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.

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.

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.

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.

Comparison of Diagnostic Performance of Semi-Quantitative Knee Ultrasound and Knee Radiography with MRI: Oulu Knee Osteoarthritis Study.

Osteoarthritis (OA) is a common degenerative musculoskeletal disease highly prevalent in aging societies worldwide. Traditionally, knee OA is diagnosed using conventional radiography. However, structural changes of articular cartilage or menisci cannot be directly evaluated using this method. On the other hand, ultrasound is a promising tool able to provide direct information on soft tissue degeneration. The aim of our study was to systematically determine the site-specific diagnostic performance of semi-quantitative ultrasound grading of knee femoral articular cartilage, osteophytes and meniscal extrusion, and of radiographic assessment of joint space narrowing and osteophytes, using MRI as a reference standard. Eighty asymptomatic and 79 symptomatic subjects with mean age of 57.7 years were included in the study. Ultrasound performed best in the assessment of femoral medial and lateral osteophytes, and medial meniscal extrusion. In comparison to radiography, ultrasound performed better or at least equally well in identification of tibio-femoral osteophytes, medial meniscal extrusion and medial femoral cartilage morphological degeneration. Ultrasound provides relevant additional diagnostic information on tissue-specific morphological changes not depicted by conventional radiography. Consequently, the use of ultrasound as a complementary imaging tool along with radiography may enable more accurate and cost-effective diagnostics of knee osteoarthritis at the primary healthcare level.

Analysis of Gene Expression and Ultrastructure of Stifle Menisci from Juvenile and Adult Pigs.

The origin of the age-associated degenerative processes in meniscal tissue is poorly understood and may be related to an imbalance of anabolic and catabolic metabolism. The aim of the current study was to compare medial menisci isolated from juvenile pigs and degenerated medial menisci from adult pigs in terms of gene expression profile and ultrastructure. Medial menisci were isolated from the knee joints of juvenile and adult pigs (n = 8 for each group). Degeneration was determined histologically according to a scoring system. In addition, the gene expression profiles of 14 genes encoding extracellular matrix proteins, catabolic matrix metalloproteinases and mediators of inflammation were analyzed. Changes in the ultrastructure of the collagen network of the meniscal tissue were analyzed by using transmission electron microscopy. The histologic analysis of menisci showed significantly higher grade of degeneration in tissue isolated from adult porcine knee joints compared with menisci isolated from juvenile knee joints. In particular, destruction of the collagen network was greater in adult menisci than in juvenile menisci. Degenerated menisci showed significantly decreased gene expression of COL1A1 and increased expression of MMP2, MMP13, and IL8. The menisci from adult porcine knee joints can serve as a model for meniscal degeneration. Degenerative changes were manifested as differences in histopathology, gene expression and ultrastructure of collagen network.

Meniscus tissue engineering using a novel combination of electrospun scaffolds and human meniscus cells embedded within an extracellular matrix hydrogel.

Meniscus injury and degeneration have been linked to the development of secondary osteoarthritis (OA). Therapies that successfully repair or replace the meniscus are, therefore, likely to prevent or delay OA progression. We investigated the novel approach of building layers of aligned polylactic acid (PLA) electrospun (ES) scaffolds with human meniscus cells embedded in extracellular matrix (ECM) hydrogel to lead to formation of neotissues that resemble meniscus-like tissue. PLA ES scaffolds with randomly oriented or aligned fibers were seeded with human meniscus cells derived from vascular or avascular regions. Cell viability, cell morphology, and gene expression profiles were monitored via confocal microscopy, scanning electron microscopy (SEM), and real-time polymerase chain reaction (PCR), respectively. Seeded scaffolds were used to produce multilayered constructs and were examined via histology and immunohistochemistry. Morphology and mechanical properties of PLA scaffolds (with and without cells) were influenced by fiber direction of the scaffolds. Both PLA scaffolds supported meniscus tissue formation with increased COL1A1, SOX9, and COMP, yet no difference in gene expression was found between random and aligned PLA scaffolds. Overall, ES materials, which possess mechanical strength of meniscus and can support neotissue formation, show potential for use in cell-based meniscus regeneration strategies.

Regional comparisons of porcine menisci.

The purpose of this study was to analyze histologic, biochemical, and biomechanical differences between zonal, regional, and anatomic locations of porcine menisci. We evaluated six menisci removed from pigs. Medial and lateral menisci were divided into three regions: anterior, middle, and posterior. In each portion, the central zone (CZ) and peripheral zone (PZ) were examined histologically (hematoxylin & eosin, safranin O/Fast green, and picrosiriusred staining), using scanning electron microscopy, biochemically (hydroxyproline assay for collagen content and dimethylmethylene blue assay for glycosaminoglycan [GAG] content), and biomechanically (compression testing). Collagen content in the CZ was lower than that in the PZ. GAG content in the CZ was higher than that in the PZ. GAG content in the PZ of the posterior portion was significantly higher than that in the anterior and middle portions. Compression strength in the CZ was higher than that in the PZ. The differences in cellular phenotype, vascular penetration, and ECM not only between CZ and PZ but also among the anterior, middle, and posterior portions were clarified in the immature porcine meniscus. This result helps further our understanding of the biological characteristic of the meniscus. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1602-1611, 2014.

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.

Biomaterials in search of a meniscus substitute.

The menisci fulfill key biomechanical functions in the tibiofemoral (knee) joint. Unfortunately meniscal injuries are quite common and most often treated by (partial) meniscectomy. However, some patients experience enduring symptoms, and, more importantly, it leads to an increased risk for symptomatic osteoarthritis. Over the past decades, researchers have put effort in developing a meniscal substitute able to prevent osteoarthritis and treat enduring clinical symptoms. Grossly, two categories of substitutes are observed: First, a resorbable scaffold mimicking biomechanical function which slowly degrades while tissue regeneration and organization is promoted. Second, a non resorbable, permanent implant which mimics the biomechanical function of the native meniscus. Numerous biomaterials with different (material) properties have been used in order to provide such a substitute. Nevertheless, a clinically applicable cartilage protecting material is not yet emerged. In the current review we provide an overview, and discuss, these different materials and extract recommendations regarding material properties for future developmental research.

Preparation of decellularized meniscal scaffolds using sonication treatment for tissue engineering.

Scaffolds play a key role in the process of regeneration and morphogenesis of tissue or organ. We have developed a novel sonication decellularization system to prepare decellularized bio-scaffolds in a short treatment time. The aim of the study is to investigate sonication decellularization condition that completely decellularize meniscus can be changed as well as to maintain the biomechanical parameters of scaffolds. The meniscus samples were decellularized using sonication treatment. The treated samples were evaluated histologically by EVG for cell removal, picrosirius red for content of collagen type I and III, and safranin-O/fast green staining for content of glycosaminoglycan, and SEM for observation of scaffold surface. Indentation apparatus was used to analyze the unconfined deformation under load of native and decellularized menisci. The load parameters which are stiffness, compression and residual force were not significantly different compare with native and sonicated scaffolds. However, the content of extracellular matrix and its fiber alignment changed significantly due to sonication treatment as observed by SEM and safranin-O/fast green staining, respectively. The removal of immunogenic cell components by sonication decellularization as well as maintain its biomechanical strength of decellularized scaffolds, so that it has potential to use as an implant material for tissue engineering of menisci.

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.

Respective interest of T2 mapping and diffusion tensor imaging in assessing porcine knee cartilage with MR at 3 Teslas.

Non-invasive quantitative assessment of articular cartilage integrity is essential for early detection and evaluation of osteoarthritis (OA) and for the follow-up of stem-cell-driven cartilage engineering. In this study, we investigated the feasibility of exploiting diffusion tensor imaging (DTI) on porcine knee joints with a clinical magnetic resonance (MR) scanner to extract micro-structural information in order to complement biochemical information quantified by T2 maps. We propose an MR protocol for quantifying T2 and cartilage microstructure with diffusion MR on a clinical scanner. Preliminary results were obtained on four pig knee joints using a 3 T GE clinical MRI scanner and an 8-channel knee coil array. The measured cartilage volume, T2 values, apparent diffusion coefficient and fractional anisotropy (FA) of femoral and tibial cartilage were respectively 9.8/2.3 mm2, 67.0/56.1 ms, 1.3/1.3×10-3 mm2/s and 0.4/0.3. This new protocol has the potential to be combined in vivo with quantitative assessment of both cartilage degradation and restoration in osteoarthritis.

[Clinical natural development in human meniscus injury].

OBJECTIVE: To investigate the changes of clinic and wound edge of the meniscus without treatment in order to provide a theoretical basis for clinical treatment. METHODS: From January 2001 to December 2011,68 patients with knee injury without diagnosis and treatment were selected in the study. According to clinical symptoms (pain,interlocking,instability, etc.) and knee MRI,32 patients were diagnosed as meniscus injury and underwent the arthroscopy. Total meniscectomy was performed in 32 cases on account of impossible repair of the meniscus. There were 21 males and 11 females,ranging in age from 15 to 49 years old with an average age of 25 years old,with an average time from diagnosis to arthroscopy for 46 weeks. Observation indexes included 1Preoperative and postoperative Lysholm scores of knee. 2Position,type and status of injury by arthroscopy. 3Observation of histology. With the procedure as follow: tissue samples were taken from different positions of the edge of the meniscus wound,and were divided into two parts. One part of sample was fixed with formalin, sliced with paraffin imbedding,and observed under an electron microscope after HE staining,and the other part of the sample was fixed with glutaraldehyde of 3%,sliced with ethoxyline imbedding ,and observed under an electron microscope after Lead Citrate staining. RESULTS: Thirty-two patients were followed up more than one year. There was significant differences in Lysholm scores bewteen preoperative and postoperative 3 months (t=15.6,P<0.01). Arthroscopy showed typical differences in 28 cases between the middle and the two ends of the wound edge and atypical differences in 4 cases. Light microscope showed typical manifestations in 26 cases, a few epithelioid cells could been seen fat the middle of the wound edge as well as cells tissue healing (such as fibroblasts) at the junction of each end,and atypical manifestations in 2 cases. Electron microscope showed typical manifestation in 25 cases and atypical manifestations in 3 cases. Typical manifestations in electron microscope showed the atrophic state tions in 25 cases and atypical manifestations in 3 cases. Typical manifestations electron microscope showed the atrophic state of nuclei and kytoplasm of cell (isogenous cells and epithelioid cells) at the middle of the wound edge; at the either junction of the wound edge, the fibroblasts exhibited an enlarged volume with many protuberances; the nuclei also increased in size, and the cytoplasm contained major rough endoplasmic reticulum, free ribosomes and Golgi complex; chondrocytes were round or oval with a large,round nucleus ; a large amount of rough endoplasmic reticulum and many free ribosomes could be observed in the cytoplasm;cartilage lacunae were observed surrounding chondrocytes. CONCLUSION: Weight loading activities with meniscus injury without treatment or before healing will increase the length of the wound and aggravate clinical symptoms. These findings indicate that early diagnosis and treatment combined with timely and effective immobilization is a key to the healing of meniscus injury and avoiding further surgery. The recent clinical effect of total meniscectomy is satisfacory in treating impossible repair meniscus.

Organized nanofibrous scaffolds that mimic the macroscopic and microscopic architecture of the knee meniscus.

The menisci are crescent-shaped fibrocartilaginous tissues whose structural organization consists of dense collagen bundles that are locally aligned but show a continuous change in macroscopic directionality. This circumferential patterning is necessary for load transmission across the knee joint and is a key design parameter for tissue engineered constructs. To address this issue we developed a novel electrospinning method to produce scaffolds composed of circumferentially aligned (CircAl) nanofibers, quantified their structure and mechanics, and compared them with traditional linearly aligned (LinAl) scaffolds. Fibers were locally oriented in CircAl scaffolds, but their orientation varied considerably as a function of position (P<0.05). LinAl fibers did not change in orientation over a similar length scale (P>0.05). Cell seeding of CircAl scaffolds resulted in a similar cellular directionality. Mechanical analysis of CircAl scaffolds revealed significant interactions between scaffold length and region (P<0.05), with the tensile modulus near the edge of the scaffolds decreasing with increasing scaffold length. No such differences were detected in LinAl specimens (P>0.05). Simulation of the fiber deposition process produced "theoretical" fiber populations that matched the fiber organization and mechanical properties observed experimentally. These novel scaffolds, with spatially varying local orientations and mechanics, will enable the formation of functional anatomic meniscus constructs.

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.

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.

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.

Características biométricas de los meniscos en rodillas de individuos Chilenos / Biometric characteristics of knee meniscus in Chilean individuals

Los meniscos son láminas cartilaginosas que contribuyen a ampliar la escasa concavidad de las superficies articulares de la tibia para recibir a los convexos cóndilos femorales. Satisfacen una necesidad funcional, lo que queda demostrado con su reconstitución después de su extirpación total. Con el propósito de aportar datos sobre sus características biométricas, se registraron diámetros anteroposteriores, transversal y de espesor a diversos niveles, para complementar la anatomía de los mismos en nuestra población. Para ello, se realizó un estudio en 20 meniscos, 10 laterales y 10 mediales, extraídos de cadáveres formolizados, de individuos adultos, chilenos, en los Laboratorios de Anatomía de la Universidad de La Frontera, los cuales se prepararon para efectuar las diversas mediciones, efectuando una limpieza de las estructuras fibrosas que están asociadas a estos cartílagos. Los registros fueron realizados con un caliper digital Mitutoyo de precisión 0,05 mm. El menisco lateral tuvo un promedio para la distancia anteroposterior entre las partes más prominentes del margen anterior del cuerno anterior y el posterior del cuerno posterior de 31,7 mm. La abertura meniscal fue de 11,5 mm; la distancia entre los márgenes anterior y posterior de su cuerno anterior fue de 10,9 mm; la de su cuerno posterior fue de 10,9 mm; el espesor periférico de su parte anterior fue de 3,8 mm, de su parte posterior fue de 6,1 mm y a nivel de su parte media fue de 5,9 mm. El menisco medial tuvo un promedio para la distancia anteroposterior entre las zonas más cercanas a la abertura meniscal de sus extremidades de 43,2 mm. La abertura fue de 26,1 mm; la distancia entre los margenes anterior y posterior de su cuerno anterior fue de 9,2 mm; la de su cuerno posterior fue de 13,5 mm; el espesor periférico a nivel de su parte anterior fue de 4,5 mm, de su parte posterior fue de 6,0 mm y a nivel de su parte media fue de 5,5 mm. Los datos aportados complementarán la anatomía ...

The menisci are cartilage plates that help to expand low concavity of articular surfaces of the tibia to receive the convex femoral condyles. They satisfy functional needs, as has been demonstrated with reconstitution following total excision. In order to provide biometric data on its characteristics, anteroposterior diameters, transverse and thickness at various levels were recorded to complement anatomy of the same in our population. We conducted a study in 20 menisci, 10 lateral and 10 medial from adult Chilean cadaver specimens, in the Anatomy Laboratory, Universidad de La Frontera. The samples were prepared to carry out various measurements cleaning the fibrous structures associated with these menisci. Recordings were registered with a Mitutoyo digital caliper with a 0.05 mm accuracy. Lateral meniscus showed an average anteroposterior distance between horns of 31.7 mm, meniscal opening was 11.5 mm, distance between anterior- posterior borders of the anterior horn was 10.9 mm, of the posterior horn 10.9 mm. The peripheral thickness of its anterior part was 3.8 mm, of its posterior part was 6.1 mm and in its middle section 5.9 mm.The medial meniscus had an average anteroposterior distance between anterior an posterior horn of 43.2 mm. Meniscal opening was 26.1 mm, distance between anterior and posterior borders of the anterior horn was 0.2 mm, in its posterior horn 13.5 mm, peripheral thickness in its anterior part was 4.5 mm, its posterior part was 6.0 mm and at the middle section of the meniscus was 5.5 mm. The data provided will complement anatomy of the menisci in Chilean population.