Nuclear factor κB overactivation in the intervertebral disc leads to macrophage recruitment and severe disc degeneration.

Persistent inflammation has been associated with severe disc degeneration (DD). This study investigated the effect of prolonged nuclear factor κB (NF-κB) activation in DD. Using an inducible mouse model, we genetically targeted cells expressing aggrecan, a primary component of the disc extra cellular matrix, for activation of the canonical NF-κB pathway. Prolonged NF-κB activation led to severe structural degeneration accompanied by increases in gene expression of inflammatory molecules (Il1b, Cox2, Il6, and Nos2), chemokines (Mcp1 and Mif), and catabolic enzymes (Mmp3, Mmp9, and Adamts4). Increased recruitment of proinflammatory (F4/80+,CD38+) and inflammatory resolving (F4/80+,CD206+) macrophages was observed within caudal discs. We found that the secretome of inflamed caudal disc cells increased macrophage migration and inflammatory activation. Lumbar discs did not exhibit phenotypic changes, suggestive of regional spinal differences in response to inflammatory genetic overactivation. Results suggest prolonged NF-κB activation can induce severe DD through increases in inflammatory cytokines, chemotactic proteins, catabolic enzymes, and the recruitment and activation of macrophage cell populations.

Bullying, harassment, and discrimination of musculoskeletal researchers and the impact of the COVID-19 pandemic: an international study.

PURPOSE: Bullying, harassment, and discrimination (BHD) are prevalent in academic, scientific, and clinical departments, particularly orthopedic surgery, and can have lasting effects on victims. As it is unclear how BHD affects musculoskeletal (MSK) researchers, the following study assessed BHD in the MSK research community and whether the COVID-19 pandemic, which caused hardships in other industries, had an impact. METHODS: A web-based anonymous survey was developed in English by ORS Spine Section members to assess the impact of COVID-19 on MSK researchers in North America, Europe, and Asia, which included questions to evaluate the personal experience of researchers regarding BHD. RESULTS: 116 MSK researchers completed the survey. Of respondents, 34.5% (n = 40) focused on spine, 30.2% (n = 35) had multiple areas of interest, and 35.3% (n = 41) represented other areas of MSK research. BHD was observed by 26.7% (n = 31) of respondents and personally experienced by 11.2% (n = 13), with mid-career faculty both observing and experiencing the most BHD. Most who experienced BHD (53.8%, n = 7) experienced multiple forms. 32.8% (n = 38) of respondents were not able to speak out about BHD without fear of repercussions, with 13.8% (n = 16) being unsure about this. Of those who observed BHD, 54.8% (n = 17) noted that the COVID-19 pandemic had no impact on their observations. CONCLUSIONS: To our knowledge, this is the first study to address the prevalence and determinants of BHD among MSK researchers. MSK researchers experienced and observed BHD, while many were not comfortable reporting and discussing violations to their institution. The COVID-19 pandemic had mixed-effects on BHD. Awareness and proactive policy changes may be warranted to reduce/eliminate the occurrence of BHD in this community.

Race, But Not Gender, Is Associated With Admissions Into Orthopaedic Residency Programs.

BACKGROUND: Orthopaedic surgery is one of the most competitive but least diverse surgical specialties, with ever-increasing academic achievements (such as test scores) shown by applicants. Prior research shows that white applicants had higher United States Medical Licensing Exam (USMLE) Step 1 and Step 2 Clinical Knowledge scores as well as higher odds of Alpha Omega Alpha status compared with Black, Hispanic, and other applicant groups. Yet, it still remains unknown whether differences in application metrics by race/ethnicity sufficiently explain the underrepresentation of certain racial or ethnic minority groups in orthopaedic residency programs. QUESTIONS/PURPOSES: In this study, we sought to determine (1) the relative weight of academic variables for admission into orthopaedic residency, and (2) whether race and gender are independently associated with admission into an orthopedic residency. METHODS: The Electronic Residency Application System (ERAS) data from the Association of American Medical Colleges (AAMC) and the National Board of Medical Examiners (NBME) of first-time MD applicants (n = 8966) for orthopaedic surgery residency positions in the United States and of admitted orthopaedic residents (n = 6218) from 2005 to 2014 were reviewed. This dataset is the first and most comprehensive of its kind to date in orthopaedic surgery. Academic metrics, such as USMLE Step 1 and Step 2 Clinical Knowledge scores, number of publications, Alpha Omega Alpha status, volunteer experiences, work experience, as well as race and gender, were analyzed using hierarchical logistic regression models. The first model analyzed the association of academic metrics with admission into orthopaedic residency. In the second model, we added race and gender and controlled for metrics of academic performance. To determine how well the models simulated the actual admissions data, we computed the receiver operating characteristics (ROC) including the area under curve (AUC), which measures the model's ability to simulate which applicants were admitted or not admitted, with an AUC = 1.0 representing a perfect simulation. The odds ratio and confidence interval of each variable were computed. RESULTS: When only academic variables were analyzed in the first model, Alpha Omega Alpha status (odds ratio 2.12 [95% CI 1.80 to 2.50]; p < 0.001), the USMLE Step 1 score (OR 1.04 [95% CI 1.03 to 1.04]; p < 0.001), the USMLE Step 2 Clinical Knowledge score (OR 1.01 [95% CI 1.01 to 1.02]; p < 0.001), publication count (OR 1.04 [95% CI 1.03 to 1.05]; p < 0.001), and volunteer experience (OR 1.03 [95% CI 1.01 to 1.04]; p < 0.001) were associated with admissions into orthopaedics while work and research experience were not. This model yielded a good prediction of the results with an AUC of 0.755. The second model, in which the variables of race and gender were added to the academic variables, also had a good prediction of the results with an AUC of 0.759. This model indicates that applicant race, but not gender, is associated with admissions into orthopaedic residency. Applicants from Asian (OR 0.78 [95% CI 0.67 to 0.92]), Black (OR 0.63 [95% CI 0.51 to 0.77], Hispanic (OR 0.48 [95% CI 0.36 to 0.65]), or other race groups (OR 0.65 [95% CI 0.55 to 0.77]) had lower odds of admission into residency compared with white applicants. CONCLUSION: Minority applicants, but not women, have lower odds of admission into orthopaedic surgery residency, even when accounting for academic performance metrics. Changes in the residency selection processes are needed to eliminate the lower admission probability of qualified minority applicants in orthopaedic residency and to improve the diversity and inclusion of orthopaedic surgery. Changes including increasing the diversity of the selection committee, bias training, blinding applications before review, removal of metrics with history of racial disparities from an interviewer's candidate profile before an interview, and use of holistic application review (where an applicants' experiences, attributes, and academic metrics are all considered) can improve the diversity landscape in training. In addition, cultivating an environment of inclusion will be necessary to address these long-standing trends in orthopaedic surgery. CLINICAL RELEVANCE: Race, but not gender, is associated with the odds of acceptance into orthopaedic surgery residency despite equivalent academic metrics. Changes in residency selection processes are suggested to eliminate the lower admission probability of qualified minority applicants into orthopaedic residency and to improve the diversity and inclusion of orthopaedic surgery.

Oral Administration of a Chemically Modified Curcumin, TRB-N0224, Reduced Inflammatory Cytokines and Cartilage Erosion in a Rabbit ACL Transection Injury Model.

OBJECTIVE: To evaluate the effects of TRB-N0224, a chemically modified curcumin (CMC) with zinc binding properties and improved pharmacokinetics, in a rabbit anterior cruciate ligament (ACL) transection injury-induced model of osteoarthritis (OA). DESIGN: Thirty-eight skeletally mature New Zealand white rabbits were studied in 4 groups: a sham with arthrotomy (n = 6), control with ACL transection (n = 6), and 2 treatment groups with ACL transection and administration of TRB-N0224 at low (25 mg/kg/day) (n = 13) and high (50 mg/kg/day) (n = 13) doses. After euthanization at 12 weeks, outcomes were measured by post-necropsy gross morphology, biomechanics, and cartilage and synovium histology. Rabbit blood ELISA quantified cytokine and matrix metalloproteinase (MMP) concentrations at 0, 4, 8, and 12 weeks. RESULTS: Both treatment doses had fewer distal femoral condyle erosive defects than the control; the low dose demonstrated a mean 78% decrease (P < 0.01). Histologically, the low- and high-dose treatment groups had fewer cartilage pathologic changes and less severe synovitis than the control. CMC alone did not have a major effect on the biomechanics of healthy cartilage or cartilage in the ACL transection model, as demonstrated in 5 of the 6 measured properties/regions (P < 0.05). ELISA results suggested that the key mediators of OA, (interleukin) IL-1ß, IL-6, TNFα (tumor necrosis factor-α), MMP-9, and MMP-13, had decreased concentrations with TRB-N0224 treatment at different time points between weeks 4 to 12 (P < 0.05). CONCLUSIONS: In the pathogenesis of OA, an imbalance exists between catabolic and anabolic mediators. These results suggest the potential of TRB-N0224 to modulate MMP and cytokine levels, slowing the macroscopic and histopathological progression of OA.

Actomyosin contractility confers mechanoprotection against TNFα-induced disruption of the intervertebral disc.

Inflammation triggers degradation of intervertebral disc extracellular matrix (ECM), a hallmark of disc degeneration that contributes to back pain. Mechanosensitive nucleus pulposus cells are responsible for ECM production, yet the impact of a proinflammatory microenvironment on cell mechanobiology is unknown. Using gain- and loss-of-function approaches, we show that tumor necrosis factor-α (TNFα)-induced inflammation alters cell morphology and biophysical properties (circularity, contractility, cell stiffness, and hydraulic permeability) in a mechanism dependent on actomyosin contractility in a three-dimensional (3D) culture. We found that RhoA activation rescued cells from TNFα-induced mechanobiological disruption. Using a novel explant-in-hydrogel culture system, we demonstrate that nuclear factor kappa-B nuclear translocation and transcription are mechanosensitive, and its downstream effects on ECM degradation are regulated by actomyosin contractility. Results define a scaling relationship between circularity, contractility, and hydraulic permeability that is conserved from healthy to inflammatory microenvironments and is indicative of cell mechanobiological control across scales in 3D.

Academic Metrics Do Not Explain the Underrepresentation of Women in Orthopaedic Training Programs.

BACKGROUND: Among medical specialties, orthopaedic surgery persistently has one of the lowest representations of women in residency programs. This study examined whether differences exist in the academic metrics of the orthopaedic residency applicants and enrolled candidates by sex, which may be contributing to the persistent underrepresentation of women. Differences in enrollment rate in orthopaedic residency programs also were analyzed. We hypothesized that academic metrics were similar for female and male applicants and thus do not explain the underrepresentation of women in training programs. METHODS: Academic data of first-time applicants (n = 9,133) and candidates who enrolled in an orthopaedic residency (n = 6,381) in the U.S. from 2005 to 2014 were reviewed. The United States Medical Licensing Examination (USMLE) Step-1 and Step-2 Clinical Knowledge (CK) scores, Alpha Omega Alpha (AΩA) Honor Medical Society status, number of publications, and volunteer experiences were compared by sex and were analyzed over time. RESULTS: From 2005 to 2014, representation of female applicants increased from 12.6% to 16.0%, corresponding with an increase in the percentage of enrolled female residents (from 12.9% to 16.1%); 70.3% of male and 67.1% of female applicants to orthopaedic residency enrolled as residents (p = 0.082). Mean academic metrics increased significantly over time for applicants and enrolled candidates, irrespective of sex. Comparing by sex, the mean USMLE Step-1 scores of male applicants and enrolled candidates were approximately 2% higher than those of female applicants (p < 0.0001). Volunteer experiences of female applicants and enrolled candidates were 12% higher compared with male applicants (p < 0.0001). There was no significant difference in USMLE Step-2 CK scores, number of publications, or AΩA status by sex. CONCLUSIONS: The enrollment rate of male and female applicants in orthopaedic residencies was similar and did not change during the 10-year study period. The academic metrics of applicants and enrolled candidates have increased significantly. The academic metrics were found to be comparable by sex; the differences in USMLE Step-1 scores and volunteer experiences were small relative to the magnitude of accomplishments that these values represent. The growth rate of the proportion of women in orthopaedic residencies lags other surgical subspecialties but appears to be independent of academic metrics.

Underrepresented Minority Applicants Are Competitive for Orthopaedic Surgery Residency Programs, but Enter Residency at Lower Rates.

INTRODUCTION: Orthopaedic surgery residency programs have the lowest representation of ethnic/racial minorities compared with other specialties. This study compared orthopaedic residency enrollment rates and academic metrics of applicants and matriculated residents by race/ethnicity. METHODS: Data on applicants from US medical schools for orthopaedic residency and residents were analyzed from 2005 to 2014 and compared between race/ethnic groups (White, Asian, Black, Hispanic, and Other). RESULTS: Minority applicants comprised 29% of applicants and 25% of enrolled candidates. Sixty-one percent of minority applicants were accepted into an orthopaedic residency versus 73% of White applicants (P < 0.0001). White and Asian applicants and residents had higher USMLE Step 1. White applicants and matriculated candidates had higher Step 2 Clinical Knowledge scores and higher odds of Alpha Omega Alpha membership compared with Black, Hispanic, and Other groups. Publication counts were similar in all applicant groups, although Hispanic residents had significantly more publications. Black applicants had more volunteer experiences. CONCLUSIONS: In orthopaedic surgery residency, minority applicants enrolled at a lower rate than White and Asian applicants. The emphasis on USMLE test scores and Alpha Omega Alpha membership may contribute to the lower enrollment rate of minority applicants. Other factors such as conscious or unconscious bias, which may contribute, were not evaluated in this study.

High mobility group box-1 induces pro-inflammatory signaling in human nucleus pulposus cells via toll-like receptor 4-dependent pathway.

Intervertebral disc (IVD) degeneration (DD) is associated with low back pain, the leading cause of disability worldwide. Damage-associated molecular patterns (DAMPs) that contribute to inflammation and trigger DD have not been well characterized. Extracellular high mobility group box-1 (HMGB1) protein has been implicated as a potent DAMP and pro-inflammatory stimulus in the immune system. In this study, we show that HMGB1 and IL-6 levels increase in patients with advanced DD in comparison to early DD. This study further tested the hypothesis that HMGB1 promotes inflammatory signaling driving DD in human nucleus pulposus (NP) cells and tissue. Immunofluorescence and western blot analysis confirmed the expression of HMGB1 and its extracellular release by NP cells under cell stress. Gene expression and protein quantification indicate that HMGB1 stimulates the expression IL-6 and MMP-1 in a dose-dependent manner. The contributions of toll-like receptor (TLR) -2, -4 and receptor for advanced glycation end products (RAGE) as receptors mediating HMGB1 signaling was examined using small molecule inhibitors. Inhibition of TLR-4 signaling, with TAK-242, completely abrogated HMGB1 induced IL-6 and MMP-1 expression, whereas inhibition of TLR-2, with O-vanillin, or RAGE, with FPS-ZM1, had mild inhibitory effects. HMGB1 stimulation activated NF-ĸB signaling while TAK-242 co-treatment abrogated it. Lastly, effects of HMGB1 on matrix deposition was evaluated in a 3D culture system of human NP cells. These results implicate HMGB1 as a potent DAMP that promotes inflammation in NP cells and degradation of NP tissues. TLR4-HMGB1 axis is a potential major pathway to alleviate disc inflammation and mitigate DD. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Dynamic Hydrostatic Pressure Regulates Nucleus Pulposus Phenotypic Expression and Metabolism in a Cell Density-Dependent Manner.

Dynamic hydrostatic pressure (HP) loading can modulate nucleus pulposus (NP) cell metabolism, extracellular matrix (ECM) composition, and induce transformation of notochordal NP cells into mature phenotype. However, the effects of varying cell density and dynamic HP magnitude on NP phenotype and metabolism are unknown. This study examined the effects of physiological magnitudes of HP loading applied to bovine NP cells encapsulated within three-dimensional (3D) alginate beads. Study 1: seeding density (1 M/mL versus 4 M/mL) was evaluated in unloaded and loaded (0.1 MPa, 0.1 Hz) conditions. Study 2: loading magnitude (0, 0.1, and 0.6 MPa) applied at 0.1 Hz to 1 M/mL for 7 days was evaluated. Study 1: 4 M/mL cell density had significantly lower adenosine triphosphate (ATP), glycosaminoglycan (GAG) and collagen content, and increased lactate dehydrogenase (LDH). HP loading significantly increased ATP levels, and expression of aggrecan, collagen I, keratin-19, and N-cadherin in HP loaded versus unloaded groups. Study 2: aggrecan expression increased in a dose dependent manner with HP magnitude, whereas N-cadherin and keratin-19 expression were greatest in low HP loading compared to unloaded. Overall, the findings of the current study indicate that cell seeding density within a 3D construct is a critical variable influencing the mechanobiological response of NP cells to HP loading. NP mechanobiology and phenotypic expression was also found to be dependent on the magnitude of HP loading. These findings suggest that HP loading and culture conditions of NP cells may require complex optimization for engineering an NP replacement tissue.

Timing of mesenchymal stem cell delivery impacts the fate and therapeutic potential in intervertebral disc repair.

Cell-based therapies offer a promising approach to treat intervertebral disc (IVD) degeneration. The impact of the injury microenvironment on treatment efficacy has not been established. This study used a rat disc stab injury model with administration of mesenchymal stromal cells (MSCs) at 3, 14, or 30 days post injury (DPI) to evaluate the impact of interventional timing on IVD biochemistry and biomechanics. We also evaluated cellular localization within the disc with near infrared imaging to track the time and spatial profile of cellular migration after in vivo delivery. Results showed that upon injection into a healthy disc, MSCs began to gradually migrate outwards over the course of 14 days, as indicated by decreased signal intensity from the disc space and increased signal within the adjacent vertebrae. Cells administered 14 or 30 DPI also tended to migrate out 14 days after injection but cells injected 3 DPI were retained at a significantly higher amount versus the other groups (p < 0.05). Correspondingly the 3 DPI group, but not 14 or 30 DPI groups, had a higher GAG content in the MSC injected discs (p = 0.06). Enrichment of MSCs and increased GAG content in 3 DPI group did not lead to increased compressive biomechanical properties. Findings suggest that cell therapies administered at an early stage of injury/disease progression may have greater chances of mitigating matrix loss, possibly through promotion of MSC activity by the inflammatory microenvironment associated with injury. Future studies will evaluate the mode and driving factors that regulate cellular migration out of the disc. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:32-40, 2017.

Serum levels of the proinflammatory cytokine interleukin-6 vary based on diagnoses in individuals with lumbar intervertebral disc diseases.

BACKGROUND: Many intervertebral disc diseases cause low back pain (LBP). Proinflammatory cytokines and matrix metalloproteinases (MMPs) participate in disc pathology. In this study, we examined levels of serum cytokines and MMPs in human subjects with diagnoses of disc herniation (DH), spinal stenosis (SS), or degenerative disc disease (DDD) relative to levels in control subjects. Comparison between subjects with DH and those with other diagnoses (Other Dx, grouped from SS and DDD) was performed to elaborate a pathological mechanism based on circulating cytokine levels. METHODS: Study participants were recruited from a spine neurosurgery practice (n = 80), a back pain management practice (n = 27), or a control cohort (n = 26). Serum samples were collected before treatment and were assayed by multiplex assays for levels of interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-γ, tumor necrosis factor-α, MMP-1, MMP-3, and MMP-9. Inflammatory and degradative mediator levels were compared for subjects with LBP and control subjects, by diagnosis and by treatment groups, controlling for effects of sex, age, and reported history of osteoarthritis. Spearman's correlation coefficient was used to examine relationships with age, body mass index (BMI), symptom duration, and smoking history. RESULTS: Serum levels of IL-6 were significantly higher in subjects with LBP compared with control subjects. Participants with LBP due to Other Dx had significantly higher levels of IL-6 than DH and controls. Serum levels of MMP-1 were significantly lower in LBP subjects, specifically those with DH, than in control subjects. Positive correlations were found between IL-6 levels and BMI, symptom duration, and age. MMP-1 levels were positively correlated with age. CONCLUSIONS: The findings of the present clinical study are the results of the first examination of circulating cytokine levels in DDD and SS and provide evidence for a more extensive role of IL-6 in disc diseases, where patients with DDD or SS have higher serum cytokine levels than those with DH or control subjects. These findings suggest that LBP subjects have low-grade systemic inflammation, and biochemical profiling of circulating cytokines may assist in refining personalized diagnoses of disc diseases.

Exploratory study for identifying systemic biomarkers that correlate with pain response in patients with intervertebral disc disorders.

Molecular events that drive disc damage and low back pain (LBP) may precede clinical manifestation of disease onset and can cause detrimental long-term effects such as disability. Biomarkers serve as objective molecular indicators of pathological processes. The goal of this study is to identify systemic biochemical factors as predictors of response to treatment of LBP with epidural steroid injection (ESI). Since inflammation plays a pivotal role in LBP, this pilot study investigates the effect of ESI on systemic levels of 48 inflammatory biochemical factors (cytokines, chemokines, and growth factors) and examines the relationship between biochemical factor levels and pain or disability in patients with disc herniation (DH), or other diagnoses (Other Dx) leading to low back pain, which included spinal stenosis (SS) and degenerative disc disease (DDD). Study participants (n = 16) were recruited from a back pain management practice. Pain numerical rating score (NRS), Oswestry Disability Index (ODI), and blood samples were collected pre- and at 7 to 10 days post-treatment. Blood samples were assayed for inflammatory mediators using commercial multiplex assays. Mediator levels were compared pre- and post-treatment to investigate the potential correlations between clinical and biochemical outcomes. Our results indicate that a single ESI significantly decreased systemic levels of SCGF-ß and IL-2. Improvement in pain in all subjects was correlated with changes in chemokines (MCP-1, MIG), hematopoietic progenitor factors (SCGF-ß), and factors that participate in angiogenesis/fibrosis (HGF), nociception (SCF, IFN-α2), and inflammation (IL-6, IL-10, IL-18, TRAIL). Levels of biochemical mediators varied based on diagnosis of LBP, and changes in pain responses and systemic mediators from pre- to post-treatment were dependent on the diagnosis cohort. In the DH cohort, levels of IL-17 and VEGF significantly decreased post-treatment. In the Other Dx cohort, levels of IL-2Rα, IL-3, and SCGF-ß significantly decreased post-treatment. In order to determine whether mediator changes were related to pain, correlations between change in pain scores and change in mediator levels were performed. Subjects with DH demonstrated a profile signature that implicated hematopoiesis factors (SCGF-ß, GM-CSF) in pain response, while subjects with Other Dx demonstrated a biomarker profile that implicated chemokines (MCP-1, MIG) and angiogenic factors (HGF, VEGF) in pain response. Our findings provide evidence that systemic biochemical factors in patients with LBP vary by diagnosis, and pain response to treatment is associated with a unique profile of biochemical responses in each diagnosis group. Future hypothesis-based studies with larger subject cohorts are warranted to confirm the findings of this pilot exploratory study.

Inflammation induces irreversible biophysical changes in isolated nucleus pulposus cells.

Intervertebral disc degeneration is accompanied by elevated levels of inflammatory cytokines that have been implicated in disease etiology and matrix degradation. While the effects of inflammatory stimulation on disc cell metabolism have been well-studied, their effects on cell biophysical properties have not been investigated. The hypothesis of this study is that inflammatory stimulation alters the biomechanical properties of isolated disc cells and volume responses to step osmotic loading. Cells from the nucleus pulposus (NP) of bovine discs were isolated and treated with either lipopolysaccharide (LPS), an inflammatory ligand, or with the recombinant cytokine TNF-α for 24 hours. We measured cellular volume regulation responses to osmotic loading either immediately after stimulation or after a 1 week recovery period from the inflammatory stimuli. Cells from each group were tested under step osmotic loading and the transient volume-response was captured via time-lapse microscopy. Volume-responses were analyzed using mixture theory framework to investigate two biomechanical properties of the cell, the intracellular water content and the hydraulic permeability. Intracellular water content did not vary between treatment groups, but hydraulic permeability increased significantly with inflammatory treatment. In the 1 week recovery group, hydraulic permeability remained elevated relative to the untreated recovery control. Cell radius was also significantly increased both after 24 hours of treatment and after 1 week recovery. A significant linear correlation was observed between hydraulic permeability and cell radius in untreated cells at 24 hours and at 1-week recovery, though not in the inflammatory stimulated groups at either time point. This loss of correlation between cell size and hydraulic permeability suggests that regulation of volume change is disrupted irreversibly due to inflammatory stimulation. Inflammatory treated cells exhibited altered F-actin cytoskeleton expression relative to untreated cells. We also found a significant decrease in the expression of aquaporin-1, the predominant water channel in disc NP cells, with inflammatory stimulation. To our knowledge, this is the first study providing evidence that inflammatory stimulation directly alters the mechanobiology of NP cells. The cellular biophysical changes observed in this study are coincident with documented changes in the extracellular matrix induced by inflammation, and may be important in disease etiology.

Nanocomposite scaffold for chondrocyte growth and cartilage tissue engineering: effects of carbon nanotube surface functionalization.

The goal of this study was to assess the long-term biocompatibility of single-wall carbon nanotubes (SWNTs) for tissue engineering of articular cartilage. We hypothesized that SWNT nanocomposite scaffolds in cartilage tissue engineering can provide an improved molecular-sized substrate for stimulation of chondrocyte growth, as well as structural reinforcement of the scaffold's mechanical properties. The effect of SWNT surface functionalization (-COOH or -PEG) on chondrocyte viability and biochemical matrix deposition was examined in two-dimensional cultures, in three-dimensional (3D) pellet cultures, and in a 3D nanocomposite scaffold consisting of hydrogels+SWNTs. Outcome measures included cell viability, histological and SEM evaluation, GAG biochemical content, compressive and tensile biomechanical properties, and gene expression quantification, including extracellular matrix (ECM) markers aggrecan (Agc), collagen-1 (Col1a1), collagen-2 (Col2a1), collagen-10 (Col10a1), surface adhesion proteins fibronectin (Fn), CD44 antigen (CD44), and tumor marker (Tp53). Our findings indicate that chondrocytes tolerate functionalized SWNTs well, with minimal toxicity of cells in 3D culture systems (pellet and nanocomposite constructs). Both SWNT-PEG and SWNT-COOH groups increased the GAG content in nanocomposites relative to control. The compressive biomechanical properties of cell-laden SWNT-COOH nanocomposites were significantly elevated relative to control. Increases in the tensile modulus and ultimate stress were observed, indicative of a tensile reinforcement of the nanocomposite scaffolds. Surface coating of SWNTs with -COOH also resulted in increased Col2a1 and Fn gene expression throughout the culture in nanocomposite constructs, indicative of increased chondrocyte metabolic activity. In contrast, surface coating of SWNTs with a neutral -PEG moiety had no significant effect on Col2a1 or Fn gene expression, suggesting that the charged nature of the -COOH surface functionalization may promote ECM expression in this culture system. The results of this study indicate that SWNTs exhibit a unique potential for cartilage tissue engineering, where functionalization with bioactive molecules may provide an improved substrate for stimulation of cellular growth and repair.

Dose-response effect of an intra-tendon application of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) in a rat Achilles tendinopathy model.

The purpose of this study was to assess whether intra-tendon delivery of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) would improve Achilles tendon repair in a rat collagenase-induced tendinopathy model. Seven days following collagenase induction of tendinopathy, one of four intra-tendinous treatments was administered: (i) Vehicle control (sodium acetate buffer), (ii) 1.02 µg rhPDGF-BB, (iii) 10.2 µg rhPDGF-BB, or (iv) 102 µg rhPDGF-BB. Treated tendons were assessed for histopathological (e.g., proliferation, tendon thickness, collagen fiber density/orientation) and biomechanical (e.g., maximum load-to-failure and stiffness) outcomes. By 7 days post-treatment, there was a significant increase in cell proliferation with the 10.2 and 102 µg rhPDGF-BB-treated groups (p=0.049 and 0.015, respectively) and in thickness at the tendon midsubstance in the 10.2 µg of rhPDGF-BB group (p=0.005), compared to controls. All groups had equivalent outcomes by Day 21. There was a dose-dependent effect on the maximum load-to-failure, with no significant difference in the 1.02 and 102 µg rhPDGF-BB doses but the 10.2 µg rhPDGF-BB group had a significant increase in load-to-failure at 7 (p=0.003) and 21 days (p=0.019) compared to controls. The rhPDGF-BB treatment resulted in a dose-dependent, transient increase in cell proliferation and sustained improvement in biomechanical properties in a rat Achilles tendinopathy model, demonstrating the potential of rhPDGF-BB treatment in a tendinopathy application. Consequently, in this model, data suggest that rhPDGF-BB treatment is an effective therapy and thus, may be an option for clinical applications to treat tendinopathy.

Toll-Like Receptor 4 (TLR4) expression and stimulation in a model of intervertebral disc inflammation and degeneration.

STUDY DESIGN: We measured the expression and responses of Toll-Like Receptor 4 (TLR4) activation in the intervertebral disc (IVD) in vitro and in vivo. We hypothesize that stimulation of the IVD with the TLR4 ligand lipopolysaccharide (LPS) results in upregulation of a coordinated set of proinflammatory mediators and inhibition of matrix expression, both consistent with a molecular profile of degeneration. OBJECTIVE: To characterize early inflammatory and morphological changes induced by TLR4 activation in the IVD. SUMMARY OF BACKGROUND DATA: TLR4 is a pattern recognition receptor activated in innate immunity that has been implicated in disease mechanisms of inflammatory cartilaginous degeneration. However, no study to date has examined the expression and responses of TLR4 in the IVD. METHODS: IVD cells were stimulated with LPS in a dose-dependent manner, and inflammatory cytokine levels were measured by quantitative reverse transcription-polymerase chain reaction. Histological and inflammatory changes due to in vivo injection of LPS into the rat caudal IVD were measured by enzyme-linked immunosorbent assay and immunoblotting. RESULTS: Baseline TLR4 expression in IVD tissue varied according to cell type. LPS stimulation resulted in significant increases in tumor necrosis factor α (TNF)-α, interleukin (IL)-1ß, IL-6, and nitric oxide levels and significant inhibition in aggrecan and collagen-2. Intradiscal injection of LPS was found to cause moderate degenerative changes in the IVD, with increases in tissue levels of IL-1ß, TNF-α, high mobility group box 1 protein (HMGB1), and macrophage migration inhibitory factor (MIF). CONCLUSION: This study provides the first evidence that IVD cells express TLR4 and are responsive to TLR4 activation by upregulating a coordinated set of inflammatory cytokines. This study suggests that intradiscal injection of LPS offers a model for triggering inflammation of the IVD, demonstrating that inflammatory insults alone may potentially trigger degenerative changes of the IVD.

Toward engineering a biological joint replacement.

Osteoarthritis is a major cause of disability and pain for patients in the United States. Treatments for this degenerative disease represent a significant challenge considering the poor regenerative capacity of adult articular cartilage. Tissue-engineering techniques have advanced over the last two decades such that cartilage-like tissue can be cultivated in the laboratory for implantation. Even so, major challenges remain for creating fully functional tissue. This review article overviews some of these challenges, including overcoming limitations in nutrient supply to cartilage, improving in vitro collagen production, improving integration of engineered cartilage with native tissue, and exploring the potential for engineering full articular surface replacements.

Tendon repair augmented with a novel circulating stem cell population.

Tendon ruptures are common sports-related injuries that are often treated surgically by the use of sutures followed by immobilization. However, tendon repair by standard technique is associated with long healing time and often suboptimal repair. Methods to enhance tendon repair time as well as the quality of repair are currently unmet clinical needs. Our hypothesis is that the introduction of a unique stem cell population at the site of tendon transection would result in an improved rate and quality of repair. Achilles tendons of fifty-one Sprague-Dawley rats were transected and suture-repaired. In half of the rats, a biodegradable scaffold seeded with allogenic circulating stem cells was placed as an onlay to the defect site in addition to the suture repair. The other half was treated with suture alone to serve as the control group. Animals were randomized to a two-, four-, or six-week time group. At the time of necropsy, tendons were harvested and prepared for either biomechanical or histological analysis. Histological slides were evaluated in a blinded fashion with the use of a grading scale. By two weeks, the experimental group demonstrated a significant improvement in repair compared to controls with no failures. Average histological scores of 0.6 and 2.6 were observed for the experimental and control group respectively. The experimental group demonstrated complete bridging of the transection site with parallel collagen fiber arrangement. By four weeks, both groups showed a continuing trend of healing, with the scaffold group exceeding the histological quality of the tissue repaired with suture alone. Biomechanically, the experimental group had a decreasing cross-sectional area with time which was also associated with a significant increase in the ultimate tensile strength of the tendons, reaching 4.2MPa by six weeks. The experimental group also achieved a significantly higher elastic toughness by six weeks and saw an increase in the tensile modulus, reaching 31Mpa by six weeks. The use of circulating stem cells as an adjunct in tendon repair demonstrates superior biomechanical properties and an improved level of histological organization, when compared to the suture alone control group. These improvements were not previously observed when gene therapy, protein therapy, or current tissue engineering technologies were used.

New methods to diagnose and treat cartilage degeneration.

Lesions in articular cartilage can result in significant musculoskeletal morbidity and display unique biomechanical characteristics that make repair difficult, at best. Several surgical procedures have been devised in an attempt to relieve pain, restore function, and delay or stop the progression of cartilaginous lesions. Advanced MRI and ultrasonography protocols are currently used in the evaluation of tissue repair and to improve diagnostic capability. Other nonoperative modalities, such as injection of intra-articular hyaluronic acid or supplementary oral glucosamine and chondroitin sulfate, have shown potential efficacy as anti-inflammatory and symptom-modifying agents. The emerging field of tissue engineering, involving the use of a biocompatible, structurally and mechanically stable scaffold, has shown promising early results in cartilage tissue repair. Scaffolds incorporating specific cell sources and bioactive molecules have been the focus in this new exciting field. Further work is required to better understand the behavior of chondrocytes and the variables that influence their ability to heal articular lesions. The future of cartilage repair will probably involve a combination of treatments in an attempt to achieve a regenerative tissue that is both biomechanically stable and, ideally, identical to the surrounding native tissues.