Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be a... more Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson's Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution. How to cite this article Warnock et al. (2014), Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study. PeerJ 2:e353; DOI 10.7717/peerj.353
Osteoarthritis (OA) is a costly and debilitating condition that is typically not diagnosed early ... more Osteoarthritis (OA) is a costly and debilitating condition that is typically not diagnosed early enough to prevent progression of disease. The purpose of this study was to evaluate synovial fluid from knees with and without OA for potential markers of joint inflammation and degradation and to correlate these findings with radiographic severity of disease. With Institutional Review Board approval, synovial fluid samples were collected before the patient undergoing total knee arthroplasty. Control knees (n = 3) were patients younger than 30 years of age with no history of anterior cruciate ligament, posterior cruciate ligament, or meniscal injury, and no surgical history for either knee. Weight-bearing, anterior-posterior radiographic views were used to determine radiographic OA severity using the modified Kellgren and Lawrence scale. Synovial fluid samples from 18 patients (21 knees) were analyzed using a multiplex assay. Matrix metalloproteinase (MMP)-1 (p < 0.001), interleukin (...
Translational models of posttraumatic osteoarthritis (PTOA) that accurately represent clinical pa... more Translational models of posttraumatic osteoarthritis (PTOA) that accurately represent clinical pathology need to be developed. This study assessed a novel canine model for PTOA using impact injury. Impacts were delivered to the medial femoral condyle of dogs using a custom-designed impactor at 20, 40, or 60 MPa. Functional assessments and magnetic resonance imaging (MRI) were performed at 2 and 12 weeks, and arthroscopic and histologic assessments were performed at 12 weeks after injury. At 2 and 12 weeks, dogs had observable lameness, knee pain, effusion, loss in range of motion (ROM) and dysfunction in both hindlimbs with severity correlated strongly (r > 0.77) to impact level. At 12 weeks, function, pain, effusion, and ROM were significantly (p < 0.049) worse in knees impacted at 40 and 60 MPa compared with 20 MPa. MRI showed consistent cartilage and subchondral bone marrow lesions, and arthroscopy revealed synovitis and cartilage destruction in impacted knees, with increas...
Journal of the American Veterinary Medical Association, 2003
To evaluate the use of a polymerase chain reaction (PCR) assay in detecting bovine leukosis virus... more To evaluate the use of a polymerase chain reaction (PCR) assay in detecting bovine leukosis virus (BLV) in adult dairy cows. Prospective study. 223 adult dairy cows. Cows were tested for BLV status by use of an ELISA and a PCR assay. Sensitivity, specificity, predictive values of positive and negative tests, and the percentage of cows correctly classified by PCR assay were calculated. Ninety-five percent confidence intervals were calculated for sensitivity and specificity. Sensitivity and specificity were 0.672 and 1.00, respectively. Prevalence of BLV in this herd was 0.807. Predictive value of a positive test was 1.00, and predictive value of a negative test was 0.421. The percentage of cows correctly classified by PCR assay was 73.5%. A positive PCR assay result provided definitive evidence that a cow was infected with BLV. Sensitivity and negative predictive value for PCR assay were low. Consequently, PCR assay alone is unreliable for routine detection of BLV in herds with high ...
To perform a controlled laboratory study to evaluate the effect of bupivacaine and morphine on ch... more To perform a controlled laboratory study to evaluate the effect of bupivacaine and morphine on chondrocytes and synovium in a coculture model of diarthrodial joints. Methods: A proven coculture model that allows for the assessment of cartilage and synovium exists. The model allows for simulation of the diarthrodial joint in both health and disease by using culture media with or without the addition of interleukin-1. Effects of the presence of bupivacaine and morphine were evaluated by measuring media concentration of glycosamino glycans (GAG), nitric oxide (NO), and prostaglandin E 2 (PGE 2 ), and tissue concentration of GAG, water, and collagen. Cell viability was determined through the use of confocal microscopy on days 1 and 2. Results: Cell viability 2 days after exposure to 0.5% bupivacaine was significantly less in the presence of bupivacaine than in the other groups, nearing a 100% decrease in viability. There was little effect of bupivacaine on cartilage water content or the tissue concentration of GAG and collagen. Morphine and bupivacaine both inhibited the expected rise in NO and PGE 2 when interleukin-1 was added to the media. Conclusions: Continuous 0.5% bupivacaine exposure has a clear detrimental effect on chondrocytes in this in vitro study. Both bupivacaine and morphine appear to have anti-inflammatory effects. Continuous morphine exposure does not cause gross chondrotoxicity in vitro and presents itself as a potential alternative intra-articular analgesic. Clinical Relevance: Intra-articular bupivacaine infusion is an effective analgesic strategy and is frequently used in both office and outpatient surgical settings. This study provides evidence that the continued usage of postoperative bupivacaine continuous infusion pumps may have a detrimental effect on chondrocytes. Morphine has been shown to be an effective intra-articular analgesic, and its anti-inflammatory role seen in this study makes it a potential alternative to bupivacaine.
This translational animal model study was designed to assess function, bone ingrowth and integrat... more This translational animal model study was designed to assess function, bone ingrowth and integration, and joint pathology associated with two different synthetic, bilayered osteochondral implants over a 3-month period after implantation into the femoral condyles of dogs. SynACart-Titanium (n = 6) and SynACart-PEEK (n = 6) (Arthrex, Naples, FL, and Sites Medical, Columbia City, IN) implants were press-fit into the lateral or medial femoral condyle (alternating location) of purpose-bred adult research dogs. Dogs were humanely euthanized 3 months after surgery and the operated knees were assessed radiographically, arthroscopically, grossly, and histologically. Based on all assessments, both types of implants were well tolerated and safe with no evidence for infection, migration, or rejection. Half of the SynACart-PEEK implants showed radiographic and histologic evidence of poor incorporation with all of these being in the lateral femoral condyle. SynACart-Titanium implants were conside...
Journal of shoulder and elbow surgery / American Shoulder and Elbow Surgeons ... [et al.], Jan 25, 2015
A hyperosmolar irrigation solution may decrease fluid extravasation during arthroscopic procedure... more A hyperosmolar irrigation solution may decrease fluid extravasation during arthroscopic procedures. Demonstrating the safety of a hyperosmolar irrigation solution with respect to chondrocyte viability and cartilage water content was deemed necessary before designing a clinical efficacy study. We designed a translational animal model study in which hyperosmolar arthroscopy irrigation fluid (1.8%, 600 mOsm/L) was compared with normal saline (0.9%, 300 mOsm/L). Purpose-bred research dogs (n = 5) underwent bilateral shoulder arthroscopy. Irrigation fluid was delivered to each shoulder joint (n = 10) at 40 mm Hg for 120 minutes using standard ingress and egress portals. The percentage change in shoulder girth was documented at the completion of 120 minutes. Articular cartilage sections from the glenoid and humeral head were harvested from both shoulders. Chondrocyte viability and tissue water content were evaluated. Differences between groups and compared with time 0 controls were determ...
Meniscal pathology is an extremely prevalent problem, which inevitably leads to osteoarthritis an... more Meniscal pathology is an extremely prevalent problem, which inevitably leads to osteoarthritis and associated pain, swelling, and disability. Relatively little data are available regarding the molecular, biochemical, and histologic aspects of meniscal disease. This study characterizes meniscal pathology in the presence of symptomatic osteoarthritis and correlates clinical and basic science data in an attempt to delineate clinically relevant mechanisms of disease. Twenty-seven knees from 23 patients who underwent total knee arthroplasty comprised the affected group and 6 aged nonsymptomatic knees were used as controls. All meniscal tissues were harvested and subjectively scored for gross and histologic pathology. Biochemical analyses were performed to determine glycosaminoglycan (GAG) content, collagen (hydroxyproline) content, and water content. Real-time polymerase chain reaction analysis was conducted for genes involved in synthesis (collagens [col] 1, 2, 3, and 6), degradation (m...
While magnetic resonance imaging (MRI) is often considered the "gold standard" diagnostic imaging... more While magnetic resonance imaging (MRI) is often considered the "gold standard" diagnostic imaging modality for detection of meniscal abnormalities, it is associated with misdiagnosis in as high as 47% of cases, is costly, and is not readily available to a large number of patients. Ultrasonographic examination of the knee has been reported to be an effective diagnostic tool for this purpose with the potential to overcome many of the shortcomings of MRI. The purpose of this study is to determine the clinical usefulness of ultrasonography for diagnosis of meniscal pathology in patients with acute knee pain and compare its diagnostic accuracy to MRI in a clinical setting. With Institutional Review Board approval, patients (n ¼ 71) with acute knee pain were prospectively enrolled with informed consent. Preoperative MRI (1.5 T) was performed on each affected knee using the hospital's standard equipment and protocols and read by faculty radiologists trained in musculoskeletal MRI. Ultrasonographic assessments of each affected knee were performed by one of two faculty members trained in musculoskeletal ultrasonography using a 10 to 14 MHz linear transducer. Arthroscopic evaluation of affected knees was performed by one of three faculty orthopedic surgeons to assess and record all joint pathology, which served as the reference standard for determining presence, type, and severity of meniscal pathology. All evaluators for each diagnostic modality were blinded to all other data. Data were collected and compared by a separate investigator to determine sensitivity (Sn), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), correct classification rate (CCR), likelihood ratios (LR[þ] and LR[À]), and odds ratios. Preoperative ultrasonographic assessment of meniscal pathology was associated with Sn ¼ 91.2%, Sp
Osteoarthritis is one of the most common, debilitating, musculoskeletal diseases; 12% associated ... more Osteoarthritis is one of the most common, debilitating, musculoskeletal diseases; 12% associated with traumatic injury resulting in post-traumatic osteoarthritis (PTOA). Our objective was to develop a single impact model with cartilage &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;injury level&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; defined in terms of controlled combinations of strain rate to a maximum strain (both independent of cartilage load resistance) to study their sensitivity to articular cartilage cell viability and potential PTOA biomarkers. A servo-hydraulic test machine was used to measure canine humeral head cartilage explant thickness under repeatable pressure, then subject it (except sham and controls) to a single impact having controlled constant velocity V=1 or 100mm/s (strain rate 1.82 or 182/s) to maximum strain ε=10%, 30%, or 50%. Thereafter, explants were cultured in media for twelve days, with media changed at day 1, 2, 3, 6, 9, 12. Explant thickness was measured at day 0 (pre-injury), 6 and 12 (post-injury). Cell viability, and tissue collagen and glycosaminoglycan (GAG) were analyzed immediately post-injury and day 12. Culture media were tested for biomarkers: GAG, collagen II, chondroitin sulfate-846, nitric oxide, and prostaglandin E2 (PGE2). Detrimental effects on cell viability, and release of GAG and PGE2 to the media were primarily strain-dependent, (PGE2 being more prolonged and sensitive at lower strains). The cartilage injury model appears to be useful (possibly superior) for investigating the relationship between impact severity of injury and the onset of PTOA, specifically for discovery of biomarkers to evaluate the risk of developing clinical PTOA, and to compare effective treatments for arthritis prevention.
Osteoarthritis (OA) is a progressive and debilitating disease that often develops from a focal le... more Osteoarthritis (OA) is a progressive and debilitating disease that often develops from a focal lesion and may take years to clinically manifest to a complete loss of joint structure and function. Currently, there is not a cure for OA, but early diagnosis and initiation of treatment may dramatically improve the prognosis and quality of life for affected individuals. This study was designed to determine the feasibility of analyzing changes in gene expression of articular cartilage using the Pond-Nuki model two weeks after ACL-transection in dogs, and to characterize the changes observed at this time point. The ACL of four dogs was completely transected arthroscopically, and the contralateral limb was used as the non-operated control. After two weeks the dogs were euthanatized and tissues harvested from the tibial plateau and femoral condyles of both limbs. Two dogs were used for histologic analysis and Mankin scoring. From the other two dogs the surface of the femoral condyle and tibi...
The menisci are integral components within the knee for ensuring optimal joint function. The over... more The menisci are integral components within the knee for ensuring optimal joint function. The overall goal of this study was to identify proteomic markers of meniscal disease within synovial fluid samples obtained from control knees versus knees affected with varying degrees of meniscal injury and osteoarthritis. Joint fluid samples were collected before the patient underwent an arthroscopic knee procedure or total knee arthroplasty. Normal controls included patients younger than 30 years with no history of anterior cruciate ligament, posterior cruciate ligament, or meniscal injury. A total of 21 joint fluid aspirates were analyzed using mass spectrometry, and a total of 296 proteins were identified. Among these, 50 proteins were determined to be of interest as potential biomarkers based on initial analysis and known functions in articular metabolism. Further statistical analysis comparing protein concentrations among clinical groups identified 13 proteins with significant difference...
The meniscus is a complex tissue and is integral to knee joint health and function. Although the ... more The meniscus is a complex tissue and is integral to knee joint health and function. Although the meniscus has been studied for years, a relatively large amount of basic science data on meniscal health and disease are unavailable. Genomic and proteomic analyses of meniscal pathology could greatly improve our understanding of etiopathogenesis and the progression of meniscal disease, yet these analyses are lacking in the current literature. Therefore, the objective of this study was to use microarray and proteomic analyses to compare aged-normal and pathologic meniscal tissues. Meniscal tissue was collected from the knees of five patient groups (n = 3/group). Cohorts included patients undergoing meniscectomy with or without articular cartilage pathology, patients undergoing total knee arthroplasty with mild or moderate-severe osteoarthritis, and aged-normal controls from organ donors. Tissue sections were collected from the white/white and white/red zones of posterior medial menisci. E...
Intra-articular injection of local anesthetic and/or corticosteroid is an adjunct treatment for a... more Intra-articular injection of local anesthetic and/or corticosteroid is an adjunct treatment for arthritic and inflammatory orthopedic conditions. Despite potential benefits, there is growing concern that these medications may cause significant morbidity, including potential toxicity to intra-articular chondrocytes and synoviocytes. Twenty dogs underwent intra-articular injection of the shoulder joint using ultrasound guidance, with the following injectates (n = 5 each): negative control (saline), methylprednisolone/1.0% lidocaine, triamcinolone/1.0% lidocaine, and triamcinolone/0.0625% bupivacaine. The dogs were euthanized 24 hours postinjection for reasons unrelated to this study. Synovium/cartilage explants were harvested under sterile conditions and assessed immediately or cultured for 7 days. Synoviocyte and chondrocyte viability was determined on day 1 and day 7 using Calcien AM and Sytox Blue live/dead fluorescent stains, and cell metabolism determined on day 2 using the alamar blue additive test. Results were compared statistically. On day 1 synovium exposed to 1%L/M demonstrated a significant decrease in cell metabolism (P = 0.0107) and subjective synoviocyte viability scores (P = 0.013) compared with the negative control. Cartilage exposed to 1%L/M demonstrated decreased chondrocyte viability and cell metabolism versus all other groups, although not significantly. After 7 days of culture, cartilage viable cell density in the 1%L/M group was significantly (P ≤ 0.001) lower than the negative control. Subjective synoviocyte viability scores was significantly lower in the 1%L/M (P = 0.013), 1%L/T (P ≤ 0.001), and 0.0625%B/T groups (P = 0.006) compared with the negative control. This study suggests potential negative effects of combination local anesthetic/corticosteroid on intra-articular cell viability and cell metabolism. Further study is needed before determining definitive clinical recommendations.
Osteochondral injury elevates the risk for developing posttraumatic osteoarthritis (PTOA). Theref... more Osteochondral injury elevates the risk for developing posttraumatic osteoarthritis (PTOA). Therefore, our objective was to evaluate the relationship between impact severity during injury to cell viability and biomarkers possibly involved in PTOA. Osteochondral explants (6 mm, n = 72) were harvested from cadaveric femoral condyles (N = 6). Using a test machine, each explant (except for No Impact) was subjected to mechanical impact at a velocity of 100 mm/s to 0.25, 0.5, 0.75, 1.0, or 1.25 mm maximum compression corresponding to Low, Low-Moderate, Moderate, Moderate-High, or High impact groups. Cartilage cell viability, collagen content, and proteoglycan content were assessed at either day 0 or after 12 days of culture. Culture media were assessed for prostaglandin E2 (PGE2); nitric oxide; granulocyte macrophage colony-stimulating factor (GM-CSF); interferon gamma (IFNγ); interleukin (IL)-2, -4, -6, -7, -8, -10, -15, -18; interferon gamma-induced protein 10 (IP-10); keratinocyte-derived chemoattractant (KC); monocyte chemoattractant protein-1 (MCP-1); tumor necrosis factor alpha (TNFα); and matrix metalloproteinase-2, -3, -8, -9, -13. There was increased impact energy absorbed for the High group compared with the Moderate-High group, Moderate group, and Low-Moderate group (p = 0.011, 0.048, 0.008, respectively). At day 0, there was decreased area cell viability for the High group compared with the Low-Moderate group (p = 0.035). At day 1, PGE2 was increased for the High group compared with the Moderate, Low-Moderate, Low, and No Impact groups (p ≤ 0.01). Cumulative PGE2 was increased for the Moderate-High and High groups compared with the Moderate, Low-Moderate, Low, and No Impact groups (p ≤ 0.036). At day 1, MCP-1 was increased for the Moderate-High and High groups compared with the Low and No Impact groups (p ≤ 0.032). Impact to osteochondral explants resulted in multiple levels of severity. PGE2 was sensitive to impact severity which may justify its use as a clinically measurable biomarker after joint injury for monitoring early PTOA.
Arthroscopy: The Journal of Arthroscopic & Related Surgery, 2015
The aim of this study was to assess the potential detrimental effects of the operating room envir... more The aim of this study was to assess the potential detrimental effects of the operating room environment on exposed healthy articular cartilage and to evaluate tissue hydration treatment strategies for preserving chondrocyte viability and extracellular matrix composition in this environment. With institutional Animal Care and Use Committee approval, femoral and tibial condyles (n = 36; 6 per specimen) were harvested from canine cadavers (n = 6) immediately after euthanasia and placed on a draped operating table under standard surgical lighting for a timed 2-hour period. Each condyle was randomly assigned to one of 6 groups (n = 6 per group): no-treatment control, hyaluronic acid (HA), saline sponge, saline drip, culture media (Dulbecco&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s modified Eagle&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s medium [DMEM]) sponge, or culture media drip. Full-thickness cartilage sections were collected from each specimen immediately after harvest (time 0) and immediately after 2-hour exposure (time 2H), and processed to determine chondrocyte viability, tissue water content, and extracellular matrix composition (glycosaminoglycan [GAG] and collagen content). Chondrocyte viability was significantly lower (P = .03) after the 2-hour exposure in the control group. HA, saline sponge, and saline drip treatment groups all had significantly higher (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .043) chondrocyte viability compared with controls at time 2H. Water content was significantly lower (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .01) after the 2-hour exposure in the control group. Further, the water content in the control group was significantly lower than all treatment groups at time 2H (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .001). No significant differences in tissue collagen or GAG content were observed within groups between time points or among groups at either time point. Canine articular cartilage did not demonstrate any reduction in chondrocyte viability or tissue water content at 2 hours when treated with hyaluronic acid, saline drip, saline-soaked sponge, or DMEM-soaked sponge compared with untreated exposed cartilage. Surgeons should consider the use of a hydrating solution for the treatment of exposed articular cartilage during open joint surgery of 2 hours or longer duration.
Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be a... more Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson's Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution. How to cite this article Warnock et al. (2014), Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study. PeerJ 2:e353; DOI 10.7717/peerj.353
Osteoarthritis and Cartilage - OSTEOARTHRITIS CARTILAGE, 2007
Osteoarthritis and Cartilage Vol. 15, Supplement C C125 synthesis and in PGE2 release elicited by... more Osteoarthritis and Cartilage Vol. 15, Supplement C C125 synthesis and in PGE2 release elicited by IL-1 in chondrocytes. Furthermore, in synoviocytes, diacerhein not only did not inhibit the release of these mediators but even super-induced their expression and synthesis. NSAIDs diminished until almost basal levels the PGE2 release, and, unexpectedly, these drugs also diminished COX-2 synthesis in both cell types. In relation to the increase in NO synthesis evoked by IL-1 in chondrocytes, only diacerhein was able to prevent it, while no effect was noted for the NSAIDs. Diacerhein did not modify the synthesis of MMPs induced by IL-1 in chondrocytes, but it decreased MMP-13 accumulation in synoviocytes. In contrast, NSAIDs induced in both cell types a further increase in MMP1 in IL-1 treated cells, as well as an enhancement of MMP-13 accumulation in chondrocytes. Conclusions: Diacerhein inhibited the activation of NF-kB induced by IL-1 in both cell types and the synthesis of NO in OA chondrocytes, whereas it failed in the control of PGE2, COX-2 and MMPs in any of the cell types. Taken together these results would anticipate a weak clinical response to diacerhein in OA. In turn, the NSAIDs inhibited NF-kB activation and PGE2 release in these cells, but they were unable to control MMP activation, in accordance with their presumed "symptom relieving" but not "disease modifying" role in the treatment of OA.
Objective: To determine the effects of uniaxial cyclic tensile load amplitude and duration on gen... more Objective: To determine the effects of uniaxial cyclic tensile load amplitude and duration on gene expression in cranial cruciate ligamentocytes cultured in monolayer. Study Design: In vitro experimental study. Animals: Adult dogs (n = 9) weighing 20-35 kg. Methods: Cranial cruciate ligaments (CCL, n = 18) were aseptically collected, diced, digested using clostridial collagenase, and primary monolayer cultures were established. CCL cells were seeded at a concentration of 3 Â 10 5 cells/mL onto a specialized collagen membrane. After 24 hours to allow attachment, ligamentocytes were subjected to 0%, 4%, or 8% uniaxial strain for 24 or 48 hours using a sinusoidal strain profile at 0.5 Hz. At the end of each time point, the ligamentocytes were harvested and analyzed for collagen 1 (COL1), collagen 3 (COL3), and matrix metalloproteinase-3 (MMP-3) gene expression using reverse transcriptase real-time polymerase chain reaction.
Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be a... more Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson's Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution. How to cite this article Warnock et al. (2014), Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study. PeerJ 2:e353; DOI 10.7717/peerj.353
Osteoarthritis (OA) is a costly and debilitating condition that is typically not diagnosed early ... more Osteoarthritis (OA) is a costly and debilitating condition that is typically not diagnosed early enough to prevent progression of disease. The purpose of this study was to evaluate synovial fluid from knees with and without OA for potential markers of joint inflammation and degradation and to correlate these findings with radiographic severity of disease. With Institutional Review Board approval, synovial fluid samples were collected before the patient undergoing total knee arthroplasty. Control knees (n = 3) were patients younger than 30 years of age with no history of anterior cruciate ligament, posterior cruciate ligament, or meniscal injury, and no surgical history for either knee. Weight-bearing, anterior-posterior radiographic views were used to determine radiographic OA severity using the modified Kellgren and Lawrence scale. Synovial fluid samples from 18 patients (21 knees) were analyzed using a multiplex assay. Matrix metalloproteinase (MMP)-1 (p < 0.001), interleukin (...
Translational models of posttraumatic osteoarthritis (PTOA) that accurately represent clinical pa... more Translational models of posttraumatic osteoarthritis (PTOA) that accurately represent clinical pathology need to be developed. This study assessed a novel canine model for PTOA using impact injury. Impacts were delivered to the medial femoral condyle of dogs using a custom-designed impactor at 20, 40, or 60 MPa. Functional assessments and magnetic resonance imaging (MRI) were performed at 2 and 12 weeks, and arthroscopic and histologic assessments were performed at 12 weeks after injury. At 2 and 12 weeks, dogs had observable lameness, knee pain, effusion, loss in range of motion (ROM) and dysfunction in both hindlimbs with severity correlated strongly (r > 0.77) to impact level. At 12 weeks, function, pain, effusion, and ROM were significantly (p < 0.049) worse in knees impacted at 40 and 60 MPa compared with 20 MPa. MRI showed consistent cartilage and subchondral bone marrow lesions, and arthroscopy revealed synovitis and cartilage destruction in impacted knees, with increas...
Journal of the American Veterinary Medical Association, 2003
To evaluate the use of a polymerase chain reaction (PCR) assay in detecting bovine leukosis virus... more To evaluate the use of a polymerase chain reaction (PCR) assay in detecting bovine leukosis virus (BLV) in adult dairy cows. Prospective study. 223 adult dairy cows. Cows were tested for BLV status by use of an ELISA and a PCR assay. Sensitivity, specificity, predictive values of positive and negative tests, and the percentage of cows correctly classified by PCR assay were calculated. Ninety-five percent confidence intervals were calculated for sensitivity and specificity. Sensitivity and specificity were 0.672 and 1.00, respectively. Prevalence of BLV in this herd was 0.807. Predictive value of a positive test was 1.00, and predictive value of a negative test was 0.421. The percentage of cows correctly classified by PCR assay was 73.5%. A positive PCR assay result provided definitive evidence that a cow was infected with BLV. Sensitivity and negative predictive value for PCR assay were low. Consequently, PCR assay alone is unreliable for routine detection of BLV in herds with high ...
To perform a controlled laboratory study to evaluate the effect of bupivacaine and morphine on ch... more To perform a controlled laboratory study to evaluate the effect of bupivacaine and morphine on chondrocytes and synovium in a coculture model of diarthrodial joints. Methods: A proven coculture model that allows for the assessment of cartilage and synovium exists. The model allows for simulation of the diarthrodial joint in both health and disease by using culture media with or without the addition of interleukin-1. Effects of the presence of bupivacaine and morphine were evaluated by measuring media concentration of glycosamino glycans (GAG), nitric oxide (NO), and prostaglandin E 2 (PGE 2 ), and tissue concentration of GAG, water, and collagen. Cell viability was determined through the use of confocal microscopy on days 1 and 2. Results: Cell viability 2 days after exposure to 0.5% bupivacaine was significantly less in the presence of bupivacaine than in the other groups, nearing a 100% decrease in viability. There was little effect of bupivacaine on cartilage water content or the tissue concentration of GAG and collagen. Morphine and bupivacaine both inhibited the expected rise in NO and PGE 2 when interleukin-1 was added to the media. Conclusions: Continuous 0.5% bupivacaine exposure has a clear detrimental effect on chondrocytes in this in vitro study. Both bupivacaine and morphine appear to have anti-inflammatory effects. Continuous morphine exposure does not cause gross chondrotoxicity in vitro and presents itself as a potential alternative intra-articular analgesic. Clinical Relevance: Intra-articular bupivacaine infusion is an effective analgesic strategy and is frequently used in both office and outpatient surgical settings. This study provides evidence that the continued usage of postoperative bupivacaine continuous infusion pumps may have a detrimental effect on chondrocytes. Morphine has been shown to be an effective intra-articular analgesic, and its anti-inflammatory role seen in this study makes it a potential alternative to bupivacaine.
This translational animal model study was designed to assess function, bone ingrowth and integrat... more This translational animal model study was designed to assess function, bone ingrowth and integration, and joint pathology associated with two different synthetic, bilayered osteochondral implants over a 3-month period after implantation into the femoral condyles of dogs. SynACart-Titanium (n = 6) and SynACart-PEEK (n = 6) (Arthrex, Naples, FL, and Sites Medical, Columbia City, IN) implants were press-fit into the lateral or medial femoral condyle (alternating location) of purpose-bred adult research dogs. Dogs were humanely euthanized 3 months after surgery and the operated knees were assessed radiographically, arthroscopically, grossly, and histologically. Based on all assessments, both types of implants were well tolerated and safe with no evidence for infection, migration, or rejection. Half of the SynACart-PEEK implants showed radiographic and histologic evidence of poor incorporation with all of these being in the lateral femoral condyle. SynACart-Titanium implants were conside...
Journal of shoulder and elbow surgery / American Shoulder and Elbow Surgeons ... [et al.], Jan 25, 2015
A hyperosmolar irrigation solution may decrease fluid extravasation during arthroscopic procedure... more A hyperosmolar irrigation solution may decrease fluid extravasation during arthroscopic procedures. Demonstrating the safety of a hyperosmolar irrigation solution with respect to chondrocyte viability and cartilage water content was deemed necessary before designing a clinical efficacy study. We designed a translational animal model study in which hyperosmolar arthroscopy irrigation fluid (1.8%, 600 mOsm/L) was compared with normal saline (0.9%, 300 mOsm/L). Purpose-bred research dogs (n = 5) underwent bilateral shoulder arthroscopy. Irrigation fluid was delivered to each shoulder joint (n = 10) at 40 mm Hg for 120 minutes using standard ingress and egress portals. The percentage change in shoulder girth was documented at the completion of 120 minutes. Articular cartilage sections from the glenoid and humeral head were harvested from both shoulders. Chondrocyte viability and tissue water content were evaluated. Differences between groups and compared with time 0 controls were determ...
Meniscal pathology is an extremely prevalent problem, which inevitably leads to osteoarthritis an... more Meniscal pathology is an extremely prevalent problem, which inevitably leads to osteoarthritis and associated pain, swelling, and disability. Relatively little data are available regarding the molecular, biochemical, and histologic aspects of meniscal disease. This study characterizes meniscal pathology in the presence of symptomatic osteoarthritis and correlates clinical and basic science data in an attempt to delineate clinically relevant mechanisms of disease. Twenty-seven knees from 23 patients who underwent total knee arthroplasty comprised the affected group and 6 aged nonsymptomatic knees were used as controls. All meniscal tissues were harvested and subjectively scored for gross and histologic pathology. Biochemical analyses were performed to determine glycosaminoglycan (GAG) content, collagen (hydroxyproline) content, and water content. Real-time polymerase chain reaction analysis was conducted for genes involved in synthesis (collagens [col] 1, 2, 3, and 6), degradation (m...
While magnetic resonance imaging (MRI) is often considered the "gold standard" diagnostic imaging... more While magnetic resonance imaging (MRI) is often considered the "gold standard" diagnostic imaging modality for detection of meniscal abnormalities, it is associated with misdiagnosis in as high as 47% of cases, is costly, and is not readily available to a large number of patients. Ultrasonographic examination of the knee has been reported to be an effective diagnostic tool for this purpose with the potential to overcome many of the shortcomings of MRI. The purpose of this study is to determine the clinical usefulness of ultrasonography for diagnosis of meniscal pathology in patients with acute knee pain and compare its diagnostic accuracy to MRI in a clinical setting. With Institutional Review Board approval, patients (n ¼ 71) with acute knee pain were prospectively enrolled with informed consent. Preoperative MRI (1.5 T) was performed on each affected knee using the hospital's standard equipment and protocols and read by faculty radiologists trained in musculoskeletal MRI. Ultrasonographic assessments of each affected knee were performed by one of two faculty members trained in musculoskeletal ultrasonography using a 10 to 14 MHz linear transducer. Arthroscopic evaluation of affected knees was performed by one of three faculty orthopedic surgeons to assess and record all joint pathology, which served as the reference standard for determining presence, type, and severity of meniscal pathology. All evaluators for each diagnostic modality were blinded to all other data. Data were collected and compared by a separate investigator to determine sensitivity (Sn), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), correct classification rate (CCR), likelihood ratios (LR[þ] and LR[À]), and odds ratios. Preoperative ultrasonographic assessment of meniscal pathology was associated with Sn ¼ 91.2%, Sp
Osteoarthritis is one of the most common, debilitating, musculoskeletal diseases; 12% associated ... more Osteoarthritis is one of the most common, debilitating, musculoskeletal diseases; 12% associated with traumatic injury resulting in post-traumatic osteoarthritis (PTOA). Our objective was to develop a single impact model with cartilage &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;injury level&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; defined in terms of controlled combinations of strain rate to a maximum strain (both independent of cartilage load resistance) to study their sensitivity to articular cartilage cell viability and potential PTOA biomarkers. A servo-hydraulic test machine was used to measure canine humeral head cartilage explant thickness under repeatable pressure, then subject it (except sham and controls) to a single impact having controlled constant velocity V=1 or 100mm/s (strain rate 1.82 or 182/s) to maximum strain ε=10%, 30%, or 50%. Thereafter, explants were cultured in media for twelve days, with media changed at day 1, 2, 3, 6, 9, 12. Explant thickness was measured at day 0 (pre-injury), 6 and 12 (post-injury). Cell viability, and tissue collagen and glycosaminoglycan (GAG) were analyzed immediately post-injury and day 12. Culture media were tested for biomarkers: GAG, collagen II, chondroitin sulfate-846, nitric oxide, and prostaglandin E2 (PGE2). Detrimental effects on cell viability, and release of GAG and PGE2 to the media were primarily strain-dependent, (PGE2 being more prolonged and sensitive at lower strains). The cartilage injury model appears to be useful (possibly superior) for investigating the relationship between impact severity of injury and the onset of PTOA, specifically for discovery of biomarkers to evaluate the risk of developing clinical PTOA, and to compare effective treatments for arthritis prevention.
Osteoarthritis (OA) is a progressive and debilitating disease that often develops from a focal le... more Osteoarthritis (OA) is a progressive and debilitating disease that often develops from a focal lesion and may take years to clinically manifest to a complete loss of joint structure and function. Currently, there is not a cure for OA, but early diagnosis and initiation of treatment may dramatically improve the prognosis and quality of life for affected individuals. This study was designed to determine the feasibility of analyzing changes in gene expression of articular cartilage using the Pond-Nuki model two weeks after ACL-transection in dogs, and to characterize the changes observed at this time point. The ACL of four dogs was completely transected arthroscopically, and the contralateral limb was used as the non-operated control. After two weeks the dogs were euthanatized and tissues harvested from the tibial plateau and femoral condyles of both limbs. Two dogs were used for histologic analysis and Mankin scoring. From the other two dogs the surface of the femoral condyle and tibi...
The menisci are integral components within the knee for ensuring optimal joint function. The over... more The menisci are integral components within the knee for ensuring optimal joint function. The overall goal of this study was to identify proteomic markers of meniscal disease within synovial fluid samples obtained from control knees versus knees affected with varying degrees of meniscal injury and osteoarthritis. Joint fluid samples were collected before the patient underwent an arthroscopic knee procedure or total knee arthroplasty. Normal controls included patients younger than 30 years with no history of anterior cruciate ligament, posterior cruciate ligament, or meniscal injury. A total of 21 joint fluid aspirates were analyzed using mass spectrometry, and a total of 296 proteins were identified. Among these, 50 proteins were determined to be of interest as potential biomarkers based on initial analysis and known functions in articular metabolism. Further statistical analysis comparing protein concentrations among clinical groups identified 13 proteins with significant difference...
The meniscus is a complex tissue and is integral to knee joint health and function. Although the ... more The meniscus is a complex tissue and is integral to knee joint health and function. Although the meniscus has been studied for years, a relatively large amount of basic science data on meniscal health and disease are unavailable. Genomic and proteomic analyses of meniscal pathology could greatly improve our understanding of etiopathogenesis and the progression of meniscal disease, yet these analyses are lacking in the current literature. Therefore, the objective of this study was to use microarray and proteomic analyses to compare aged-normal and pathologic meniscal tissues. Meniscal tissue was collected from the knees of five patient groups (n = 3/group). Cohorts included patients undergoing meniscectomy with or without articular cartilage pathology, patients undergoing total knee arthroplasty with mild or moderate-severe osteoarthritis, and aged-normal controls from organ donors. Tissue sections were collected from the white/white and white/red zones of posterior medial menisci. E...
Intra-articular injection of local anesthetic and/or corticosteroid is an adjunct treatment for a... more Intra-articular injection of local anesthetic and/or corticosteroid is an adjunct treatment for arthritic and inflammatory orthopedic conditions. Despite potential benefits, there is growing concern that these medications may cause significant morbidity, including potential toxicity to intra-articular chondrocytes and synoviocytes. Twenty dogs underwent intra-articular injection of the shoulder joint using ultrasound guidance, with the following injectates (n = 5 each): negative control (saline), methylprednisolone/1.0% lidocaine, triamcinolone/1.0% lidocaine, and triamcinolone/0.0625% bupivacaine. The dogs were euthanized 24 hours postinjection for reasons unrelated to this study. Synovium/cartilage explants were harvested under sterile conditions and assessed immediately or cultured for 7 days. Synoviocyte and chondrocyte viability was determined on day 1 and day 7 using Calcien AM and Sytox Blue live/dead fluorescent stains, and cell metabolism determined on day 2 using the alamar blue additive test. Results were compared statistically. On day 1 synovium exposed to 1%L/M demonstrated a significant decrease in cell metabolism (P = 0.0107) and subjective synoviocyte viability scores (P = 0.013) compared with the negative control. Cartilage exposed to 1%L/M demonstrated decreased chondrocyte viability and cell metabolism versus all other groups, although not significantly. After 7 days of culture, cartilage viable cell density in the 1%L/M group was significantly (P ≤ 0.001) lower than the negative control. Subjective synoviocyte viability scores was significantly lower in the 1%L/M (P = 0.013), 1%L/T (P ≤ 0.001), and 0.0625%B/T groups (P = 0.006) compared with the negative control. This study suggests potential negative effects of combination local anesthetic/corticosteroid on intra-articular cell viability and cell metabolism. Further study is needed before determining definitive clinical recommendations.
Osteochondral injury elevates the risk for developing posttraumatic osteoarthritis (PTOA). Theref... more Osteochondral injury elevates the risk for developing posttraumatic osteoarthritis (PTOA). Therefore, our objective was to evaluate the relationship between impact severity during injury to cell viability and biomarkers possibly involved in PTOA. Osteochondral explants (6 mm, n = 72) were harvested from cadaveric femoral condyles (N = 6). Using a test machine, each explant (except for No Impact) was subjected to mechanical impact at a velocity of 100 mm/s to 0.25, 0.5, 0.75, 1.0, or 1.25 mm maximum compression corresponding to Low, Low-Moderate, Moderate, Moderate-High, or High impact groups. Cartilage cell viability, collagen content, and proteoglycan content were assessed at either day 0 or after 12 days of culture. Culture media were assessed for prostaglandin E2 (PGE2); nitric oxide; granulocyte macrophage colony-stimulating factor (GM-CSF); interferon gamma (IFNγ); interleukin (IL)-2, -4, -6, -7, -8, -10, -15, -18; interferon gamma-induced protein 10 (IP-10); keratinocyte-derived chemoattractant (KC); monocyte chemoattractant protein-1 (MCP-1); tumor necrosis factor alpha (TNFα); and matrix metalloproteinase-2, -3, -8, -9, -13. There was increased impact energy absorbed for the High group compared with the Moderate-High group, Moderate group, and Low-Moderate group (p = 0.011, 0.048, 0.008, respectively). At day 0, there was decreased area cell viability for the High group compared with the Low-Moderate group (p = 0.035). At day 1, PGE2 was increased for the High group compared with the Moderate, Low-Moderate, Low, and No Impact groups (p ≤ 0.01). Cumulative PGE2 was increased for the Moderate-High and High groups compared with the Moderate, Low-Moderate, Low, and No Impact groups (p ≤ 0.036). At day 1, MCP-1 was increased for the Moderate-High and High groups compared with the Low and No Impact groups (p ≤ 0.032). Impact to osteochondral explants resulted in multiple levels of severity. PGE2 was sensitive to impact severity which may justify its use as a clinically measurable biomarker after joint injury for monitoring early PTOA.
Arthroscopy: The Journal of Arthroscopic & Related Surgery, 2015
The aim of this study was to assess the potential detrimental effects of the operating room envir... more The aim of this study was to assess the potential detrimental effects of the operating room environment on exposed healthy articular cartilage and to evaluate tissue hydration treatment strategies for preserving chondrocyte viability and extracellular matrix composition in this environment. With institutional Animal Care and Use Committee approval, femoral and tibial condyles (n = 36; 6 per specimen) were harvested from canine cadavers (n = 6) immediately after euthanasia and placed on a draped operating table under standard surgical lighting for a timed 2-hour period. Each condyle was randomly assigned to one of 6 groups (n = 6 per group): no-treatment control, hyaluronic acid (HA), saline sponge, saline drip, culture media (Dulbecco&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s modified Eagle&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s medium [DMEM]) sponge, or culture media drip. Full-thickness cartilage sections were collected from each specimen immediately after harvest (time 0) and immediately after 2-hour exposure (time 2H), and processed to determine chondrocyte viability, tissue water content, and extracellular matrix composition (glycosaminoglycan [GAG] and collagen content). Chondrocyte viability was significantly lower (P = .03) after the 2-hour exposure in the control group. HA, saline sponge, and saline drip treatment groups all had significantly higher (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .043) chondrocyte viability compared with controls at time 2H. Water content was significantly lower (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .01) after the 2-hour exposure in the control group. Further, the water content in the control group was significantly lower than all treatment groups at time 2H (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .001). No significant differences in tissue collagen or GAG content were observed within groups between time points or among groups at either time point. Canine articular cartilage did not demonstrate any reduction in chondrocyte viability or tissue water content at 2 hours when treated with hyaluronic acid, saline drip, saline-soaked sponge, or DMEM-soaked sponge compared with untreated exposed cartilage. Surgeons should consider the use of a hydrating solution for the treatment of exposed articular cartilage during open joint surgery of 2 hours or longer duration.
Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be a... more Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson's Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution. How to cite this article Warnock et al. (2014), Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study. PeerJ 2:e353; DOI 10.7717/peerj.353
Osteoarthritis and Cartilage - OSTEOARTHRITIS CARTILAGE, 2007
Osteoarthritis and Cartilage Vol. 15, Supplement C C125 synthesis and in PGE2 release elicited by... more Osteoarthritis and Cartilage Vol. 15, Supplement C C125 synthesis and in PGE2 release elicited by IL-1 in chondrocytes. Furthermore, in synoviocytes, diacerhein not only did not inhibit the release of these mediators but even super-induced their expression and synthesis. NSAIDs diminished until almost basal levels the PGE2 release, and, unexpectedly, these drugs also diminished COX-2 synthesis in both cell types. In relation to the increase in NO synthesis evoked by IL-1 in chondrocytes, only diacerhein was able to prevent it, while no effect was noted for the NSAIDs. Diacerhein did not modify the synthesis of MMPs induced by IL-1 in chondrocytes, but it decreased MMP-13 accumulation in synoviocytes. In contrast, NSAIDs induced in both cell types a further increase in MMP1 in IL-1 treated cells, as well as an enhancement of MMP-13 accumulation in chondrocytes. Conclusions: Diacerhein inhibited the activation of NF-kB induced by IL-1 in both cell types and the synthesis of NO in OA chondrocytes, whereas it failed in the control of PGE2, COX-2 and MMPs in any of the cell types. Taken together these results would anticipate a weak clinical response to diacerhein in OA. In turn, the NSAIDs inhibited NF-kB activation and PGE2 release in these cells, but they were unable to control MMP activation, in accordance with their presumed "symptom relieving" but not "disease modifying" role in the treatment of OA.
Objective: To determine the effects of uniaxial cyclic tensile load amplitude and duration on gen... more Objective: To determine the effects of uniaxial cyclic tensile load amplitude and duration on gene expression in cranial cruciate ligamentocytes cultured in monolayer. Study Design: In vitro experimental study. Animals: Adult dogs (n = 9) weighing 20-35 kg. Methods: Cranial cruciate ligaments (CCL, n = 18) were aseptically collected, diced, digested using clostridial collagenase, and primary monolayer cultures were established. CCL cells were seeded at a concentration of 3 Â 10 5 cells/mL onto a specialized collagen membrane. After 24 hours to allow attachment, ligamentocytes were subjected to 0%, 4%, or 8% uniaxial strain for 24 or 48 hours using a sinusoidal strain profile at 0.5 Hz. At the end of each time point, the ligamentocytes were harvested and analyzed for collagen 1 (COL1), collagen 3 (COL3), and matrix metalloproteinase-3 (MMP-3) gene expression using reverse transcriptase real-time polymerase chain reaction.
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