William D. Bugbee

Prolonged Storage Effects on the Articular Cartilage of Fresh Human Osteochondral Allografts

BACKGROUND Fresh osteochondral allograft transplantation is a well-established technique for the treatment of cartilage defects of the knee. It is believed that the basic paradigm of the technique is that the transplantation of viable chondrocytes maintains the articular cartilage matrix over time. Allograft tissue is typically transplanted up to forty-two days after the death of the donor, but it is unknown how the conditions and duration of storage affect the properties of fresh human osteochondral allografts. This study examined the quality of human allograft cartilage as a function of storage for a duration of one, seven, fourteen, and twenty-eight days. We hypothesized that chondrocyte viability, chondrocyte metabolic activity, and the biochemical and biomechanical properties of articular cartilage would remain unchanged after storage for twenty-eight days. METHODS Sixty osteochondral plugs were harvested from ten fresh human femoral condyles within forty-eight hours after the death of the donor and were stored in culture medium at 4 degrees C. At one, seven, fourteen, and twenty-eight days after harvest, the osteochondral plugs were analyzed for (1) viability and viable cell density by confocal microscopy, (2) proteoglycan synthesis by quantification of (35)SO(4) incorporation, (3) glycosaminoglycan content, (4) indentation stiffness, (5) compressive modulus and hydraulic permeability by static and dynamic compression testing, and (6) tensile modulus by equilibrium tensile testing. RESULTS Chondrocyte viability and viable cell density remained unchanged after storage for seven and fourteen days (p > 0.7) and then declined at twenty-eight days (p < 0.001). Proteoglycan synthesis remained unchanged at seven days (p > 0.1) and then declined at fourteen days (p < 0.01) and twenty-eight days (p < 0.001). No significant differences were detected in glycosaminoglycan content (p > 0.8), indentation stiffness (p > 0.4), compressive modulus (p > 0.05), permeability (p > 0.3), or equilibrium tensile modulus after storage for twenty-eight days (p > 0.9). CONCLUSIONS These data demonstrate that fresh human osteochondral allograft tissue stored for more than fourteen days undergoes significant decreases in chondrocyte viability, viable cell density, and metabolic activity, with preservation of glycosaminoglycan content and biomechanical properties. The cartilage matrix is preserved during storage for twenty-eight days, but the chondrocytes necessary to maintain the matrix after transplantation decreased over that time-period.

Allografts in articular cartilage repair

Hyaline articular cartilage is an avascular and insensate tissue with a distinct structural organization, which provides a low-friction and wear-resistant interface for weight-bearing surface articulation in diarthrodial joints. Ideally, articular cartilage is maintained in homeostasis over the lifetime of an individual, with its biomechanical properties inherently suited to transmit a variety of physiologic loads through a functional range of motion. However, in the skeletally mature individual, articular cartilage does not heal effectively when injured. Although several restorative options for biomimetic replacement in acquired articular cartilage defects do exist, fresh osteochondral allografting currently remains the only technique that restores anatomically appropriate, mature hyaline cartilage in large articular defects. The fundamental paradigm of fresh osteochondral allografting is the transplantation of mature orthotopic hyaline cartilage, with viable chondrocytes that survive hypothermic storage and subsequent transplantation while maintaining their metabolic activity and sustaining the surrounding collagen matrix. Fresh osteochondral allografts have application in the treatment of a wide spectrum of articular pathology, particularly conditions that include both an osseous and a chondral component. The surgical procedure for femoral condyle lesions is straightforward but demands precision to achieve reproducible results and to minimize early graft failures related to surgical technique. As with other cartilage-restorative procedures, the indications for use of fresh osteochondral allografts are still being expanded. Many clinical and basic scientific studies support the theoretical foundation and efficacy of small fragment allografting, although more scientific validation of empirical clinical practice is still needed.

Long-Term Clinical Consequences of Stress-Shielding after Total Hip Arthroplasty without Cement*

Remodeling of the femur, or so-called stress-shielding, was observed on the two-year postoperative radiographs of forty-eight (23 per cent) of 207 hips that were part of a consecutive, non-selected series of 223 hips that had had a primary arthroplasty with use of the anatomic medullary locking hip system. Three patients (three hips) died within ten years after the arthroplasty, leaving forty-four patients (forty-five hips) who had a minimum of ten years of clinical follow-up. At the time of the latest follow-up, thirty-eight patients (86 per cent) reported that they had either no or mild pain related to the hip, forty-two (95 per cent) had less pain than they had had preoperatively, and forty-one (93 per cent) were satisfied with the results of the arthroplasty. Two patients had a reoperation, but neither procedure involved the femoral component; specifically, one patient had a revision of a loose acetabular component and one had an exchange of a polyethylene liner. No femoral component was associated with clinical or radiographic evidence of loosening. Femoral osteolysis, confined to zones 1 and 7 of Gruen et al., was observed on the ten-year radiographs of four of the thirty-three hips for which such radiographs were available. Stress-shielding (defined as evidence of pronounced femoral bone-remodeling on the two-year radiographs) had not adversely affected the outcome for these four hips by the time of the latest follow-up. The findings regarding postoperative pain, function, and over-all satisfaction for the forty-four patients (forty-five hips) who were included in the present study were similar to those reported for our larger (parent) series of patients who had been managed with the anatomic medullary locking hip system and to those reported for a similar series of patients who were followed for 9.5 years after the insertion of a porous-coated anatomic prosthesis. In addition, the prevalence of acetabular and femoral osteolysis (four [12 per cent] of thirty-three hips) and that of revision of the femoral component (zero [0 per cent] of forty-five hips) were lower than those for our larger (parent) series (fifty-four [39 per cent] of 137 hips and three [1 per cent] of 201 hips, respectively) as well as those for the series of patients who had been managed with the porous-coated anatomic prosthesis (thirty-five [45 per cent] and four [5 per cent] of seventy-eight hips, respectively).

The effects of storage on fresh human osteochondral allografts.

Historically, fresh human osteochondral allografts have been stored in lactated Ringer’s solution at 4° C and then transplanted as quickly as possible, generally within 2 to 5 days, to ensure delivery of a high level of viable chondrocytes. Recently, allograft distribution companies have begun to provide fresh osteochondral allografts that are stored in a proprietary culture medium usually for at least 2 weeks before delivery to the surgeon for implantation. The effects of such storage on human cartilage have not been well-defined. In the current study the effects of storage in lactated Ringer’s solution and in culture media were assessed. After 7 days of storage in lactated Ringer’s solution, a significant decline in chondrocyte viability and metabolic activity was seen. Culture media provided significantly better preservation of the cartilage with viability and metabolic activity remaining essentially unchanged from baseline for as many as 14 days. The biochemical and biomechanical properties of the extracellular matrix remained stable with storage in both solutions with time. These data suggest that osteochondral allografts stored under traditional conditions in lactated Ringer’s solution should continue to be implanted as quickly as possible and certainly within 7 days of donor death. If kept in culture media, the storage duration may be extended to approximately 2 weeks.

Quantitative biomarker analysis of synovial gene expression by real-time PCR

Synovial biomarker analysis in rheumatoid arthritis can be used to evaluate drug effect in clinical trials of novel therapeutic agents. Previous studies of synovial gene expression for these studies have mainly relied on histological methods including immunohistochemistry and in situ hybridization. To increase the reliability of mRNA measurements on small synovial tissue samples, we developed and validated real time quantitative PCR (Q-PCR) methods on biopsy specimens. RNA was isolated from synovial tissue and cDNA was prepared. Cell-based standards were prepared from mitogen-stimulated peripheral blood mononuclear cells. Real time PCR was performed using TaqMan chemistry to quantify gene expression relative to the cell-based standard. Application of the cellular standard curve method markedly reduced intra- and inter-assay variability and corrected amplification efficiency errors compared with the C(t) method. The inter-assay coefficient of variation was less than 25% over time. Q-PCR methods were validated by demonstrating increased expression of IL-1β and IL-6 expression in rheumatoid arthritis synovial samples compared with osteoarthritis synovium. Based on determinations of sampling error and coefficient of variation, twofold differences in gene expression in serial biopsies can be detected by assaying approximately six synovial tissue biopsies from 8 to 10 patients. These data indicate that Q-PCR is a reliable method for determining relative gene expression in small synovial tissue specimens. The technique can potentially be used in serial biopsy studies to provide insights into mechanism of action and therapeutic effect of new anti-inflammatory agents.

Fresh Osteochondral Allografts

Twenty knees in 18 patients were treated (mean age, 42 years; range, 19-64 years) with fresh osteochondral allografting limited to the patellofemoral joint. The knees were analyzed retrospectively to determine the rate of successful outcomes. The trochlea and patella were treated in 12 patients and the patella was treated in eight patients. There were 11 women and seven men. The primary outcome measures were revision allografting, arthrodesis, or arthroplasty, and clinical scoring using a modified Merle D’Aubigné-Postel 18-point scale. Radiographs were available for 12 knees. There were five failures. For the remaining knees, the clinical scores increased from a mean of 11.7 points (range, 7-15 points) to 16.3 points (range, 12-18 points). Of the knees evaluated radiographically, four had no evidence of patellofemoral arthrosis, and six had only mild arthrosis. Fresh osteochondral allografting is a salvage procedure for the young, active patient with severe articular cartilage disease of the patellofemoral joint. The results of this procedure are comparable to results of described other techniques in the literature. If allograft incorporation does occur, the procedure is associated with improved pain, function, range of motion, and a low risk of progressive arthritis. Level of Evidence: Level IV (case series-no, or historical control group). See the Guidelines for Authors for a complete description of levels of evidence.

Analysis of Stored Osteochondral Allografts at the Time of Surgical Implantation

Background To date, the morphological, biochemical, and biomechanical characteristics of articular cartilage in osteochondral allografts that have been stored have not been fully described. Hypothesis Osteochondral allografts procured and stored commercially for a standard period as determined by tissue banking protocol will have compromised chondrocyte viability but preserved extracellular matrix quality. Study Design Controlled laboratory study. Methods Unused cartilage from 16 consecutive osteochondral allografts was sampled during surgery after tissue bank processing and storage. Ten grafts were examined for cell viability and viable cell density using confocal microscopy, proteoglycan synthesis via 35SO4 uptake, and glycosaminoglycan content and compared with fresh cadaveric articular cartilage. Biomechanical assessment was performed on the 6 remaining grafts by measuring the indentation stiffness of the cartilage. Results The mean storage time for the transplanted specimens was 20.3 ± 2.9 days. Chondrocyte viability, viable cell density, and 35SO4 uptake were significantly lower in allografts at implantation when compared to fresh, unstored controls, whereas matrix characteristics, specifically glycosaminoglycan content and biomechanical measures, were unchanged. In addition, chondrocyte viability in the stored allografts was preferentially decreased in the superficial zone of cartilage. Conclusion Human osteochondral allografts stored for a standard period (approximately 3 weeks) before implantation undergo decreases in cell viability, especially in the critically important superficial zone, as well as in cell density and metabolic activity, whereas matrix and biomechanical characteristics appear conserved. The exact clinical significance of these findings, however, is unknown, as there are no prospective studies examining clinical outcomes using grafts stored for extended periods. Clinical Relevance Surgeons who perform this procedure should understand the cartilage characteristics of the graft after 21 days of commercial storage in serum-free media.

Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis.

Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during DNA replication. Key members of the MMR system include MutSα (hMSH2 and hMSH6) and MutSβ (hMSH2 and hMSH3). To provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer sequences for five key microsatellites. Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis tissue. Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS. When FLS were cultured with S-nitroso-N-acetylpenicillamine, the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6). Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.

Analysis of Cartilage Tissue on a Cellular Level in Fresh Osteochondral Allograft Retrievals

Background Fresh human osteochondral allografting is a biological cartilage replacement technique used to treat articular and osteoarticular defects in the knee. A small number of grafts fail, and we analyzed every retrieved graft during a 4-year period in order to learn more about the potential causes of failure. Hypothesis A large percentage of chondrocytes still remain viable many years after fresh osteochondral allografting. Study Design Descriptive laboratory study. Methods Retrieval specimens were obtained at the time of revision surgery and immediately analyzed. Chondrocyte viability and viable cell density were determined using a live/dead staining technique followed by confocal microscopy. Glycosaminoglycan content was a measure of the cartilage matrix. Radiolabeled sulfate uptake served as a biochemical marker of chondrocyte metabolic activity. Cartilage and subchondral bone were examined histologically. Results Fourteen patients yielded a total of 26 retrieval specimens that had been originally implanted as individual fresh osteochondral allografts. Average graft survival was 42 months. Chondrocyte viability was 82% ± 17%, and chondrocyte viable cell density was 15 590 ± 5900 viable cells/mm3. Retrieved tissue demonstrated radiolabeled sulfate uptake of 437 ± 270 counts per minute and 3.5% ± 0.8% hexosamine per dry weight. Histologically, all specimens showed some degree of cartilage fibrillation. There was evidence of bone allograft incorporation in most specimens, as well as pannus formation in 4 specimens, but no evidence of immune rejection. Conclusion A small percentage of fresh osteochondral allografts fail, but the precise cause is unknown. The main theories for failure investigated here include immunologic rejection, failure of bony incorporation, and chondrocyte death causing breakdown of the cartilage matrix. We show that chondrocytes remain viable many years after transplantation, allograft bone incorporates, and immune rejection does not seem to play a primary role in failure. Clinical Relevance Fresh osteochondral allografting is becoming more common in the treatment of articular cartilage defects in the knee. Our findings support the paradigm of fresh osteochondral allografting, the transplantation of hyaline cartilage with biological incorporation of the underlying bone scaffold. The reasons for failure of a small percentage of grafts remain unclear.

Expression and regulation of cryopyrin and related proteins in rheumatoid arthritis synovium.

Background: Rheumatoid arthritis (RA) synovium is characterised by enhanced NF-κB activity and proinflammatory cytokines. Cryopyrin (CIAS-1, NALP-3, PYPAF-1) has been shown to regulate NF-κB and caspase-1 activation. Objective: To study the expression of cryopyrin, its effector molecule ASC, and its putative antagonist pyrin in RA and osteoarthritis (OA) synovium, and the main two cellular constituents of synovial lining, cultured fibroblast-like synoviocytes (FLS) and macrophages. Methods: FLS and macrophages were cultured in the presence of inflammatory mediators. Real time polymerase chain reaction was used to quantify message levels in synovial biopsy specimens and cells. In situ hybridisation was employed to localise expression of cryopyrin mRNA. Results: Cryopyrin mRNA was raised in RA synovium and detected in both lining and sublining regions. FLS from RA and OA tissue expressed low baseline levels of cryopyrin transcripts that were induced by tumour necrosis factor α (TNFα). In contrast, macrophages differentiated in vitro expressed relatively high cryopyrin levels, which were further induced by TNFα, but not by interleukin 1β. ASC mRNA levels were comparable in RA and OA tissue, FLS, and macrophages, and were depressed by TNFα in macrophages. Pyrin expression was higher in RA synovium than in OA tissue, and virtually undetectable in FLS but high in macrophages where it was unchanged by TNFα treatment. Conclusion: These results suggest that enhanced cryopyrin levels in RA synovium are due to a greater numbers of tissue macrophages, and demonstrate transcriptional regulation of cryopyrin in a chronic inflammatory disease.