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.

Bony Ingrowth of Coil-Type Open-Architecture Anchors Compared With Screw-Type PEEK Anchors for the Medial Row in Rotator Cuff Repair: A Randomized Controlled Trial.

PURPOSE: To evaluate outcomes of screw-type and coil-type open-architecture suture anchors with respect to bony ingrowth, release of biological markers, and patient-reported outcome measures when used in rotator cuff repair (RCR). METHODS: Forty patients undergoing arthroscopic RCR for full-thickness rotator cuff tears were enrolled and prospectively randomized to receive a screw-type (19 patients) or coil-type (21 patients) suture anchor for the medial row during repair. All repairs used a transosseous-equivalent configuration with footprint anchors laterally. Marrow elements released during surgery were evaluated for 9 cytokine markers (insulin-like growth factor 1, fibroblast growth factor 2, bone morphogenetic proteins 7 and 2, platelet-derived growth factors AA and BB, epidermal growth factor, transforming growth factor beta1, and vascular endothelial growth factor). Postoperative computed tomography scans were performed at 6 months. Range of motion, strength, and validated patient-reported outcome measures (Simple Shoulder Test, Single Assessment Numeric Evaluation, visual analog scale, and American Shoulder and Elbow Surgeons scores) were gathered before the operation and at 6 months and 1 year postoperatively. RESULTS: Bone mineral density surrounding the coil-type anchor was significantly greater than that surrounding the screw-type anchor (P = .005). Bone mineral density values within the coil-type and screw-type anchors were comparable (P = .527); however, a larger amount of total bone mineral mass (in milligrams) was shown within the coil-type anchor owing to its larger volume (P < .01). Marrow elements released at the repair site were similar between groups (P > .05). Postoperatively, no statistically significant difference was found between groups for clinical outcome measures at 6 months or 1 year. Retear and complication rates were similar between groups (P > .05). CONCLUSIONS: Both the coil-type and screw-type anchors can be reliably used for RCR and produce similar clinical outcomes. The coil-type anchor resulted in superior bony growth surrounding the anchor and a larger total bone mineral mass within the anchor owing to its larger volume. LEVEL OF EVIDENCE: Level II, randomized prospective comparative study.

Agili-C implant promotes the regenerative capacity of articular cartilage defects in an ex vivo model.

PURPOSE: Osteochondral implants are currently adopted for the treatment of symptomatic full-thickness chondral and osteochondral defects. Agili-C™ is a cell-free aragonite-based scaffold which aims to reproduce the original structure and function of the articular joint while directing the growth and regeneration of both cartilage and its underlying subchondral bone. The goal of the present study was to investigate the ex vivo mechanisms of action (MOA) of the Agili-C™ implant in the repair of full-thickness cartilage defects. In particular, we tested whether Agili-C™ implant has the potential to stimulate cartilage ingrowth through chondrocytes migration into the 3D interconnected porous structure of the scaffold, along with maintaining their viability and phenotype and the deposition of hyaline cartilage matrix. METHODS: Articular cartilage samples were collected through the Gift of Hope Organ and Tissue Donor Network (Itasca, IL) within 24 h from death. For this study, cartilage from a total of 14 donors was used. To model a chondral defect, donut-shaped cartilage explants were prepared from each tissue specimen. The chondral phase of the Agili-C™ implant was placed inside the tissue in full contact and press fit manner. Cartilage explants with the Agili-C™ implant inside were cultured for 60 days. As a control, the same donut-shaped cartilage explants were cultured without Agili-C™, under the same culture conditions. RESULTS: Using fresh human cadaveric articular cartilage tissue in a 60-day culture, it was demonstrated that chondrocytes were able to migrate into the Agili-C™ scaffold and contribute to the deposition of the extracellular matrix (ECM) rich in collagen type II and aggrecan, and lacking collagen type I. Additionally, we were able to show the formation of a layer populated by progenitor-like cells on the articular surface of the implant. CONCLUSIONS: The analysis of samples taken from knee and ankle joints of human donors with a wide age range and both genders supports the potential of Agili-C™ scaffold to stimulate cartilage regeneration and repair. Based on these results, the present scaffold can be used in the clinical practice as a one-step procedure to treat full-thickness chondral defects.

Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes.

Oxidative stress-mediated post-translational modifications of redox-sensitive proteins are postulated as a key mechanism underlying age-related cellular dysfunction and disease progression. Peroxiredoxins (PRX) are critical intracellular antioxidants that also regulate redox signaling events. Age-related osteoarthritis is a common form of arthritis that has been associated with mitochondrial dysfunction and oxidative stress. The objective of this study was to determine the effect of aging and oxidative stress on chondrocyte intracellular signaling, with a specific focus on oxidation of cytosolic PRX2 and mitochondrial PRX3. Menadione was used as a model to induce cellular oxidative stress. Compared with chondrocytes isolated from young adult humans, chondrocytes from older adults exhibited higher levels of PRX1-3 hyperoxidation basally and under conditions of oxidative stress. Peroxiredoxin hyperoxidation was associated with inhibition of pro-survival Akt signaling and stimulation of pro-death p38 signaling. These changes were prevented in cultured human chondrocytes by adenoviral expression of catalase targeted to the mitochondria (MCAT) and in cartilage explants from MCAT transgenic mice. Peroxiredoxin hyperoxidation was observedin situin human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism.

Junior Investigators Thinking About Quitting Research: A Survey.

OBJECTIVE: Junior tenure-track faculty report high levels of stress and low satisfaction; the increasingly competitive funding environment compounds this discontent. We examined factors associated with junior investigators who were thinking about quitting research. METHOD: Data were collected as part of a program evaluation of an interdisciplinary research mentoring program in an academic medical center. RESULTS: Of the 62 mentees, 44 responded to the survey (71%). When asked "In the past year, have you considered quitting research?" 39 mentees answered the question; 17 (44%) answered in the affirmative. Those who had considered quitting had lower scores on the Clinical Research Appraisal Inventory-12 (CRAI-12) and job satisfaction and higher scores on burnout. In a regression model, we found that only CRAI-12 scores were significantly, inversely associated with thinking about quitting. CONCLUSION: Factors associated with thinking about quitting included lower confidence in research skills, reduced job satisfaction, and higher levels of burnout.

Assessment of glenoid chondral healing: comparison of microfracture to autologous matrix-induced chondrogenesis in a novel rabbit shoulder model.

BACKGROUND: Management of glenohumeral arthrosis in young patients is a considerable challenge, with a growing need for non-arthroplasty alternatives. The objectives of this study were to develop an animal model to study glenoid cartilage repair and to compare surgical repair strategies to promote glenoid chondral healing. METHODS: Forty-five rabbits underwent unilateral removal of the entire glenoid articular surface and were divided into 3 groups--untreated defect (UD), microfracture (MFx), and MFx plus type I/III collagen scaffold (autologous matrix-induced chondrogenesis [AMIC])--for the evaluation of healing at 8 weeks (12 rabbits) and 32 weeks (33 rabbits) after injury. Contralateral shoulders served as unoperated controls. Tissue assessments included 11.7-T magnetic resonance imaging (long-term healing group only), equilibrium partitioning of an ionic contrast agent via micro-computed tomography (EPIC-µCT), and histologic investigation (grades on International Cartilage Repair Society II scoring system). RESULTS: At 8 weeks, x-ray attenuation, thickness, and volume did not differ by treatment group. At 32 weeks, the T2 index (ratio of T2 values of healing to intact glenoids) was significantly lower for the MFx group relative to the AMIC group (P = .01) whereas the T1ρ index was significantly lower for AMIC relative to MFx (P = .01). The micro-computed tomography-derived repair tissue volume was significantly higher for MFx than for UD. Histologic investigation generally suggested inferior healing in the AMIC and UD groups relative to the MFx group, which exhibited improvements in both integration of repair tissue with subchondral bone and tidemark formation over time. DISCUSSION: Improvements conferred by AMIC were limited to magnetic resonance imaging outcomes, whereas MFx appeared to promote increased fibrous tissue deposition via micro-computed tomography and more hyaline-like repair histologically. The findings from this novel model suggest that MFx promotes biologic resurfacing of full-thickness glenoid articular injury.

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.

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.

Synovial fluid from patients with early osteoarthritis modulates fibroblast-like synoviocyte responses to toll-like receptor 4 and toll-like receptor 2 ligands via soluble CD14.

OBJECTIVE: Synovial inflammation, a feature of both osteoarthritis (OA) and meniscal injury, is hypothesized to be triggered in part via stimulation of Toll-like receptors (TLRs). We undertook this study to test whether a TLR-2- or TLR-4-stimulating factor in synovial fluid (SF) from patients with early knee OA with meniscal injury could lead to inflammatory activation of synoviocytes. METHODS: SF was obtained from patients with early OA cartilage damage undergoing arthroscopic meniscal procedures. SF was used to stimulate primary cultures of fibroblast-like synoviocytes (FLS) and cell lines transfected with TLR-2 or TLR-4. SF was used either alone or in combination with a TLR-2 stimulus (palmitoyl-3-cysteine-serine-lysine-4 [Pam3CSK4]) or a TLR-4 stimulus (lipopolysaccharide [LPS]). In blocking experiments, SF was preincubated with anti-CD14 antibody. RESULTS: SF from these patients did not stimulate interleukin-8 (IL-8) release from TLR transfectants. Compared with SF on its own, SF (at concentrations of 0.09-25%) in combination with TLR-2 or TLR-4 ligands resulted in significant augmentation of IL-8 release from both transfectants and primary FLS. Soluble CD14 (sCD14), a coreceptor for TLRs, was measured in SF from patients with early OA at levels comparable to those in patients with advanced OA and patients with rheumatoid arthritis. Blockade with anti-CD14 antibody abolished the ability of SF to augment IL-8 production in response to LPS, and diminished Pam3CSK4 responses. CONCLUSION: SF augments FLS responses to TLR-2 and TLR-4 ligands. This effect was largely due to sCD14. Our results demonstrate that sCD14 in the setting of OA and meniscal injury sensitizes FLS to respond to inflammatory stimuli such as TLR ligands.

Rotator cuff healing after continuous subacromial bupivacaine infusion: an in vivo rabbit study.

BACKGROUND: The objective of this study was to evaluate the effects of continuous subacromial bupivacaine infusion on supraspinatus muscle and rotator cuff tendon healing using gross, biomechanical, and histologic analyses. MATERIALS AND METHODS: Thirty-three New Zealand White rabbits underwent unilateral supraspinatus transection and rotator cuff repair (RCR). Rabbits were assigned to 1 of 3 groups: (1) RCR only, (2) RCR with continuous saline infusion for 48 hours, or (3) RCR with continuous 0.25% bupivacaine with epinephrine (1:200,000) infusion for 48 hours. Rabbits were euthanized postoperatively at 2 weeks (for histologic assessment) or 8 weeks (for biomechanical and histologic assessment). RESULTS: Tensile testing showed a significantly higher load to failure in intact tendons compared with repaired tendons (P < .01); however, no statistical differences were detected among RCR only, RCR saline, and RCR bupivacaine groups. Histologically, the enthesis of repaired tendons showed increased cellularity and disorganized collagen fibers compared with intact tendons, with no differences between treatment groups. Muscle histology demonstrated scattered degenerative muscle fibers at 2 weeks in RCR saline and RCR bupivacaine groups, but no degeneration was noted at 8 weeks. CONCLUSIONS: The healing supraspinatus tendons exposed to bupivacaine infusion showed similar histologic and biomechanical characteristics compared with untreated and saline-infused RCR groups. Muscle histology showed fiber damage at 2 weeks for the saline and bupivacaine-treated groups, with no apparent disruption at 8 weeks, suggesting a recovery process. Therefore, subacromial bupivacaine infusion in this rabbit rotator cuff model does not appear to impair muscle or tendon after acute injury and repair.

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.

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.

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.

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.

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.

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).

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.

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.