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Background: Meniscal allograft transplantation (MAT) has been developed as a treatment for meniscal deficiency. Despite promising outcomes, there are no real-time methods to evaluate graft survivorship and predict functional outcomes. Hypothesis: Assessment of serum and urine biomarkers could be used to develop biomarker panels—prognostic (1- and 3-month postsurgical time points) and diagnostic (6-month time point)—based on strong associations with clinically relevant outcome metrics obtained 6 months after surgery. Study Design: Descriptive laboratory study. Methods: Twelve adult purpose-bred research hounds were included and underwent medial meniscal release to induce meniscal deficiency. Three months after meniscal release surgery, medial menisci were replaced with fresh-frozen meniscus (n = 4), fresh meniscus (n = 4), or fresh meniscotibial osteochondral allograft (n = 4) such that a spectrum of pain and functional outcomes could be anticipated. Serum and urine from all dogs were collected preoperatively and at 1, 3, and 6 months after MAT surgery. Dogs were assessed for pain-related and functional outcomes at the same time points. To develop a prognostic panel of biomarkers, biomarker data from the 1- and 3-month post-MAT surgery time points were used to model 6-month clinical outcomes. A diagnostic panel of biomarkers was developed using data from the 6-month post-MAT surgery to model 6-month clinical outcomes. Primary outcomes for pain and function were visual analog scale (VAS) and operated limb percentage total pressure index (%TPI), respectively. Using random subject effects, linear mixed models were used to develop prognostic biomarker panels, and linear fixed-effect models were used to develop diagnostic biomarker panels, with variance explained for each panel reported ( R2) along with individual biomarker relationships. Results: Across prognostic biomarker panels, a panel including serum IL-6, IL-8, IL-10, and IL-18 was fit for the primary functional outcome, operated limb %TPI ( R2 = 0.450), whereas a panel including serum CTX-II and OPG was fit for the primary pain-related outcome, VAS ( R2 = 0.516). Across diagnostic biomarker panels, a panel including serum MMP-1 and MMP-3 and urine PINP and TIMP-1 was fit for %TPI ( R2 = 0.863). Separately, a panel including urine CTX-I, CTX-II, IL-8, MMP-2, and TIMP-1 was fit as diagnostic biomarkers for the VAS for pain ( R2 = 0.438). Conclusion: Biomarker panels of selected serum and/or urine proteins can model clinically relevant metrics for function and pain in a preclinical model of MAT. Clinical Relevance: Biomarker panels could be used to provide real-time diagnostic and prognostic data regarding outcomes after MAT.

Anterior cruciate ligament (ACL) injuries are common knee ligament injuries. While generally successful, ACL reconstruction that uses a tendon graft to stabilize the knee is still associated with a notable percentage of failures and long-term morbidities. Preclinical research that uses small laboratory species (i.e., mice, rats, and rabbits) to model ACL reconstruction are important to evaluate factors that can impact graft incorporation or posttraumatic osteoarthritis after ACL reconstruction. Small animal ACL reconstruction models are also used for proof-of-concept studies for the development of emerging biological strategies aimed at improving ACL reconstruction healing. The objective of this review is to provide an overview on the use of common small animal laboratory species to model ACL reconstruction. The review includes a discussion on comparative knee anatomy, technical considerations including types of tendon grafts employed amongst the small laboratory species (i.e., mice, rats, and rabbits), and common laboratory evaluative methods used to study healing and outcomes after ACL reconstruction in small laboratory animals. The review will also highlight common research questions addressed with small animal models of ACL reconstruction.

The purpose of this study was to determine the effects of a multidrug injectate containing morphine, ropivacaine, epinephrine, and ketorolac, commonly referred to as the "Orthococktail," on cartilage tissue viability and metabolic responses using an established in vitro model.With institutional review board approval and informed patient consent, tissues normally discarded after total knee arthroplasty (TKA) were recovered. Full-thickness cartilage explants (n = 72, Outerbridge grade 1 to 3) were created and bisected. Paired explant halves were treated with either 1 mL Orthococktail or 1 mL of saline and cultured for 8 hours at 37°C, with 0.5 mL of the treatment being removed and replaced with tissue culture media every hour. Explants were cultured for 6 days, and media were changed and collected on days 3 and 6. After day 6, tissues were processed for cell viability, weighed, and processed for histologic grading. Outcome measures were compared for significant differences between treated and untreated samples.There were no significant differences in cartilage viability between control and Orthococktail-treated samples across a spectrum of cartilage pathologies. Orthococktail treatment consistently resulted in a significant decrease in the release of PGE2, MCP-1, MMP-7, and MMP-8 on day 3 of culture and PGE2, MMP-3, MMP-7, and MMP-8 on day 6 of culture, compared with saline controls.The results of the present study indicate that an Orthococktail injection composed of morphine, ropivacaine, epinephrine, and ketorolac is associated with a transient decrease in degradative and inflammatory mediators produced by more severely affected articular cartilage and may mitigate perioperative joint pain such that postoperative narcotic drug use could be reduced.The Orthococktail solution used in this study may be a safe intraoperative, intra-articular injection option for patients undergoing joint arthroplasty and other joint preservation surgical procedures.

Bone defects often require operative intervention with the use of bone graft. Two sources of autologous bone graft include reamer-irrigator-aspirate (RIA) and bone marrow aspirate concentrate (BMC). Osteoprogenitor cells and osteoconductive proteins have been identified in both sources. This study collected samples of these cells and proteins from a canine model and cultured them on human cancellous allograft bone blocks. Findings suggest that BMC may be preferred for indications that allow for delivery via injection, saturation of the patient's tissues, or an implanted scaffold, whereas RIA may be preferred when the biologic augment is delivered as a scaffold or graft.

The aim of the study was to compare the acute effects of walking the golf course versus using a golf cart during a round of golf on biological markers of joint disease, joint pain, and cardiovascular parameters in individuals with knee osteoarthritis.Participants with knee OA (n = 10) older than 50 yrs were recruited for this crossover designed study in which they completed two 18-hole rounds of golf: (1) walking the course and (2) using a golf cart. Five control participants (n = 5) performed the walking condition only. Step count, heart rate, rating of perceived exertion and pain using the Numeric Pain Rating Scale were measured during the round. Serum was collected at baseline, 9th hole (halfway), and 18th hole (completion) and tested for biomarkers associated with tissue turnover (cartilage oligomeric matrix protein), inflammation (tumor necrosis factor α, interleukin 1β, interleukin 6), and degradative enzyme production (matrix metalloproteinase 3, matrix metalloproteinase 13).In knee OA participants, walking the course was associated with significantly higher step count and duration of moderate/vigorous physical activity (72.2% vs. 32.6% of the round) but did lead to a significant increase in knee joint pain (P0.05). Both conditions caused cartilage oligomeric matrix protein and matrix metalloproteinase 13 concentration increases from baseline to completion (P0.05), but inflammatory markers (tumor necrosis factor α, interleukin 6, and interleukin 1β, P0.05) only increased when walking the course. Biomarker concentrations did not increase in control participants.Walking the course optimizes the duration of moderate/vigorous activity during a round of golf, but the golf cart is a beneficial option in those with exacerbated joint pain and inflammation that would otherwise limit participation.

Posttraumatic osteoarthritis (PTOA) significantly affects patients with pilon fractures even after adequate anatomical reduction, and treatment strategies targeting the biologic mediators of PTOA are needed. This study was designed to determine the effects of intra-articular injection of platelet-rich plasma (PRP) on synovial fluid (SF) biomarkers for patients undergoing open reduction and internal fixation (ORIF) of pilon fractures. Patients undergoing staged management of pilon fractures were enrolled in a prospective, double-blinded, randomized, and placebo-controlled clinical trial to determine the effects of a single intra-articular injection of leukocyte-reduced PRP on SF biomarkers. Arthrocentesis of the injured and uninjured ankles was performed at the time of external skeletal fixation (ESF) and ORIF. Patients were randomized to receive either autogenous leukocyte-reduced PRP or saline (control) via intra-articular injection into the injured ankle at the time of ESF. SF biomarker concentrations were compared-uninjured, injured pretreatment, and saline-injected or PRP-injected. Eleven patients (PRP, n = 5; saline, n = 6) completed the study. Twenty-one uninjured, and 11 injured pretreatment, five PRP-treated, and six saline-treated SF samples were analyzed. PRP-treated SF contained significantly higher levels of PDGF-AA (p = 0.046) and significantly lower levels of MMP-3 (p = 0.042), MMP-9 (p = 0.009), IL-1β (p = 0.049), IL-6 (p < 0.01), IL-8 (p = 0.048), and PGE2 (p < 0.04). This study provided mechanistic data to suggest that a single intraarticular injection of leukocyte-reduced PRP is associated with anti-inflammatory, anti-degradative, and anabolic responses compared with saline control. These findings provide the impetus for investigating long-term clinical outcomes after PRP injection as an orthobiologic adjunct to ORIF for mitigating the incidence and severity of PTOA after pilon fractures.

Treatment options that result in consistently successful outcomes for young and active patients with joint disorders are needed. This article summarizes two decades of rigorous research using a bedside-to-bench- to-bedside translational approach based on the One Health – One Medicine concept that culminated in successful clinical use of biologic joint restoration options for treatment of knee, hip, ankle, and shoulder problems in this growing patient population.

OBJECTIVE To determine the following: (1) whether an irrigation solution that is hyperosmolar (HYPER) relative to synovial fluid decreases tissue extravasation during an arthroscopic protocol when compared to a relatively hypoosmolar solution, (2) the safety of a HYPER solution based on viability of joint tissues following joint irrigation, and (3) if the use of a HYPER solution decreases water content in stifle joint tissue. ANIMALS 8 adult horses. PROCEDURES A prospective, blinded, randomized controlled trial was performed to compare lactated Ringer’s solution (LRS; 273 mOsm/L) and a HYPER (600 mOsm/L) irrigation solution for routine medial femorotibial joint (MFTJ) arthroscopy. Primary outcomes included quantification of periarticular fluid retention based on measured changes in defined stifle joint girth and ultrasonographic (US) criteria. Water content of tissue samples was assessed. The viability of articular cartilage was determined using a microscopic fluorescent cell viability staining system. RESULTS No significant difference in postprocedural joint swelling was observed between LRS and HYPER treatment groups. Percent increments in femorotibial joint dimensions (mean ± SD) were seen in both treatment groups based on US (LRS, 83.9 ± 84.6%; HYPER, 131.2 ± 144.9%) and caliper measurements (LRS 5.5 ± 4.3%; HYPER 7.5 ± 5.8%) (P ≤ .05). Chondrocyte viability and tissue water content were maintained in both treatment groups, and differences were not statistically significant. CLINICAL RELEVANCE Doubling the osmolarity of an irrigation solution used routinely for arthroscopy does not result in detrimental effects on chondrocyte viability or tissue water content. However, use of a relatively HYPER irrigation solution did not attenuate procedural tissue swelling of the equine stifle joint.

Objective To evaluate differences in pro-inflammatory and degradative mediator production from osteoarthritic knee articular cartilage explants treated with a hyperosmolar saline solution supplemented with anti-inflammatory components (l-glutamine, ascorbic acid, sodium pyruvate, epigallocatechin gallate [EGCG], and dexamethasone) or normal saline using an in vitro model for knee arthroscopy. Design Full-thickness 6 mm articular cartilage explants ( n = 12/patient) were created from femoral condyle and tibial plateau samples collected from patients who received knee arthroplasty. One explant half was treated for 3 hours with hyperosmolar saline (600 mOsm/L) supplemented with anti-inflammatory components and the corresponding half with normal saline (308 mOsm/L). Explants were cultured for 3 days and then collected for biomarker analyses. Media biomarker concentrations were normalized to the wet weight of the tissue (mg) and were analyzed by a paired t test with significance set at P < 0.05. Results Cartilage was collected from 9 females and 2 males (mean age = 68 years). Concentrations of MCP-1 ( P < 0.001), IL-8 ( P = 0.03), GRO-α ( P = 0.02), MMP-1 ( P < 0.001), MMP-2 ( P < 0.001), and MMP-3 ( P < 0.001) were significantly lower in explant halves treated with the enhanced hyperosmolar solution. When considering only those cartilage explants in the top tercile of tissue metabolism, IL-6 ( P = 0.005), IL-8 ( P = 0.0001), MCP-1 ( P < 0.001), GRO-α ( P = 0.0003), MMP-1 ( P < 0.001), MMP-2 ( P < 0.001), MMP-3 ( P < 0.001), and GAG expression ( P = 0.0001) was significantly lower in cartilage explant halves treated with the enhanced hyperosmolar solution. Conclusions Treatment of cartilage explants with a hyperosmolar saline arthroscopic irrigation solution supplemented with anti-inflammatory components was associated with significant decreases in inflammatory and degradative mediator production and mitigation of proteoglycan loss.

Objective Evaluate serum and urine biomarker panels for their capabilities in discriminating between individuals (13- to 34-years-olds) with healthy hips versus those with developmental dysplasia of the hip (DDH) prior to diagnosis of secondary hip osteoarthritis (OA). Design Urine and serum were collected from individuals (15-33 years old) with DDH, prior to and following diagnosis of hip OA, and from age-matched healthy-hip controls. Samples were analyzed for panels of protein biomarkers with potential for differentiation of hip status using receiver operator characteristic curve (area under curve [AUC]) assessments. Results Multiple urine and serum biomarker panels effectively differentiated individuals with DDH from healthy-hip controls in a population at risk for developing secondary hip OA with the best performing panel demonstrating an AUC of 0.959. The panel comprised of two serum and two urinary biomarkers provided the highest combined values for sensitivity, 0.85, and specificity, 1.00, while a panel of four serum biomarkers provided the highest sensitivity, 0.93, while maintaining adequate specificity, 0.71. Conclusion Results of this study indicate that panels of protein biomarkers measured in urine and serum may be able to differentiate young adults with DDH from young adults with healthy hips. These data suggest the potential for clinical application of a routine diagnostic method for cost-effective and timely screening for DDH in at-risk populations. Further development and validation of these biomarker panels may result in highly sensitive and specific tools for early diagnosis, staging, and prognostication of DDH, as well as treatment decision making and monitoring capabilities. Level of Evidence: III

Background:Different tendons are chosen for anterior cruciate ligament (ACL) reconstruction based on perceived advantages and disadvantages, yet there is a relative paucity of information regarding biologic responsiveness of commonly used tendon grafts to mechanical strain.Purpose:To evaluate the in vitro responses of graft fibroblasts derived from tendons used for ACL reconstruction to clinically relevant strain levels.Study Design:Controlled laboratory study.Methods:Twelve quadriceps tendons (QTs), 12 patellar tendons (PTs), and 9 hamstring tendons (HTs) were harvested from skeletally mature dogs (n = 16). Tendon fibroblasts were isolated and seeded onto BioFlex plates (1 × 105cells/well). Cells were subjected to 3 strain conditions (stress deprivation, 0%; physiologic, 4%; high, 10%) for 5 days. Media were collected for proinflammatory and metabolic assays. RNA was extracted for gene expression analysis using real-time reverse transcription polymerase chain reaction.Results:Stress deprivation elicited significantly higher metabolic activity from HT and PT cells than from QT cells ( P < .001 and P = .001, respectively). There were no differences in metabolic activity among all 3 graft fibroblasts at physiologic and high strain. COL-1 expression was significantly higher in PT versus HT during physiologic strain ( P = .007). No significant differences with COL-3 expression were seen. TIMP-1 ( P = .01) expression was higher in PT versus HT under physiologic strain. Scleraxis expression was higher in PT versus HT ( P = .007) under physiologic strain. A strain-dependent increase in PGE2 levels occurred for all grafts. At physiologic strain conditions, HT produced significantly higher levels of PGE2 versus QT ( P < .001) and PT ( P = .005).Conclusion:Fibroblasts from common ACL graft tissues exhibited different metabolic responses to mechanical strain. On the basis of these data, we conclude that early production of extracellular matrix and proinflammatory responses from ACL grafts are dependent on mechanical loading and graft source.Clinical Relevance:Graft-specific differences in ACL reconstruction outcomes are known to exist. Our results suggest that there are differences in the biologic responsiveness of cells from the tendon grafts used in ACL reconstruction, which are dependent on strain levels and graft source. The biologic properties of the tissue used for ACL reconstruction should be considered when selecting graft source.

Many factors contribute to the development and progression of intervertebral disc (IVD) degeneration. This study was designed to assess the effects of compressive load magnitude on IVD metabolism. It was hypothesized that as load magnitude increased, there would be a significant increase in release of proinflammatory and degradative biomarkers, and a significant decrease in tissue proteoglycan (GAG) and collagen contents compared with unloaded controls. IVD whole organ functional spinal units (FSU) consisting of cranial and caudal body halves, cartilage endplates, and IVD (n = 36) were harvested from the tails of six Sprague Dawley rats, and FSUs were cultured at 0.0 MPa, 0.5 MPa, or 1.0 MPa at 0.5 Hz for 3 days. After culture, media were collected for biomarker analysis and FSUs were analyzed for extracellular matrix composition. Significant differences were determined using a one-way analysis of variance or Kruskal-Wallis test and post hoc analyses. Media concentrations of IFN-γ, IL-6, IL-1β, and MMP-8 were significantly higher in the 0.5 MPa compared with the 0.0 MPa group. Media concentrations of PGE2 and TIMP-1 were significantly higher in the 1.0 MPa group compared with the 0.0 MPa group, and media PGE2 was significantly higher in the 1.0 MPa group compared with the 0.5 MPa group. Media GAG content was significantly higher in the 1.0 MPa group compared with the 0.0 MPa group, and percent GAG in the tissue was significantly lower in 0.5 MPa and 1.0 MPa groups compared with the 0.0 MPa group. Clinical Significance: These data suggest that there are magnitude-dependent inflammatory and degradative IVD responses to cyclic loading, which may contribute to IVD degeneration.

Objective This study tests the elution capabilities of a magnesium phosphate cement (MPC). Study objectives were to quantify the passive release of magnesium ions from MPC and to assess the effects of antibiotic-loaded MPC on bacterial growth and osteoblast viability. Methods MPC constructs were created and incubated in fetal bovine serum (FBS). At 2, 4, and 17 weeks, a sample was collected for magnesium ion concentration analysis. Control and vancomycin-loaded (vanc) MPC beads were also created. Zone of inhibition was measured after incubating beads on Staphylococcus aureus agar plates for 24 h. Osteoblasts were seeded onto control and vanc beads and cultured for 9 days. Metabolic activity was measured via a resazurin assay. ANOVA with Tukey HSD post-hoc tests and t-tests were performed. Results Magnesium ions were eluted at 2 and 4-week time points without significant difference, but demonstrated a significant spike at the 17-week time point. Zones of inhibition for the bacterial species was observed for Vanc-MPC beads, but not control beads. No cytotoxic effects on osteoblasts were noted. Conclusion MPC has potential to improve bone regeneration based on its ability to passively elute magnesium. Additionally, antibiotic-loaded MPC inhibits bacterial growth while avoiding osteoblast cytotoxicity.

Background This study was designed to test the hypothesis that biologic scaffold augmentation of articular-sided partial-thickness supraspinatus tendon tears would be associated with superior functional, imaging, biomechanical, and histologic properties compared with untreated tears in a preclinical canine model. Methods With Institutional Animal Care and Use Committee approval, dogs (n = 16) underwent half-thickness resection of the articular portion of the supraspinatus tendon (SST). Defects were treated by debridement (DB) (n = 8) or scaffold augmentation on the bursal side using amnion matrix cord scaffold (AM) (n = 8), decellularized human dermal allograft (AF) (n = 8), or bovine collagen patch (RMP) (n = 8). Control dogs (n = 4; 8 normal shoulders) were included. Assessments included lameness, function, comfortable shoulder range of motion (CROM), pain, ultrasonography, magnetic resonance imaging (MRI), arthroscopy, gross examination, biomechanical testing, and histopathology. Results At 3 months, CROM was significantly lower and pain significantly higher in DB compared with all other groups. At 6 months, CROM was significantly lower and pain significantly higher in RMP compared with AM and AF, and AM and AF showed significantly less thickening than DB and RMP. AF had the least severe MRI pathology and AM had significantly less MRI pathology than DB. AF SSTs and biceps tendons showed the least severe histopathology, and AM SSTs showed significantly less histopathology than DB and RMP SSTs. Conclusion Biologic scaffolds can be effective in augmenting healing of articular-sided partial-thickness SST tears when compared with debridement in a preclinical canine model. Decellularized human dermal allograft and amnion matrix cord may have advantages over the bovine collagen patch for use in this indication.

There is a critical unmet need in the clinical implementation of valid preventative and therapeutic strategies for patients with articular cartilage pathology based on the significant gap in understanding of the relationships between diagnostic data, disease progression, patient-related variables, and symptoms. In this article, the current state of classification and categorization for articular cartilage pathology is discussed with particular focus on machine learning methods and the authors propose a bedside–bench–bedside approach with highly quantitative techniques as a solution to these hurdles. Leveraging computational learning with available data toward articular cartilage pathology patient phenotyping holds promise for clinical research and will likely be an important tool to identify translational solutions into evidence-based clinical applications to benefit patients. Recommendations for successful implementation of these approaches include using standardized definitions of articular cartilage, to include characterization of depth, size, location, and number; using measurements that minimize subjectivity or validated patient-reported outcome measures; considering not just the articular cartilage pathology but the whole joint, and the patient perception and perspective. Application of this approach through a multistep process by a multidisciplinary team of clinicians and scientists holds promise for validating disease mechanism-based phenotypes toward clinically relevant understanding of articular cartilage pathology for evidence-based application to orthopaedic practice.

Articular cartilage injuries are a common source of joint pain and dysfunction. We hypothesized that pulsed electromagnetic fields (PEMFs) would improve growth and healing of tissue-engineered cartilage grafts in a direction-dependent manner. PEMF stimulation of engineered cartilage constructs was first evaluated in vitro using passaged adult canine chondrocytes embedded in an agarose hydrogel scaffold. PEMF coils oriented parallel to the articular surface induced superior repair stiffness compared to both perpendicular PEMF (p = .026) and control (p = .012). This was correlated with increased glycosaminoglycan deposition in both parallel and perpendicular PEMF orientations compared to control (p = .010 and .028, respectively). Following in vitro optimization, the potential clinical translation of PEMF was evaluated in a preliminary in vivo preclinical adult canine model. Engineered osteochondral constructs (∅ 6 mm × 6 mm thick, devitalized bone base) were cultured to maturity and implanted into focal defects created in the stifle (knee) joint. To assess expedited early repair, animals were assessed after a 3-month recovery period, with microfracture repairs serving as an additional clinical control. In vivo, PEMF led to a greater likelihood of normal chondrocyte (odds ratio [OR]: 2.5, p = .051) and proteoglycan (OR: 5.0, p = .013) histological scores in engineered constructs. Interestingly, engineered constructs outperformed microfracture in clinical scoring, regardless of PEMF treatment (p < .05). Overall, the studies provided evidence that PEMF stimulation enhanced engineered cartilage growth and repair, demonstrating a potential low-cost, low-risk, noninvasive treatment modality for expediting early cartilage repair.

Articular cartilage defects are a common source of joint pain and dysfunction. We hypothesized that sustained low-dose dexamethasone (DEX) delivery via an acellular osteochondral implant would have a dual pro-anabolic and anti-catabolic effect, both supporting the functional integrity of adjacent graft and host tissue while also attenuating inflammation caused by iatrogenic injury. An acellular agarose hydrogel carrier with embedded DEX-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres (DLMS) was developed to provide sustained release for at least 99 days. The DLMS implant was first evaluated in an in vitro pro-inflammatory model of cartilage degradation. The implant was chondroprotective, as indicated by maintenance of Young's modulus (EY) (p = 0.92) and GAG content (p = 1.0) in the presence of interleukin-1β insult. In a subsequent preliminary in vivo experiment, an osteochondral autograft transfer was performed using a pre-clinical canine model. DLMS implants were press-fit into the autograft donor site and compared to intra-articular DEX injection (INJ) or no DEX (CTL). Functional scores for DLMS animals returned to baseline (p = 0.39), whereas CTL and INJ remained significantly worse at 6 months (p Statement of significance Articular cartilage defects are a common source of joint pain and dysfunction. Effective treatment of these injuries may prevent the progression of osteoarthritis and reduce the need for total joint replacement. Dexamethasone, a potent glucocorticoid with concomitant anti-catabolic and pro-anabolic effects on cartilage, may serve as an adjuvant for a variety of repair strategies. Utilizing a dexamethasone-loaded osteochondral implant with controlled release characteristics, we demonstrated in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes following osteochondral repair. These improved outcomes were correlated with superior histological cartilage scores and minimal-to-no comorbidity, which is a risk with high dose dexamethasone injections. Using this model of cartilage restoration, we have for the first time shown the application of targeted, low-dose dexamethasone for improved healing in a preclinical model of focal defect repair.

Purpose To provide an initial characterization of relevant bacterial DNA profiles for patients undergoing closed-fracture fixation or total joint arthroplasties. Patients and methods Swabs were collected and analyzed using Polymerase Chain Reaction from adult patients undergoing closed-fracture fixation or total shoulder, knee, or hip arthroplasties. Results Bacterial DNA profiles varied across the different orthopaedic patient populations, and produced uncharacteristic profile shifts with direct relevance to each clinical infection. Conclusion Findings provide a foundational dataset regarding bacterial colonization of relevant anatomic sites that can act as sources of surgical site infections for patients.

This study characterizes outcomes associated with subchondroplasty (SCP) versus SCP enhanced with platelet-rich plasma (PRP) or bone marrow aspirate concentrate (BMC) treatment of impact-induced subchondral bone marrow lesions (BML) using a validated preclinical canine model. With IACUC approval, purpose-bred research hounds (n = 24) underwent arthroscopic impact injury (40 N) to both medial femoral condyles. At 3 months, functional assessments, arthroscopy, and magnetic resonance imaging (MRI) were performed. One knee in each dog (n = 24; n = 12 per endpoint) was randomly assigned to SCP with the other knee randomly assigned to SCP + PRP, SCP + BMC or sham injection (control) (n = 8 per group; n = 4 per endpoint). Dogs were evaluated at 6 and 12 months after treatment using functional assessments, radiography, arthroscopy, and MRI and humanely euthanatized at 6 or 12 months after treatment for histologic assessments. At 6 months post-treatment, comfortable range-of-motion (CROM) was higher (p < 0.04) in SCP + PRP and SCP + BMC knees compared with controls. At 1 year post-treatment, %Total Pressure Index was higher (p = 0.036) in SCP + BMC compared with controls, pain was lower (p < 0.05) in SCP + BMC and SCP + PRP compared with SCP and controls, and CROM was higher (p < 0.05) in SCP + BMC and SCP + PRP compared with SCP and controls. Knees treated with SCP + PRP and SCP + BMC had better (p < 0.05) MRI grades than SCP and controls. No statistically significant differences in arthroscopic or histologic pathology were noted. Clinical significance: Biologics added to SCP treatment may further enhance its beneficial effects by improving range-of-motion, pain severity, and limb loading through 1 year after treatment. However, these benefits must be considered alongside cost, logistics, and treatment availability. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:740-746, 2020.

Purpose: The objective of this study was to determine the responses of normal meniscus to collagenase activity. It was hypothesized that meniscal explants exposed to collagenase would significantly increase release of pro-inflammatory cytokines and degradative enzymes, in a dose-dependent manner, compared to control.Methods: Menisci were harvested from adult dogs (n = 6) euthanized for reasons unrelated to this study. Meniscal explants were created from the central portion of lateral and medial meniscus. Explants were injected with 100 µl collagenase at a concentration of 50 µg/ml, 5 µg/ml, or 0 µg/ml of collagenase. Explants were cultured for 12 days, and media were changed and collected every 3 days for biomarker analyses. Differences among collagenase concentrations were determined by a three factor ANOVA with adjustment for multiple comparisons, with pre-adjustment statistical significance set at p < 0.05.Results: When data from all explants were compared, the 50 µg group released significantly higher IL-6 and PGE2, and the 5 µg group released significantly higher levels of MMP-3 and CTX-II compared to the 0 µg group. Explants from the medial meniscus released significantly more MMP-1, MMP-2, MMP-3, and MMP-13 in response to stimulation with 5 µg/ml of collagenase compared to explants from the lateral meniscus.Discussion: The data from this study indicate that in response to localized degradative enzyme activity, the meniscus increases the release of pro-inflammatory and degradative biomarkers in a dose-dependent manner. Further, these data indicate potential differences in metabolic responses of lateral versus medial menisci to collagenase insult.

Symptomatic chondral and osteochondral defects affect a large and growing number of patients. A safe and effective surgical treatment for large articular defects is osteochondral allograft (OCA) transplantation. One of the major causes of failure for OCA transplantation is loss of essential chondrocyte viability during the preservation and storage period. It is also possible that metabolic responses of the OCA when transitioning from storage temperature to body temperature may contribute to mechanisms causing failure. The present study was designed to compare MOPSSM -preserved OCAs to those stored using the current standard of care (SOC) method with respect to metabolic responses when rewarmed for transplantation to and maintenance at body temperature (37°C). It was theorized that grafts stored using the MOPSSM protocol would maintain significantly higher chondrocyte viability and produce significantly lower levels of inflammatory mediators and degradative enzymes, and significantly higher levels of chemokines compared to grafts stored using the SOC protocol. Left over SOC and MOPSSM -stored OCA tissues were collected after surgery, and cartilage explants were cultured for 6 days. Media was analyzed for biomarkers using commercially available assays. Cartilage from SOC grafts released significantly higher levels of PGE2, MMP-1, MMP-2, and MMP-13, and significantly lower levels of IL-8 and Gro-α, compared to cartilage from MOPSSM -stored grafts. Clinical significance: These data suggest that OCAs stored using the MOPSSM protocol have potentially less detrimental initial inflammatory and degradative responses to re-warming for transplantation compared to OCAs stored using the current tissue bank protocols. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1530-1536, 2019.

OBJECTIVE: To evaluate differences in pro-inflammatory and degradative mediator production from osteoarthritic knee articular cartilage explants treated with a hyperosmolar saline solution supplemented with anti-inflammatory components (l-glutamine, ascorbic acid, sodium pyruvate, epigallocatechin gallate [EGCG], and dexamethasone) or normal saline using an in vitro model for knee arthroscopy. DESIGN: Full-thickness 6 mm articular cartilage explants (n = 12/patient) were created from femoral condyle and tibial plateau samples collected from patients who received knee arthroplasty. One explant half was treated for 3 hours with hyperosmolar saline (600 mOsm/L) supplemented with anti-inflammatory components and the corresponding half with normal saline (308 mOsm/L). Explants were cultured for 3 days and then collected for biomarker analyses. Media biomarker concentrations were normalized to the wet weight of the tissue (mg) and were analyzed by a paired t test with significance set at P < 0.05. RESULTS: Cartilage was collected from 9 females and 2 males (mean age = 68 years). Concentrations of MCP-1 (P < 0.001), IL-8 (P = 0.03), GRO-α (P = 0.02), MMP-1 (P < 0.001), MMP-2 (P < 0.001), and MMP-3 (P < 0.001) were significantly lower in explant halves treated with the enhanced hyperosmolar solution. When considering only those cartilage explants in the top tercile of tissue metabolism, IL-6 (P = 0.005), IL-8 (P = 0.0001), MCP-1 (P < 0.001), GRO-α (P = 0.0003), MMP-1 (P < 0.001), MMP-2 (P < 0.001), MMP-3 (P < 0.001), and GAG expression (P = 0.0001) was significantly lower in cartilage explant halves treated with the enhanced hyperosmolar solution. CONCLUSIONS: Treatment of cartilage explants with a hyperosmolar saline arthroscopic irrigation solution supplemented with anti-inflammatory components was associated with significant decreases in inflammatory and degradative mediator production and mitigation of proteoglycan loss.

Despite the growing success for osteochondral allograft (OCA) transplantation in treating large articular cartilage lesions in multiple joints, associated revision and failure rates are still higher than desired. While immunorejection responses have not been documented, the effects of the host's immune responses on OCA transplantation failures have not been thoroughly characterized. The objective of this study was to systematically review clinically relevant peer-reviewed evidence pertaining to the immunology of OCAs to elucidate theragnostic strategies for improving functional graft survival and outcomes for patients undergoing OCA transplantation. This systematic review of Cochrane Central Register of Controlled Trials, the Cochrane Database of Systematic Reviews, MEDLINE, PubMed, and EMBASE suggests that host immune responses play key roles in incorporation and functional survival of OCA transplants. OCA rejection has not been reported; however, graft integration through creeping substitution is reliant on host immune responses. Prolonged inflammation, diminished osteogenic potential for healing and incorporation, and relative bioburden are mechanisms that may be influenced by the immune system and contribute to undesirable outcomes after OCA transplantation. Based on the safety and efficacy of OCA transplantation and its associated benefits to a large and growing patient population, basic, preclinical, and clinical osteoimmunological studies on OCA transplantation that comprehensively assess and correlate cellular, molecular, histologic, biomechanical, biomarkers, diagnostic imaging, arthroscopic, functional, and patient-reported outcome measures are of high interest and importance.

Study Design:Controlled laboratory study.Objective:To investigate the impact of exposure to physiologically relevant caffeine concentrations on intervertebral disc (IVD) cell viability and extracellular matrix composition (ECM) in a whole organ culture model as potential contributing mechanisms in development and progression of IVD disorders in humans. Primary outcome measures were IVD viable cell density (VCD) and ECM composition.Methods:A total of 190 IVD whole organ explants from tails of 16 skeletally mature rats—consisting of cranial body half, endplate, IVD, endplate, and caudal body half—were harvested. IVD explants were randomly assigned to 1 of 2 groups: uninjured (n = 90) or injured (20G needle disc puncture/aspiration method, n = 100). Explants from each group were randomly assigned to 1 of 3 treatment groups: low caffeine (LCAF: 5 mg/L), moderate caffeine (MCAF: 10 mg/L), and high caffeine (HCAF: 15 mg/L) concentrations.Results:Cell viability was significantly higher in the low-caffeine group compared with the high-caffeine group at day 7 ( P = .037) and in the low-caffeine group compared with the medium- and high-caffeine groups at day 21 ( P ≤ .004). Analysis of ECM showed that all uninjured and control groups had significantly higher ( P < .05) glycosaminoglycan concentrations compared with all injured groups. Furthermore, we observed a temporal, downward trend in proteoglycan to collagen ratio for the caffeine groups.Conclusions:Caffeine intake may be a risk factor for IVD degeneration, especially in conjunction with disc injury. Mechanisms for caffeine associated disc degeneration may involve cell and ECM, and further studies should elucidate mechanistic pathways and potential benefits for caffeine restriction.

Neck and low back pain are common among the adult human population and impose large social and economic burdens on health care and quality of life. Spine‐related disorders are also significant health concerns for canine companions with etiopathogeneses, clinical presentations, and diagnostic and therapeutic options that are very similar to their human counterparts. Historically, induced and spontaneous pathology in laboratory rodents, dogs, sheep, goats, pigs, and nonhuman primates have been used for study of human spine disorders. While each of these can serve as useful preclinical models, they all have inherent limitations. Spontaneously occurring spine disorders in dogs provide highly translatable data that overcome many of the limitations of other models and have the added benefit of contributing to veterinary healthcare as well. For this scoping review, peer‐reviewed manuscripts were selected from PubMed and Google Scholar searches using keywords: “intervertebral disc,” “intervertebral disc degeneration,” “biomarkers,” “histopathology,” “canine,” and “mechanism.” Additional keywords such as “injury,” “induced model,” and “nucleus degeneration” were used to further narrow inclusion. The objectives of this review were to (a) outline similarities in key features of spine disorders between dogs and humans; (b) describe relevant canine models; and (c) highlight the applicability of these models for advancing translational research and clinical application for mechanisms of disease, diagnosis, prognosis, prevention, and treatment, with a focus on intervertebral disc degeneration. Best current evidence suggests that dogs share important anatomical, physiological, histological, and molecular components of spinal disorders in humans, such that induced and spontaneous canine models can be very effective for translational research. Taken together, the peer‐reviewed literature supports numerous advantages for use of canine models for study of disorders of the spine when the potential limitations and challenges are addressed., The spontaneously‐occurring canine models described in the present review article are amenable to this comprehensive and correlative approach while also corresponding directly to in vitro, ex vivo, and induced‐disease canine models. Taken together, preclinical studies using the full breadth of canine models can guide targeted research toward developing valid and effective tools for early diagnosis, prevention, and treatments both for human and canine patients.

Osteoarthritis (OA) is a complex disease with biologic, biomechanical, and clinical heterogeneity among patients. Relationships among OA tissue metabolism, histopathology, and extracellular matrix (ECM) composition have not been well characterized. It was hypothesized that moderate (r = .4-.69) to strong (r > .7) correlations exist among these different measures of disease severity in osteochondral tissues from OA knees. Joint surfaces were obtained from patients (n = 6) undergoing total knee arthroplasty. Osteochondral explants (n = 136) were created and cultured for 3 days. Culture media were collected for biomarker analyses, and tissue was assessed for viability, histological scoring, and ECM composition. Correlations among media biomarker concentrations, histological scoring, ECM composition, and viability were determined using a Spearman correlation. GRO-α, IL-6, IL-8, and MCP-1 showed strong positive correlations to each other, and moderate positive correlations to NO, PGE2, and MMP-2. Total MMP activity, MMP-9, and MMP-13 had strong positive correlations to each other, and moderate positive correlations to MMP-1. MMP-2 had a moderate to strong positive correlations to histological scores (total and cartilage structure) and collagen content. MMP-2, IL-6, IL-8, and MCP-1 had moderate negative correlations, and MMP-9 had a moderate positive correlation, to viability. GRO-α, IL-6, IL-8, and MCP-1 had moderate positive correlations to collagen content. MMP-9, MMP-13, and total MMP activity had moderate negative correlations to tissue GAG. The data suggest links among proinflammatory and degradative pathways are present in OA osteochondral tissues. Further characterization of these links have the potential to delineate mechanisms of disease and diagnostic and therapeutic targets for knee OA.

Objectives: Common autografts for ACL reconstruction include patellar (PT), hamstring (HT), and quadriceps tendons (QT). Various adverse outcomes, including osteoarthritis and revision risk, have been associated with different graft choices. While the time-zero material properties of autograft tissues are known, there is limited information on the biologic properties of autografts. In addition, the early differential responses of autograft fibroblasts to the biomechanical environment after surgery are not known. Our study objective was therefore to evaluate the in vitro responses of common ACL graft fibroblasts to clinically relevant strain levels. We hypothesized that fibroblasts from common autografts would exhibit differential gene expression and production of inflammatory biomarkers in response to cyclic strain. Methods: 12 QT, 12 PT, and 9 HT were harvested from skeletally mature dogs (n=12) (ACUC #9163, 9164). Tissues were digested and ACL graft fibroblasts were seeded onto BioFlex® plates (1 × 105 cells/well), and cultured for 48h prior to application of strain. Cells were subjected to mechanical stimulation (2-s strain, 10-s relaxation, 0.5 Hz frequency) with a biaxial sinusoidal waveform with 3 strains (stress deprivation-0%, physiologic-4%, and high-10%) for 5 days using a Flexcell. On day 5, media was collected for inflammatory biomarker analysis (PGE2, IL-8, KC, MCP-1) and RNA was extracted from cells for gene expression analysis with RT-PCR (COL1, COL2, COL3, MMP2, MMP3, MMP13, ELASTIN, SCX). Significant differences among graft types were set at pResults: Gene expression: QT had higher COL1 expression at 0% strain (pConclusion: Our study showed that fibroblasts from common ACL graft tissues exhibited different cellular responses to strain levels. At 4% strain, PT tended to have higher expression of extracellular matrix (ECM) genes and may have a more robust remodeling response under physiologic strain. Furthermore, the data from this study indicates that all autografts have a similar inflammatory metabolism at 4% strain, but the QT and PT may have a higher inflammatory response in stress deprivation (0%) and over strain (10%) conditions. Based on these data, we conclude that production of ECM and inflammatory response from ACL grafts are not only dependent on the mechanical environment, but also graft source.

Osteochondral allograft (OCA) transplantation can restore large articular defects in the knee. Bipolar OCA transplantations for partial and whole joint resurfacing often have less favorable results than single-surface transplants. This study was designed to use a large animal model to test the hypothesis that unicompartmental bipolar osteochondral and meniscal allograft transplantation (BioJoint) would be as or more effective for treatment of medial compartment osteoarthritis (OA) compared to standard-of-care nonoperative treatment. OA was induced in one knee of each research hound (n = 8) using a meniscal release model and pretreatment assessments were performed. After 3 months, dogs were randomly assigned to either the control group (n = 4, no surgical intervention, daily nonsteroidal antiinflammatory drugs [NSAIDs]) or the BioJoint group (n = 4). Clinical, radiographic, and arthroscopic assessments were performed longitudinally and histopathology was evaluated at the 6-month endpoint. At study endpoint, functional, pain, and total pressure index measures, as well as radiographic and arthroscopic grading of graft appearance and joint health, demonstrated superior outcomes for BioJoints compared to NSAID controls. Furthermore, histologic assessments showed that osteochondral and meniscal transplants maintain integrity and integrated into host tissues. Clinical significance: The results support the safety and efficacy of unicompartmental bipolar osteochondral and meniscal allograft transplantation in a preclinical model with highly functional outcomes without early OA progression.

The joint is an organ with each tissue playing critical roles in health and disease. Intact articular cartilage is an exquisite tissue that withstands incredible biologic and biomechanical demands in allowing movement and function, which is why hyaline cartilage must be maintained within a very narrow range of biochemical composition and morphologic architecture to meet demands while maintaining health and integrity. Unfortunately, insult, injury, and/or aging can initiate a cascade of events that result in erosion, degradation, and loss of articular cartilage such that joint pain and dysfunction ensue. Importantly, articular cartilage pathology affects the health of the entire joint and therefore should not be considered or addressed in isolation. Treating articular cartilage lesions is challenging because left alone, the tissue is incapable of regeneration or highly functional and durable repair. Nonoperative treatments can alleviate symptoms associated with cartilage pathology but are not curative or lasting. Current surgical treatments range from stimulation of intrinsic repair to whole-surface and whole-joint restoration. Unfortunately, there is a relative paucity of prospective, randomized controlled, or well-designed cohort-based clinical trials with respect to cartilage repair and restoration surgeries, such that there is a gap in knowledge that must be addressed to determine optimal treatment strategies for this ubiquitous problem in orthopedic health care. This review article discusses the basic science rationale and principles that influence pathology, symptoms, treatment algorithms, and outcomes associated with articular cartilage defects in the knee.

A thorough understanding of the relationship between the biological and mechanical functions of articular cartilage is necessary to develop diagnostics and treatments for arthritic diseases. A key step in developing this understanding is the establishment of models which utilize large numbers of biomarkers to create comprehensive models of the interplay between cartilage biology and biomechanics, which will more accurately demonstrate the complex etiology and progression of tissue adaptation and degradation. It is the goal of this study to demonstrate the ability of artificial neural networks (ANNs) to utilize biomarkers to create predictive models of articular cartilage biomechanics, which will provide a basis for more sophisticated research in the future. Osteochondral plugs were collected from patients undergoing total knee arthroplasty, cultured, then analyzed to collect proteomic, compositional, and histologic biomarker data. Samples were subjected to stress relaxation testing as well as computational simulations using finite element analysis (FEA) modeling and optimization to determine key mechanical properties. The acquired data was fed into an ANN to generate a model which predicts the biomechanical properties of cartilage from given biomarkers. Using all significant inputs, the developed neural network predicted the ground substance modulus with a moderate degree of accuracy, but had difficulty predicting the collagen fiber modulus and cartilage permeability. Using only clinically attainable biomarkers, the best-performing model produced comparably accurate and more consistent predictions of all three mechanical properties. These models demonstrate the potential for ANNs to be included in clinical studies of articular cartilage.

Meniscal allograft transplantation (MAT) can be a safe, effective treatment for meniscal deficiency resulting in knee dysfunction, leading to osteoarthritis (OA) without proper treatment with 5-year functional success rates (75%-90%). While different grafts and techniques have generally proven safe and effective, complications include shrinkage, extrusion, progression of joint pathology, and failure. The objective of this study was to assess the functional outcomes after MAT using three different clinically-relevant methods in a preclinical canine model. The study was designed to test the hypothesis that fresh meniscal-osteochondral allograft transplantation would be associated with significantly better function and joint health compared with fresh-viable or fresh-frozen meniscus-only allograft transplantations. Three months after meniscal release to induce meniscus-deficient medial compartment disease, research hounds (n = 12) underwent MAT using meniscus allografts harvested from matched dogs. Three MAT conditions (n = 4 each) were compared: frozen meniscus-fresh-frozen meniscal allograft with menisco-capsular suture repair; fresh meniscus-fresh viable meniscal allograft (Missouri Osteochondral Preservation System (MOPS)-preservation for 30 days) with menisco-tibial ligament repair; fresh menisco-tibial-fresh, viable meniscal-tibial-osteochondral allografts (MOPS-preservation for 30 days) with menisco-tibial ligament preservation and autogenous bone marrow aspirate concentrate on OCA bone. Assessment was performed up to 6 months after MAT. Pain, comfortable range of motion, imaging, and arthroscopic scores as well histological and cell viability findings were superior (P < .05) for the fresh menisco-tibial group compared with the two other groups. Novel meniscal preservation and implantation techniques with fresh, MOPS-preserved, viable meniscal-osteochondral allografts with menisco-tibial ligament preservation appears to be safe and effective for restoring knee function and joint health in this preclinical model. This has the potential to significantly improve outcomes after MAT.

This study characterizes long-term outcomes associated with subchondroplasty (SCP) treatment for impact-induced subchondral bone marrow lesions (BML) using a validated pre-clinical canine model. With IACUC approval, purpose-bred research hounds (n = 16) underwent arthroscopic impact injury (40N) to both medial femoral condyles. At 3 months, functional assessments, arthroscopy, and MRI were performed and knees (n = 32) were randomly assigned to SCP (3 ml fluoroscopically guided percutaneous injection of AccuFill BSM into BML bone defects) or sham injection (Control). Dogs were assessed at 3, 6, 12, and 24 months after treatment using functional assessments, radiographic evaluation, arthroscopy, and MRI. Dogs were humanely euthanatized at 3, 6, 12, or 24 months after treatment for gross, microCT, and histologic assessments. All knees had focal articular cartilage defects with associated subchondral BMLs, as well as clinical dysfunction, 3 months after injury. At the 3 and 6 months, SCP knees showed more functional impairment than Control knees, however, these differences were not statistically significant. At 1- and 2-year post-treatment, function in SCP knees was better than in Control knees with range of motion being significantly (p < 0.05) better for SCP. Radiographic, arthroscopic, MRI, gross, microCT, and histologic findings matched the functional assessments well with Control being associated with better results at the two early time points and SCP being associated with better results at 1 and 2 years. Clinical significance: SCP treatment using calcium phosphate bone void filler was associated with an initial increase in pain and dysfunction followed by symptomatic benefits for up to 2 years after treatment for post-traumatic femoral condyle BMLs in a preclinical canine model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2709-2717, 2018.

This study was designed to characterize metabolic responses of meniscal tissue explants to injury and inflammation. We hypothesized that impact injury and interleukin (IL-1β) stimulation of meniscal explants would result in significant increases in matrix metalloproteinase (MMP) activity and relevant cytokine production compared to controls. Mature canine meniscal explants (n = 9/group) were randomly assigned to: (i) IL-1β (0.1 ng/ml) treated (IL); (ii) 25% strain (25); (iii) 75% strain (75); (iv) 25% + IL-1β (25IL); (v) 75% + IL-1β (75IL); or (vi) 0% + no IL-1β control (NC). Explants were impacted at 100 mm/s to 0%, 25%, or 75% strain and then cultured for 12 days with or without 0.1 ng/ml rcIL-1β. Media were refreshed every 3 days and analyzed for MMP activity, ADAMTS-4 activity, MMP-1, MMP-2, MMP-3, GAG, NO, PGE2 , IL-6, IL-8, MCP-1, and KC concentrations. Treatment with IL-1β alone significantly increased NO, PGE2, general MMP activity, IL-6, IL-8, KC, and MCP-1 media concentrations compared to negative controls. Impact at 75% significantly increased PGE2, IL-6, IL-8, and KC media concentrations compared to negative controls. The combination of IL-1β and 75% strain significantly increased production of PGE2 compared to IL-1β or 75% strain alone. Impact injury to meniscal explants ex vivo is associated with increased production of pro-inflammatory mediators and degradative enzyme activity, which are exacerbated by stimulation with IL-1β. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2657-2663, 2018.

Articular defects are a major problem with few effective treatment options. Osteochondral allograft (OCA) transplantation can be an effective treatment; however, lack of OCA bone integration can cause failure. This controlled laboratory study was designed to compare clinically applicable methods for marrow element removal and enhanced delivery of bone marrow aspirate concentrate (BMC) to OCA bone. We hypothesized that compressed carbon dioxide (CO2) treatment of OCA bone would result in significantly better marrow element removal, significantly more retention and distribution of viable osteoprogenitor cells, and significantly higher osteoinductive protein elution from OCAs compared with other preimplantation treatments. Fresh humeral heads (n = 24) were harvested and stored for 14 days, then randomly assigned to treatment based on marrow element removal and bone treatment: (standard of care [SOC]) (n = 4) – SOC high-pulse saline lavage, no BMC; (BMC) (n = 5) – saline lavage then canine BMC; (Drill + BMC) (n = 5) – 1.1 mm drill-hole immediately subchondral then saline lavage then BMC injection through drill hole; (Carb + BMC) (n = 5) – saline lavage then CO2 then BMC; or (Saline-Carb + BMC) (n = 5) – saline lavage and CO2 together then BMC. Treated OCAs were cultured for 14 days. On day 3, media were collected, centrifuged to isolate cells, and replaced. Cells were cultured for 11 days for colony forming unit (CFU) determination. OCA media were collected on days 7 and 14 of culture for analysis. On day 14, each graft was assessed for viable cell retention and distribution, and bone marrow element removal. BMC had significantly higher (p = 0.001) viable cell distribution compared with the SOC, Drill + BMC, Carb + BMC, and Saline-Carb + BMC groups. BMC and Drill + BMC had significantly higher (p

This article identifies the potential mechanisms of action for meniscal degeneration in response to joint inflammation and potential contributions of the meniscus to the development and progression of osteoarthritis (OA). It was hypothesized that interleukin-1β (IL-1β) stimulation of meniscal explants would result in significant increases in nitric oxide (NO), matrix metalloproteinase (MMP) production and activity, and relevant cytokine production compared with controls. Canine meniscal explants (4 mm) were cultured for 21 days with (IL-1) or without (negative control [NC]) 50 ng/mL rcIL-1β (n = 6/group). Media were changed every 3 days and analyzed for MMP activity, ADAMTS-4 activity, MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, NO, prostaglandin E2 (PGE2), IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and keratinocyte-derived chemokine (KC) concentrations. Media NO and PGE2 concentrations were significantly higher in the IL-1 group at all time points except for days 9 and 12. The concentrations of MMP-13 were significantly higher in the IL-1 group at days 3, 6, 9, and 12. The production of MMP-2 was significantly lower in the IL-1 group on days 3 through 15. ADAMTS4 activity was significantly higher in the IL-1 group on days 6 through 18. MMP-3 concentrations and general MMP activity were significantly higher in the IL-1 group at all time points. Concentrations of IL-6, IL-8, MCP-1, and KC were significantly higher in the IL-1 group at most time points. Glycosaminoglycans (GAG) content decreased significantly (p = 0.009) in the IL-1 group compared with the NC group. Proinflammatory mediators appear to directly influence degradative processes in the meniscus, which in turn contribute to development and progression of OA by production of proinflammatory and degradative mediators. These findings have important clinical implications for the management of the degenerative meniscus and the osteoarthritic knee.

Background: Fresh osteochondral allografts (OCAs) are limited in availability. The Missouri Osteochondral Allograft Preservation System (MOPS) has been reported to effectively preserve OCAs twice as long as current tissue bank protocols in preclinical studies. Hypothesis: The viable chondrocyte density (VCD) in OCAs preserved for up to 70 days using the MOPS will not be significantly different from day 0, and the VCD in MOPS-preserved OCAs will be significantly higher than for standard tissue bank preservation. Media changes during preservation will significantly improve the VCD. Study Design: Controlled laboratory study. Methods: Femoral condyles harvested from qualified donors (n = 12) were quartered (n = 48), assigned to 1 of 4 treatment groups (tissue bank protocol at 4°C or MOPS at 25°C, with or without media changes), and preserved for 0, 28, 56, or 70 days and assessed for the VCD and histopathological characteristics. In addition, osteochondral explants were created from the femoral condyles of 12 donors (n = 36 explants), assigned to the same groups and time points, and tested for biomechanical properties. Results: MOPS-preserved OCAs maintained the day 0 VCD through 56 days. OCAs stored using current tissue bank protocols had a significantly lower VCD compared with day 0 and the MOPS by day 28. OCA histological and biomechanical properties did not significantly change from day 0 for any group. Conclusion: The MOPS preserved essential OCA viability and quality at significantly higher levels than current tissue bank protocols for at least 56 days after procurement. Clinical Relevance: Improving the viability and duration of OCA preservation provides potential benefits to tissue banks, donor families, surgeons, and patients with respect to tissue use, financial costs, and outcomes.

Fresh osteochondral allograft (OCA) transplantation is an attractive treatment option for symptomatic articular cartilage lesions in young, healthy patients. Since a lack of OCA bone integration can be a cause of treatment failure, methods for speeding and enhancing OCA bone integration to mitigate this potential complication are highly desirable. This study sought to determine and compare the potential of bone marrow aspirate concentrate (BMC) and leukoreduced platelet rich plasma (PRP) to repopulate the osseous portion of an OCA with cells and deliver osteogenic proteins. It was hypothesized that BMC would have significantly higher colony forming units (CFUs)/mL and seed the osseous portion of OCA with more cells than PRP. Finally, we hypothesized that the media of BMC and PRP treated OCAs would have significantly higher concentrations of osteogenic proteins compared with negative control OCAs. Cylindrical OCAs (n = 36) created from tissue stored for 21 days were treated with BMC (n = 12) or PRP (n = 12) obtained for 6 dogs, or left untreated as a negative control (n = 12). After treatment, OCAs were cultured for 7 or 14 days. Media were collected for analysis of osteogenic biomarker concentration. Samples of each BMC and PRP were tested for CFU concentration. On day 7 or 14, the grafts were assessed for cell surface adhesion and penetration using fluorescent microscopy. Significant differences in CFU and media biomarker concentration between the groups were determined using one-way analysis of variance (ANOVA) and Tukey's post-hoc test with the significance set at p

Summary Background/Objective The purpose of this study was to evaluate supraspinatus tenocyte viability and metabolism in explants exposed to various local anaesthetics and corticosteroids. Our hypothesis was that the tendons exposed to these common injectates would have significantly decreased cell viability and metabolism compared with controls. Methods Supraspinatus tendon explants were obtained from dogs, placed in a culture media, and randomly assigned to one of the following groups: culture media only (control), 1% lidocaine, 0.5% lidocaine, 0.25% bupivacaine, 0.125% bupivacaine, 0.0625% bupivacaine, betamethasone acetate (5 mg), methylprednisolone acetate (40 mg), or triamcinolone acetonide (40 mg). Cell viability was determined on Days 1 and 7 after culture treatment using calcein AM (live cell) and Sytox Blue (dead cell) stains. Tissue metabolism was assessed on Days 1 and 7 using the resazurin blue metabolic assay. Significant differences were evaluated using a one-way analysis of variance with Tukey post hoc analysis. Results Compared with the controls, there were significant decreases in cell viability noted at Days 1 and 7 in tenocytes exposed to 1% lidocaine, betamethasone, and methylprednisolone. Significant decreases in cell metabolism were also noted at Days 1 and 7 in those groups. Treatment with 0.125% bupivacaine, 0.0625% bupivacaine, and triamcinolone demonstrated no decrease in cell viability or metabolism when compared with controls at any time point. Conclusion This data confirms that peritendinous injection of commonly used local anaesthetics and corticosteroids results in significant supraspinatus tenotoxicity in vitro. Further in vivo studies are required before making definitive clinical recommendations.

The dog is the most commonly used large animal model for the study of osteoarthritis. Optimizing methods for assessing cartilage health would prove useful in reducing the number of dogs needed for a valid study of osteoarthritis and cartilage repair. Twelve beagles had critical-sized osteochondral defects created in the medial femoral condyle of both knees. Eight dogs had T1ρ and T2 magnetic resonance imaging (MRI) performed approximately 6 months after defect creation. Following MRI evaluations, all 12 dogs were humanely euthanatized and cartilage samples were obtained from the medial and lateral femoral condyles, medial and lateral tibial plateaus, trochlear groove, and patella for proteoglycan and collagen quantification. Equilibrium partitioning of an ionic contrast (EPIC)-µCT was then performed followed by the histologic assessment of the knees. Correlations between T1ρ, T2, EPIC-µCT and proteoglycan, collagen, and histology scores were assessed using a multivariate analysis accounting for correlations from samples within the same knee and in the same dog. Pearson's correlation coefficients were calculated to assess the strength of significant relationships. Correlations between µCT values and biochemical or histologic assessment were weak to moderately strong (0.09-0.41; p < 0.0001-0.66). There was a weak correlation between the T2 values and cartilage proteoglycan (-0.32; p = 0.04). The correlation between T1ρ values and cartilage proteoglycan were moderately strong (-0.38; p < 0.05) while the strongest correlation was between the T1ρ values and histological assessment of cartilage with a correlation coefficient of 0.58 (p < 0.0001). These data suggest that T1ρ shows promise for possible utility in the translational study of cartilage health and warrants further development in this species. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:368-377, 2020.

The knee is a fascinating yet complex joint. Researchers and clinicians agree that the joint is an organ comprised of highly specialized intrinsic and extrinsic tissues contributing to both health and disease. Key to the function and movement of the knee are the menisci, exquisite fibrocartilage structures that are critical structures for maintaining biological and biomechanical integrity of the joint. The biological/physiological functions of the menisci must be understood at the tissue, cellular and even molecular levels in order to determine clinically relevant methods for assessing it and influencing it. By investigating normal and pathological functions at the basic science level, we can begin to translate data to patients. The objective of this article is to provide an overview of this translational pathway so that progression toward improved diagnostic, preventative, and therapeutic strategies can be effectively pursued. We have thoroughly examined the pathobiological, biomarker, and imaging aspects of meniscus research. This translational approach can be effective toward optimal diagnosis, prevention, and treatment for the millions of patients who suffer from meniscal disorders each year.

Purpose Femoral reaming using a Reamer Irrigator Aspirator (RIA) can produce greater than three liters of waste water per procedure, which contains cells and proteins that could promote bone healing. This purpose of this study was to determine the protein profile of RIA waste water and compare protein synthesis by cells harvested via RIA versus iliac crest (IC) bone graft. Method Bone graft was collected from 30 patients—15 using RIA from the femur and 15 harvested from the iliac crest. Waste water collected during the RIA procedure was analyzed in 12 patients. Cells from each graft were cultured in monolayer using growth media for 14 days and inductive media for the next 14 days. Media samples were collected on days 14, 21, and 28. Proteins for analysis were chosen based on their potential in bone healing, pro-inflammatory, and anti-inflammatory processes. Results Proteins present in RIA waste water indicate the potential for clinical use of this filtrate as an adjunct for enhancing bone production, healing, and remodeling. Similarly, cells cultured from RIA bone graft harvests compared favorably to those from iliac crest bone grafts with respect to their potential to aid in bone healing. Conclusion RIA waste water has potential to serve as an autogenic and allogenic enhancer for bone healing. Continued development of processing protocols for viable commercial use of the waste water and pre-clinical studies designed to evaluate RIA waste water products for bone healing are ongoing.

Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring Ø3–4 mm × 2.3 mm, with cell seeding densities ranging from 15 to 90 × 106 cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young’s moduli of up to ~250 kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950 kPa (0.01 Hz), and contained 5.7 percent wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5 %/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes.

Due to the degradation of osteoarthritic (OA) cartilage in post-traumatic OA (PTOA), these tissues are challenging to study and manipulate in vitro. In this study, chondrocytes isolated from either PTOA (meniscal-release (MR) model) or normal (contralateral limb) cartilage of canine knee joints were used to form micropellets to assess the maintenance of the OA chondrocyte phenotype in vitro. Media samples from the micropellet cultures were used to measure matrix metalloproteinase (MMP), chemokine, and cytokine concentrations. Significant differences in matrix synthesis were observed as a function of disease with OA chondrocytes generally synthesizing more extracellular matrix with increasing time in culture. No donor dependent differences were detected. Luminex multiplex analysis of pellet culture media showed disease and time-dependent differences in interleukin (IL)-8, keratinocyte chemoattractant (KC)-like protein, MMP-1, MMP-2, and MMP-3, which are differentially expressed in OA. This memory of their diseased phenotype persists for the first 2 weeks of culture. These results demonstrate the potential to use chondrocytes from an animal model of OA to study phenotype alterations during the progression and treatment of OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:829-836, 2017.