Dispase/collagenase cocktail allows for coisolation of satellite cells and fibroadipogenic progenitors from human skeletal muscle.

Satellite cells (SCs) and fibroadipogenic progenitors (FAPs) are progenitor populations found in muscle that form new myofibers postinjury. Muscle development, regeneration, and tissue-engineering experiments require robust progenitor populations, yet their isolation and expansion are difficult given their scarcity in muscle, limited muscle biopsy sizes in humans, and lack of methodological detail in the literature. Here, we investigated whether a dispase and collagenase type 1 and 2 cocktail could allow dual isolation of SCs and FAPs, enabling significantly increased yield from human skeletal muscle. Postdissociation, we found that single cells could be sorted into CD56 + CD31-CD45- (SC) and CD56-CD31-CD45- (FAP) cell populations, expanded in culture, and characterized for lineage-specific marker expression and differentiation capacity; we obtained ∼10% SCs and ∼40% FAPs, with yields twofold better than what is reported in current literature. SCs were PAX7+ and retained CD56 expression and myogenic fusion potential after multiple passages, expanding up to 1012 cells. Conversely, FAPs expressed CD140a and differentiated into either fibroblasts or adipocytes upon induction. This study demonstrates robust isolation of both SCs and FAPs from the same muscle sample with SC recovery more than two times higher than previously reported, which could enable translational studies for muscle injuries.NEW & NOTEWORTHY We demonstrated that a dispase/collagenase cocktail allows for simultaneous isolation of SCs and FAPs with 2× higher SC yield compared with other studies. We provide a thorough characterization of SC and FAP in vitro expansion that other studies have not reported. Following our dissociation, SCs and FAPs were able to expand by up to 1012 cells before reaching senescence and maintained differentiation capacity in vitro demonstrating their efficacy for clinical translation for muscle injury.

Ultrasound-guided percutaneous peripheral nerve stimulation: neuromodulation of the suprascapular nerve and brachial plexus for postoperative analgesia following ambulatory rotator cuff repair. A proof-of-concept study.

BACKGROUND AND OBJECTIVES: Percutaneous peripheral nerve stimulation (PNS) is an analgesic modality involving the insertion of a lead through an introducing needle followed by the delivery of electric current. This modality has been reported to treat chronic pain as well as postoperative pain following knee and foot surgery. However, it remains unknown if this analgesic technique may be used in ambulatory patients following upper extremity surgery. The purpose of this proof-of-concept study was to investigate various lead implantation locations and evaluate the feasibility of using percutaneous brachial plexus PNS to treat surgical pain following ambulatory rotator cuff repair in the immediate postoperative period. METHODS: Preoperatively, an electrical lead (SPR Therapeutics, Cleveland, Ohio) was percutaneously implanted to target the suprascapular nerve or brachial plexus roots or trunks using ultrasound guidance. Postoperatively, subjects received 5 min of either stimulation or sham in a randomized, double-masked fashion followed by a 5 min crossover period, and then continuous stimulation until lead removal postoperative days 14-28. RESULTS: Leads (n=2) implanted at the suprascapular notch did not appear to provide analgesia, and subsequent leads (n=14) were inserted through the middle scalene muscle and placed to target the brachial plexus. Three subjects withdrew prior to data collection. Within the recovery room, stimulation did not decrease pain scores during the first 40 min of the remaining subjects with brachial plexus leads, regardless of which treatment subjects were randomized to initially. Seven of these 11 subjects required a single-injection interscalene nerve block for rescue analgesia prior to discharge. However, subsequent average resting and dynamic pain scores postoperative days 1-14 had a median of 1 or less on the Numeric Rating Scale, and opioid requirements averaged less than 1 tablet daily with active stimulation. Two leads dislodged during use and four fractured on withdrawal, but no infections, nerve injuries, or adverse sequelae were reported. CONCLUSIONS: This proof-of-concept study demonstrates that ultrasound-guided percutaneous PNS of the brachial plexus is feasible for ambulatory shoulder surgery, and although analgesia immediately following surgery does not appear to be as potent as local anesthetic-based peripheral nerve blocks, the study suggests that this modality may provide analgesia and decrease opioid requirements in the days following rotator cuff repair. Therefore, it suggests that a subsequent, large, randomized clinical trial with an adequate control group is warranted to further investigate this therapy in the management of surgical pain in the immediate postoperative period. However, multiple technical issues remain to be resolved, such as the optimal lead location, insertion technique, and stimulating protocol, as well as preventing lead dislodgment and fracture. TRIAL REGISTRATION NUMBER: NCT02898103.

Ultrasound-Guided Percutaneous Peripheral Nerve Stimulation: Neuromodulation of the Femoral Nerve for Postoperative Analgesia Following Ambulatory Anterior Cruciate Ligament Reconstruction: A Proof of Concept Study.

OBJECTIVES: The purpose of this prospective proof of concept study was to investigate the feasibility of using percutaneous peripheral nerve stimulation of the femoral nerve to treat pain in the immediate postoperative period following ambulatory anterior cruciate ligament reconstruction with a patellar autograft. MATERIALS AND METHODS: Preoperatively, an electrical lead (SPRINT, SPR Therapeutics, Inc., Cleveland, OH, USA) was percutaneously implanted with ultrasound guidance anterior to the femoral nerve caudad to the inguinal crease. Within the recovery room, subjects received 5 min of either stimulation or sham in a randomized, double-masked fashion followed by a 5-min crossover period, and then continuous active stimulation until lead removal postoperative Day 14-28. Statistics were not applied to the data due to the small sample size of this feasibility study. RESULTS: During the initial 5-min treatment period, subjects randomized to stimulation (n = 5) experienced a slight downward trajectory (decrease of 7%) in their pain over the 5 min of treatment, while those receiving sham (n = 5) reported a slight upward trajectory (increase of 4%) until their subsequent 5-min stimulation crossover, during which time they also experienced a slight downward trajectory (decrease of 11% from baseline). A majority of subjects (80%) used a continuous adductor canal nerve block for rescue analgesia (in addition to stimulation) during postoperative Days 1-3, after which the median resting and dynamic pain scores remained equal or less than 1.5 on the numeric rating scale, respectively, and the median daily opioid consumption was less than 1.0 tablet. CONCLUSIONS: This proof of concept study demonstrates that percutaneous femoral nerve stimulation is feasible for ambulatory knee surgery; and suggests that this modality may be effective in providing analgesia and decreasing opioid requirements following anterior cruciate ligament reconstruction. clinicaltrials.gov: NCT02898103.

Failed healing of rotator cuff repair correlates with altered collagenase and gelatinase in supraspinatus and subscapularis tendons.

BACKGROUND: Despite improvements in arthroscopic rotator cuff repair technique and technology, a significant rate of failed tendon healing persists. Improving the biology of rotator cuff repairs may be an important focus to decrease this failure rate. The objective of this study was to determine the mRNA biomarkers and histological characteristics of repaired rotator cuffs that healed or developed persistent defects as determined by postoperative ultrasound. HYPOTHESIS: Increased synovial inflammation and tendon degeneration at the time of surgery are correlated with the failed healing of rotator cuff tendons. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: Biopsy specimens from the subscapularis tendon, supraspinatus tendon, glenohumeral synovium, and subacromial bursa of 35 patients undergoing arthroscopic rotator cuff repair were taken at the time of surgery. Expression of proinflammatory cytokines, tissue remodeling genes, and angiogenesis factors was evaluated by quantitative real-time polymerase chain reaction. Histological characteristics of the affected tissue were also assessed. Postoperative (>6 months) ultrasound was used to evaluate the healing of the rotator cuff. General linear modeling with selected mRNA biomarkers was used to predict rotator cuff healing. RESULTS: Thirty patients completed all analyses, of which 7 patients (23%) had failed healing of the rotator cuff. No differences in demographic data were found between the defect and healed groups. American Shoulder and Elbow Surgeons shoulder scores collected at baseline and follow-up showed improvement in both groups, but there was no significant difference between groups. Increased expression of matrix metalloproteinase 1 (MMP-1) and MMP-9 was found in the supraspinatus tendon in the defect group versus the healed group (P = .006 and .02, respectively). Similar upregulation of MMP-9 was also found in the subscapularis tendon of the defect group (P = .001), which was consistent with the loss of collagen organization as determined by histological examination. From a general linear model, the upregulation of MMP-1 and MMP-9 was highly correlated with failed healing of the rotator cuff (R(2) = .656). CONCLUSION: The upregulation of tissue remodeling genes in the torn rotator cuff at the time of surgery provides a snapshot of the biological environment surrounding the torn rotator cuff that is closely related to the healing of repaired rotator cuffs.

Alterations in internal rotation and alpha angles are associated with arthroscopic cam decompression in the hip.

BACKGROUND: Symptomatic labral tears of the hip are associated with bony abnormalities of the femoral head and acetabulum, resulting in impingement. These patients have characteristic internal rotation limitations, which can result in compensatory athletic injury patterns around the hip, pelvis, and lumbar spine. HYPOTHESIS: Patients undergoing arthroscopic cam decompression will have improvement in internal rotation after decompression. Patients with decreased femoral neck anteversion will have decreased preoperative internal rotation of the hip and show less improvement after cam decompression. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Patients undergoing arthroscopic decompression of cam and pincer lesions of the hip and treatment of labral injury were evaluated for range of motion and bony anatomy by preoperative computed tomography and pre- and postoperative radiographs. Patients were excluded for age older than 40 years, arthritic changes of the joint, and revision setting. RESULTS: Fifty-five patients (56 hips) were treated with selective labral debridement with functional labral preservation (33/56) or selective labral debridement with labral refixation (23/56) and cam decompression. Fifty-one of the 56 had resection of associated pincer lesions. Patients were divided into femoral anteversion subgroups: normal (5°-20°, 34 patients), increased (>20°, 8 patients), and decreased (<5°, 13 patients). Mean patient age was 24.7 ± 6.3 years (range, 14-39 years). Alpha angle, a measure of the head-neck offset, decreased from 68.0° ± 10.0° preoperatively to 43.4° ± 4.0° after decompression (P < .001). Internal rotation of the hip increased from 9.9° ± 6.6° preoperatively to 27.6° ± 6.4° after decompression (P < .001) and 30.1° ± 5.3° at 3 months (P < .001). Hip flexion was not significantly different immediately after decompression but was significantly improved from 115.7° ± 13.3° preoperatively to 127.9° ± 6.6° at 3 months postoperatively (P < .003). Although improvement in internal rotation after decompression increased independent of femoral version, patients with abnormal version had altered internal rotation with increased values associated with increased anteversion (15.7° ± 5.4°/34.3° ± 6.7°) and decreased with relative retroversion (7.1° ± 8.3°/25.2° ± 4.9°; P < .05). CONCLUSION: Arthroscopic decompression results in improvement of the radiographic alpha angle and normalization of internal rotation in impingement-related disease of the hip. Internal rotation improvements can be achieved even in the setting of femoral retroversion.

Return to athletic activity after osteochondral allograft transplantation in the knee.

BACKGROUND: Fresh-stored osteochondral allografts have been used successfully to resurface large chondral and osteochondral defects of the knee. However, there are limited data available for the return to athletic activity. PURPOSE: To review the rate of return to athletic activity after osteochondral allograft transplantation in the knee and to identify any potential risk factors for not returning to sport. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Forty-three athletes were treated with fresh-stored osteochondral allograft transplantation for symptomatic large chondral or osteochondral defects of the knee from 2000 to 2010. The average age of the athletes (30 men, 13 women) was 32.9 years (range, 18-49 years). Patients were prospectively evaluated by International Knee Documentation Committee (IKDC), activities of daily living scale of the Knee Injury and Osteoarthritis Outcome Score (KOOS), Marx Activity Rating Scale, and Cincinnati Sports Activity Scale scores. A multivariable regression analysis was performed to identify potential risk factors for failure to return to sport at the preinjury level. RESULTS: At an average 2.5-year follow-up, limited return to sport was possible in 38 of 43 athletes (88%), with full return to the preinjury level achieved in 34 of 43 athletes (79%). In these 34 athletes, time to return to sport was 9.6 ± 3.0 months. Age ≥25 years (P = .04) and preoperative duration of symptoms greater than 12 months (odds ratio, 37; P = .003) negatively affected the ability to return to sport. In the athletes who returned to their previous level of competition, IKDC (P < .001), KOOS (P = .02), and Marx Activity Rating Scale (P < .001) scores were all significantly greater than in those athletes who did not return to sport. CONCLUSION: Osteochondral allograft transplantation in an athletic population for chondral and osteochondral defects in the knee allows for a high rate of return to sport. Risk factors for not returning to sport included age ≥25 years and preoperative duration of symptoms ≥12 months.

Allograft use in anterior cruciate ligament reconstruction.

Anterior cruciate ligament (ACL) reconstruction is one of the most common procedures performed by orthopedic surgeons. While autograft reconstruction remains the gold standard, allograft tissues have become a controversial option for ACL reconstruction. No data currently exist regarding recent trends in graft choices, and no consensus exists over which graft type is most appropriate for which patient. In this article, we examine trends in ACL graft choice at our institution, and review the pertinent information a surgeon must consider when making this decision. We reviewed operating room records from 2002 to 2008 to determine trends in graft choice for primary single bundle ACL reconstruction. Total number of procedures performed, graft choices, and patient ages were recorded. Patients were divided into the following age groups: less than 16, 16 to 20, 21 to 30, 31 to 40, 41 to 50, and over 50. Percent of ACL reconstructions using allograft was calculated for each year, as well as for each age group. Data were analyzed for trends in ACL graft choice over this time period as well as for trends in graft choice by age. We hypothesized that the rate of allograft use in primary ACL reconstruction had increased over time and that allograft use was associated with higher patient age. We also review the risks, safety, and standards for tissue procurement. Allograft use increased significantly (p < 0.001) from 2002 (17%) to 2008 (46%). There was also a significant difference (p < 0.001) in average age of patients receiving allografts (40.4 years) and autografts (26.4 years). Allograft use was significantly associated with higher patient age (p < 0.05) and increased with each successive age group from a rate of 9.9% in patients under 16 to 79.9% in patients over 50. Our study found that allograft use in primary ACL reconstruction has significantly increased from 2002 to 2008 and is significantly more common in older patients.

Role of apoptotic and matrix-degrading genes in articular cartilage and meniscus of mature and aged rabbits during development of osteoarthritis.

OBJECTIVE: To determine expression patterns of apoptotic and matrix-degrading genes during aging and development of osteoarthritis (OA), using a rabbit model of induced OA. METHODS: Six mature and 6 aged rabbits underwent anterior cruciate ligament transection and were killed 4 and 8 weeks after surgery, respectively, to create early-grade and advanced-grade OA. RNA from articular cartilage and menisci was examined for expression of the genes caspase 8, Fas, Fas ligand, p53, aggrecanase, matrix metalloproteinase 1 (MMP-1), and MMP-3. A second cohort of animals that had undergone no intervention in the joint was also killed. Parametric data were analyzed with analysis of variance and Student's t-tests, while nonparametric data were assessed with the Mann-Whitney U test. RESULTS: Expression levels of Fas, caspase 8, FasL, and MMP-1 were significantly higher (>100%) in aged cartilage compared with mature cartilage (P < 0.05). After induction of OA, expression of apoptotic genes in aged rabbits remained high, while significant up-regulation of Fas and caspase 8 (nearly 150% increase) was observed in mature rabbits (P < 0.05). No significant up-regulation of these genes was observed in the menisci of aged or mature rabbits prior to or after induction of OA. Development of OA occurred more rapidly in aged cartilage compared with mature cartilage (P < 0.05). CONCLUSION: Differential expression of apoptotic and matrix-degrading genes occurs in aged compared with mature cartilage, both at baseline and during development of OA. This may be responsible for faster degradation of aged cartilage and its predisposition for developing OA.

Prolonged storage of osteochondral allografts: does the addition of fetal bovine serum improve chondrocyte viability?

Osteochondral plugs were harvested from eight fresh human femoral condyles within 96 hours of donor death. The plugs were either stored in a serum-free media containing glucose, salts, and amino acids or 10% fetal bovine serum at 4 degrees C. After 28 days of storage, the osteochondral plugs were analyzed for chondrocyte viability and viable cell density using confocal microscopy, proteoglycan synthesis by (35)SO4 incorporation, and glycosaminoglycan content. Chondrocyte viability and cell density were significantly lower in grafts stored in serum-free media compared to fetal bovine serum, 27% versus 68% (P < .001) and 3250 cells/mm3 versus 8960 cells/mm3, respectively (P < .001). The metabolic activity determined by proteoglycan synthesis was significantly better in the specimens stored in fetal bovine serum (P < .01). No significant difference was detected between the glycosaminoglycan content in any of the specimens. These data suggest that the quality of osteochondral allografts as measured by chondrocyte viability, viable cell density, and proteoglycan synthesis is superior after storage in fetal bovine serum versus serum-free media. These results must be taken cautiously, however, as the clinical ramifications of storage in fetal bovine serum, including potential infectious disease transmission risks and immunogenic factors, have yet to be studied.

Meniscal debridement with an arthroscopic radiofrequency wand versus an arthroscopic shaver: comparative effects on menisci and underlying articular cartilage.

PURPOSE: Meniscal debridement with an arthroscopic radiofrequency (RF) wand versus an arthroscopic shaver and their comparative effects on menisci and underlying articular cartilage were studied. METHODS: When repair is not feasible, degenerative or post-traumatic meniscal tears often need debridement. Six fresh bovine knees were harvested, the tibial plateau was dissected free from the femoral articulation and placed in a saline bath at 28 degrees C, with 10% to 15% of the posterior horn of menisci debrided arthroscopically, and the surfaces debrided using a basket punch plus shaver, punch plus RF wand, RF wand alone, and untreated control. Treatment time of each case was 24 seconds at wand power 7. We characterized an injury zone, as well as viability and metabolic activity of meniscal cells and tibial articular cartilage chondrocytes. RESULTS: Chondrocyte viability of the tibial articular surface was 96% to 98%. We saw no differences in viability or injury zone (0 to 150 microm) among debrided groups or versus the control for any experimental surface, with no significant difference in metabolic activity in menisci debrided surfaces versus control. Meniscal viability was variable with analyses showing substantial levels (150 to 500 microm) of cell death in debrided and control groups. Metabolic activity in treated meniscus was lower than in cartilage specimens. No significant differences were observed among treatment groups versus control. CONCLUSIONS: Focal areas of chondrocyte cell death were not seen. Meniscal samples showed cell death (150 to 500 mum) throughout the tissue. CLINICAL RELEVANCE: Debridement of menisci with a bipolar RF wand produces levels of cell injury and death similar to those of debridement with a basket punch mechanical shaver. The RF wand did not harm underlying articular surfaces and produced a precise cut to the meniscal surface.

Upregulation of apoptotic and matrix-related gene expression during fresh osteochondral allograft storage.

We identified changes in proapoptotic and extracellular matrix-related gene expression with prolonged storage of fresh osteochondral allografts using gene array analysis to better understand the process of graft degradation during storage. Six human distal femurs were obtained according to standard organ harvesting protocol and stored in serum-free allograft media. Each was examined at baseline (within 72 hours postmortem), 21 days (average time of implantation), and 35 days (maximum time to implantation) for proapoptotic and extracellular matrix-related gene expression using two 100-gene microarrays, cell viability using confocal microscopy, and proteoglycan synthesis via SO4 incorporation. We found numerous genes showing upregulation associated with increased storage time, including CD30, CD30 ligand, Fas, Fas ligand, tumor necrosis factor-alpha, and several caspases. Cell viability and proteoglycan synthesis also were significantly decreased with increased storage. Loss of chondrocytes via apoptosis is likely a key determinant of osteochondral allograft viability during storage, whereas extracellular matrix degeneration may occur at a later stage. These findings provide targets for future media modulation. Improved graft viability and the potential for lengthened storage periods through improved storage conditions may improve clinical outcomes and availability of fresh osteochondral allografts.

Does subchondral bone affect the fate of osteochondral allografts during storage?

BACKGROUND: Osteochondral allografts currently are hypothermically stored for a minimum of 14 days to a maximum of 28 days before surgical implantation, making storage conditions increasingly important. Previous studies have suggested that graft deterioration during storage may result from degradative factors and residual marrow elements in the subchondral bone. HYPOTHESIS: Allografts stored with large bone-to-cartilage ratios will be compromised after prolonged storage compared with grafts with minimal bone. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral plugs were harvested from 16 fresh human femoral condyles and randomly assigned to 1 of 3 groups based on bone-to-cartilage ratios: 1:1, 5:1, or 10:1. These ratios were considered on the basis that the 1:1 ratio is the minimum bone necessary to press-fit an allograft and 10:1 is the present ratio used by tissue banks for allograft storage. After 14 and 28 days of storage at 4 degrees C, the specimens were assessed for viability and viable cell density using confocal microscopy, proteoglycan synthesis by (35)SO4 incorporation, and glycosaminoglycan content. RESULTS: All grafts underwent a significant decline in viable cell density, proteoglycan synthesis, and chondrocyte viability (particularly in the superficial region) after 14 days of storage, but no differences were observed between the 1:1, 5:1, or 10:1 ratio groups at either day 14 or day 28. In addition, no significant difference was noted in the glycosaminoglycan content in any of the groups. CONCLUSION: Osteochondral allografts stored with a 10:1 bone-to-cartilage ratio, similar to tissue-banking ratios, performed no worse than allografts stored with minimal bone, suggesting that the bone-to-cartilage ratio plays little to no role in the degradation of allografts during prolonged storage. CLINICAL RELEVANCE: As the practice of osteochondral allograft resurfacing becomes more commonplace, it is important that surgeons understand the factors that affect graft quality.

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.

Characterization of mature vs aged rabbit articular cartilage: analysis of cell density, apoptosis-related gene expression and mechanisms controlling chondrocyte apoptosis.

OBJECTIVE: The prevalence of osteoarthritis (OA) is increased in aged individuals and a direct correlation between chondrocyte apoptosis and cartilage degradation secondary to OA has been demonstrated. To address the question of whether age predisposes articular cartilage to apoptosis, the objective of the present study was to characterize and compare in aged and mature non-OA rabbit articular cartilage, cell density and expression levels of specific genes associated with apoptosis. Mechanistic studies on the inhibition of induced apoptosis were also carried out. METHODS: Grade I (non-OA) femoral condyles and tibial plateaus from mature and aged rabbits were taken for assessment of viable cell density (VCD) and mRNA (reverse transcription-polymerase chain reaction) expression levels of the pro-apoptotic genes, Fas, Fas ligand (FasL), caspase-8, inducible nitric oxide synthase (iNOS) and p53. In vitro insulin-like growth factor (IGF-1)-mediated inhibition of nitric oxide (NO)-induced apoptosis was also examined using sodium nitroprusside (SNP) as NO donor. RESULTS: VCD was decreased 50-70% in aged articular cartilage relative to mature cartilage. mRNA expression levels of Fas, FasL, caspase-8 and p53 were higher in aged cartilage than in mature cartilage. iNOS expression was unchanged. IGF-1-mediated inhibition of NO-induced apoptosis was dose-dependent and reversed with addition of phosphatidylinositol-3 kinase inhibitor. CONCLUSIONS: This controlled animal model study demonstrates that age predisposes articular cartilage to changes in VCD and expression levels of specific pro-apoptotic genes. It is significant that these findings were demonstrated on cartilage that showed no prior signs of OA; it is also possible that such changes are a prelude to the age-related development of OA.