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PubMed2006

Magnetization transfer analysis of cartilage repair tissue: a preliminary study.

PURPOSE: To evaluate the magnetization transfer ratio (MTR) after two different cartilage repair procedures, and to compare these data with the MTR of normal cartilage. DESIGN AND PATIENTS: Twenty-seven patients with a proven cartilage defect were recruited: 13 were treated with autologous chondrocyte implantation (ACI) and 14 were treated with the microfracture technique (MFR). All patients underwent MRI examinations with MT-sequences before the surgical treatment, after 12 months (26 patients) and after 24 months (11 patients). Eleven patients received a complete follow-up study at all three time points (five of the ACI group and six of the MFR group). All images were transferred to a workstation to calculate MTR images. For every MT image set, different ROIs were delineated by two radiologists. Means were calculated per ROI type in the different time frames and in both groups of cartilage repair. The data were analyzed with unpaired t- and ANOVA tests, and by calculating Pearson's correlation coefficient. RESULTS: No significant differences were found in the MTR of fatty bone marrow, muscle and normal cartilage in the different time frames. There was a significant but small difference between the MTR of normal cartilage and the cartilage repair area after 12 months for both procedures. After 24 months, the MTR of ACI repaired cartilage (0.31+/-0.07) was not significantly different from normal cartilage MTR (0.34+/-0.05). The MTR of MFR repaired cartilage (0.28+/-0.02), still showed a significant difference from normal cartilage. CONCLUSION: The differences between damaged and repaired cartilage MTR are too small to enable MT-imaging to be a useful tool for postoperative follow-up of cartilage repair procedures. There is, however, an evolution towards normal MTR-values in the cartilage repair tissue (especially after ACI repair).

Adult↗
PubMed2005

Tissue engineering of human cartilage and osteochondral composites using recirculation bioreactors.

Chondrocytes isolated from human foetal epiphyseal cartilage were seeded dynamically into polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to produce tissue-engineered cartilage. Several culture techniques with the potential to provide endogenous growth factors and other conditions beneficial for de novo cartilage synthesis were investigated. Osteochondral composite constructs were generated by seeding separate PGA scaffolds with either foetal chondrocytes or foetal osteoblasts then suturing the scaffolds together before bioreactor cultivation. This type of co-culture system provided direct contact between the tissue-engineered cartilage and developing tissue-engineered bone and yielded significant improvements in cartilage quality. In the cartilage section of the composites, the concentrations of glycosaminoglycan (GAG) and total collagen were increased by 55% and 2.5-fold, respectively, compared with control cartilage cultures, while levels of collagen type II were similar to those in the controls. The osteochondral composites were harvested from the bioreactors as single units with good integration between the cartilage and bone tissues. Only the cartilage layer contained GAG while only the bone layer was mineralised. In other experiments, co-culture of tissue-engineered cartilage with pieces of ex-vivo cartilage or ex-vivo bone did not improve the quality of the cartilage relative to control cultures. Addition of 10(-6) M diacerein to the culture medium also had no effect on the properties of engineered cartilage. This work demonstrates the beneficial effects of generating cartilage tissues in contact with developing bone. It also demonstrates the feasibility of producing composite osteochondral constructs for clinical application using recirculation column bioreactors.

Bioreactors↗
PubMed2004

The use of photooxidized, mushroom-structured osteochondral grafts for cartilage resurfacing--a comparison to photooxidized cylindrical grafts in an experimental study in sheep.

OBJECTIVE: This article addresses the problem of structural design with osteochondral grafts used for cartilage resurfacing. METHODS: Photooxidized cylindrical or mushroom-shaped grafts were surgically implanted in the weight bearing area of the medial and lateral femoral condyles of eight sheep (condyles: N=8/group). Both types of photooxidized grafts contained no viable chondrocytes at the time of implantation. Results were evaluated at 2 and 6 months after surgical implantation of the grafts. Qualitative and quantitative evaluation of the subchondral bone area was performed using plastic embedded sections of non-decalcified bone and cartilage specimens and placing emphasis on graft anchorage, cyst-like lesions at the base of the cartilage junction and at the base of the graft in the subchondral bone region. Cartilage morphology was studied qualitatively focusing on viability of the graft and adjacent host cartilage, while a score system was developed for semi-quantitative evaluation of the overall articular cartilage performance. The semiquantitative scores and histomorphometrical measurements were subjected to statistical analysis using a factorial analysis of variance (ANOVA-test). RESULTS: The photooxidized mushroom-shaped grafts developed less fibrous tissue and cyst-like lesions in the subchondral bone area at 2 and 6 months compared to the cylindrical grafts. Areas of endochondral ossification and bone remodeling were noticeable in the mushroom structured grafts at 2 months, and also bone remodeling was more complete at 6 months than with the cylindrical grafts. Increased numbers of cells were seen in the basal remodeling zones of both graft types increased from the 2 months to the 6 months specimens, but mushroom structured grafts showed better results. In both graft types, however, the midzone of the cartilage matrix was still acellular at 6 months. Cells from the subchondral bone area started to penetrate the calcified cartilage zone and tide mark at 2 months and repopulated the old photooxidized cartilage matrix already at 6 months after implantation. Cartilage repopulation was dependent on a stable subchondral bone area in both types of grafts. Matrix degradation of the adjacent host cartilage was minimal at 2 and 6 months. At 6 months a junction between host and graft cartilage was already noticed in some of the mushroom-shaped grafts. CONCLUSION: This study confirmed the importance of the subchondral bone area for osteochondral graft survival. In addition it demonstrated that the structure of the graft influences considerably the architecture of the subchondral bone, and with this the possibility for the repopulation of the old cartilage matrix including the junction between the host and graft cartilage matrix.

Analysis of Variance↗
PubMed1997

Cartilage fibronectin isoforms: in search of functions for a special population of matrix glycoproteins.

Fibronectins are a part of the repertoire of matrix molecules produced by the chondrocyte in order to assemble a functional cartilage matrix. They are encoded by a single gene, but significant protein heterogeneity results from alternative RNA splicing. The population of fibronectin isofroms in adult cartilage is significantly different from fibronectins in other tissues and includes relatively high levels (20-30%) of ED-B(+) fibronectins and high levels (50-80%) of the cartilage specific (V + C)- isoform which lacks the V, III-15 and I-10 segments. Less than 4% of the fibronectins in cartilage are ED-A(+). The synthesis and accumulation of cartilage fibronectins are modulated in response to matrix pathology and to biochemical and mechanical mediators. In addition, alternative splicing patterns are altered when chondrocytes are allowed to dedifferentiate in monolayer culture such that the (V + C)- isoform is lost but the ED-A(+) isoform is reexpressed at high levels. Cartilage fibronectins have the potential to participate in cell signalling via integrin mediated pathways and to interact with other cartilage matrix macromolecules. The tissue-specific splicing pattern gives rise to a unique population of fibronectins within the cartilage. Together, this points to a critical role for cartilage fibronectins in chondrocyte cell biology and the organization of a biomechanically sound matrix. However, the precise function (or functions) of the cartilage fibronectins has (or have) not been defined. This minireview examines current information about the structure, synthesis and interactions of cartilage fibronectins. When possible, potential consequences of the inclusion of the ED-B segment or the exclusion of the V, III-15 and I-10 segments are discussed. The goal is to stimulate critical thought and discussion in the field about cartilage fibronectin isoforms, their function(s) in normal cartilage, and their role(s) in the pathogenesis of cartilage diseases.

Alternative Splicing↗
PubMed2001

Histologic analysis of the implanted cartilage in an exact-fit osteochondral transplantation model.

PURPOSE: Osteochondral transplantation is one of the useful treatments for articular cartilage defect. However, the histologic change of the implanted cartilage has not been reported in detail. We investigated the histology of exact-fit osteochondral transplants used to repair articular cartilage defects in an animal model. TYPE OF STUDY: This was a nonrandomized control study using an animal model. METHODS: Sixteen skeletally mature female Japanese white rabbits were used in the study. The region of the femoral groove was selected as the site for the osteochondral defect. A full-thickness cylindrical defect (7 mm in diameter and 7 mm in depth) through the articular cartilage and into the subchondral bone was made using the Osteochondral Autograft Transfer System (Arthrex, Naples, FL). The entire osteochondral fragment was removed and then returned to its original site in the femoral condyle precisely. Thus, the defect was repaired with an autogenous osteochondral transplantation of exactly the same size and configuration as the defect. Specimens were obtained 2, 4, 12, and 24 weeks postoperatively and were analyzed both macroscopically and histologically. RESULTS: Macroscopically, there was smooth continuity of the articular surface and the integration of the graft to the normal host cartilage. However, histologic examination showed that the layer of the grafted cartilage was thicker than that of the normal host cartilage and the extracellular matrix of the implanted cartilage exhibited a stronger staining pattern with safranin-O fast green than the normal cartilage. Cell density was higher in the grafted cartilage, particularly in the middle and the deep zones. Round and polygonal hypertrophic clusters of chondrocytes were observed in the middle and deep zones of the grafted cartilage. CONCLUSIONS: The histologic properties of the exact-fit implanted cartilage were different from that of normal articular cartilage. Further investigation of mechanical and structural properties of grafted cartilage is necessary to verify the long-term effects of osteochondral transplantation.

Animals↗
PubMed2001

Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specific manner.

UNLABELLED: It was hypothesized that cartilage macro-molecular characteristics are influenced by exercise intensity and by location within a joint. OBJECTIVE: To determine the macromolecular characteristics of carpal articular cartilage at common and uncommon sites of pathology in horses undergoing high or low intensity exercise, and to compare this composition between exercise groups. DESIGN: Twelve horses (19.3+/-0.9 years) were assigned to exercise groups. Each group underwent 19 weeks high-intensity treadmill training (N=6) or low-intensity exercise (N=6). Dorsal and palmar test sites were identified on radial, intermediate and third carpal articular surfaces after euthanasia. Cartilage was collected from each site, freeze-dried and assessed for water content. Hydroxyproline, glycosaminoglycan and DNA analyses were performed on cartilage from each test site. Adjacent cartilage underwent histological preparation and assessment for chondrocyte numerical density at each site and proteoglycan distribution through the depth of cartilage. RESULTS: Dorsal cartilage had a higher collagen content, DNA content, and chondrocyte numerical density, but lower glycosaminoglycan content than palmar cartilage. Cartilage from horses undergoing high-intensity training had a significantly higher glycosaminoglycan content than cartilage from horses undergoing low-intensity exercise, with maximal difference being observed in cartilage from dorsal radial and dorsal intermediate carpal articular surfaces. Overall no effect of exercise on collagen was observed, but at sites predisposed to clinical lesions cartilage from horses undergoing high-intensity training contained significantly less collagen than from horses undergoing low-intensity exercise. Distribution of proteoglycan was non-uniform in 52% of the sections examined, with superficial loss of toluidine blue staining primarily at dorsal sites and in the high-intensity exercise group. CONCLUSIONS: These results indicate that topographical and exercise related differences exist in carpal cartilage composition, and that the effect of exercise on overall composition and distribution within the cartilage was maximal at sites predisposed to clinical lesions. These findings could indicate that the combined effect of exercise and local load variations within a joint may lead to a risk of exceeding the physiologic threshold at high load sites that are predisposed to clinical injury.

Animals↗
PubMed2002

Ultrastructural localization of type VI collagen in normal adult and osteoarthritic human articular cartilage.

OBJECTIVE: Type VI collagen is a major component of the pericellular matrix compartment in articular cartilage and shows severe alterations in osteoarthritic cartilage degeneration. In this study, we analysed the exact localization of type VI collagen in its relationship to the chondrocyte and the (inter)territorial cartilage matrix. Additionally, we were interested in its ultrastructural appearance in normal and osteoarthritic cartilage. DESIGN: Distribution and molecular appearance was investigated by conventional immunostaining, by multilabeling confocal scanning microscopy, conventional transmission, and immunoelectron microscopy. RESULTS: Our analysis confirmed the pericellular concentration of type VI collagen in normal and degenerated cartilage. Type VI collagen formed an interface in between the cell surface and the type II collagen network. The type VI collagen and the type II collagen networks appeared to have a slight physical overlap in both normal and diseased cartilage. Additionally, some epitope staining was observed in the cell-associated interterritorial cartilage matrix, which did not appear to have an immediate relation to the type II collagen fibrillar network as evaluated by immunoelectron microscopy. In osteoarthritic cartilage, significant differences were found compared with normal articular cartilage: the overall dimension of the lacunar volume increased, and a significantly increased type VI collagen epitope staining was observed in the interterritorial cartilage matrix. Also, the banded isoform of type VI collagen was found around many chondrocytes. CONCLUSIONS: Our study confirms the close association of type VI collagen with both, the chondrocyte cell surface and the territorial cartilage matrix. They show severe alterations in type VI collagen distribution and appearance in osteoarthritic cartilage. Our immunohistochemical and ultrastructural data are compatible with two ways of degradation of type VI collagen in osteoarthritic cartilage: (1) the pathologically increased physiological molecular degradation leading to the complete loss of type VI collagen filaments from the pericellular chondrocyte matrix and (2) the transformation of the fine filaments to the band-like form of type VI collagen. Both might implicate a significant loss of function of the pericellular microenvironment in osteoarthritic cartilage.

Adult↗
PubMed1973

Release of cartilage mucopolysaccharide-degrading neutral protease from human leukocytes.

The granule fraction of human leukocytes contains neutral protease capable of degrading the noncollagenous protein mucopolysaccharide matrix of cartilage at neutral pH in physiological salt solution. Cartilage degradation was monitored by quantitating the release of (35)S from labeled rabbit ear cartilage. Degradation of cartilage matrix occurs when intact viable human leukocytes are incubated with cartilage opsonized with aggregated human gamma globulin (AHGG). During a similar 4 h incubation period cells did not degrade uncoated cartilage or cartilage coated with nonaggregated gamma globulin. Cells remain viable during the enzyme release process as evidenced by the absence of a cytoplasmic enzyme marker (lactic dehydrogenase) in the supernatant and dye exclusion studies. The release of (35)S from labeled cartilage by human leukocytes in the presence of cartilage coated with AHGG (nonphagocytic enzyme release) was compared with the cartilage degrading activity of the supernatant from the same number of cells preincubated with a suspension of AHGG (phagocytic enzyme release). Nonphagocytic enzyme release by 5 x 10(6) cells provoked two to four times more (35)S and beta-glucuronidase (beta-G) release from cartilage than phagocytic enzyme release conditions. beta-glucuronidase was used as an indicator of the release of lysosomal granule enzymes. By the use of selected pharmacological agents it was possible to dissociate the enzyme release process from intrinsic enzyme (neutral protease) activity. Neutral protease and beta-G release by human cells in the presence of AHGG-coated cartilage was inhibited by 10(-5)M colchicine, whereas the protease activity, but not the release process, was inhibited by 10(-6)M gold thiomalate and 10% human serum. It is suggested that the release of a cartilage degrading neutral protease by viable human cells when exposed to AHGG might be a relevant model for the study of cartilage destruction as it occurs in rheumatoid arthritis.

Cartilage↗
PubMed2002

Image processing techniques for noise removal, enhancement and segmentation of cartilage OCT images.

Osteoarthritis, whose hallmark is the progressive loss of joint cartilage, is a major cause of morbidity worldwide. Recently, optical coherence tomography (OCT) has demonstrated considerable promise for the assessment of articular cartilage. Among the most important parameters to be assessed is cartilage width. However, detection of the bone cartilage interface is critical for the assessment of cartilage width. At present, the quantitative evaluations of cartilage thickness are being done using manual tracing of cartilage-bone borders. Since data is being obtained near video rate with OCT, automated identification of the bone-cartilage interface is critical. In order to automate the process of boundary detection on OCT images, there is a need for developing new image processing techniques. In this paper we describe the image processing techniques for speckle removal, image enhancement and segmentation of cartilage OCT images. In particular, this paper focuses on rabbit cartilage since this is an important animal model for testing both chondroprotective agents and cartilage repair techniques. In this study, a variety of techniques were examined. Ultimately, by combining an adaptive filtering technique with edge detection (vertical gradient, Sobel edge detection), cartilage edges can be detected. The procedure requires several steps and can be automated. Once the cartilage edges are outlined, the cartilage thickness can be measured.

Animals↗
PubMed2000

Cell-based tissue-engineered allogeneic implant for cartilage repair.

The potential for using of allogeneic cartilage chips, transplanted in a biologic polymer with articular chondrocytes, as a tool for articular cartilage repair was studied. Small lyophilized articular cartilage chips were mixed with a cell/fibrinogen solution and thrombin to obtain implantable constructs made of fibrin glue, chondrocytes, and cartilage chips. Specimens were implanted in the subcutaneous tissue on the backs of nude mice (experimental group A). Three groups of controls (groups B, C, and D) were also prepared. Group B consisted of fibrin glue and cartilage chips without chondrocytes. Group C consisted of fibrin glue and chondrocytes without cartilage chips, and group D was composed solely of fibrin glue. All samples were carefully weighed before implantation in the mice. The constructs were harvested from the animals at 6, 9, and 12 weeks, examined grossly, and weighed. The samples were then processed and stained with hematoxylin and eosin for histological examination. Gross evaluation and weight analysis of the constructs at the time of retrieval showed retention of the original mass in the samples made of fibrin glue, chondrocytes, and cartilage chips (group A) and demonstrated a cartilaginous consistency upon probing. Specimens from constructs of fibrin glue and cartilage chips without chondrocytes (control group B) retained most of their volume, but were statistically lighter than specimens from group A and were much softer and more pliable than those in group A. Samples of specimens from constructs of fibrin glue and chondrocytes (groups C) and fibrin glue alone (group D) both showed a substantial reduction of their original masses over the experimental time periods when compared to the samples in groups A and B, although specimens from group C demonstrated new cartilage matrix formation. Histological analysis of specimens in experimental group A demonstrated the presence of cartilage chips surrounded by newly formed cartilaginous matrix, while specimens of control group B showed only fibrotic tissue surrounding the devitalized cartilage pieces. Cartilaginous matrix was also observed in control group C, in which cartilage chips were absent, whereas only fibrin glue debris was observed in control group D. This study demonstrated that a composite of fibrin glue and devitalized cartilage can serve as a scaffold for chondrocyte transplantation, preserve the original phenotype of the chondrocytes, and maintain the original mass of the implant. This may represent a valid option for addressing the problem of articular cartilage repair.

Animals↗
PubMed1998

Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel.

Full-thickness articular cartilage defects are a major clinical problem; however, presently there is no treatment available to regeneratively repair these lesions. The current therapeutic approach is to drill the base of the defect to expose the subchondral bone with its cells and growth factors. This usually results in a repair tissue of fibrocartilage that functions poorly in the loaded joint environment. The use of phenotypically appropriate chondrocytes embedded in a collagen gel delivery vehicle may provide a method that could be used to repair full-thickness articular cartilage defects with functionally satisfactory hyaline cartilage. Allograft articular chondrocytes embedded in a type I collagen gel were transplanted into large (6 x 3 x 3 mm), full-thickness articular cartilage defects in condylar and patellar weight-bearing surfaces to develop clinically applicable methods to repair articular cartilage defects. Chondrocytes were isolated from the articular cartilage of 4-week-old New Zealand rabbits and embedded in type I collagen gels. This composite was transplanted into a full-thickness defect on the medial femoral condyle and patellar groove of adolescent host rabbits. The repair cartilage was assessed histologically by a semiquantitative scoring system and biomechanically with a microindentation technique of specimens 4-48 weeks after chondrocyte transplantation. Defects in both locations were repaired with histologically apparent hyaline cartilage observed from as early as 4 weeks until 48 weeks after transplantation. The repair cartilage in the medial femoral condyle was more irregular than in the patellar groove, but in all other respects was similar. The grafted tissue did not remodel and differentiate into the morphological zones seen in normal articular cartilage. No tidemark or subchondral bony plate formed even 48 weeks after transplantation. Biomechanically, the repaired cartilage demonstrated indentation values similar to normal articular cartilage 12 weeks after transplantation and remained the same 48 weeks after transplantation. By contrast, the control (i.e., empty) defects healed with tissue that exhibited very poor metachromatic staining and exhibited very high indentation values. Incomplete bonding of the repair tissue to the normal cartilage was seen, and the surface was significantly irregular with major discontinuities. These observations provide the basis for considering the use of allograft articular chondrocytes to repair articular cartilage defects in the weight-bearing regions of the knee.

Animals↗
PubMedDate not supplied

Regeneration of canine tracheal cartilage by slow release of basic fibroblast growth factor from gelatin sponge.

We investigated the efficiency of basic fibroblast growth factor (b-FGF) released from a gelatin sponge in the regeneration of tracheal cartilage. A 1-cm gap was made in the midventral portion of each of 10 consecutive cervical tracheal cartilages (rings 4 to 13) in 15 experimental dogs. In the control group (n = 5), the resulting gap was left blank. In the gelatin group (n = 5), a gelatin sponge alone was implanted in the gap. In the b-FGF group (n = 5), a gelatin sponge containing 100 mug b-FGF solution was implanted in the gap. We euthanatized one of the five dogs in each group at 1 month after implantation and one at 3 months and examined the implant sites macroscopically and microscopically. In the control and gelatin groups, no regenerated cartilage was observed in the tracheal cartilage gap at 1 or 3 months. The distances between the cartilage stumps had shrunk. In the b-FGF group, fibrous cartilage had started to regenerate from both host cartilage stumps at 1 month. At 3 months, regenerated fibrous cartilage filled the gap and had connected each of the stumps. The regenerated cartilage was covered with regenerated perichondrium originating from the host perichondrium. Shrinkage of the distance between the host cartilage stumps was not observed in the b-FGF group. We succeeded in inducing cartilage regeneration in the gaps in canine tracheal cartilage rings by using the slow release of b-FGF from a gelatin sponge. The regenerated cartilage induced by b-FGF was fibrous cartilage.

Animals↗
PubMed2002

The dual role of perichondrium in cartilage wound healing.

Cartilage structures from the head and neck possess a certain but limited capacity to heal after injury. This capacity is accredited to the perichondrium. In this study, the role of the inner (cambium) and the outer (fibrous) layers of the perichondrium in cartilage wound healing in vitro is investigated. For the first time, the possibility of selectively removing the outer perichondrium layer is presented. Using rabbit ears, three different conditions were created: cartilage explants with both perichondrium layers intact, cartilage explants with only the outer perichondrium layer dissected, and cartilage explants with both perichondrium layers removed. The explants were studied after 0, 3, 7, 14, and 21 days of in vitro culturing using histochemistry and immunohistochemistry for Ki-67, collagen type II, transforming growth factor beta 1 (TGFbeta1), and fibroblast growth factor 2 (FGF2). When both perichondrium layers were not disturbed, fibrous cells grew over the cut edges of the explants from day 3 of culture on. New cartilage formation was never observed in this condition. When only the outer perichondrium layer was dissected from the cartilage explants, new cartilage formation was observed around the whole explant at day 21. When both perichondrium layers were removed, no alterations were observed at the wound surfaces. The growth factors TGFbeta1 and FGF2 were expressed in the entire perichondrium immediately after explantation. The expression gradually decreased with time in culture. However, the expression of TGFbeta1 remained high in the outer perichondrium layer and the layer of cells growing over the explant. This indicates a role for TGFbeta1 in the enhancement of fibrous overgrowth during the cartilage wound-healing process. The results of this experimental in vitro study demonstrate the dual role of perichondrium in cartilage wound healing. On the one hand, the inner layer of the perichondrium, adjacent to the cartilage, provides (in time) cells for new cartilage formation. On the other hand, the outer layer rapidly produces fibrous overgrowth, preventing the good cartilage-to-cartilage connection necessary to restore the mechanical function of the structure.

Animals↗
PubMed2004

Validating the subcutaneous model of injectable autologous cartilage using a fibrin glue scaffold.

PURPOSE: To create and validate an injectable model for autologous in vivo cartilage engineering with ultimate clinical applicability in human subjects. HYPOTHESIS: Cartilage can be generated subcutaneously using fibrin glue and autologous chondrocyte components. BACKGROUND: To date, cartilage engineering studies have been limited by several factors. Immunocompromised animals and nonautologous chondrocytes have been successfully used to create cartilage, but results using identical designs failed in immunocompetent subjects. Recent studies using more biocompatible tissues and matrices have been performed with both in vitro and in vivo steps. Although successful, several problems are notable. In vitro cartilage displays a poor modulus of elasticity, even after in vivo implantation. Variable deformation and volume loss occurs when in vitro specimens are matured in vivo. These concerns limit the clinical utility of these methods. We therefore set out to create autologous cartilage using a model that was clinically feasible, easy to create, and could be performed with very low patient harvest morbidity. MATERIALS AND METHODS: Eight New Zealand white rabbits underwent a unilateral harvest of ear cartilage. Samples were then digested using standard methods. Cell counts and survival assays were performed before implantation. One sample of fibrin glue (Tisseel) and chondrocytes was injected subcutaneously into each donor rabbit and then left in situ for 3 months. A second sample with both basic fibroblast growth factor (b-FGF) and insulin-like growth factor (IGF)-1 in the injection suspension was also assessed (for a total of 16 samples). After harvest, analysis of overall volume, histology, and chondrocyte drop out counts was performed. RESULTS: Cartilage formation occurred in 8 of 14 (57%) specimens that were obtained at the time of sacrifice. Of note, 6 of 7 (85%) non-growth-factor containing samples yielded positive results. Comparison with the success rate using concomitant growth factors (2/7) showed a negative effect on cartilage yield (P = .015). Chondrocyte survival, based on chondrocyte dropout counts, was not effected. Angiogenesis appeared to correlate with cartilage formation in the central regions of the implant. Alcian blue demonstrated the presence of active matrix deposition, and elastin Verhoff-van Geison (EVG) stains were positive, showing an elastic cartilage phenotype. Very limited osteoid formation was seen in successful implants. Failed implants demonstrated avascular necrosis, giant cell reactions, and inflammatory infiltrates. CONCLUSIONS: This study validates the subcutaneous site as a recipient bed for the engineering of autologous cartilage in vivo. It also represents the first subcutaneous implantation of fibrin glue and chondrocytes in an immunocompetent host as well as the first published report of elastic cartilage generation in vivo. Although the model needs to be further streamlined to increase yields and overall volume, this study clearly demonstrates the feasibility of in vivo chondrogenesis (85% success). The addition of FGF and IGF-1 at the concentrations used negatively influenced cartilage yield. However, extrapolation of these results to other combinations or concentrations can not be done, and this issue deserves further investigation.

Animals↗
PubMed1999

The effect of training on the calcified zone of equine middle carpal articular cartilage.

Carpal osteochondral injury is frequently observed in strenuously trained horses. It is clear that the integrity of articular cartilage and subchondral bone are intimately related, although there is controversy about which component is the most important. Calcified cartilage provides the mechanical link between soft hyaline cartilage and stiff subchondral bone so it is essential to understand the tissue's response to exercise. Middle carpal calcified and uncalcified (hyaline) cartilage thickness was investigated in horses undergoing high- and low-intensity exercise. Twelve untrained horses (18-21 months) were paired, and randomly assigned to an exercise group. Group 1 underwent 19 weeks progressive high-intensity training on a high-speed treadmill. Group 2 underwent walking exercise only. Osteochondral specimens were obtained from 8 test sites per carpus immediately after euthanasia. Histomorphometric measurements of total cartilage, hyaline layer, and calcified zone thickness were obtained from decalcified and undecalcified samples. Mean +/- s.d. (micron) calcified cartilage thickness for dorsal cartilage from Group 1 was 271 +/- 73 and from Group 2 was 163 +/- 49; for palmar cartilage from Group 1 was 195 +/- 42 and Group 2 was 150 +/- 52. Group 1 horses had significantly thicker total cartilage (P < 0.0001) and calcified zone (P < 0.0001) than Group 2, but there was no difference in hyaline layer. Within each group all dorsal sites had thicker calcified cartilage (P < 0.003) than palmar sites, but no difference in hyaline or total cartilage. These findings indicate that high-intensity exercise leads to greater calcified zone depth without alteration in hyaline cartilage thickness, and that this response is maximal at sites that withstand high, intermittent loads. Increasing calcified cartilage thickness with exercise may maintain the articular surface stiffness gradient in the face of alterations in hyaline cartilage and/or subchondral bone stiffness.

Analysis of Variance↗
PubMed2004

T2 relaxation time of cartilage at MR imaging: comparison with severity of knee osteoarthritis.

PURPOSE: To evaluate differences in T2 values in femoral and tibial cartilage at magnetic resonance (MR) imaging in patients with varying degrees of osteoarthritis (OA) compared with healthy subjects and to develop a mapping and display method based on calculation of T2 z scores for visual grading and assessment of cartilage heterogeneity in patients with OA. MATERIALS AND METHODS: Knee cartilage was evaluated in 55 subjects who were categorized with radiography as healthy (n = 7) or as having mild OA (n = 20) or severe OA (n = 28). Cartilage regions were determined with manual segmentation of an MR image acquired with spoiled gradients and fat suppression. The segmentation was applied to a map of T2 relaxation time and was analyzed in four knee cartilage compartments (ie, the medial and lateral tibia and femur). Differences between cartilage compartment T2 values and subject groups were analyzed with analysis of covariance. Correlations of cartilage T2 values with clinically reported symptoms and cartilage thickness and volume were examined. Cartilage T2 values were converted to z scores per voxel on the basis of normal population values in the same cartilage compartment to better interpret cartilage heterogeneity and variation from normal. RESULTS: Healthy subjects had mean T2 values of 32.1-35.0 msec, while patients with mild and severe OA had mean T2 values of 34.4-41.0 msec. All cartilage compartments except the lateral tibia showed significant (P <.05) increases in T2 relaxation time between healthy and diseased knees; however, no significant difference was found between patients with mild and severe OA. Correlation of T2 values with clinical symptoms and cartilage morphology was found predominantly in medial compartments. CONCLUSION: Femoral and medial tibial cartilage T2 values increase with the severity of OA.

Adult↗
PubMed1999

Development and aging of the articular cartilage of the rabbit knee joint: Distribution of biglycan, decorin, and matrilin-1.

We determined the distributions of the small proteoglycans biglycan and decorin and the glycoprotein matrilin-1 (cartilage matrix protein) during development and aging of articular cartilage in the rabbit knee joint. Before cavitation, the matrices of the interzone and the adjacent epiphyseal cartilage do not contain biglycan or decorin, but some chondrocytes express their mRNAs. Matrilin-1 is found only in the deeper epiphyseal cartilage. After cavitation, biglycan and decorin are detected in the presumptive articular cartilage, but there is no matrilin-1. All are present in the underlying epiphyseal cartilage. In the neonate, the epiphyseal cartilage is ossified and the articular cartilage becomes a discrete layer. Biglycan and decorin accumulate in the articular cartilage, but matrilin-1 remains confined to the residual epiphyseal cartilage. In the adult, the distributions of biglycan and decorin are highly variable. Decorin tends to be confined to the central region; matrilin-1 is absent. The findings indicate that the articular and epiphyseal cartilages are different from the earliest developmental stages. The epiphyseal cartilage can be identified by its possession of matrilin-1. Epiphyseal cartilage is removed during development to leave the articular cartilage. The relationships between the distributions of decorin and matrilin-1 and the fibrillar collagens are discussed. (J Histochem Cytochem 47:1603-1615, 1999)

Aging↗
PubMed1993

Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens.

The collagens of growth plate and articular cartilage from 5-6 month old commercial pigs were characterised. Growth plate cartilage was found to contain less total collagen than articular cartilage as a proportion of the dry weight. Collagen types I, II, VI, IX and XI are present in both growth plate and articular cartilage whereas type X is found exclusively in growth plate cartilage. Types III and V collagen could not be detected in either cartilage. Type I collagen makes up at least 10% of the collagenous component of both cartilages. There are significant differences in the ratios of the quantifiable collagen types between growth plate and articular cartilage. Collagen types I, II, and XI were less readily extracted from growth plate than from articular cartilage following pepsin treatment, although growth plate cartilage contains less of the mature collagen cross-links, hydroxylysyl-pyridinoline and lysyl-pyridinoline. Both cartilages contain significant amounts of the divalent reducible collagen cross-links, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine. Immunofluorescent localisation indicated that type I collagen is located predominantly at the surface of articular cartilage but is distributed throughout the matrix in growth plate. Types II and XI are located in the matrix of both cartilages whereas type IX is predominantly pericellular in the calcifying region of articular cartilage and the hypertrophic region of the growth plate. Collagen type VI is located primarily as a diffuse area at the articular surface.

Animals↗