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.

[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.

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.

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.

Role of mesenchymal stem cells in tissue engineering of meniscus.

Tissue engineering is a promising approach for the treatment of tissue defects. Mesenchymal stem cells are of potential use as a source of repair cells or of important growth factors for tissue engineering. The purpose of this study was to examine the role of mesenchymal stem cells in meniscal tissue repair. This was tested using several cell and biomaterial-based treatment options for repair of defects in the avascular zone of rabbit menisci. Circular meniscal punch defects (2 mm) were created in the avascular zone of rabbit menisci and left empty or filled with hyaluronan-collagen composite matrices without cells, loaded with platelet-rich plasma, autologous bone marrow, or autologous mesenchymal stem cells. In some experiments, matrices with stem cells were precultured in chondrogenic medium for 14 days before implantation. Rabbits were then allowed free cage movement after surgery for up to 12 weeks. Untreated defects and defects treated with cell-free implants had muted fibrous healing responses. Neither bone marrow nor platelet-rich plasma loaded in matrices produced improvement in healing compared with cell-free implants. The implantation of 14 days precultured chondrogenic stem cell-matrix constructs resulted in fibrocartilage-like repair tissue, which was only partially integrated with the native meniscus. Non-precultured mesenchymal stem cells in hyaluronan-collagen composite matrices stimulated the development of completely integrated meniscus-like repair tissue. The study shows the necessity of mesenchymal stem cells for the repair of meniscal defects in the avascular zone. Mesenchymal stem cells seem to fulfill additional repair qualities besides the delivery of growth factors.

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.

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.

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.

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.

Freezing causes changes in the meniscus collagen net: a new ultrastructural meniscus disarray scale.

Alterations in meniscal permeability leading to nutritional deficit have been suggested as a cause of shrinkage in meniscal transplantation. The purpose of this study was to ascertain how freezing, one of the most common procedures used to preserve meniscal allografts, alters the collagen's architecture. Twenty-six fresh human external menisci were analyzed with transmission electron microscopy. Thirteen of them were previously frozen at -80 degrees C while the rest were used as controls. A new scale of the collagen meniscal architecture was proposed according to the collagen's periodicity and degree of disruption, loss of banding, degree of collagen packing, fibril size variability and its intrafibrilar oedema. Each meniscus was scored from 0 to 7. Subsequently they were classified in grades ranging from a normal state (grade I; 0-2 points) to severe disarray (grade III; 5-7 points). The fibril collagen diameters of those menisci which had been previously frozen showed an average size in the longitudinal section of 14.26 nm, whereas it was 17.28 nm in the menisci used as controls (p=0.019). In the transverse section, the frozen menisci averaged 13.14 and 16.93 nm in the controls (p=0.003). Samples of the 13 previously frozen menisci were classified as grade III in 61.54% of the cases. In the control group, all the menisci were classified either as grade I or II. The frozen menisci averaged 4.85 points, whereas the control group did so at 2.46 (p<0.001). The fibril diameters in frozen menisci showed a thinner diameter and had a higher degree of disarray. Therefore, the results suggest that the freezing process alters the menisci's collagen net. This could partially explain the pathological changes found in shrunken menisci after transplantation.

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.

Knee cartilage thickness measurements using MRI: a 4(1/2)-month longitudinal study in the meniscectomized guinea pig model of OA.

OBJECTIVE: The aim of this study was to follow, over a 4(1/2)-month period, the medial tibia cartilage thickness on a meniscectomy (MNX) guinea pig osteoarthritis (OA) model and to compare with control animals, using three-dimensional high-resolution magnetic resonance imaging (3D HR-MRI). METHODS: MRI experimentations were performed in vivo at 7 T on guinea pig knee joints. 3D HR-MR images were acquired in 60 controls (SHAM) and 45 osteoarthritic animals (MNX) at four time-points (15, 45, 90 and 135 days) after surgery. Medial tibial cartilage thickness was measured from MRI images using in-house dedicated 3D software followed by a statistical analysis. At each time-point 15 SHAM and 15 MNX animals were sacrificed for histomorphometric assessments. RESULTS: No significant difference of mean cartilage thickness between the groups was found at early stage (D45) using MRI; however, significant differences were found between the groups at D90 (P<0.001) and D135 (P<0.001). Histomorphometry data confirmed the pathological status of the animals and was well correlated with MRI at D15 (r=0.79, P<0.01), D45 (r=0.67, P<0.01), and D135 (r=0.39, P<0.05) for SHAM, and at D45 (r=0.63, P<0.01), and D135 (r=0.81, P<0.01) for MNX. CONCLUSION: Medial tibial cartilage measurement based on HR-MR images enables the monitoring of longitudinal cartilage thickness changes. This technique showed significant differences between SHAM and MNX as from D90 after surgery. It could be used as a noninvasive and reproducible tool to monitor therapeutic response in this OA model.

Diacerhein versus glucosamine in a rat model of osteoarthritis

PURPOSE: The purpose of this study was to compare the chondroprotective effect of diacerein and glucosamine regarding degenerative changes and articular stiffness in an experimental model of arthritis. METHODS: Twenty rats underwent medial meniscectomy on the right knee. Ten animals were given diacerhein, and 10 were given glucosamine, from day 1 to the third month postoperatively, when all of them were killed. Histological and functional analysis of the knees were performed (measurement of maximum extension). RESULTS: All operated knees showed more limited extension values and more degenerative changes as compared to nonoperated contralateral sides. A comparison of the two drugs showed that the degree of articular stiffness was significantly lower with diacerein, although degenerative changes were similar. CONCLUSIONS: 1) Prophylactic use of diacerein leads to lower degree of articular stiffness when compared to glucosamine; 2) The prophylactic chondroprotective effects of diacerein and glucosamine are histologically similar.

OBJETIVO: O trabalho foi realizado com o objetivo de comparar o efeito condroprotetor da diacereína em relação ao da glicosamina quanto às alterações degenerativas e à rigidez articular num modelo experimental de artrose. MÉTODOS: Vinte ratos foram submetidos à meniscectomia medial do joelho direito. Dez animais receberam diacereína, e dez glicosamina, todos do primeiro dia ao terceiro mês pós-operatório, quando foram sacrificados. Foram realizadas análise histológica e funcional (medida da extensão máxima) dos joelhos. RESULTADOS: Todos os joelhos operados apresentaram amplitude de extensão mais limitada e maiores alterações degenerativas, em relação ao lado contra-lateral não operado. Ao compararmos as duas drogas, a rigidez articular foi significantemente menor com a diacereína, e as alterações degenerativas foram semelhantes. CONCLUSÕES: 1- O uso profilático da diacereína leva à menor rigidez articular em relação a glicosamina. 2- O efeito condroprotetor profilático da diacereína é semelhante, histologicamente, ao da glicosamina.

Evaluation of the neurosensory function of the medial meniscus in humans.

PURPOSE: Menisci are known to have receptors mainly concentrated at the anterior and posterior horns. Although they are purported to send afferent impulses to the central nervous system, this function has not been thoroughly evaluated. The purpose of the study was to investigate whether stimulation of the menisci initiates a cortical response. The reaction of the end organ to the reflex arc is also evaluated. TYPE OF STUDY: Prospective case series. METHODS: Fourteen patients with normal medial menisci were included in the study. Different parts of the knee joint (the posterior horn and the body of the medial meniscus, the medial femoral condyle, the capsule, and the joint space) were electrically stimulated by a probe during arthroscopy. The cortical response was monitored with somatosensory-evoked potentials (SEPs). The compound muscle action potentials (CMAPs) of the semimembranosus, quadriceps, and biceps femoris muscles were also monitored with electroneuromyography (ENMG). RESULTS: Among the stimulated parts, only the posterior horn of the meniscus produced cortical responses. No response was obtained with stimulation of the medial femoral condyle, the body of the medial meniscus, the capsule, or the joint space. Stimulation of the posterior horn of the medial meniscus produced a measurable amount of CMAP latency for the semimembranosus muscle, but not for the quadriceps and biceps femoris muscles. CONCLUSIONS: Stimulation of the posterior horn of the medial meniscus produces reproducible cortical SEPs and results in ENMG-verified response of the semimembranosus muscle where no response of the semimembranosus muscle is detected with stimulation of the other parts of the knee. CLINICAL RELEVANCE: The knowledge that only the horns of the medial meniscus have mechanoreceptors in the medial compartment of the knee helps to understand patients' signs and symptoms in medial compartment disease.

Evaluation of human knee meniscus biopsies with near-infrared, reflectance confocal microscopy. A pilot study.

Knee cartilage biopsy is used to confirm the pathology in both clinical and experimental conditions and often guides diagnosis and therapeutic strategies. Current histopathological techniques are time consuming, induce tissue artefacts and often prevent further evaluation, once the tissue has been fixed. Hence, there is a potential need for a fast and nondestructive imaging technique for unfixed tissue. Near-infrared, reflectance confocal microscopy (CM) allows real-time, virtual sectioning of unstained, bulk tissue samples. This pilot study evaluates the use of CM in the assessment of meniscus histopathology in a series of 26 freshly-excised human meniscus samples compared to standard light microscopy of stained sections. CM images of the meniscus show cell and matrix detail, depicting morphologic features of collagen and elastic fibres, vessels and nerve endings. In addition, crystal deposits of gout and pseudogout are also demonstrable. Thus, CM is a novel imaging technique that could enable the pathologist to make a rapid microscopic evaluation of cartilage in a fresh and unfixed fashion.

Histology and ultrastructure of a tissue-engineered collagen meniscus before and after implantation.

The collagen meniscus implant (CMI) is a tissue-engineering technique designed to stimulate regeneration of meniscus-like tissue in cases of irreparable tears or previous meniscectomy. CMI morphology was investigated before and after implantation by light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). In a case series biopsy specimens were harvested from four patients who underwent a second arthroscopic look 6 months after placement of the CMI. CMI sections appeared composed of parallel connective laminae of 10-30 microm, connected by smaller bundles (5-10 microm). This connective network formed lacunae with diameters between 40 and 60 microm. At greater magnification, the walls of the lacunae demonstrated tightly packed and randomly distributed collagen fibrils, with diameters ranging from 73 to 439 nm. In the biopsy specimens, the lacunae were filled with connective tissue that contained newly formed vessels and fibroblast-like cells, presenting an abundant rough endoplasmic reticulum and several mitochondria. In the extracellular matrix, the collagen fibrils showed uniform diameters (126 nm +/- 32 nm). The original structure of CMI was still recognizable, and no inflammatory cells were detected within the implant. The morphological findings of this case series demonstrate that CMI provides a three-dimensional scaffold suitable for colonization by precursor cells and vessels and leading to the formation of a fully functional tissue.

[Possibility of using cartilage cultured in centrifuge tube as a substitute for meniscus].

OBJECTIVE: To compare biological characteristics between articular chondrocyte and meniscal fibrochondrocyte cultured in vitro and to investigate the possibility of using cultured cartilage as a substitute for meniscus. METHODS: Chondrocytes isolated from articular cartilage and meniscus of rabbits aged 3 weeks were respectively passaged in monolayer and cultured in centrifuge tube. Cartilages cultured in centrifuge tube and meniscus of rabbit aged 6 weeks were detected by histological examination and transmission electron microscopy. Growth curves of articular chondrocytes and meniscal fibrochondrocytes were compared; meanwhile, cell cycles of articular chondrocytes and meniscal fibrochondrocytes in passage 2 and 4 were separately measured by flow cytometry. RESULTS: Articular chondrocytes in passage 4 were dedifferentiated. Articular chondrocytes formed cartilage 2 weeks after cultivation in centrifuge tube, but meniscal fibrochondrocytes could not generate cartilage. The differences in ultrastructure and histology obviously existed between cultured cartilage and meniscus; moreover, apoptosis of chondrocytes appeared in cultured cartilage. Proportion of subdiploid cells in articular chondrocytes passage 2 and 4 was markedly higher than that in passage 2 and 4 fibrochondrocytes (P < 0.05). CONCLUSION: Meniscal fibrochondrocytes can not form cartilage after cultivation in centrifuge tube, while cartilage cultured in centrifuge tube from articular chondrocytes can not be used as graft material for meniscus. Articular cartilage is markedly different from meniscus.

[Ultrastructure of cultured cartilage, articular cartilage, growth plate and meniscus].

OBJECTIVE: To investigate possibility of cartilage cultured in centrifuge tube as graft materials. METHODS: Articular chondrocytes isolated from a 3-week-old rabbit formed cartilage after cultivation for 2 weeks. Articular cartilage of humeral head, growth plate of proximal tibia and meniscus were collected from a 6-week-old rabbit. The ultrastructure of chondrocytes and extracellular matrix in the three kinds of cartilages and cultured cartilage were observed by transmission electronic microscopy. RESULTS: Cartilage cultured in centrifuge tube possessed unique ultrastructure and was similar to articular cartilage and growth plate, but it was markedly different from meniscus. The four kinds of cartilages were characteristic of respectively different chondrocytes and extracellular matrix. Cultured cartilage showed typical apoptosis of chondrocytes and "dark chondrocytes" appeared in growth plate. Condrocyte apoptosis was not seen in articular cartilage and meniscus. CONCLUSION: Cartilage cultured in centrifuge tube has unique ultrastructure and may be used as graft materials for articular cartilage and growth plate.

Quantitative assessment of articular cartilage and subchondral bone histology in the meniscectomized guinea pig model of osteoarthritis.

OBJECTIVE: A new image analysis system was employed to quantify the main histological parameters reflecting osteoarthritic features, at the cartilage and bone levels, in the meniscectomized guinea pig model of osteoarthritis (OA). METHODS: Meniscectomized (MNX) and sham-operated (SH) guinea pigs were studied 1 and 3 months after partial meniscectomy at the medial side of the left knee (n=10 to 12 animals/group). The left proximal tibias were included in methylmethacrylate. Sections were cut and stained with safranin O or Goldner trichrome. Parameters were quantified using special programs of a Biocom image analyser. The following parameters were evaluated at the medial side of the tibia: cartilage thickness (CT); fibrillation index (FI); proteoglycan content ratio based on safranin O staining intensities (PC); chondrocyte density (CD); bone volume (BV) and subchondral bone plate thickness (SBPT). The degree of user interaction varied from manually tracing objects to almost complete computer automation. RESULTS: Meniscectomy resulted in significant variations of these reproducible histomorphometric parameters both after 1 month (FI: +522%, P<0.01) and 3 months (FI: +162%, P<0.001; PC: -36.7%, P<0.001; CD: -31.8%, P<0.001; SBPT: +8.7%, P<0.05) post-operation (results expressed as percentage variation of MNX vs SH). The linear correlation analysis including data from SH and/or MNX animals at the two grouped time points revealed significant r values, in particular between cartilage (CT) and subchondral bone parameters (SBPT) (r=-0.41, P<0.01). CONCLUSIONS: Contrary to scoring evaluation, this system allowed to show the time-dependent impact of the pathology with an early fibrillation of the medial tibial cartilage appearing as soon as 1 month post-surgery, and the close relationship between bone and cartilage parameters during the progression of OA.