Impact loading of articular cartilage during transplantation of osteochondral autograft

Surgical reconstruction of articular surfaces by transplantation of osteochondral autografts has shown considerable promise in the treatment of focal articular lesions. During mosaicplasty, each cylindrical osteochondral graft is centred over the recipient hole and delivered by impacting the articular surface. Impact loading of articular cartilage has been associated with structural damage, loss of the viability of chondrocytes and subsequent degeneration of the articular cartilage. We have examined the relationship between single-impact loading and chondrocyte death for the specific confined-compression boundary conditions of mosaicplasty and the effect of repetitive impact loading which occurs during implantation of the graft on the resulting viability of the chondrocytes. Fresh bovine and porcine femoral condyles were used in this experiment. The percentage of chondrocyte death was found to vary logarithmically with single-impact energy and was predicted more strongly by the mean force of the impact rather than by the number of impacts required during placement of the graft. The significance of these results in regard to the surgical technique and design features of instruments for osteochondral transplantation is discussed.     

Effects of cartilage impact with and without fracture on chondrocyte viability and the release of inflammatory markers

Post‐traumatic arthritis (PTA) frequently develops after intra‐articular fracture of weight bearing joints. Loss of cartilage viability and post‐injury inflammation have both been implicated as possible contributing factors to PTA progression. To further investigate chondrocyte response to impact and fracture, we developed a blunt impact model applying 70%, 80%, or 90% surface‐to‐surface compressive strain with or without induction of an articular fracture in a cartilage explant model. Following mechanical loading, chondrocyte viability, and apoptosis were assessed. Culture media were evaluated for the release of double‐stranded DNA (dsDNA) and immunostimulatory activity via nuclear factor kappa B (NF‐κB) activity in Toll‐like receptor (TLR) ‐expressing Ramos‐Blue reporter cells. High compressive strains, with or without articular fracture, resulted in significantly reduced chondrocyte viability. Blunt impact at 70% strain induced a loss in viability over time through a combination of apoptosis and necrosis, whereas blunt impact above 80% strain caused predominantly necrosis. In the fracture model, a high level of primarily necrotic chondrocyte death occurred along the fracture edges. At sites away from the fracture, viability was not significantly different than controls. Interestingly, both dsDNA release and NF‐κB activity in Ramos‐Blue cells increased with blunt impact, but was only significantly increased in the media from fractured cores. This study indicates that the mechanism of trauma determines the type of chondrocyte death and the potential for post‐injury inflammation. (c) 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1283–1292, 2013     

Autologous chondrocyte implantation drives early chondrogenesis and organized repair in extensive full‐ and partial‐thickness cartilage defects in an equine model

Autologous chondrocyte implantation (ACI) has been used clinically for over 15 years and yet definitive evidence of chondrocyte persistence and direct impact on cartilage repair in full‐thickness lesions is scant and no data are available on ACI in partial‐thickness defects in any animal model. This study assessed the effect of chondrocytes secured using periosteal overlay in partial‐ and full‐thickness cartilage defects in the equine model. Paired cartilage defects 15 mm in diameter were made in the patellofemoral joint of 16 horse and repaired with ACI or periosteal flap alone. Response was assessed at 8 weeks by clinical, microradiographic, and histologic appearance, and by collagen type II immunohistochemistry, and proteoglycan and DNA quantification. ACI improved histologic scores in partial‐ and full‐thickness cartilage defects, including defect filling, attachment to the underlying subchondral bone, and presence of residual chondrocyte accumulations. For partial‐thickness defects chondrocyte predominance, collagen type II content, and toluidine stained matrix were enhanced, and attachment to the surrounding cartilage improved. DNA and PG content of grafted partial‐thickness defects was improved by chondrocyte implantation. Periosteal patches alone did not induce cartilage repair. This study indicated implantation of chondrocytes to cartilage defects improved healing with a combination of persisting chondrocyte regions, enhanced collagen type II formation, and better overall cartilage healing scores. Use of ACI in the more challenging partial‐thickness defects also improved histologic indices and biochemical content. The equine model of cartilage healing closely resembles cartilage repair in man, and results of this study confirm cell persistence and improved early cartilage healing events after ACI. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1121–1130, 2011     

In vitro model of full-thickness cartilage defect healing.

Integration of the host-graft interface is implicated as one of the significant reasons for lack of complete healing in osteochondral grafting procedures for the treatment of cartilage lesions. We developed an in vitro model of cartilage healing in an osteochondral setting to study the effect of developmental age and collagenase treatment. Circular full-thickness vertical surgical incisions were made in the cartilaginous portion of cylindrical bovine osteochondral specimens. Two age groups were selected: Young (1-2 months old) and Older (6-8 months old). Cartilage integration across the surgical incisions was assessed by histologic analysis and by mechanical push-out testing at 2 and 4 weeks in culture. Histologic integration as well as peak push-out shear stress was significantly higher in older calf cartilage than in the young calf. Collagenase pretreatment in the older calf samples increased push-out strength at 4 weeks. Histologic integration correlated well with the mechanical push-out strength. Developmental age and time after injury affected the response to collagenase pretreatment. This osteochondral cartilage integration model can be useful to study factors that modulate healing of surgical replacement procedures in vitro, which may aid the development of newer approaches to promote the healing of cartilage defects.     

Long-term viability and mechanical behavior following laser cartilage reshaping

OBJECTIVE: To investigate the long-term in vivo effect of laser dosimetry on rabbit septal cartilage integrity, viability, and mechanical behavior. METHODS: Nasal septal cartilage specimens (control and irradiated pairs) were harvested from 18 rabbits. Specimens were mechanically deformed and irradiated with an Nd:YAG laser across a broad dosimetry range (4-8 W and 6-16 seconds). Treated specimens and controls were autologously implanted into a subperichondrial auricular pocket. Specimens were harvested an average +/- SD of 208 +/- 35 days later. Tissue integrity, histology, chondrocyte viability, and mechanical property evaluations were performed. Tissue damage results were compared with Monte Carlo simulation models. RESULTS: All laser-irradiated specimens demonstrated variable tissue resorption and calcification, which increased with increased dosimetry. Elastic moduli of the specimens were significantly either lower or higher than controls (all P<.05). Viability assays illustrated a total loss of viable chondrocytes within the laser-irradiated zones in all treated specimens. Histologic examination confirmed these findings. Experimental results were consistent with damage profiles determined using numerical simulations. CONCLUSION: The loss of structural integrity and chondrocyte viability observed across a broad dosimetry range underscores the importance of spatially selective heating methods prior to initiating application in human subjects.     

Mitoprotective therapy preserves chondrocyte viability and prevents cartilage degeneration in an ex vivo model of posttraumatic osteoarthritis

No disease‐modifying osteoarthritis (OA) drugs are available to prevent posttraumatic osteoarthritis (PTOA). Mitochondria (MT) mediate the pathogenesis of many degenerative diseases, and recent evidence indicates that MT dysfunction is a peracute (within minutes to hours) response of cartilage to mechanical injury. The goal of this study was to investigate cardiolipin‐targeted mitoprotection as a new strategy to prevent chondrocyte death and cartilage degeneration after injury. Cartilage was harvested from bovine knee joints and subjected to a single, rapid impact injury (24.0 ±1.4 MPa, 53.8 ± 5.3 GPa/s). Explants were then treated with a mitoprotective peptide, SS‐31 (1µM), immediately post‐impact, or at 1, 6, or 12 h after injury, and then cultured for up to 7 days. Chondrocyte viability and apoptosis were quantified in situ using confocal microscopy. Cell membrane damage (lactate dehydrogenase activity) and cartilage matrix degradation (glycosaminoglycan loss) were quantified in cartilage‐conditioned media. SS‐31 treatment at all time points after impact resulted in chondrocyte viability similar to that of un‐injured controls. This effect was sustained for up to a week in culture. Further, SS‐31 prevented impact‐induced chondrocyte apoptosis, cell membrane damage, and cartilage matrix degeneration. Clinical Significance: This study is the first investigation of cardiolipin‐targeted mitoprotective therapy in cartilage. These results suggest that even when treatment is delayed by up to 12 h after injury, mitoprotection may be a useful strategy in the prevention of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2147–2156, 2018.

     

Articular cartilage injuries

The acute and repetitive impact and torsional joint loading that occurs during participation in sports can damage articular surfaces causing pain, joint dysfunction, and effusions. In some instances, this articular surface damage leads to progressive joint degeneration. Three classes of chondral and osteochondral injuries can be identified based on the type of tissue damage and the repair response: (1) damage to the joint surface that does not cause visible mechanical disruption of the articular surface, but does cause chondral damage and may cause subchondral bone injury; (2) mechanical disruption of the articular surface limited to articular cartilage; and (3) mechanical disruption of articular cartilage and subchondral bone. In most instances, joints can repair damage that does not disrupt the articular surface if they are protected from additional injury. Mechanical disruption of articular cartilage stimulates chondrocyte synthetic activity, but it rarely results in repair of the injury. Disruption of subchondral bone stimulates chondral and bony repair, but it rarely restores an articular surface that duplicates the biologic and mechanical properties of normal articular cartilage. In selected patients, surgeons have used operative treatments including penetrating subchondral bone, soft tissue grafts, and cell transplants and osteochondral autografts and allografts to restore articular surfaces after chondral injuries. Experimental studies indicate that use of artificial matrices and growth factors also may promote formation of a new joint surface. However, an operative treatment of an articular surface injury that will benefit patients must not just provide a new joint surface, it must produce better long-term joint function than would be expected if the injury was left untreated or treated by irrigation and debridement alone. Therefore, before selecting a treatment for a patient with an articular cartilage injury, the surgeon should define the type of injury and understand its likely natural history.     

Damage and degenerative changes in menisci‐covered and exposed tibial osteochondral regions after simulated landing impact compression—a porcine study

The capacity of menisci‐covered and exposed tibial osteochondral regions in resisting impact‐induced damage and degeneration is not fully understood. This study sought to evaluate damage and degenerative changes in these regions upon a single simulated landing impact. We hypothesized that the menisci‐covered regions are more susceptible to damage and degeneration than their exposed counterparts. Menisci‐covered and exposed tibial osteochondral explants were extracted from fresh porcine hind legs and placed in culture up to 14 days. Impact compression, based on a single 10‐Hz haversine, was performed at Day 1. Control (non‐impact) and impacted explants were randomly selected for cell viability assessment, glycoaminoglycan and collagen content assays, histology, immunohistochemistry, and micro‐computed tomography. When subjected to 2‐mm displacement compression, exposed explants achieved a significantly higher peak impact stress (p < 0.05) than menisci‐covered explants. No significant difference in cell viability, glycoaminoglycan and collagen content, and Mankin scores (p > 0.05) was observed between both explant groups. Both groups were observed with reduced proteoglycan and type II collagen staining at Day 14; the exposed group was noted with increased cartilage volume at Days 7–14. The inferior resistance of menisci‐covered regions, against impact‐induced damage and degeneration, is a potential factor that may contribute to the meniscectomy model of osteoarthritis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1100–1108, 2009     

Inhibition of cell‐matrix adhesions prevents cartilage chondrocyte death following impact injury

Focal adhesions are transmembrane protein complexes that attach chondrocytes to the pericellular cartilage matrix and in turn, are linked to intracellular organelles via cytoskeleton. We previously found that excessive compression of articular cartilage leads to cytoskeleton‐dependent chondrocyte death. Here we tested the hypothesis that this process also requires integrin activation and signaling via focal adhesion kinase (FAK) and Src family kinase (SFK). Osteochondral explants were treated with FAK and SFK inhibitors (FAKi, SFKi, respectively) for 2 h and then subjected to a death‐inducing impact load. Chondrocyte viability was assessed by confocal microscopy immediately and at 24 h post‐impact. With no treatment immediate post‐impact viability was 59%. Treatment with 10 µM SFKi, 10 μM, or 100 µM FAKi improved viability to 80%, 77%, and 82%, respectively (p < 0.05). After 24 h viability declined to 34% in controls, 48% with 10 µM SFKi, 45% with 10 µM FAKi, and 56% with 100 µM FAKi (p < 0.01) treatment. These results confirmed that most of the acute chondrocyte mortality was FAK‐ and SFK‐dependent, which implicates integrin‐cytoskeleton interactions in the death signaling pathway. Together with previous findings, these data support the hypothesis that the excessive tissue strains accompanying impact loading induce death via a pathway initiated by strain on cell adhesion receptors. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:448–454, 2014.     

Genipin crosslinking of cartilage enhances resistance to biochemical degradation and mechanical wear

Collagen crosslinking enhances many beneficial properties of articular cartilage, including resistance to chemical degradation and mechanical wear, but many crosslinking agents are cytotoxic. The purpose of this study was to evaluate the effectiveness of genipin, a crosslinking agent with favorable biocompatibility and cytotoxicity, as a potential treatment to prevent the degradation and wear of articular cartilage. First, the impact of genipin concentration and treatment duration on the viscoelastic properties of bovine articular cartilage was quantified. Next, two short‐term (15 min) genipin crosslinking treatments were chosen, and the change in collagenase digestion, cartilage wear, and the friction coefficient of the tissue with these treatments was measured. Finally, chondrocyte viability after exposure to these genipin treatments was assessed. Genipin treatment increased the stiffness of healthy, intact cartilage in a dose‐dependent manner. The 15‐min crosslinking treatments improved cartilage's resistance to both chemical degradation, particularly at the articular surface, and to damage due to mechanical wear. These enhancements were achieved without sacrificing the low coefficient of friction of the tissue and at a genipin dose that preserved chondrocyte viability. The results of this study suggest that collagen crosslinking via genipin may be a promising preventative treatment to slow the degradation of cartilage. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1571–1579, 2015.     

Rotenone prevents impact‐induced chondrocyte death

Mechanical insult to articular cartilage kills chondrocytes, an event that may increase the risk of posttraumatic osteoarthritis. Recent reports indicate that antioxidants decrease impact‐induced chondrocyte death, but the source(s) of oxidants, the time course of oxidant release, and the identity of the oxidative species generated in response to injury are unknown. A better understanding of these processes could lead to new treatments of acute joint injuries. To that end, we studied the kinetics and distribution of oxidant production in osteochondral explants subjected to a single, blunt‐impact injury. We followed superoxide production by measuring the time‐dependent accumulation of chondrocyte nuclei stained with the superoxide‐sensitive probe dihydroethidium. The percentage of chondrocytes that were dihydroethidium‐positive was 35% above baseline 10 min after impact, and 65% above baseline 60 min after impact. Most positive cells were found within and near areas contacted directly by the impact platen. Rotenone, an electron transport chain inhibitor, was used to test the hypothesis that mitochondria contribute to superoxide release. Rotenone treatment significantly reduced dihydroethidium staining, which remained steady at 15% above baseline for up to 60 min postimpact. Moreover, rotenone reduced chondrocyte death in impact sites by more than 40%, even when administered 2 h after injury (p < 0.001). These data show that much of the acute chondrocyte mortality caused by in vitro impact injuries results from superoxide release from mitochondria, and suggest that brief exposure to free radical scavengers could significantly improve chondrocyte viability following joint injury. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1057–1063, 2010     

Cell death after cartilage impact occurs around matrix cracks.

The damage from rapid high energy impacts to cartilage may contribute to the development of osteoarthritis (OA). Understanding how and when cells are damaged during and after the impact may provide insight into how these lesions progress. Mature bovine articular cartilage on the intact patella was impacted with a flat impacter to 53 MPa in 250 ms. Cell viability was determined by culturing the cartilage with nitroblue tetrazolium for 18 h or for 4 days in medium containing 5% serum before labeling (5-day sample) and compared to adjacent, non-impacted tissue as viable cells per area. There was a decrease in viable cell density only in specimens with macroscopic cracks and the loss was localized primarily near matrix cracks, which were in the upper 25% of the tissue. This was confirmed using confocal microscopy with a fluorescent live/dead assay, using 5'-chloromethylfluorescein diacetate and propidium iodide. Cell viability in the impacted regions distant from visible cracks was no different than the non-impacted control. At 5 days, viable cell density decreased in the surface layer in both the control and impacted tissue, but there was no additional impact-related change. In summary, cell death after the impaction of cartilage on bone occurred around impact induced cracks, but not in impacted areas without cracks. If true in vivo, early stabilization of the damaged area may prevent late sequelae that lead to OA.     

Cytoskeletal dissolution blocks oxidant release and cell death in injured cartilage

The mechanisms by which articular surface impact causes post‐traumatic osteoarthritis are not well understood, but studies of cartilage explants implicate the mitochondrial electron transport chain as a source of oxidants that cause chondrocyte death from mechanical injury. The linkage of mitochondria to the cytoskeleton suggests that they might release oxidants in response to mechanical strain, an effect that disrupting the cytoskeleton would prevent. To test this we investigated the effects of agents that promote the dissolution of microfilaments (cytochalasin B) or microtubules (nocodazole) on oxidant production and chondrocyte death following impact injury. Osteochondral explants treated with cytochalasin B or nocodazole for 4 h were impacted (7 J/cm²) and stained for oxidant production directly after impact and for cell viability 24 h after impact. Surfaces within and outside impact sites were then imaged by confocal microscopy. Both agents significantly reduced impact‐induced oxidant release (p < 0.05); however, cytochalasin B was more effective than nocodazole (>60% reduction vs. 40% reduction, respectively). Both agents also prevented impact induced cell death. Dissolution of the cytoskeleton by both drugs was confirmed by phalloidin staining and confocal microscopy. These findings show that chondrocyte mortality from impact injury depends substantially on mitochondrial–cytoskeletal linkage, suggesting new approaches to stem mechanically induced cartilage degeneration. © 2011 Orthopaedic Research Society. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:593–598, 2012     

Mechanical properties and structure–function relationships in articular cartilage repaired using IGF‐I gene‐enhanced chondrocytes

Several studies have demonstrated the benefits of IGF‐I gene therapy in enhancing the histologic and biochemical content of cartilage repaired by chondrocyte transplantation. However, there is little to no data on the mechanical performance of IGF‐I augmented cartilage grafts. This study evaluated the compressive properties of full‐thickness chondral defects in the equine femur repaired with and without IGF‐I gene therapy. Animals were randomly assigned to one of three study cohorts based on chondrocyte treatment provided in each defect: (i) IGF‐I gene delivered by recombinant adeno‐associated virus (rAAV)‐5; (ii) AAV‐5 delivering GFP as a reporter; (iii) naïve cells without virus. In each case, the opposite limb was implanted with a fibrin carrier without cells. Samples were prepared for confined compression testing to measure the aggregate modulus and hydraulic permeability. All treatment groups, regardless of cell content or transduction, had mechanical properties inferior to native cartilage. Overexpression of IGF‐I increased modulus and lowered permeability relative to other treatments. Investigation of structure–property relationships revealed that Ha and k were linearly correlated with GAG content but logarithmically correlated with collagen content. This provides evidence that IGF‐I gene therapy can improve healing of articular cartilage and can greatly increase the mechanical properties of repaired grafts. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:149–153, 2016.     

Impact insertion of osteochondral grafts: Interference fit and central graft reduction affect biomechanics and cartilage damage

An osteochondral graft (OCG) is an effective treatment for articular cartilage and osteochondral defects. Impact of an OCG during insertion into the osteochondral recipient site (OCR) can cause chondrocyte death and matrix damage. The aim of the present study was to analyze the effects of graft‐host interference fit and a modified OCG geometry on OCG insertion biomechanics and cartilage damage. The effects of interference fit (radius of OCG ‐ radius of OCR), loose (0.00 mm), moderate (0.05 mm), tight (0.10 mm), and of a tight fit with OCG geometry modification (central region of decreased radius), were analyzed for OCG cylinders and OCR blocks from adult bovine knee joints with an instrumented drop tower apparatus. An increasingly tight (OCG ‐ OCR) interference fit led to increased taps for insertion, peak axial force, graft cartilage axial compression, cumulative and total energy delivery to cartilage, lower time of peak axial force, lesser graft advancement during each tap, higher total crack length in the cartilage surface, and lower chondrocyte viability. The modified OCG, with reduction of diameter in the central area, altered the biomechanical insertion variables and biological consequences to be similar to those of the moderate interference fit scenario. Micro‐computed tomography confirmed structural interference between the OCR bone and both the proximal and distal bone segments of the OCGs, with the central regions being slightly separated for the modified OCGs. These results clarify OCG insertion biomechanics and mechanobiology, and introduce a simple modification of OCGs that facilitates insertion with reduced energy while maintaining a structural interference fit. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:377–386, 2018.     

Effects of Loading Conditions on Articular Cartilage in a Metal‐on‐Cartilage Pairing

The aim of this in vitro study was to investigate the response of articular cartilage to frictional load when sliding against a metal implant, and identify potential mechanisms of damage to articular cartilage in a metal‐on‐cartilage pairing. Bovine osteochondral cylinders were reciprocally slid against metal cylinders (cobalt–chromium–molybdenum alloy) with several variations of load and sliding velocity using a microtribometer. The effects of different loads and velocities, and the resulting friction coefficients on articular cartilage, were evaluated by measuring histological and metabolic outcomes. Moreover, the biotribocorrosion of the metal was determined. Chondrocytes stimulated with high load and velocity showed increased metabolic activity and cartilage‐specific gene expression. In addition, higher load and velocity resulted in biotribocorrosion of the metal implant and damage to the surface of the articular cartilage, whereas low velocity and a high coefficient of friction increased the expression of catabolic genes. Articular cartilage showed particular responses to load and velocity when sliding against a metal implant. Moreover, metal implants showed tribocorrosion. Therefore, corrosion particles may play a role in the mechano‐biochemical wear of articular cartilage after implantation of a metal implant. These findings may be useful to surgeons performing resurfacing procedures and total knee arthroplasty. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 37:2531–2539, 2019     

Wnt/β‐catenin signaling of cartilage canal and osteochondral junction chondrocytes and full thickness cartilage in early equine osteochondrosis

The objective of this study was to elucidate gene and protein expression of Wnt signaling molecules in chondrocytes of foals having early osteochondrosis (OC) versus normal controls. The hypothesis was that increased expression of components of Wnt signaling pathway in osteochondral junction (OCJ) and cartilage canal (CC) chondrocytes would be found in early OC when compared to controls. Paraffin‐embedded osteochondral samples (7 OC, 8 normal) and cDNA from whole cartilage (7 OC, 10 normal) and chondrocytes surrounding cartilage canals and osteochondral junctions captured with laser capture microdissection (4 OC, 6 normal) were obtained from femoropatellar joints of 17 immature horses. Equine‐specific Wnt signaling molecule mRNA expression levels were evaluated by two‐step real‐time qPCR. Spatial tissue protein expression of β‐catenin, Wnt‐11, Wnt‐4, and Dkk‐1 was determined by immunohistochemistry. There was significantly decreased Wnt‐11 and increased β‐catenin, Wnt‐5b, Dkk‐1, Lrp6, Wif‐1, Axin1, and SC‐PEP gene expression in early OC cartilage canal chondrocytes compared to controls. There was also significantly increased β‐catenin gene expression in early OC osteochondral junction chondrocytes compared to controls. Based on this study, abundant gene expression differences in OC chondrocytes surrounding cartilage canals suggest pathways associated with catabolism and inhibition of chondrocyte maturation are targeted in early OC pathogenesis. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1433–1438, 2015.     

Oxidant conditioning protects cartilage from mechanically induced damage

Articular cartilage degeneration in osteoarthritis has been linked to abnormal mechanical stresses that are known to cause chondrocyte apoptosis and metabolic derangement in in vitro models. Evidence implicating oxidative damage as the immediate cause of these harmful effects suggests that the antioxidant defenses of chondrocytes might influence their tolerance for mechanical injury. Based on evidence that antioxidant defenses in many cell types are stimulated by moderate oxidant exposure, we hypothesized that oxidant preconditioning would reduce acute chondrocyte death and proteoglycan depletion in cartilage explants after exposure to abnormal mechanical stresses. Porcine cartilage explants were treated every 48 h with tert‐butyl hydrogen peroxide (tBHP) at nonlethal concentrations (25, 100, 250, and 500 µM) for a varying number of times (one, two, or four) prior to a bout of unconfined axial compression (5 MPa, 1 Hz, 1800 cycles). When compared with untreated controls, tBHP had significant positive effects on post‐compression viability, lactate production, and proteoglycan losses. Overall, the most effective regime was 100 µM tBHP applied four times. RNA analysis revealed significant effects of 100 µM tBHP on gene expression. Catalase, hypoxia‐inducible factor‐1alpha (HIF‐1α), and glyceraldehyde 6‐phosphate dehydrogenase (GAPDH) were significantly increased relative to untreated controls in explants treated four times with 100 µM tBHP, a regime that also resulted in a significant decrease in matrix metalloproteinase‐3 (MMP‐3) expression. These findings demonstrate that repeated exposure of cartilage to sublethal concentrations of peroxide can moderate the acute effects of mechanical stress, a conclusion supported by evidence of peroxide‐induced changes in gene expression that could render chondrocytes more resistant to oxidative damage. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:914–920, 2010     

Intra‐articular autologous uncultured adipose‐derived stromal cell transplantation inhibited the progression of cartilage degeneration

The role of uncultured adipose‐derived stromal cells for osteoarthritis treatment remains unclear despite sporadic reports supporting their use in clinical settings. This study aimed to evaluate the therapeutic effects of autologous uncultured adipose‐derived stromal cell transplantation in a rabbit osteoarthritis model. Uncultured adipose‐derived stromal cells isolated from rabbits were administered via intra‐articular injection into the knees after osteoarthritis onset. Animals were sacrificed at 8 and 12 weeks after osteoarthritis onset to compare the macroscopic, histological, and immunohistochemical characteristics between the uncultured adipose‐derived stromal cell and control groups. Co‐culture assay was also performed. The chondrocytes isolated from the model were co‐cultured with adipose‐derived stromal cells. The cell viability of chondrocytes and expression of chondrocyte‐specific genes in the co‐culture (uncultured adipose‐derived stromal cell) group were compared with the mono‐culture (control; chondrocytes only) group. In macroscopic and histological analyses, the uncultured adipose‐derived stromal cell group showed less damage to the cartilage surface than the control group at 8 and 12 weeks after osteoarthritis onset. In immunohistochemical and co‐culture assay, the uncultured adipose‐derived stromal cell group showed higher expression of collagen type II and SRY box‐9 and lower expression of matrix metalloproteinase‐13 than the control group. The cell viability of chondrocytes in the uncultured adipose‐derived stromal cell group was higher than that in the control group. Intra‐articular autologous uncultured adipose‐derived stromal cell transplantation inhibited the progression of cartilage degeneration in a rabbit osteoarthritis model by regulating chondrocyte viability and secreting chondrocyte‐protecting cytokines or growth factors, which promote anabolic factors and inhibit catabolic factors. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1376–1386, 2019.     

Effect of impact on chondrocyte viability during insertion of human osteochondral grafts

BACKGROUND: Osteochondral grafts, used to treat chondral and osteochondral defects, require high insertional forces that may affect the viability of chondrocytes in the graft. The objectives of this study were to (1) measure the loading impact during insertion of osteochondral grafts, (2) evaluate the effect of insertional loading on chondrocyte viability, and (3) assess this effect on chondrocyte apoptosis and activation of caspase-3. METHODS: The distal parts of twelve fresh femora from six adult human cadavers were harvested within seventy-two hours after the death of the donor. From each femur, four 15-mm-diameter cylindrical osteochondral grafts were isolated; two of these grafts (a total of twenty-four grafts in the study) were transplanted with standard impact insertion into recipient sockets in the other condyle of the ipsilateral femur. The other two grafts served as unloaded controls. Loads were measured during the insertion of ten of the twenty-four transplanted grafts. Full-thickness cartilage disks were then removed from the grafts, incubated for up to forty-eight hours, and analyzed for cell viability, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive reactivity, and caspase-3 activation, each as a function of the depth from the articular surface. RESULTS: The insertion of an osteochondral graft was characterized, on the average (and standard deviation), by 10 +/- 4 impacts, each generating 2.4 +/- 0.9 kN of load and 13.3 +/- 4.9 MPa of stress for a duration of 0.57 +/- 0.13 ms with a 0.62 +/- 0.25 N.s impulse. Impact insertion increased cell death in the superficial 500 mum to 21% at one hour (p < 0.001) and 47% at forty-eight hours (p < 0.001) and also increased cell death in deeper layers at forty-eight hours. Some cell death was due to apoptosis, as indicated by an increase in caspase-3 activation at eight hours (p < 0.01) and TUNEL-positive cells at forty-eight hours (p < 0.05) in the superficial 500 mum of impacted cartilage. CONCLUSIONS: Impact insertion of osteochondral grafts generates damaging loads that cause chondrocyte death, particularly in the superficial zone, mainly as a result of apoptosis mediated by the activation of caspases. CLINICAL RELEVANCE: Chondrocyte death that occurs during impact insertion of osteochondral grafts may lead to compromised function. Understanding the mechanisms and consequences of such impact loading may provide insights into potential therapeutic interventions, or lead to changes in the insertion technique, to decrease the cell injury associated with impact loading.