Charge-Reversed Exosomes for Targeted Gene Delivery to Cartilage for Osteoarthritis Treatment.

Gene therapy has the potential to facilitate targeted expression of therapeutic proteins to promote cartilage regeneration in osteoarthritis (OA). The dense, avascular, aggrecan-glycosaminoglycan (GAG) rich negatively charged cartilage, however, hinders their transport to reach chondrocytes in effective doses. While viral vector mediated gene delivery has shown promise, concerns over immunogenicity and tumorigenic side-effects persist. To address these issues, this study develops surface-modified cartilage-targeting exosomes as non-viral carriers for gene therapy. Charge-reversed cationic exosomes are engineered for mRNA delivery by anchoring cartilage targeting optimally charged arginine-rich cationic motifs into the anionic exosome bilayer by using buffer pH as a charge-reversal switch. Cationic exosomes penetrated through the full-thickness of early-stage arthritic human cartilage owing to weak-reversible ionic binding with GAGs and efficiently delivered the encapsulated eGFP mRNA to chondrocytes residing in tissue deep layers, while unmodified anionic exosomes do not. When intra-articularly injected into destabilized medial meniscus mice knees with early-stage OA, mRNA loaded charge-reversed exosomes overcame joint clearance and rapidly penetrated into cartilage, creating an intra-tissue depot and efficiently expressing eGFP; native exosomes remained unsuccessful. Cationic exosomes thus hold strong translational potential as a platform technology for cartilage-targeted non-viral delivery of any relevant mRNA targets for OA treatment.

Use of a novel magnetically actuated compression system to study the temporal dynamics of axial and lateral strain in human osteochondral plugs.

The high water content of articular cartilage allows this biphasic tissue to withstand large compressive loads through fluid pressurization. The system presented here, termed the "MagnaSquish", provides new capabilities for quantifying the effect of rehydration on cartilage behavior during cyclic loading. An imbalanced rate of fluid exudation during load and fluid re-entry during recovery can lead to the accumulation of strain during successive loading cycles - a phenomenon known as ratcheting. Typical experimental systems for cartilage biomechanics use continuous contact between the platen and sample, which may affect tissue rehydration by compressing the top layer of cartilage and slowing fluid re-entry. To address this limitation, we developed a magnetically actuated device that provides full lift-off of the platen in between loading cycles. We investigated strain accumulation in cadaveric human osteochondral plugs during 750 loading cycles, with two dimensional profiles of the cartilage captured at 30 frames per second throughout loading and 10 min of additional free swelling recovery. Axial and lateral strain measurements were extracted from the tissue profiles using a UNet-based deep learning algorithm to circumvent manual tracing. We observed increased axial strain accumulation with shorter inter-cycle recovery, with static loading serving as the extreme case of zero recovery. The loading waveform during the 750 cycles dictated the pace of the recovery during the extended free swelling period, as shorter inter-cycle recovery led to more persistent axial strain accumulation for up to five minutes. This work showcases the importance of fluid re-entry in resisting strain accumulation during cyclical compression.

Sirtuin 6 activation rescues the age-related decline in DNA damage repair in primary human chondrocytes.

While advanced age is widely recognized as the greatest risk factor for osteoarthritis (OA), the biological mechanisms behind this connection remain unclear. Previous work has demonstrated that chondrocytes from older cadaveric donors have elevated levels of DNA damage as compared to chondrocytes from younger donors. The purpose of this study was to determine whether a decline in DNA repair efficiency is one explanation for the accumulation of DNA damage with age, and to quantify the improvement in repair with activation of Sirtuin 6 (SIRT6). After acute damage with irradiation, DNA repair was shown to be more efficient in chondrocytes from young (≤45 years old) as compared to middle-aged (50-65 years old) or older (>70 years old) cadaveric donors. Activation of SIRT6 with MDL-800 improved the repair efficiency, while inhibition with EX-527 reduced the rate of repair and increased the percentage of cells that retain high levels of damage. In addition to affecting repair after acute damage, treating chondrocytes from older donors with MDL-800 for 48 hours significantly reduced the amount of baseline DNA damage. Chondrocytes isolated from the knees of mice between 4 months and 22 months of age revealed both an increase in DNA damage with aging, and a decrease in DNA damage following MDL-800 treatment. Lastly, treating murine cartilage explants with MDL-800 lowered the percentage of chondrocytes with high p16 promoter activity, which supports the concept that using SIRT6 activation to maintain low levels of DNA damage may prevent the initiation of senescence.

Redox-active endosomes mediate α5ß1 integrin signaling and promote chondrocyte matrix metalloproteinase production in osteoarthritis.

Mechanical cues sensed by integrins induce cells to produce proteases to remodel the extracellular matrix. Excessive protease production occurs in many degenerative diseases, including osteoarthritis, in which articular cartilage degradation is associated with the genesis of matrix protein fragments that can activate integrins. We investigated the mechanisms by which integrin signals may promote protease production in response to matrix changes in osteoarthritis. Using a fragment of the matrix protein fibronectin (FN) to activate the α5ß1 integrin in primary human chondrocytes, we found that endocytosis of the integrin and FN fragment complex drove the production of the matrix metalloproteinase MMP-13. Activation of α5ß1 by the FN fragment, but not by intact FN, was accompanied by reactive oxygen species (ROS) production initially at the cell surface, then in early endosomes. These ROS-producing endosomes (called redoxosomes) contained the integrin-FN fragment complex, the ROS-producing enzyme NADPH oxidase 2 (NOX2), and SRC, a redox-regulated kinase that promotes MMP-13 production. In contrast, intact FN was endocytosed and trafficked to recycling endosomes without inducing ROS production. Articular cartilage from patients with osteoarthritis showed increased amounts of SRC and the NOX2 complex component p67phox. Furthermore, we observed enhanced localization of SRC and p67phox at early endosomes, suggesting that redoxosomes could transmit and sustain integrin signaling in response to matrix damage. This signaling mechanism not only amplifies the production of matrix-degrading proteases but also establishes a self-perpetuating cycle that contributes to the ongoing degradation of cartilage matrix in osteoarthritis.

Development of a computational-experimental framework for enhanced mechanical characterization and cross-species comparison of the articular cartilage superficial zone.

To provide a better understanding of the contribution of specific constituents (i.e. proteoglycan, collagen, fluid) to the mechanical behavior of the superficial zone of articular cartilage, a complex biological tissue with several time-dependent properties, a finite element model was developed. Optimization was then used to fit the model to microindentation experiments. We used this model to compare superficial zone material properties of mature human vs. immature bovine articular cartilage. Non-linearity and stiffness of the fiber-reinforced component of the model differed between human and bovine tissue. This may be due to the more complex collagen architecture in mature tissue and is of interest to investigate in future work.

Adipocytes regulate fibroblast function, and their loss contributes to fibroblast dysfunction in inflammatory diseases.

Fibroblasts play critical roles in tissue homeostasis, but in pathologic states can drive fibrosis, inflammation, and tissue destruction. In the joint synovium, fibroblasts provide homeostatic maintenance and lubrication. Little is known about what regulates the homeostatic functions of fibroblasts in healthy conditions. We performed RNA sequencing of healthy human synovial tissue and identified a fibroblast gene expression program characterized by enhanced fatty acid metabolism and lipid transport. We found that fat-conditioned media reproduces key aspects of the lipid-related gene signature in cultured fibroblasts. Fractionation and mass spectrometry identified cortisol in driving the healthy fibroblast phenotype, confirmed using glucocorticoid receptor gene ( NR3C1 ) deleted cells. Depletion of synovial adipocytes in mice resulted in loss of the healthy fibroblast phenotype and revealed adipocytes as a major contributor to active cortisol generation via Hsd11 ß 1 expression. Cortisol signaling in fibroblasts mitigated matrix remodeling induced by TNFα- and TGFß, while stimulation with these cytokines repressed cortisol signaling and adipogenesis. Together, these findings demonstrate the importance of adipocytes and cortisol signaling in driving the healthy synovial fibroblast state that is lost in disease.

Age and oxidative stress regulate Nrf2 homeostasis in human articular chondrocytes.

OBJECTIVE: The purpose of this study was to investigate the effect of age and oxidative stress on regulation of nuclear factor erythroid-2-related factor 2 (Nrf2) in young, old, and osteoarthritic (OA) human articular chondrocytes. DESIGN: Levels of Nrf2 in primary human chondrocytes isolated from young, old, and OA donors were measured by immunoblotting, qPCR, and immunohistochemistry. Effects on levels of Nrf2, antioxidant proteins regulated by Nrf2, as well as p65, and the anabolic response to insulin-like growth factor-1 (IGF-1) were evaluated after induction of oxidative stress with menadione, Nrf2 knockdown with siRNA, and/or Nrf2 activation with RTA-408. RESULTS: Nrf2 protein levels were significantly lower in older adult chondrocytes (∼0.59 fold; p = 0.034) and OA chondrocytes (∼0.50 fold; p = 0.016) compared to younger cells. Menadione significantly increased Nrf2 protein levels in young chondrocytes by just under four-fold without changes in old chondrocytes. Nrf2 knockdown and activation differentially regulated levels of anti-oxidant proteins including sulfiredoxin and NAD(P)H quinone dehydrogenase 1. Nrf2 activation with RTA-408 also decreased basal p65 phosphorylation, increased aggrecan and type II collagen gene expression, and increased production of proteoglycans in OA chondrocytes treated with IGF-1. CONCLUSIONS: Targeted therapeutic strategies aimed at maintaining Nrf2 activity could be useful in maintaining chondrocyte homeostasis through maintenance of intracellular antioxidant function and redox balance.

SIRT6 activation rescues the age-related decline in DNA damage repair in primary human chondrocytes.

While advanced age has long been recognized as the greatest risk factor for osteoarthritis (OA), the biological mechanisms behind this connection remain unclear. Previous work has demonstrated that chondrocytes from older cadaveric donors have elevated levels of DNA damage as compared to chondrocytes from younger donors. The purpose of this study was to determine whether a decline in DNA repair efficiency is one explanation for the accumulation of DNA damage with age, and to quantify the improvement in repair with activation of Sirtuin 6 (SIRT6). Using an acute irradiation model to bring the baseline level of all donors to the same starting point, this study demonstrates a decline in repair efficiency during aging when comparing chondrocytes from young (≤45 years old), middle-aged (50-65 years old), or older (>70 years old) cadaveric donors with no known history of OA or macroscopic cartilage degradation at isolation. Activation of SIRT6 in middle-aged chondrocytes with MDL-800 (20 µM) improved the repair efficiency, while inhibition with EX-527 (10 µM) inhibited the rate of repair and the increased the percentage of cells that retained high levels of damage. Treating chondrocytes from older donors with MDL-800 for 48 hours significantly reduced the amount of DNA damage, despite this damage having accumulated over decades. Lastly, chondrocytes isolated from the proximal femurs of mice between 4 months and 22 months of age revealed both an increase in DNA damage with aging, and a decrease in DNA damage following MDL-800 treatment.

Effects of dexamethasone and dynamic loading on cartilage of human osteochondral explants challenged with inflammatory cytokines.

Post-traumatic osteoarthritis (PTOA), characterized by articular cartilage degradation initiated in an inflammatory environment after traumatic joint injury, can lead to alterations in cartilage biomechanical properties. Low dose dexamethasone (Dex) shows chondroprotection in cartilage challenged with inflammatory cytokines, but little is known about the structural biomechanical response of human cartilage to Dex in such a diseased state. This study examined changes in the biomechanical properties and biochemical composition of the cartilage within human osteochondral explants in response to treatment with exogenous cytokines, Dex, and a regimen of cyclic loading at the start and end of culture. Osteochondral explants were harvested from five pairs of human ankle talocrural joints (Collins grade 0-1) and cultured for 10 days with/without exogenous cytokines (100 ng/mL TNFα, 50 ng/mL IL-6, 250 ng/mL sIL-6R) ± Dex (100 nM). Biomechanical testing on day-0 and day-10 enabled estimation of the unconfined compression equilibrium modulus (Ey), dynamic stiffness (Ed) and hydraulic permeability (kp) of cartilage excised from bone, accompanied by biochemical assessment of media and cartilage tissue. Dex preserved chondrocyte cell viability and decreased sulfated glycosaminoglycan (sGAG) loss and nitric oxide release, but did not alter Ey, Ed and kp (before or after loading) on day-10. In the cytokine/cytokine+Dex treated groups, sGAG content exhibited a weaker correlation with Ey and Ed than at baseline, suggesting an important role for structural rather than biochemical changes in producing biomechanical alterations in response to cytokines and Dex. These findings aid in forming a more complete profile of potential clinical effects of Dex for use in OA/PTOA treatment regimens.

Comet assay for quantification of the increased DNA damage burden in primary human chondrocytes with aging and osteoarthritis.

It is known that chondrocytes from joints with osteoarthritis (OA) exhibit high levels of DNA damage, but the degree to which chondrocytes accumulate DNA damage during "normal aging" has not been established. The goal of this study was to quantify the DNA damage present in chondrocytes obtained from cadaveric donors of a wide age range, and to compare the extent of this damage to OA chondrocytes. The alkaline comet assay was used to measure the DNA damage in normal cartilage from the ankle (talus) and the knee (femur) of cadaveric donors, as well as in OA chondrocytes obtained at the time of total knee replacement. Chondrocytes from younger donors (<45 years) had less DNA damage than older donors (>70 years) as assessed by the percentage of DNA in the comet "tail". In donors between 50 and 60 years old, there was increased DNA damage in chondrocytes from OA cartilage as compared to cadaveric. Talar chondrocytes from 23 donors between the ages of 34 and 78 revealed a linear increase in DNA damage with age (R2  = 0.865, p < 0.0001). A "two-tailed" comet assay was used to demonstrate that most of the accumulated damage is in the form of strand breaks as opposed to alkali-labile base damage. Chondrocytes from young donors required 10 Gy irradiation to recapitulate the DNA damage present in chondrocytes from older donors. Given the potential for DNA damage to contribute to chondrocyte dysfunction and senescence, this study supports the investigation of mechanisms by which hypo-replicative cell types accumulate high levels of damage.

Effect of Mechanical Mincing on Minimally Manipulated Articular Cartilage for Surgical Transplantation.

BACKGROUND: Point-of-care treatment options for medium to large symptomatic articular cartilage defects are limited. Minced cartilage implantation is an encouraging single-stage option, providing fresh viable autologous tissue with minimal morbidity and cost. PURPOSE: To determine the histological properties of mechanically minced versus minimally manipulated articular cartilage. STUDY DESIGN: Controlled laboratory study. METHODS: Remnant articular cartilage was collected from fresh femoral condylar allografts. Cartilage samples were divided into 4 groups: cartilage explants with or without fibrin glue and mechanically minced cartilage with or without fibrin glue. Samples were cultured for 42 days. Chondrocyte viability was assessed using live/dead assay. Cellular migration and outgrowth were monitored using bright-field microscopy. Extracellular matrix deposition was assessed via histological staining. Proteoglycan content and synthesis were assessed using dimethylmethylene blue assay and radiolabeled 35S-sulfate, respectively. Type II collagen (COL2A1) gene expression was analyzed via polymerase chain reaction. RESULTS: The mean viability of minced cartilage particles (34% ± 14%) was not significantly reduced compared with baseline (46% ± 13%) on day 0 (P = .90). After culture, no significant difference in the percentage of live cells was appreciated between mechanically minced (58% ± 23%) and explant (73% ± 14%) cartilage in the presence of fibrin glue (P = .52). The addition of fibrin glue did not significantly affect the viability of cartilage samples. The qualitative assessment revealed comparable cellular migration and outgrowth between groups. Proteoglycan synthesis was not significantly different between groups. Histological analysis findings were positive for COL2A1 in all groups, and matrix formation was appreciated in all groups. COL2A1 expression in minced cartilage (1.72 ± 1.88) was significantly higher than in explant cartilage (0.15 ± 0.07) in the presence of fibrin glue (P = .01). CONCLUSION: Mechanically minced articular cartilage remained viable after 42 days of culture in vitro and was comparable with cartilage explants with regard to cellular migration, outgrowth, and extracellular matrix synthesis. CLINICAL RELEVANCE: Mechanically minced articular cartilage is an encouraging intervention for the treatment of symptomatic cartilage defects. Further translational work is warranted to determine the viability of minced cartilage implantation as a single-stage therapeutic intervention in vivo.

Tissue catabolism and donor-specific dexamethasone response in a human osteochondral model of post-traumatic osteoarthritis.

BACKGROUND: Post-traumatic osteoarthritis (PTOA) does not currently have clinical prognostic biomarkers or disease-modifying drugs, though promising candidates such as dexamethasone (Dex) exist. Many challenges in studying and treating this disease stem from tissue interactions that complicate understanding of drug effects. We present an ex vivo human osteochondral model of PTOA to investigate disease effects on cartilage and bone homeostasis and discover biomarkers for disease progression and drug efficacy. METHODS: Human osteochondral explants were harvested from normal (Collins grade 0-1) ankle talocrural joints of human donors (2 female, 5 male, ages 23-70). After pre-equilibration, osteochondral explants were treated with a single-impact mechanical injury and TNF-α, IL-6, and sIL-6R ± 100 nM Dex for 21 days and media collected every 2-3 days. Chondrocyte viability, tissue DNA content, and glycosaminoglycan (sGAG) percent loss to the media were assayed and compared to untreated controls using a linear mixed effects model. Mass spectrometry analysis was performed for both cartilage tissue and pooled culture medium, and the statistical significance of protein abundance changes was determined with the R package limma and empirical Bayes statistics. Partial least squares regression analyses of sGAG loss and Dex attenuation of sGAG loss against proteomic data were performed. RESULTS: Injury and cytokine treatment caused an increase in the release of matrix components, proteases, pro-inflammatory factors, and intracellular proteins, while tissue lost intracellular metabolic proteins, which was mitigated with the addition of Dex. Dex maintained chondrocyte viability and reduced sGAG loss caused by injury and cytokine treatment by 2/3 overall, with donor-specific differences in the sGAG attenuation effect. Biomarkers of bone metabolism had mixed effects, and collagen II synthesis was suppressed with both disease and Dex treatment by 2- to 5-fold. Semitryptic peptides associated with increased sGAG loss were identified. Pro-inflammatory humoral proteins and apolipoproteins were associated with lower Dex responses. CONCLUSIONS: Catabolic effects on cartilage tissue caused by injury and cytokine treatment were reduced with the addition of Dex in this osteochondral PTOA model. This study presents potential peptide biomarkers of early PTOA progression and Dex efficacy that can help identify and treat patients at risk of PTOA.

Intramural Grant Program to Promote Research Activity Among Early-Career Faculty Members.

PROBLEM: Early grant support for junior faculty members appears to positively influence their career trajectory. The authors sought to determine whether provision of grant support that enables early-career faculty members to conduct clinical, basic science, or educational research improves their academic success and enhances retention. APPROACH: The authors compared career development and retention among 30 Cohn Fellowship recipients and 31 nonrecipients who participated in the same mentoring program. An award of $20,000 to the fellowship recipients ensured protected time for research for 1 year. Academic productivity of both groups was monitored for 6 years. OUTCOMES: The authors found statistically significant differences between the 2 groups regarding research funding and scholarly productivity. The Cohn Fellowship recipients received a total of $14.7 million in external funding vs $3.7 million for nonrecipients, reflecting mean funding of $588,116 and $196,658 per person, respectively ( P < .01). Recipients published a total of 174 peer-reviewed articles vs 26 for nonrecipients, reflecting a mean of 7 and 1 per person, respectively ( P < .01). Recipients gave a total of 268 presentations vs 25 for nonrecipients, with a mean of 11 and 1 per person, respectively ( P < .01). Furthermore, 8 of the 25 recipients who stayed at Rush University (32%) were promoted to associate professor compared with 2 of the 19 (11%) nonrecipients ( P = .15). A majority of the Cohn Fellows (25; 83%) stayed at Rush University during the study compared with 61% of nonrecipients ( P = .06). These findings suggest that even small amounts of research support received early in a career can benefit the faculty and the university as a whole. NEXT STEPS: We plan to continue gathering data to increase sample size and analyze outcomes for specific variables (e.g., time, rank, gender, promotion, retention).

Sirtuin 6 (SIRT6) regulates redox homeostasis and signaling events in human articular chondrocytes.

The nuclear localized protein deacetylase, SIRT6, has been identified as a crucial regulator of biological processes that drive aging. Among these processes, SIRT6 can promote resistance to oxidative stress conditions, but the precise mechanisms remain unclear. The objectives of this study were to examine the regulation of SIRT6 activity by age and oxidative stress and define the role of SIRT6 in maintaining redox homeostasis in articular chondrocytes. Although SIRT6 levels did not change with age, SIRT6 activity was significantly reduced in chondrocytes isolated from older adults. Using dimedone-based chemical probes that detect oxidized cysteines, we identified that SIRT6 is oxidized in response to oxidative stress conditions, an effect that was associated with reduced SIRT6 activity. Enhancement of SIRT6 activity through adenoviral SIRT6 overexpression specifically increased the basal levels of two antioxidant proteins, peroxiredoxin 1 (Prx1) and sulfiredoxin (Srx) and decreased the levels of an inhibitor of antioxidant activity, thioredoxin interacting protein (TXNIP). Conversely, in chondrocytes derived from mice with cartilage specific Sirt6 knockout, Sirt6 loss decreased Prx1 levels and increased TXNIP levels. SIRT6 overexpression decreased nuclear-generated H2O2 levels and oxidative stress-induced accumulation of nuclear phosphorylated p65. Our data demonstrate that SIRT6 activity is altered with age and oxidative stress conditions associated with aging. SIRT6 contributes to chondrocyte redox homeostasis by regulating specific members of the Prx catalytic cycle. Targeted therapies aimed at preventing the age-related decline in SIRT6 activity may represent a novel strategy to maintain redox balance in joint tissues and decrease catabolic signaling events implicated in osteoarthritis (OA).

Proteomic Clustering Reveals the Kinetics of Disease Biomarkers in Bovine and Human Models of Post-Traumatic Osteoarthritis.

Objectives: In this study, we apply a clustering method to proteomic data sets from bovine and human models of post-traumatic osteoarthritis (PTOA) to distinguish clusters of proteins based on their kinetics of release from cartilage and examined these groups for PTOA biomarker candidates. We then quantified the effects of dexamethasone (Dex) on the kinetics of release of the cartilage media proteome. Design: Mass spectrometry was performed on sample medium collected from two separate experiments using juvenile bovine and human cartilage explants (3 samples/treatment condition) during 20- or 21-day treatment with inflammatory cytokines (TNF-α, IL-6, sIL-6R) with or without a single compressive mechanical injury. All samples were incubated with or without 100 nM Dex. Clustering was performed on the correlation between normalized averaged release vectors for each protein. Results: Our proteomic method identified the presence of distinct clusters of proteins based on the kinetics of their release over three weeks of culture. Clusters of proteins with peak release after one to two weeks had biomarker candidates with increased release compared to control. Dex rescued some of the changes in protein release kinetics the level of control, and in all conditions except control, there was late release of immune-related proteins. Conclusions: We demonstrate a clustering method applied to proteomic data sets to identify and validate biomarkers of early PTOA progression and explore the relationships between the release of spatially related matrix components. Dex restored the kinetics of release to many matrix components, but not all factors that contribute to cartilage homeostasis.

CCL2 induces articular chondrocyte MMP expression through ERK and p38 signaling pathways.

Objective: In previous studies, we determined an association between increased serum and articular cartilage levels of CCL2 with osteoarthritis (OA) progression, cartilage damage and increased MMP13 in cartilage. Here we analyzed CCL2 downstream signaling mediators that lead to gene expression of cartilage catabolic markers, in healthy and OA human articular chondrocytes. Design: Human articular chondrocytes obtained from healthy or OA subjects were treated with or without recombinant human CCL2; cell lysates or mRNA were collected for immunoblotting or qRT-PCR. For pathway analysis, chondrocytes were pre-incubated with an inhibitor of CCR2 (the unique CCL2 receptor), ERK inhibitor or p38 inhibitor prior to CCL2 treatment. Results: CCL2 treatment of both healthy and OA chondrocytes activated ERK and p38 via CCR2. In healthy chondrocytes, short (6h) and prolonged (24-72h) CCL2 treatments led to Ccr2, Mmp-1, Mmp-3, Mmp-13 and Timp1 upregulation. In OA chondrocytes, CCL2 induced expression of Ccr2, Mmp-1 and Mmp-3, but not Mmp1 and Timp1, and only following longer treatments (72h). In both healthy and OA chondrocytes, the CCL2-mediated upregulation of Ccr2 and cartilage catabolic markers was mediated by ERK and p38 signaling. Conclusions: The triggering of the CCL2/CCR2 axis in articular chondrocytes activates specific MAPK pathways leading to gene expression of cartilage degrading enzymes. However, some differences in the response to CCL2 stimulation are detected in healthy vs OA chondrocytes with respect to the number of activated genes and to the time of exposure to CCL2, suggesting that CCL2 action in articular cartilage may be dependent on OA stage and severity.

Scaffolds for Knee Chondral and Osteochondral Defects: Indications for Different Clinical Scenarios. A Consensus Statement.

OBJECTIVE: To develop patient-focused consensus guidelines on the indications for the use of scaffolds to address chondral and osteochondral femoral condyle lesions. DESIGN: The RAND/UCLA Appropriateness Method (RAM) was used to develop patient-specific recommendations by combining the best available scientific evidence with the collective judgement of a panel of experts guided by a core panel and multidisciplinary discussers. A list of specific clinical scenarios was produced regarding adult patients with symptomatic lesions without instability, malalignment, or meniscal deficiency. Each scenario underwent discussion and a 2-round vote on a 9-point Likert-type scale (range 1-3 "inappropriate," 4-6 "uncertain," 7-9 "appropriate"). Scores were pooled to generate expert recommendations. RESULTS: Scaffold (chondral vs. osteochondral), patient characteristics (age and sport activity level), and lesion characteristics (etiology, size, and the presence of osteoarthritis [OA]) were considered to define 144 scenarios. The use of scaffold-based procedures was considered appropriate in all cases of chondral or osteochondral lesions when joints are not affected by OA, while OA joints presented more controversial results. The analysis of the evaluated factors showed a different weight in influencing treatment appropriateness: the presence of OA influenced 58.3% of the indications, while etiology, size, and age were discriminating factors in 54.2%, 29.2%, and 16.7% of recommendations, respectively. CONCLUSIONS: The consensus identified indications still requiring investigation, but also the convergence of the experts in several scenarios defined appropriate or inappropriate, which could support decision making in the daily clinical practice, guiding the use of scaffold-based procedures for the treatment of chondral and osteochondral knee defects.

Role of the Hypoxia-Inducible Factor Pathway in Normal and Osteoarthritic Meniscus and in Mice after Destabilization of the Medial Meniscus.

OBJECTIVE: Meniscus injury and the hypoxia-inducible factor (HIF) pathway are independently linked to osteoarthritis pathogenesis, but the role of the meniscus HIF pathway remains unclear. We sought to identify and evaluate HIF pathway response in normal and osteoarthritic meniscus and to examine the effects of Epas1 (HIF-2α) insufficiency in mice on early osteoarthritis development. METHODS: Normal and osteoarthritic human meniscus specimens were obtained and used for immunohistochemical evaluation and cell culture studies for the HIF pathway. Meniscus cells were treated with pro-inflammatory stimuli, including interleukins (IL)-1ß, IL-6, transforming growth factor (TGF)-α, and fibronectin fragments (FnF). Target genes were also evaluated with HIF-1α and HIF-2α (Epas1) overexpression and knockdown. Wild-type (n = 36) and Epas1+/- (n = 30) heterozygous mice underwent destabilization of the medial meniscus (DMM) surgery and were evaluated at 2 and 4 weeks postoperatively for osteoarthritis development using histology. RESULTS: HIF-1α and HIF-2α immunostaining and gene expression did not differ between normal and osteoarthritic meniscus. While pro-inflammatory stimulation significantly increased both catabolic and anabolic gene expression in the meniscus, HIF-1α and Epas1 expression levels were not significantly altered. Epas1 overexpression significantly increased Col2a1 expression. Both wild-type and Epas1+/- mice developed osteoarthritis following DMM surgery. There were no significant differences between genotypes at either time point. CONCLUSION: The HIF pathway is likely not responsible for osteoarthritic changes in the human meniscus. Additionally, Epas1 insufficiency does not protect against osteoarthritis development in the mouse at early time points after DMM surgery. The HIF pathway may be more important for protection against catabolic stress.

Transient stiffening of cartilage during joint articulation: A microindentation study.

As a mechanoactive tissue, articular cartilage undergoes compression and shear on a daily basis. With the advent of high resolution and sensitive mechanical testing methods, such as micro- and nanoindentation, it has become possible to assess changes in small-scale mechanical properties due to compression and shear of the tissue. However, investigations on the changes of these properties before and after joint articulation have been limited. To simulate articular loading of cartilage in the context of human gait, a previously developed bioreactor system was used. Immediately after bioreactor testing, the stiffness was measured using microindentation. Specifically, we investigated whether the mechanical response of the tissue was transient or permanent, dependent on counterface material, and an effect limited to the superficial zone of cartilage. We found that cartilage surface stiffness increases immediately after articular loading and returns to baseline values within 3 hr. Cartilage-on-cartilage stiffening was found to be higher compared to both alumina- and cobalt chromium-on-cartilage stiffening, which were not significantly different from each other. This stiffening response was found to be unique to the superficial zone, as articular loading on cartilage with the superficial zone removed showed no changes in stiffness. The findings of this study suggest that the cartilage superficial zone may adapt its stiffness as a response to articular loading. As the superficial zone is often compromised during the course of osteoarthritic disease, this finding is of clinical relevance, suggesting that the load-bearing function deteriorates over time.