ultrastructure of the DLM, focusing on its collagen fibers, and to compare the collagen net architecture between intact and torn DLMs using the Collagen Meniscal Architecture (CMA) scoring system. STUDY DESIGN: Descriptive laboratory study. METHODS: Thirty specimens were taken from 30 patients with a diagnosis of a complete DLM using a 1-piece technique. The collagen ultrastructure of the DLMs was assessed with transmission electron microscopy. To evaluate the meniscal ultrastructure, the degree of collagen disruption, intrafibrillar edema, loss of banding, degree of collagen packing, and fibril size variability were assessed and graded from 1 (normal) to 3 (severe disarray) according to the CMA scoring system. The DLM specimens were divided into 3 groups according to the intrasubstance tear: the intact group (group I) had no tear; the simple tear group (group S) had a radial, longitudinal, or horizontal tear; and the complicated tear group (group C) had a complicated horizontal tear. Intact normal meniscus specimens (group N) were used as the control group. RESULTS: There were 10 specimens in group I, 8 in group S, 12 in group C, and 13 in group N. In group I, there were 5 grade 1 and 5 grade 2 menisci; group S had 2 grade 1, 3 grade 2, and 3 grade 3 menisci; group C had 1 grade 1, 4 grade 2, and 7 grade 3 menisci; and group N had 4 grade 1, 7 grade 2, and 2 grade 3 menisci. A significant difference in the CMA score was observed between the 4 groups ( P = .009). The median CMA score was significantly lower in group I (2; range, 1-4) than in group S (4; range, 2-7) ( P = .041) and group C (4.25; range, 1.5-7) ( P = .018). No significant difference was found between groups S, C, and N. CONCLUSION: Variability existed in the collagen ultrastructure of the DLM, and some DLMs showed a nearly normal ultrastructural pattern. The degree of density and disorganization of the collagen architecture in the DLM was related to the tear. CLINICAL RELEVANCE: The study results might provide a histological background for partial meniscectomy in the treatment of a symptomatic DLM.