Articular cartilage preservation and storage

Articular cartilage slice explants were stored under various conditions, including freezing-thawing at various rates by using dimethyl sulfoxide (DMSO) as a cryoprotective agent, incubating in standard tissue culture medium (MEM Eagle:NCTC 135:15% fetal calf serum) in 5% CO₂ and air at 4°, 21°, and 37°C, and incubating in standard tissue culture medium containing 200 μg/ml α-tocopherol (vitamin E) at 37°C after first ascertaining a dose-response curve of vitamin E. Results indicated that articular cartilage slice explants did not survive freezing or storage at 4° and 21°C as measured by ³⁵S uptake. When stored at 37°C in standard tissue culture in 5% CO₂ and air, the slice explants remained viable for up to 60 days. The addition of α-tocopherol to the medium resulted in significantly less release of previously incorporated ³⁵S in stored cartilage slices and significantly less reduction of the amount of hexosamine present in the stored explants. α-Tocopherol in the medium also preserved safranin O staining. Thus, the application of tissue culture techniques to the storage of articular cartilage made it possible to preserve cartilage slice explants in a viable, biochemically “normal” state.     

Survival of Vero, myeloma and hybridoma cells during cold storage

Vero cells, SP2/O-Ag 14 myeloma cells and 4B87 hybridoma cells were stored either at refrigeration (5 degrees C) or freezing (-18 degrees C) temperatures. Cells were recovered every five days and percentages of viable cells were determined by the trypan blue exclusion staining method before the cells were incubated at 37 degrees C in a 5% CO2 atmosphere. SP2/0 cells grew after 30 days of storage at 5 degrees C. Hybridoma (4B87) cells survived 20 days of cold storage in HY medium and maintained antibody production. For each cell type, higher percentages of viable cells were observed among cells stored in HY medium than among cells stored in DMEM. Vero cells stored for 40 days at 5 degrees C grew when removed to optimal conditions of 37 degrees C and 5% CO2. There was no growth of cells recovered after storage at -18 degrees C.     

Articular cartilage preservation and storage. II. Mechanical indentation testing of viable, stored articular cartilage.

Mature rabbit articular cartilage in the form of distal femoral condyles, composite osteoarticular structures, were incubated in the presence of alpha-tocopherol (200 micrograms/ml) over a period of time. Indentation testing and 35S uptake indicate preservation of sustained load carrying capacity and viability, respectively, in the presence of alpha-tocopherol for up to 30 days in organ culture. Condylar cartilage stored in the absence of alpha-tocopherol as well as frozen cartilage demonstrated progressive inability to resist sustained loading over time. Nonoptimal synthetic function apparently occurred in these latter two groups when compared to alpha-tocopherol stored material.     

In vitro rabbit articular cartilage organ model II. 35S incorporation in various oxygen tensions

Adult rabbit articular cartilage slice and plug explants were cultured in MEM/NCTC 109/fetal calf serum at 37°C in various oxygen tensions. In both the slice and plug explants, ³⁵SO₄ incorporation exhibited a broad tolerance to oxygen tensions varying from 5 (38 mm. Hg) to 60 (456 mm Hg) per cent. Significantly (P 0.01) less ³⁵SO₄ was incorporated in 1 (7.6 mm Hg) and 90% (684 mm Hg) oxygen than in 21% (160 mm Hg) oxygen. No histologic or autoradiographic differences, and only slight histochemical differences, were discernible after seven days culture in the various oxygen tensions. It is suggested that low synovial fluid oxygen tension is detrimental to articular cartilage glycosaminoglycan metabolism.     

The effect of hydrocortisone acetate on adult human articular cartilage.

Living adult human articular cartilage has been maintained in tissue culture for eight days. Cartilage explants were cultured in one of three concentrations of hydrocortisone acetate or as control explants without this drug. The concentrations of hydrocortisone used were 0.01 microng/ml, 0.1 microng/ml and 1.0 mg/ml. The amount of proteoglycan shed into the medium was measured and no significant difference was found between the explants exposed to hydrocortisone and those that were not. These results suggest that hydrocortisone does not have a catabolic effect on the proteoglycan of articular cartilage. The widely held view that intra-articular corticosteroid injection causes cartilage damage, must remain in doubt.     

Modulation of fibroblast-mediated cartilage degradation by articular chondrocytes in rheumatoid arthritis

OBJECTIVE: To determine the role of chondrocytes and factors released from chondrocytes in cartilage destruction by fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis (RA). METHODS: RA FLS from 2 patients were implanted into SCID mice, together with fresh articular cartilage or with cartilage that had been stored for 24 hours at 4 degrees C or at 37 degrees C. The invasion of the same RA FLS into the fresh and stored cartilage was compared histologically using a semiquantitative scoring system. In addition, we investigated whether protein synthesis in chondrocytes affects the invasion of RA FLS in vitro. A 3-dimensional cartilage-like matrix formed by cultured chondrocytes was labeled with 35S. After formation of the cartilage-like matrix, protein synthesis was blocked with cycloheximide. The invasion of RA FLS from 6 patients into cycloheximide-treated and untreated matrix was assessed by measuring the released radioactivity in coculture with and without interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). RESULTS: The SCID mouse experiments showed a significant invasion of RA FLS into the cartilage (overall mean score 3.2) but revealed significant differences when the invasion of the same RA FLS into fresh and stored cartilage was compared. RA FLS that were implanted with fresh articular cartilage showed a significantly higher invasiveness than those implanted with pieces of cartilage that had been stored for 24 hours (overall mean score 2.3). Storage at 37 degrees C and 4 degrees C resulted in the same reduction of invasion (35% and 37%, respectively). In the in vitro experiments, RA FLS rapidly destroyed the cartilage-like matrix. Blocking of chondrocyte protein biosynthesis significantly decreased the invasion of RA FLS, as shown by a decreased release of radioactivity. Addition of IL-1beta, but not TNFalpha, to the cocultures partially restored the invasiveness of RA FLS. CONCLUSION: These data underline the value of the SCID mouse in vivo model of rheumatoid cartilage destruction and demonstrate that chondrocytes contribute significantly to the degradation of cartilage by releasing factors that stimulate RA FLS. Among those, IL-1beta-mediated mechanisms might be of particular importance.     

Development of a human stomach explant organ culture system to study the pathogenesis of Helicobacter pylori

These studies were undertaken to define conditions under which Helicobacter (formerly Campylobacter) pylori and viable human gastric mucosa could coexist in tissue culture with the ultimate goal of developing an in vitro experimental model which could be used to study interactions between H. pylori and gastric epithelium. Antral gastric biopsies obtained at upper endoscopy were placed in culture in either CMRL-1066 or keratinocyte growth media and incubated at 37 degrees C in either an oxygen-enriched environment (45% O2, 50% N2, 5% CO2) or a standard oxygen environment (95% air, 5% CO2). Without selective antibiotics to suppress growth of non-H.-pylori organisms, H. pylori could not be isolated from most initially positive tissue even after only 2 h in tissue culture; however, when selective antibiotics were utilized in the tissue culture media, H. pylori was isolated from 9 of 14 initially positive cases after 24-72 h in tissue culture. There was little difference in the morphology of either surface or glandular epithelium in H.-pylori-negative explants between time zero and 48-hour cultures. However, H.-pylori-positive explants after 48 h in tissue culture showed a significant increase in injury to both surface and glandular epithelium when compared to time zero specimens. These data demonstrate that viable H. pylori and human gastric epithelium can be maintained in explant organ culture and suggest that this gastric mucosal explant culture system may be useful in studying the significance of H. pylori infection of human gastric epithelia.     

Mechanisms of cartilage growth: modulation of balance between proteoglycan and collagen in vitro using chondroitinase ABC

OBJECTIVE: To examine the cartilage growth-associated effects of a disruption in the balance between the swelling pressure of glycosaminoglycans (GAGs) and the restraining function of the collagen network, by diminishing GAG content prior to culture using enzymatic treatment with chondroitinase ABC. METHODS: Immature bovine articular cartilage explants from the superficial and middle layers were analyzed immediately or after incubation in serum-supplemented medium for 13 days. Other explants were treated with chondroitinase ABC to deplete tissue GAG and also either analyzed immediately or after incubation in serum-supplemented medium for 13 days. Treatment- and incubation-associated variations in tissue volume, contents of proteoglycan and collagen network components, and tensile mechanical properties were assessed. RESULTS: Incubation in serum-supplemented medium resulted in expansive growth with a marked increase in tissue volume that was associated with a diminution of tensile integrity. In contrast, chondroitinase ABC treatment on day 0 led to a marked reduction of GAG content and enhancement of tensile integrity, and subsequent incubation led to maturational growth with minimal changes in tissue volume and maintenance of tensile integrity at the enhanced levels. CONCLUSION: The data demonstrate that a manipulation of GAG content in articular cartilage explants can distinctly alter the growth phenotype of cartilage. This may have practical utility for tissue engineering and cartilage repair. For example, the expansive growth phenotype may be useful to fill cartilage defects, while the maturational growth phenotype may be useful to induce matrix stabilization after filling defect spaces.     

Interleukin-1alpha induction of tensile weakening associated with collagen degradation in bovine articular cartilage

OBJECTIVE: To determine whether interleukin-1alpha (IL-1alpha) induces tensile weakening of articular cartilage that is concomitant with the loss of glycosaminoglycans (GAGs) or the subsequent degradation of the collagen network. METHODS: Explants of young adult bovine cartilage obtained from the superficial (including the articular surface), middle, and deep layers were cultured with or without IL-1alpha for 1 week or 3 weeks. Then, portions of the explants were analyzed for their tensile properties (ramp modulus, strength, and failure strain); other portions of explants and spent culture medium were analyzed for the amount of GAG and the amount of cleaved, denatured, and total collagen. RESULTS: The effect of IL-1alpha treatment on cartilage tensile properties and content was dependent on the duration of culture and the depth of the explant from the articular surface. The tensile strength and failure strain of IL-1alpha-treated samples from the superficial and middle layers were lower after 3 weeks of culture, but not after 1 week of culture. However, by 1 week of culture, IL-1alpha had already induced release of the majority of tissue GAGs into the medium, without detectable loss or degradation of collagen. In contrast, after 3 weeks of culture, IL-1alpha induced significant collagen degradation, as indicated by the amount of total, cleaved, or denatured collagen in the medium or in explants from the superficial and middle layers. CONCLUSION: IL-1alpha-induced degradation of cartilage results in tensile weakening that occurs subsequent to the depletion of GAG and concomitant with the degradation of the collagen network.     

Could 37 degrees C storage temperature extend homovital valve allograft viability?

BACKGROUND AND AIM OF THE STUDY: Preservation of allograft valves is the most important determinant of their durability. Unprocessed, homovital valve allografts stored at 4 degrees C in nutrient medium have provided superior mid-term results over routinely used cryopreserved or antibiotic-sterilized allografts. As storage temperature may alter viability, it was hypothesized that allograft storage at 37 degrees C may maintain greater viability over time. METHODS: Porcine aortic (n = 10) and pulmonary valve conduits (n = 10) were harvested under sterile conditions. Valve leaflets and sinus walls were separated, and each was divided into two specimens, which were stored in modified culture medium at 4 degrees C and 37 degrees C, respectively. Cell viability was tested by monitoring metabolic activity at 37 degrees C at days 1, 3, 7, 10, and 14. The proliferative ability of cells isolated from valve leaflets was assessed after 14 days by cell culture. Sterility testing of the storage medium was also carried out. RESULTS: Valve leaflet cells and sinus wall cells had significantly higher metabolic activity when stored at 37 degrees C. The median number of isolated cells at 4 degrees C was 3,231.5 (range: 422-3,844), and at 37 degrees C was 8,317.50 (range: 4,329-8,650). The storage medium was sterile in all cases. CONCLUSION: Storage at 37 degrees C significantly improved valve allograft cell metabolic activity and viability compared with storage at 4 degrees C for up to 14 days. The lower concentration of antibiotics did not affect the sterility of tissues stored at 37 degrees C.     

Amount and rates of CO2 storage in lung tissue.

The slope of the lung tissue CO2 dissociation curve and the rate of storage of CO2 in the lung tissue were studied at 22 degrees C and at 37 degrees C in 21 isolated, bloodless dog lungs with a total of 465 separate observations. Results at the two temperatures were similar. The slope of the tissue dissociation curve of lung tissue at a PCO2 of 40 torr was approximately 0.3 ml CO2 X 100 g wet tissue-1 X torr-1. Normally, this storage was 90% complete in about 5 seconds. After carbonic anhydrase inhibition by acetazolamide, the total storage capacity was unchanged, but the rate at which storage occurred decreased significantly, so that it took about 25 seconds for 90% of the storage to be completed.     

Influence of hypoxia and reoxygenation on cytokine-induced production of proinflammatory mediators in articular cartilage

OBJECTIVE: Articular cartilage is an avascular tissue that functions at a lower oxygen tension than do most tissues. With mobilization, arthritic joints may undergo cycles of hypoxia and reoxygenation. The goal of this study was to determine the effects of hypoxia and reoxygenation on cytokine-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in articular cartilage. METHODS: Porcine cartilage explants were incubated at 37 degrees C for 72 hours in either 1% O(2) (hypoxia) or 20% O(2) (normoxia) in media supplemented with interleukin-1alpha (IL-1alpha) or tumor necrosis factor alpha (TNFalpha), with or without the NO synthase 2 (NOS2) selective inhibitor 1400W. Culture media were then removed and replaced with freshly prepared media and incubated for a further 24 hours in normoxia. RESULTS: NO levels were significantly higher in explants supplemented with IL-1alpha and TNFalpha compared with controls, in both hypoxia and normoxia. Compared with normoxia, hypoxia decreased IL-1alpha- and TNFalpha-induced NO production significantly. Reoxygenation of hypoxic explants resulted in sustained significant NO production in response to either cytokine. However, comparably high levels of NO production were not sustained in explants cultured continuously in normoxia. Although IL-1alpha alone did not significantly increase PGE(2) production, significant PGE(2) superinduction occurred in cartilage stimulated with IL-1alpha and the NOS2 inhibitor 1400W compared with stimulation with IL-1alpha alone in hypoxia, but not in normoxia. CONCLUSION: Oxygen tension significantly affects cytokine-induced proinflammatory mediator production in articular cartilage. Furthermore, hypoxia alters NO mediation of PGE(2) production. Hypoxia and reoxygenation can affect cytokine-induced proinflammatory mediator production, suggesting that oxygen tension may influence inflammation associated with cartilage injury and disease.     

Stability of meropenem in normal saline solution after storage at room temperature

The bactericidal activity of meropenem is determined by the time that concentrations in tissue and serum are above the MIC for the pathogens during the dosing interval. Thus, the most effective mode of administering of meropenem is continuous infusion. However, the stability of meropenem reconstituted in solution is influenced by the storage temperature. Until now we have had no data to evaluate the stability of this drug during continuous infusion in a tropical country. The objective of this study was to provide such data. Meropenem 0.5 g and 100 ml normal saline solution were mixed together and stored at room temperature for 8 hours. Half of the solution was stored in a room with air conditioning at 20 degrees C and the other half of the solution was stored in a room without air conditioning at 32 degrees-37 degrees C. The concentrations of meropenem in the solution were measured at 0, 1, 2, 3, 4, 6 and 8 hours after the drug was reconstituted. Twelve lots of (0.5 g meropenem in normal saline) solution were evaluated in each temperature condition. The mean meropenem concentrations reconstituted in normal saline solution decreased 1.66%, 3.31% and 5.80% after 2, 4 and 8 hours storage at 20 degrees C, respectively. Drug concentrations decreased 3.14%, 5.86% and 11.85% after 2, 4 and 8 hours storage at 32 degrees-37 degrees C, respectively. Therefore, we conclude that this agent should not be administered by 8-hour continuous infusion at room temperature in a tropical country.     

The effect of synovial tissue on the synthesis of proteoglycan by the articular cartilage of young pigs

Explants of pig articular cartilage were grown in organ culture in the presence of synovial tissue; controls consisted of paired explants that were cultivated in isolation. To find whether the synovial tissue affected synthesis of sulfated proteoglycan by the cartilage, ³⁵SO₄ was added to the medium and its incorporation into the cartilage examined by both biochemical assay and autoradiography. The synovial tissue severely inhibited the uptake of ³⁵SO₄, but if the synovium was removed after 8 days cultivation and the cartilage was grown in isolation for a further 4 days, incorporation of ³⁵SO₄ equalled and sometimes surpassed that of controls which had been grown without synovium continuously for 12 days. Synovium did not prevent the formation of new cartilage on the cut surfaces of the explants, but it reduced the incidence of newly formed cartilage.     

Specific enzymatic treatment of bovine and human articular cartilage: implications for integrative cartilage repair

OBJECTIVE: Chondrocyte death in articular cartilage wound edges and the subsequent lack of matrix-producing cells in the interface area are considered to be a major cause of impaired cartilage wound healing and poor integrative cartilage repair. This study was undertaken to investigate whether enzymatic matrix digestion can be used to stimulate integrative cartilage repair via a mechanism of local increase in the amount of vital chondrocytes in cartilage wound edges. METHODS: Full-thickness bovine articular cartilage samples were cultured in vitro for 14 days in standard medium. Samples were either left untreated or treated for 48 hours with 0.3% hyaluronidase or 30 units/ml highly purified collagenase VII. Nuclear and cytoplasmic changes were analyzed to determine cell viability, and the number of vital chondrocytes in wound edges was determined. Subsequently, we investigated whether increased chondrocyte density in the lesion edges resulted in better wound healing. Finally, full-thickness human tibial plateau cartilage explants were tested with similar enzyme treatment protocols to determine the clinical value of our results. RESULTS: In bovine explants a rapid onset of chondrocyte death was observed in wound edges in all treatment groups. This led to low chondrocyte density in a band of 0-150 microm from the lesion edges in untreated and hyaluronidase-treated explants. Treatment with 30 units/ml collagenase resulted in a significant increase in chondrocyte density in this area. The integration experiments demonstrated improved integration of the lesion edges after treatment with collagenase. In human articular cartilage an increase in chondrocyte density at the lesion edges could also be achieved, but only when proteoglycans were depleted from the wound edges prior to collagenase treatment. CONCLUSION: Treatment with highly purified collagenase improves integrative cartilage repair, possibly by increasing the cell density at cartilage wound edges.     

A Staphylococcus aureus factor exclusively removes large proteoglycan monomers from explants of articular cartilage

Exposure of explants of viable, as well as freeze-thawed, bovine articular cartilage to culture filtrates of Staphylococcus aureus increased the release of proteoglycans from the tissue significantly within 24 h. The S. aureus factor exclusively releases the large, 4 M guanidinium extractable proteoglycans from the matrix; the small proteoglycans are not affected by this factor. The large proteoglycans, released by Staphylococcal culture filtrate, had a slightly smaller hydrodynamic volume compared to the proteoglycans remaining in the matrix. Purified large proteoglycans, incubated in S. aureus filtrate, lost their capacity to aggregate with hyaluronic acid. This suggests that some culture filtrates of S. aureus contain a factor that degrades the core protein of the large proteoglycans at or near the hyaluronic acid binding region.     

Effect of homologous synovial membrane on adult human articular cartilage in organ culture, and failure to influence it with D-penicillamine.

Adult human articular cartilage has been maintained in organ culture for 8 days, and the culture medium, which was changed on alternate days, was pooled. Normal and rheumatoid cartilage was obtained from patients and 4 types of culture were prepared: (1) cartilage alone; (2) cartilage + D-penicillamine; (3) cartilage + homologous synovium; (4) cartilage, synovium, and D-penicillamine. The hexosamines and hexuronic acid were measured in the cartilage explants and in the medium. The quantity released was divided by the amount measured in the original cartilage explant and the different culture variables were compared. D-penicillamine did not alter the release of cartilage proteoglycan, but the addition of synovium did. The rheumatoid cartilage released significantly more proteoglycan than normal cartilage whether or not homologous synovium was present.