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A new technique is described for increasing the nasal tip by using an en bloc autogenous ear cartilage graft in such a manner as to create, in essence, a replacement of the entire ipsilateral alar cartilage.
This study introduces an implantable scaffold-free (SF) cartilage tissue construct that is composed of chondrocytes and their self-produced extracellular matrix (ECM). Chondrocytes were isolated from the articular cartilages from knees of domestic pigs (2-week old) and monolayer-cultured for 3-4 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 microg/mL of ascorbic acid. Briefly treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), an intact chondrocytes/ECM membrane, as a cell sheet was released from the plate bottom and subsequently centrifuged into a pellet-type construct. Each was grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 14, 21, and 35 days). For in vivo implantation, full-thickness defects (n = 4) were manually created on the femoro-patellar groove of the left porcine knee and 1-week-cultured SF construct was implanted as an allograft for a month. One defect (#1) was an empty control and the remaining three received different recipes; construct only (#2) or 0.25% trypsin/EDTA-treated first and then construct and collagen gel (#3) or construct and collagen gel (#4). While the total cell numbers significantly increased by 2 weeks and then remained stable, cell viability stayed in the mid-70% range through the entire culture period. Biochemical assay found continuous glycosaminoglycan (GAG) accumulation. Histology exhibited that cell distribution was even in the construct and GAG intensity became stronger and uniform with time. Real-time reverse transcription polymerase chain reaction (RT-PCR) results showed that phenotypic stability peaked at 2 weeks, which was arable to that of freshly isolated chondrocytes. Upon analysis of the retrieved implants, some promising results were witnessed in the defects (#3) retaining not only their intact mass but also chondrocytic morphology with lacuna formation.
The modulation of interleukin 1 (IL-1) effects on proteoglycan metabolism in intact murine patellar cartilage by transforming growth factor beta (TGF-beta) was investigated in vitro and in vivo. In vitro TGF-beta (400 pmol/l) had no effect on basal proteoglycan degradation. Proteoglycan degradation induced by IL-1, however, was suppressed by TGF-beta in serum free medium alone and in medium supplemented with 0.5 micrograms/ml insulin-like growth factor 1. This suggests a specific regulatory role for TGF-beta under pathological conditions. In contrast with the suppression of breakdown, synthesis of proteoglycans was stimulated by TGF-beta for both basal and IL-1 suppressed proteoglycan synthesis in cultures without insulin-like growth factor. In the presence of insulin-like growth factor no extra effect of TGF-beta on proteoglycan synthesis was observed. With insulin-like growth factor, however, TGF-beta potentiated the ex vivo recovery of IL-1 induced suppression of proteoglycan synthesis. Analogous to the in vitro effects, TGF-beta injected intraarticularly suppressed IL-1 induced proteoglycan degradation. Furthermore, TGF-beta injected into the joint counteracted IL-1 induced suppression of proteoglycan synthesis. This indicates that in vivo also TGF-beta can ameliorate the deleterious effects of IL-1 on the cartilage matrix.
Methods for isolating chondrocytes from the craniofacial complex and culturing them in vitro were established. Chondrocytes which were isolated by collagenase digestion from mandibular condylar cartilage, nasal septal cartilage, and spheno-occipital synchondrosis grew well in vitro. All three types of chondrocytes actively synthesized glycosaminoglycans, a differentiated phenotype of chondrocytes, and responded well to parathyroid hormone. However, some different characteristics were noted among the three types of chondrocytes in culture.
PURPOSE: To determine the influence of osteochondral defect size on defect rim stress concentration, peak rim stress, and load redistribution to adjacent cartilage over the weightbearing area of the medial and lateral femoral condyles in the human knee. METHODS: Eight fresh-frozen cadaveric knees were mounted at 30 degrees of flexion in a materials testing machine. Digital electronic pressure sensors were placed in the medial and lateral compartments of the knee. Each intact knee was first loaded to 700 N and held for 5 seconds. Dynamic pressure readings were recorded throughout the loading and holding phases. Loading was repeated over circular osteochondral defects (5, 8, 10, 12, 14, 16, 18, and 20 mm) in the 30 degrees weightbearing area of the medial and lateral femoral condyles. RESULTS: Stress concentration around the rims of defects 8 mm and smaller was not demonstrated, and pressure distribution in this size range was dominated by the menisci. For defects 10 mm and greater, distribution of peak pressures followed the rim of the defect with a mean distance from the rim of 2.2 mm on the medial condyle and 3.2 mm on the lateral condyle. An analysis of variance with Bonferroni correction revealed a statistically significant trend of increasing radius of peak pressure as defect size increased for defects from 10 to 20 mm (P = .0011). Peak rim pressure values did not increase significantly as defects were enlarged from 10 to 20 mm. Load redistribution during the holding phase was also observed. CONCLUSIONS: Rim stress concentration was demonstrated for osteochondral defects 10 mm and greater in size. This altered load distribution has important implications relating to the long-term integrity of cartilage adjacent to osteochondral defects in the human knee. Although the decision to treat osteochondral lesions is certainly multifactorial, a size threshold of 10 mm, based on biomechanical data, may be a useful adjunct to guide clinical decision making.
OBJECTIVE: We sought to describe a range of in vivo observations of glycosaminoglycan distribution in knee cartilage using the delayed gadolinium-enhanced MRI of cartilage technique. CONCLUSION: The index of glycosaminoglycan distribution, T1(Gd), can exceed 500 msec (denoting high glycosaminoglycan) or can be less than 300 msec, with focal areas as low as 240 msec. Compartmental differences, as well as focal defects within the knee, were observed in patients who had sustained injuries to the ligaments and menisci of the knee or who had chronic osteoarthritis. Overall, these results suggest the need for further research into the biochemical changes seen during disease progression and the effects of therapeutic interventions.
OBJECTIVE: To quantify and compare the microscopic changes in articular cartilage (AC), zone of calcified cartilage (ZCC), and subchondral bone plate in femoral heads from clinically normal dogs and dogs with moderate or severe osteoarthritis. SAMPLE POPULATION: Femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) osteoarthritis. PROCEDURES: Femoral heads were allocated to 3 categories (normal, moderate, or severe osteoarthritis) on the basis of radiographic findings, macroscopic findings, and histologic grade determined by use of a modified Mankin scale. Equally spaced 2-mm sections were cut in each femoral head in a coronal or transverse plane. Thickness of the AC, ZCC, and subchondral bone plate was recorded. RESULTS: Mean thickness of AC was significantly greater in samples with moderate and severe osteoarthritis than those considered normal. Mean thickness of the ZCC was significantly greater in samples with moderate and severe osteoarthritis than those considered normal. Mean thickness of the subchondral bone plate in samples with severe osteoarthritis was significantly greater than those with moderate osteoarthritis and those considered normal. A significant decrease in AC thickness was detected in the proximomedial area of femoral heads with severe osteoarthritis, compared with those considered normal. CONCLUSIONS AND CLINICAL RELEVANCE: A cause and effect association between thickening of subchondral structures and thinning and loss of the overlying AC was not detected. Changes in AC were associated with changes in the subchondral bone plate, which is compatible with the theory of adaptation in response to altered load distribution.
The articular surface of the distal part of the femur was removed operatively in dogs, and the regenerating articular surface and the GTC were investigated at different stages from the 7th to the 70th postoperative days. During this period cartilage islets arose in the GTAS, while the GTC transformed to connective tissue. At 7 days the lipid content of the tissue was markedly higher than at the other stages studied. Lipids, predominantly triglycerides, were present in extracellular form as well. From the 20th to the 70th day the PL fraction became predominant and, in addition to the pre-existing lecithin, relatively large quantities of lysolecithin, sphingomyelin, phosphatidyl-ethanolamine, phosphatidyl-serine and phosphatidyl-inositol could be gradually demonstrated. Differences were noted in the time of appearance and binding of PLs between the two types of granulation tissue. As time proceeded, the proportion of saturated fatty acids decreased in favour of unsaturated ones. At 70 days, the GTAS contained fatty acids up to C18. About 50% of the fatty acids consisted of C16:1, C18:2 and C18:1. At the same stage, in the GTC C16:1, C18:1 and C20:1 were present in larger amounts. Of the free fatty acids C16:1, C16 and C18 were in predominance in the GTAS and the proportion of fatty acids having more then one double bonds increased with time. In the GTC C16 and C18:1 were in great majority. According to histochemical evidence, the tissues did not contain extracellular lipids from the 20th postoperative day. In the cells, the presence of glycerides, PLs, lipoproteins and cholesterol was demonstrated. In addition, in cartilage precursors of more advanced maturity, a considerable fatty acid positivity was noted.
A non-collagenous quantitatively prominent protein was purified from guanidine hydrochloride extracts of bovine tracheal cartilage. Purification was achieved by cesium chloride density gradient centrifugation and chromatography on DEAE-cellulose at pH 7.0 followed by CM-cellulose at pH 5.0. The protein has a marked tendency to form aggregates in denaturing solutions of high ionic strength, e.g. 6 M guanidine hydrochloride. The purified protein contains a single, Mr 36,000 polypeptide chain, with a particularly high content of leucine. It contains about 1% carbohydrate with a remarkable absence of hexosamines and sialic acid, whereas xylose, galactose, mannose, and fucose were identified in the preparation. The protein was identified in extracts of cartilage and bone and could be shown to be primarily extracellular. Tendon may contain trace amounts of the protein, whereas extracts of several other tissues showed no immunoreactivity in enzyme-linked immunosorbent assay.
We could demonstrate the existence of class I and class II antigens on the chondrocytes of the auricular cartilage in inbred strain rats (Louvain/M/Him, Fischer 344). Indirect fluorescence and related techniques were used. The cartilage was stored in Ham's nutrient medium F 12 or in the tissue culture for 8-10 days before allotransplantation. A complete disappearance of the antigens could not be achieved by means of the vital conservation. Grafting did not modify further the distribution of the antigens; in 2 experiments only renewed strong coloration of the chondrocyte-membranes and the surrounding matrix could be observed.
Some of the effects of the semi-synthetic glycosaminoglycan polysulfuric acid ester (GAGPE) were investigated on the osteoarthritis-like lesions in the Pond-Nuki dog model, in respect to histological grade of anatomical lesions, collagenolytic enzyme activity, and a parameter of collagen network integrity. Prophylactic treatment by intra-articular injections twice weekly for 4 weeks caused amelioration of canine cartilage erosions. Preliminary evidence for suppression of collagenolytic enzyme activity, as well as protection of a tight collagen network studied in the canine cartilage was obtained.
Gross morphology and histology of the craniofacial complex was studied in the offspring of two transgenic founder mice, Gly85-1 and Gly85-3, carrying several copies of a mouse type II collagen transgene that causes a single amino acid substitution (Gly-->Cys) at position 85 of the triple helix. The newborn transgenic mice had a short snout and mandible, a protruding tongue, a cleft of the secondary palate with the tongue situated between the shelves, and a doming cranial vault. Radiological examination revealed that the cranial base of the transgenic mice was shorter and its posterior part downward bent; in addition both the palate and the cribriform plate were less extended in relation to the cranial base than in the controls. Histologically the midline cartilaginous structures were composed of densely packed enlarged chondrocytes in a reduced extracellular matrix containing abnormally thick collagen fibrils. With the exception of the zone of hypertrophic chondrocytes the matrix also showed a loss of glycosaminoglycans. The cellular architecture of the basicranial synchondroses was disorganized, and the nasocerebrally oriented collagen fibrils formed unevenly distributed aggregates. The craniofacial morphology described here for the Gly85 mice shares features typical for other mouse mutations causing short limbed dwarfism. The observations indicate that defective cartilage production causes disproportionate craniofacial growth. Transgenic mice with specific mutations in cartilage-specific genes should therefore be useful for elucidating the complex mechanisms involved in determining the craniofacial growth.
Patients with a bifid, cephalically rotated, contour-deformed, bulky, overprojected, pinched-tip, alar-dislocated, and/or alar-tethered nose had primary and secondary rhinoplasties using complete lateral alar cartilage mobilization, modification, and repositioning and the cartilage disc tip-graft technique. This technique avoids the pitfalls of classic in situ subtraction rhinoplasty and provides a better way to correct the nasal shape without causing airway obstruction. This technique was performed in 30 patients in the past 6 years who had primary or secondary rhinoplasties, with satisfactory results.
In a 32 years old professional handball layer, the micro-traumata typical of this game led to retropatellar cartilage lesion, which became sympthomatic following an distortion-impact trauma. Since the patient remains symptomatic after initial conservative treatment as well as arthroscopic chondroplastic, osteo-chondral transplantation was performed. There was a good functional result 6 months after surgery.
OBJECTIVE: To establish an in vitro model for the investigation of destructive processes in rheumatoid arthritis (RA), to study the interaction between fibroblasts, macrophages, and chondrocytes, and to evaluate strategies to inhibit joint destruction in RA. METHODS: Human and bovine chondrocytes cultured in sponges pretreated with native bovine embryonic extracellular matrix produced a cartilaginous matrix reflected by the incorporation of 35S into proteoglycans. The 3-dimensional culture system was optimized for the number of chondrocytes (10(5) cells/sponge), the timing of 35S incorporation (day 21 after chondrocyte isolation), and the medium (20% fetal calf serum). RA synovial fibroblasts (RASF; 10(5)) were added, and the matrix destruction mediated by these RASF was monitored by the release of 35S. The system was modulated by the addition of monocytes (U937 cells), cytokines (interleukin-1beta [IL-1beta] and tumor necrosis factor alpha [TNFalpha]), interleukin-1 receptor antagonist (IL-1Ra), and monoclonal antibodies against IL-1beta and CD44. RESULTS: RASF destroyed the bovine cartilaginous matrix within 2 weeks (days 5-12) and the human cartilaginous matrix within 3 weeks (days 10-18). Compared with the effect of RASF alone (mean +/- SD 948 +/-180 counts per minute/week), the addition of U937 cells (a monocytic cell line), IL-1beta, or TNFalpha to the incubation medium increased the destruction of human cartilaginous matrix by at least 71% up to 90% (ranging from 1,618+/-204 cpm/week to 1,802+/-307 cpm/week). IL-1Ra and anti-IL-1beta monoclonal antibodies reduced the destruction of human matrix by 45% and 35%, respectively; this was partially reversed by the addition of U937 cells. The pretreatment of RASF with anti-CD44 monoclonal antibody (an adhesion molecule and receptor for hyaluronic acid) inhibited the destruction of the cartilaginous matrix by an average of 41% over 3 weeks. CONCLUSION: This model is envisioned to study distinct aspects of human destructive joint diseases under in vitro conditions and to replace and/or supplement animal experiments in basic research and drug testing. Based on the fact that proinflammatory cytokines enhance destruction whereas IL-1Ra and antibodies against IL-1beta and CD44 inhibit the process, it is concluded that anti-IL-1- and anti-CD44-directed therapies may help prevent cartilage destruction in RA.
The calcification of cartilage matrix in endochondral bone formation occurs in an extracellular matrix composed of fibrils of type II collagen with which type X collagen is closely associated. Also present within this matrix are the large proteoglycans containing chondroitin sulfate which aggregate with hyaluronic acid. In addition, the matrix contains matrix vesicles containing alkaline phosphatase. There is probably a concentration of calcium as a result of its binding to the many chondroitin sulfate chains. At the time of calcification, these proteoglycans become focally concentrated in sites where mineral is deposited. This would result in an even greater focal concentration of calcium. Release of inorganic phosphate, as a result of the activity of alkaline phosphatase, can lead to the displacement of proteoglycan bound calcium and its precipitation. The C-propeptide of type II collagen becomes concentrated in the mineralizing sites, prior to which it is mainly associated with type II collagen fibrils and is present in dilated cisternae of the enlarged hypertrophic chondrocytes. The synthesis of type II collagen and the C-propeptide, together with alkaline phosphatase, are regulated by the vitamin D metabolites 24,25(OH)2 cholecalciferol and 1,25 (OH)2 cholecalciferol. At the time of calcification, type X collagen remains associated with type II collagen fibrils. It may play a role in preventing the initial calcification of these fibrils focusing mineral formation in focal interfibrillar sites. This process of calcification is clearly very complex, and involves different interacting matrix molecules and is carefully regulated at the cellular level.
Cultured tissue slices from normal immature rabbit articular cartilage released latent neutral metalloproteinases into serum-free medium. On activation with 4-aminophenylmercuric acetate, these metalloproteinases could degrade collagen, proteoglycan, and gelatin. Also produced were an acid proteinase with the properties of cathepsin D and an inhibitor of the neutral metalloproteinases. The appearance of both the proteinases and the inhibitor in the culture medium could be prevented by incubation of cultures with cycloheximide. The active and latent forms of the proteinases were characterized using Ultrogel AcA 54 chromatography.
Chondrons were isolated from human and canine osteoarthritic cartilage using low-speed homogenization techniques. Changes in chondron morphology were evaluated using differential interference-contrast microscopy, phase-contrast microscopy, and histochemical and ultrastructural methods. Chondrocyte viability was assessed using fluorescein diacetate staining, and chondron metabolism was investigated using autoradiography. The results suggest that initial changes in the collagen and proteoglycan distribution within the chondron are followed by chondrocyte proliferation to form clusters. These techniques offer the potential to study cell matrix interactions in degenerative osteoarthritis.