Peripheral shift in the viable chondrocyte population of the medial femoral condyle after anterior cruciate ligament injury in the porcine knee.

Anterior cruciate ligament injuries result in posttraumatic osteoarthritis in the medial compartment of the knee, even after surgical treatment. How the chondrocyte distribution within the articular cartilage changes early in this process is currently unknown. The study objective was to investigate the chondrocyte distribution within the medial femoral condyle after an anterior cruciate ligament transection in a preclinical model. Forty-two adolescent Yucatan minipigs were allocated to receive unilateral anterior cruciate ligament surgery (n = 36) or no surgery (n = 6). Central coronal sections of the medial femoral condyle were obtained at 1- and 4 weeks after surgery, and the chondrocyte distribution was measured via whole slide imaging and a cell counting batch processing tool utilized in ImageJ. Ki-67 immunohistochemistry was performed to identify proliferating cells. Empty lacunae, karyolysis, karyorrhexis, and pyknosis were used to identify areas of irreversible cell injury. The mean area of irreversible cell injury was 0% in the intact controls, 13.4% (95% confidence interval: 6.4, 20.3) at 1-week post-injury and 19.3% (9.7, 28.9) at 4 weeks post-injury (p < .015). These areas occurred closest to the femoral intra-articular notch. The remaining areas containing viable chondrocytes had Ki-67-positive cells (p < .02) and increased cell density in the middle (p < .03) and deep zones (p = .001). For the entire section, the total chondrocyte number did not change significantly post-operatively; however, the density of cells in the peripheral regions of the medial femoral condyle increased significantly at 1- and 4 weeks post-injury relative to the intact control groups (p = .032 and .004, respectively). These data demonstrate a peripheral shift in the viable chondrocyte population of the medial femoral condyle after anterior cruciate ligament injury and further suggest that chondrocytes with the capacity to proliferate are not confined to one particular cartilage layer.

Analytic Sensitivity of 3 Nucleic Acid Detection Assays in Diagnosis of SARS-CoV-2 Infection.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription PCR is the primary method to diagnose coronavirus disease 2019 (COVID-19). However, the analytic sensitivity required is not well defined and it is unclear how available assays compare. METHODS: For the Abbott RealTime SARS-CoV-2 assay (m2000; Abbott Molecular), we determined that it could detect viral concentrations as low as 26 copies/mL, we defined the relationship between cycle number and viral concentrations, and we tested naso- and oropharyngeal swab specimens from 8538 consecutive individuals. Using the m2000 as a reference assay method, we described the distribution of viral concentrations in these patients. We then used selected clinical specimens to determine the positive percent agreement of 2 other assays with more rapid turnaround times [Cepheid Xpert Xpress (GeneXpert; Cepheid); n = 27] and a laboratory developed test on the Luminex ARIES system [ARIES LDT (Luminex); n = 50] as a function of virus concentrations, from which we projected their false-negative rates in our patient population. RESULTS: SARS-CoV-2 was detected in 27% (95% CI: 26%-28%) of all specimens. Estimated viral concentrations were widely distributed, and 17% (95% CI: 16%-19%) of positive individuals had viral concentrations <845 copies/mL. Positive percent agreement was strongly related to viral concentration, and reliable detection (i.e., ≥95%) was observed at concentrations >100 copies/mL for the GeneXpert but not the ARIES LDT, corresponding to projected false-negative rates of 4% (95% CI: 0%-21%) and 27% (95% CI: 11%-46%), respectively. CONCLUSIONS: Substantial proportions of clinical specimens have low to moderate viral concentrations and may be missed by methods with less analytic sensitivity.

Bridge-Enhanced Anterior Cruciate Ligament Repair Leads to Greater Limb Asymmetry and Less Cartilage Damage Than Untreated ACL Transection or ACL Reconstruction in the Porcine Model.

BACKGROUND: The extent of posttraumatic osteoarthritis (PTOA) in the porcine anterior cruciate ligament (ACL) transection model is dependent on the surgical treatment selected. In a previous study, animals treated with bridge-enhanced ACL repair using a tissue-engineered implant developed less PTOA than those treated with ACL reconstruction (ACLR). Alterations in gait, including asymmetric weightbearing and shorter stance times, have been noted in clinical studies of subjects with osteoarthritis. HYPOTHESIS: Animals receiving a surgical treatment that results in less PTOA (ie, bridge-enhanced ACL repair) would exhibit fewer longitudinal postoperative gait asymmetries over a 1-year period when compared with treatments that result in greater PTOA (ie, ACLR and ACL transection). STUDY DESIGN: Controlled laboratory study. METHODS: Thirty-six Yucatan minipigs underwent ACL transection and were randomized to receive (1) no further treatment, (2) ACLR, or (3) bridge-enhanced ACL repair. Gait analyses were performed preoperatively, and at 4, 12, 26, and 52 weeks postoperatively. Macroscopic cartilage assessments were performed at 52 weeks. RESULTS: Knees treated with bridge-enhanced ACL repair had less macroscopic damage in the medial tibial plateau than those treated with ACLR or ACL transection (adjusted P = .03 for both comparisons). The knees treated with bridge-enhanced ACL repair had greater asymmetry in hindlimb maximum force and impulse loading at 52 weeks than the knees treated with ACL transection (adjusted P < .05 for both comparisons). Although not significant, there was a trend that knees treated with bridge-enhanced ACL repair had greater asymmetry in hindlimb maximum force and impulse loading (adjusted P < .10 for both comparisons) compared with ACLR. CONCLUSION: Contrary to our hypothesis, the surgical treatment resulting in less macroscopic cartilage damage (ie, bridge-enhanced ACL repair) exhibited greater asymmetry in load-related gait parameters than the other surgical groups. This finding suggests that increased offloading of the surgical knee may be associated with a slower rate of PTOA development. CLINICAL RELEVANCE: Less cartilage damage at 52 weeks was found in the surgical group that continued to protect the limb from full body weight during gait. This finding suggests that protection of the knee from maximum stresses may be important in minimizing the development of PTOA in the ACL-injured knee within 1 year.

Research productivity during orthopedic surgery residency correlates with pre-planned and protected research time: a survey of German-speaking countries.

PURPOSE: The purpose of this study was to identify modifiable factors associated with research activity among residents working in orthopedic surgery and traumatology. METHODS: Residents at 796 university-affiliated hospitals in Austria, Germany, and Switzerland were invited to participate. The online survey consisted of questions that ascertained 13 modifiable and 17 non-modifiable factors associated with the residents' current research activities. Responses of 129 residents were analyzed. Univariate linear regression was used to determine the association of individual factors with the current research activity (hours per week). The impact of significant non-modifiable factors (with unadjusted p values < 0.05) was controlled for using multivariate linear regression. RESULTS: The univariate analysis demonstrated six non-modifiable factors that were significantly associated with the current research activity: a University hospital setting (p < 0.001), an A-level hospital setting (p = 0.024), Swiss residents (p = 0.0012), the completion of a dedicated research year (p = 0.007), female gender (p = 0.016), and the department's size (p = 0.048). Multivariate regression demonstrated that the number of protected research days per year (p < 0.029) and the percentage of protected days, that were known 1 week before (p < 0.001) or the day before (p < 0.001), were significantly associated with a higher research activity. CONCLUSIONS: As hypothesized, more frequent and predictable protected research days were associated with higher research activity among residents in orthopedic surgery and traumatology. LEVEL OF EVIDENCE: III.

Evolutionary Selection and Constraint on Human Knee Chondrocyte Regulation Impacts Osteoarthritis Risk.

During human evolution, the knee adapted to the biomechanical demands of bipedalism by altering chondrocyte developmental programs. This adaptive process was likely not without deleterious consequences to health. Today, osteoarthritis occurs in 250 million people, with risk variants enriched in non-coding sequences near chondrocyte genes, loci that likely became optimized during knee evolution. We explore this relationship by epigenetically profiling joint chondrocytes, revealing ancient selection and recent constraint and drift on knee regulatory elements, which also overlap osteoarthritis variants that contribute to disease heritability by tending to modify constrained functional sequence. We propose a model whereby genetic violations to regulatory constraint, tolerated during knee development, lead to adult pathology. In support, we discover a causal enhancer variant (rs6060369) present in billions of people at a risk locus (GDF5-UQCC1), showing how it impacts mouse knee-shape and osteoarthritis. Overall, our methods link an evolutionarily novel aspect of human anatomy to its pathogenesis.

Proteolysis and cartilage development are activated in the synovium after surgical induction of post traumatic osteoarthritis.

Anterior cruciate ligament (ACL) transection surgery in the minipig induces post-traumatic osteoarthritis (PTOA) in a pattern similar to that seen in human patients after ACL injury. Prior studies have reported the presence of cartilage matrix-degrading proteases, such as Matrix metalloproteinase-1 (MMP-1) and A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), in the synovial fluid of injured or arthritic joints; however, the tissue origin of these proteases is unknown. The objective of this study was to identify transcriptional processes activated in the synovium after surgical induction of PTOA with ACL transection, and to determine if processes associated with proteolysis were enriched in the synovium after ACL transection. Unilateral ACL transection was performed in adolescent Yucatan minipigs and synovium samples were collected at 1, 5, 9, and 14 days post-injury. Transcriptome-wide gene expression levels were determined using bulk RNA-Sequencing in the surgical animals and control animals with healthy knees. The greatest number of transcripts with significant changes was observed 1 day after injury. These changes were primarily associated with cellular proliferation, consistent with measurements of increased cellularity of the synovium at the two-week time point. At five to 14 days, the expression of transcripts relating to proteolysis and cartilage development was significantly enriched. While protease inhibitor-encoding transcripts (TIMP2, TIMP3) represented the largest fraction of protease-associated transcripts in the uninjured synovium, protease-encoding transcripts (including MMP1, MMP2, ADAMTS4) predominated after surgery. Cartilage development-associated transcripts that are typically not expressed by synovial cells, such as ACAN and COMP, were enriched in the synovium following ACL-transection. The upregulation in both catabolic processes (proteolysis) and anabolic processes (cartilage development) suggests that the synovium plays a complex, balancing role in the early response to PTOA induction.

Cartilage Damage Is Related to ACL Stiffness in a Porcine Model of ACL Repair.

Inferior anterior cruciate ligament (ACL) structural properties may inadequately restrain tibiofemoral joint motion following surgery, contributing to the increased risk of post-traumatic osteoarthritis. Using both a direct measure of ACL linear stiffness and an in vivo magnetic resonance imaging (MRI) T2 *-based prediction model, we hypothesized that cartilage damage and ACL stiffness would increase over time, and that an inverse relationship between cartilage damage and ACL stiffness would emerge at a later stage of healing. After either 6, 12, or 24 weeks (w) of healing after ACL repair, ACL linear stiffness was determined from the force-displacement relationship during tensile testing ex vivo and predicted in vivo from the MRI T2 *-based multiple linear regression model in 24 Yucatan minipigs. Tibiofemoral cartilage was graded postmortem. There was no relationship between cartilage damage and ACL stiffness at 6 w (R2 = 0.04; p = 0.65), 12 w (R2 = 0.02; p = 0.77), or when the data from all animals were pooled (R2 = 0.02; p = 0.47). A significant inverse relationship between cartilage damage and ACL stiffness based on both ex vivo measurement (R2 = 0.90; p < 0.001) and in vivo MRI prediction (R2 = 0.78; p = 0.004) of ACL stiffness emerged at 24 w. This result suggests that 90% of the variability in gross cartilage changes is associated with the repaired ACL linear stiffness at 6 months of healing. Clinical Significance: Techniques that provide a higher stiffness to the repaired ACL may be required to mitigate the post-traumatic osteoarthritis commonly seen after ACL injury, and MRI T2 * can be used as a noninvasive estimation of ligament stiffness. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2249-2257, 2019.

Synovial fluid proteome changes in ACL injury-induced posttraumatic osteoarthritis: Proteomics analysis of porcine knee synovial fluid.

Surgical transection of the anterior cruciate ligament (ACL) in the porcine model leads to posttraumatic osteoarthritis if left untreated. However, a recently developed surgical treatment, bridge-enhanced ACL repair, prevents further cartilage damage. Since the synovial fluid bathes all the intrinsic structures of knee, we reasoned that a comparative analysis of synovial fluid protein contents could help to better understand the observed chondroprotective effects of the bridge-enhanced ACL repair. We hypothesized that post-surgical changes in the synovial fluid proteome would be different in the untreated and repaired knees, and those changes would correlate with the degree of cartilage damage. Thirty adolescent Yucatan mini-pigs underwent unilateral ACL transection and were randomly assigned to either no further treatment (ACLT, n = 14) or bridge-enhanced ACL repair (BEAR, n = 16). We used an isotopically labeled high resolution LC MS/MS-based proteomics approach to analyze the protein profile of synovial fluid at 6 and 12 months after ACL transection in untreated and repaired porcine knees. A linear mixed effect model was used to compare the normalized protein abundance levels between the groups at each time point. Bivariate linear regression analyses were used to assess the correlations between the macroscopic cartilage damage (total lesion area) and normalized abundance levels of each of the identified secreted proteins. There were no significant differences in cartilage lesion area or quantitative abundance levels of the secreted proteins between the ACLT and BEAR groups at 6 months. However, by 12 months, greater cartilage damage was seen in the ACLT group compared to the BEAR group (p = 0.005). This damage was accompanied by differences in the abundance levels of secreted proteins, with higher levels of Vitamin K-dependent protein C (p = 0.001), and lower levels of Apolipoprotein A4 (p = 0.021) and Cartilage intermediate layer protein 1 (p = 0.049) in the ACLT group compared to the BEAR group. There were also group differences in the secreted proteins that significantly changed in abundance between 6 and 12 months in ACLT and BEAR knees. Increased concentration of Ig lambda-1 chain C regions and decreased concentration of Hemopexin, Clusterin, Coagulation factor 12 and Cartilage intermediate layer protein 1 were associated with greater cartilage lesion area. In general, ACLT knees had higher concentrations of pro-inflammatory proteins and lower concentrations of anti-inflammatory proteins than BEAR group. In addition, the ACLT group had a lower and declining synovial concentrations of CILP, in contrast to a consistently high abundance of CILP in repaired knees. These differences suggest that the knees treated with bridge-enhanced ACL repair may be maintaining an environment that is more protective of the extracellular matrix, a function which is not seen in the ACLT knees.

Myxedema Ascites: An Unusual Presentation of Uncontrolled Hypothyroidism.

We describe a case of myxedema ascites in a 64-year-old male with a history of hypothyroidism noncompliant with medical therapy who presented with syncope, hematemesis, melena, and abdominal distension. The patient received intravenous levothyroxine with a good response and improved upon discharge. This case highlights the importance of considering hypothyroidism as an etiology of unexplained ascites. The analysis of ascites from myxedema may not always have a significantly elevated protein (>2.5g/dL). Appropriate diagnosis should also rely on the clinical presentation along with a rapid and positive response to thyroid hormone replacement therapy.

Mesenchymal Stem Cells Isolated from the Anterior Cruciate Ligament: Characterization and Comparison of Cells from Young and Old Donors.

PURPOSE: Mesenchymal stem cells (MSCs) isolated from the anterior cruciate ligament (ACL) share multiple characteristics of bone marrow-derived mesenchymal stem cells (BMSCs), allowing their use for regenerative therapies. Injuries to the ACL can affect people of all ages. This study assesses whether the regenerative potential of ACL-derived MSCs (ACL-MSCs) from old donors is as high as the potential of ACL-MSCs from young donors. MATERIALS AND METHODS: ACL-MSCs were isolated from ACL tissues obtained from young and old donors at the time of ACL reconstruction or arthroplasty. Proliferative capacity, multilineage differentiation potential (chondrogenic, osteogenic, and adipogenic lineages), and transcriptome-wide gene expression were assessed and compared between young and old donors. BMSCs of middle-aged donors served as an additional comparator. RESULTS: No substantial differences between ACL-MSCs from young and old donors were observed in their proliferative capacity and multilineage differentiation potential. The latter did not substantially differ between both ACL-MSC groups and BMSCs. Differential expression of genes related to the cytoskeleton and to protein dephosphorylation amongst other pathways was detected between ACL-MSCs from young and old donors. CONCLUSIONS: Regenerative potential of ACL-MSCs from old donors was not substantially lower than that from young donors, suggesting that regenerative therapies of ACL tears are feasible in both age groups. In vivo studies of the effect of age on the efficacy of such therapies are needed.

Transcriptional profiling of synovium in a porcine model of early post-traumatic osteoarthritis.

To determine the transcriptional profile of synovium during the molecular phase of post-traumatic osteoarthritis, anterior cruciate ligament transections (ACL) were performed in 36 Yucatan minipigs. Equal numbers were randomly assigned to no further treatment, ACL reconstruction or repair. Perimeniscal synovium for histopathology and RNA-sequencing was harvested at 1 and 4 weeks post-operatively and from six healthy control animals. Microscopic synovitis scores significantly worsened at 1 (p < 0.001) and 4 weeks (p = 0.003) post-surgery relative to controls, and were driven by intimal hyperplasia and increased stromal cellularity without inflammatory infiltrates. Synovitis scores were similar between no treatment, reconstruction, and repair groups (p ≥ 0.668). Relative to no treatment at 1 week, 88 and 367 genes were differentially expressed in the reconstruction and repair groups, respectively (227 and 277 at 4 weeks). Relative to controls and with the treatment groups pooled, 1,683 transcripts were concordantly differentially expressed throughout the post-surgery time-course. Affected pathways included, proteolysis_connective tissue degradation (including upregulations of protease-encoding MMP1, MMP13, and ADAMTS4), and development_cartilage development (including upregulations of ACAN, SOX9, and RUNX2), among others. Using linear regression, significant associations of post-surgery synovial expression levels of 20 genes with the articular cartilage glycosaminoglycan loss were identified. These genes were predominantly related to embryonic skeletal system development and included RUNX2. In conclusion, this study confirmed an increased synovial expression of genes that may serve as targets to prevent cartilage degradation, including MMP1, MMP13, and ADAMTS4, in knees with microscopic synovitis and cartilage proteoglycan loss. Attractive novel targets include regulators of embryonic developmental processes in synovium. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Magnetic resonance measurements of tissue quantity and quality using T2 * relaxometry predict temporal changes in the biomechanical properties of the healing ACL.

The purpose of this study was to develop a magnetic resonance T2 * relaxometry-based multiple linear regression model to predict the structural properties of the healing anterior cruciate ligament (ACL) over a 24-week healing period following ACL repair in Yucatan minipigs. Two hypotheses were tested: (i) that a regression model based on ACL sub-volumes containing short and long T2 * relaxation times would outperform a competing model based on sub-volumes of short T2 * relaxation times only; and (ii) that an optimized regression model would be capable of predicting ACL structural properties between 6 and 24 weeks post-repair. ACLs were imaged in 24 minipigs (8/group) at either 6, 12, or 24 weeks after ACL repair. The structural properties of the ACLs were determined from tensile failure tests. Four multiple linear regression models of increasing complexity were fitted to the data. Akaike Information Criterion values and Bland-Altman tests were used to compare model performance and to test the hypotheses. The structural properties predicted from the multiple linear regression model that was based on the change in ACL sub-volumes of both the short and long T2 * relaxation times over the healing period were in closest agreement to the measured values, suggesting that the amounts of both organized and disorganized collagen, and the change in these quantities over time, are required to predict the structural properties of healing ACLs accurately. CLINICAL SIGNIFICANCE: our time-specific, T2 *-based regression model may allow us to estimate the structural properties of ACL repairs in vivo longitudinally. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1701-1709, 2018.

Transcriptional profiling of articular cartilage in a porcine model of early post-traumatic osteoarthritis.

To identify the molecular pathophysiology present in early post-traumatic osteoarthritis (PTOA), the transcriptional profile of articular cartilage and its response to surgical PTOA induction were determined. Thirty six Yucatan minipigs underwent anterior cruciate ligament (ACL) transection and were randomly assigned in equal numbers to no further treatment, reconstruction or ligament repair. Cartilage was harvested at 1 and 4 weeks post-operatively and histology and RNA-sequencing were performed and compared to controls. Microscopic cartilage scores significantly worsened at 1 (p = 0.028) and 4 weeks (p = 0.001) post-surgery relative to controls, but did not differ between untreated, reconstruction or repair groups. Gene expression after ACL reconstruction and ACL transection were similar, with only 0.03% (including SERPINB7 and CR2) and 0.2% of transcripts (including INHBA) differentially expressed at 1 and 4 weeks respectively. COL2A1, COMP, SPARC, CHAD, and EF1ALPHA were the most highly expressed non ribosomal, non mitochondrial genes in the controls and remained abundant after surgery. A total of 1,275 genes were differentially expressed between 1 and 4 weeks post-surgery. With the treatment groups pooled, 682 genes were differentially expressed at both time-points, with the most significant changes observed in MMP1, COCH, POSTN, CYTL1, and PTGFR. This study confirmed the development of a microscopic PTOA stage after ACL surgery in the porcine model. Upregulation of multiple proteases (including MMP1 and ADAMTS4) were found; however, the level of expression remained orders of magnitude below that of extracellular matrix protein-coding genes (including COL2A1 and ACAN). In summary, genes with established roles in PTOA as well as novel targets for specific intervention were identified. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:318-329, 2018.

Bench-to-bedside: Bridge-enhanced anterior cruciate ligament repair.

Anterior cruciate ligament (ACL) injuries are one of the most well-known orthopaedic injuries and are treated with one of the most common orthopaedic procedures performed in the United States. This surgical procedure, ACL reconstruction, is successful at restoring the gross stability of the knee. However, the outcomes of ACL reconstruction can be limited by short and long-term complications, including muscle weakness, graft rupture, and premature osteoarthritis. Thus, new methods of treating this injury are being explored. This review details the pathway of how a tissue engineering strategy can be used to improve the healing of the ACL in preclinical studies and then translated to patients in an FDA-approved clinical study. This review paper will outline the clinical importance of ACL injuries, history of primary repair, the pathology behind failure of the ACL to heal, pre-clinical studies, the FDA approval process for a high risk medical device, and the preliminary results from a first-in-human study. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2606-2612, 2017.

Immediate Administration of Intraarticular Triamcinolone Acetonide After Joint Injury Modulates Molecular Outcomes Associated With Early Synovitis.

OBJECTIVE: To test whether intraarticular corticosteroid injection mitigates injury-induced synovitis and collagen degradation after anterior cruciate ligament transection (ACLT) and to characterize the synovial response using a functional genomics approach in a preclinical model of posttraumatic osteoarthritis. METHODS: Yorkshire pigs underwent unilateral ACLT without subsequent corticosteroid injection (the ACLT group; n = 6) or ACLT with immediate injection of 20 mg triamcinolone acetonide (the steroid group; n = 6). A control group of pigs (the intact group; n = 6) did not undergo surgery. Total synovial membrane cellularity and synovial fluid concentration of C1,2C neoepitope-bearing collagen fragments 14 days after injury were primary end points and were compared between the ACLT, steroid, and intact groups. Cells were differentiated by histologic phenotype and counted, while RNA sequencing was used to quantify transcriptome-wide gene expression and monocyte, macrophage, and lymphocyte markers. RESULTS: In the intact group, total cellularity was 13% (95% confidence interval [95% CI] 9-16) and the C1,2C concentration was 0.24 µg/ml (95% CI 0.08-0.39). In the ACLT group, significant increases were observed in total cellularity (to 21% [95% CI 16-27]) and C1,2C concentration (to 0.49 µg/ml [95% CI 0.39-0.59]). Compared to values in the ACLT group, total cellularity in the steroid group was nonsignificantly decreased to 17% (95% CI 15-18) (P = 0.26) and C1,2C concentration in the steroid group was significantly decreased to 0.29 µg/ml (95% CI 0.23-0.35) (P = 0.04). A total of 255 protein-coding transcripts were differentially expressed between the ACLT group and the intact group. These genes mainly enriched pathways related to cellular immune response, proteolysis, and angiogenesis. Mononuclear leukocytes were the dominant cell type in cell-dense areas. MARCO, SOCS3, CCR1, IL4R, and MMP2 expression was significantly associated with C1,2C levels. CONCLUSION: Early intraarticular immunosuppression mitigated injury-induced increases in collagen fragments, an outcome better predicted by specific marker expression than by histologic measures of synovitis.

Extracellular matrix-blood composite injection reduces post-traumatic osteoarthritis after anterior cruciate ligament injury in the rat.

The objective of this study was to determine if an injection of a novel extracellular matrix scaffold and blood composite (EMBC) after anterior cruciate ligament (ACL) injury would have a mitigating effect on post-traumatic osteoarthritis (PTOA) development in rat knees. Lewis rats underwent unilateral ACL transection and were divided into three groups as follows: (1) no further treatment (ACLT; n = 10); (2) an intra-articular injection of EMBC on day 0 (INJ0; n = 11); and (3) an intra-articular injection of EMBC on day 14 (INJ14; n = 11). Ten additional animals received capsulotomy only (n = 10, SHAM group). The OARSI histology scoring of the tibial cartilage and micro-CT of the tibial epiphysis were performed after 35 days. The ratio of intact/treated hind limb forces during gait was determined using a variable resistor walkway. The OARSI cartilage degradation sum score and total degeneration width were significantly greater in the ACLT group when compared to the INJ0 (p = 0.031, and p = 0.005) and INJ14 (p = 0.022 and p = 0.04) group. Weight bearing on the operated limb only decreased significantly in the ACLT group (p = 0.048). In the rat ACL transection model, early or delayed injection of EMBC ameliorated the significant decrease in weight bearing and cartilage degradation seen in knees subjected to ACL transection without injection. The results indicate that the injection of EMBC may slow the process of PTOA following ACL injury and may provide a promising treatment for PTOA. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:995-1003, 2016.

Effect of low-temperature ethylene oxide and electron beam sterilization on the in vitro and in vivo function of reconstituted extracellular matrix-derived scaffolds.

Reconstituted extracellular matrix (ECM)-derived scaffolds are commonly utilized in preclinical tissue engineering studies as delivery vehicles for cells and growth factors. Translation into clinical use requires identifying a sterilization method that effectively removes bacteria but does not harm scaffold function. To determine effectiveness of sterilization and impact on ECM scaffold integrity and function, low-temperature ethylene oxide and 15 kGy electron beam irradiation techniques were evaluated. Scaffold sterility was assessed in accordance to United States Pharmacopeia Chapter 71. Scaffold matrix degradation was determined in vitro using enzymatic resistance tests and gel electrophoresis. Scaffold mechanics including elastic modulus, yield stress and collapse modulus were tested. Lastly, 14 Yorkshire pigs underwent ACL transection and bio-enhanced ACL repair using sterilized scaffolds. Histologic response of ligament, synovium, and lymph nodes was compared at 4, 6, and 8 weeks. Ethylene oxide as well as electron beam irradiation yielded sterile scaffolds. Scaffold resistance to enzymatic digestion and protein integrity slightly decreased after electron beam irradiation while ethylene oxide altered scaffold matrix. Scaffold elastic modulus and yield stress were increased after electron beam treatment, while collapse modulus was increased after ethylene oxide treatment. No significant changes in ACL dimensions, in vivo scaffold resorption rate, or histologic response of synovium, ligament, and lymph nodes with either terminal sterilization technique were detectable. In conclusion, this study identifies two methods to terminally sterilize an ECM scaffold. In vitro scaffold properties were slightly changed without significantly influencing the biologic responses of the surrounding tissues in vivo. This is a critical step toward translating new tissue engineering strategies to clinical trials.

Electron beam sterilization does not have a detrimental effect on the ability of extracellular matrix scaffolds to support in vivo ligament healing.

Extracellular matrix (ECM) scaffolds have been used to enhance anterior cruciate ligament (ACL) repair in large animal models. To translate this technology to clinical care, identifying a method which effectively sterilizes the material without significantly impairing in vivo function is desirable. Sixteen Yorkshire pigs underwent ACL transection and were randomly assigned to bridge-enhanced ACL repair-primary suture repair of the ACL with addition of autologous blood soaked ECM scaffold--with either (i) an aseptically processed ECM scaffold, or (ii) an electron beam irradiated ECM scaffold. Primary outcome measures included sterility of the scaffold and biomechanical properties of the scaffold itself and the repaired ligament at 8 weeks after surgery. Scaffolds treated with 15 kGy electron beam irradiation had no bacterial or fungal growth noted, while aseptically processed scaffolds had bacterial growth in all tested samples. The mean biomechanical properties of the scaffold and healing ligament were lower in the electron beam group; however, differences were not statistically significant. Electron beam irradiation was able to effectively sterilize the scaffolds. In addition, this technique had only a minimal impact on the in vivo function of the scaffolds when used for ligament healing in the porcine model.

Bio-enhanced repair of the anterior cruciate ligament.

Suture repair of the anterior cruciate ligament (ACL) has been widely abandoned in favor of ACL reconstruction, largely because of the high rates of failure and unreliability of the outcomes after suture repair. However, there have been recent basic science studies that suggest that combining a suture repair with a biological adjunct may improve the results of suture repair of the ACL, with several studies in large animal models showing equivalent strength of an ACL treated with bio-enhanced repaired to that of an ACL graft at 3, 6, and 12 months after surgery. In addition, the groups treated with bio-enhanced repair had significantly less osteoarthritis when compared with the animals undergoing ACL reconstruction. These findings have led to a renewed interest in bio-enhanced primary repair as a way to make repair of the ACL a viable option for a select group of patients in the future.