Identifying small molecules for protecting chondrocyte function and matrix integrity after controlled compressive injury.

Objective: Articular cartilage injury is central for the development of post-traumatic osteoarthritis (PTOA). With few disease-modifying therapies successful at offsetting progressive osteoarthritis (OA), our goal is to use a high throughput screening platform of cartilage injury to identify novel chondroprotective compounds. Targeting articular cartilage damage immediately after injury remains a promising therapeutic strategy to overcome irreversible tissue damage. Method: We constructed a single impact-cartilage screening method using a multi-platen system that simultaneously impacts 48 samples and makes use of engineered cartilage tissue analogs (known as CTAs). Drug libraries were screened and assessed for their ability to alter two crucial biological responses to impact injuries, namely matrix degradation and cell stress. Results: Over 500 small molecules were screened for their ability to alter proteoglycan loss, matrix metalloproteinase activity, and cell stress or death. Fifty-five compounds passed through secondary screening and were from commercial libraries of natural and redox, stem cell related compounds, as well as protease, kinase and phosphatase inhibitors. Through secondary screening, 16 promising candidates exhibited activity on one or more critical function of chondrocytes. While many are mechanistically known compounds, their function in joint diseases is not known. Conclusion: This platform was validated for screening drug activity against a tissue engineered model of PTOA. Multiple compounds identified in this manner have potential application as early protective therapy for treating PTOA, and require further study. We propose this screening platform can identify novel molecules that act on early chondrocyte responses to injury and provide an invaluable tool for therapeutic development.

Evaluating whole-genome expression differences in idiopathic and diabetic adhesive capsulitis.

BACKGROUND: Diabetic patients have a greater incidence of adhesive capsulitis (AC) and a more protracted disease course than patients with idiopathic AC. The purpose of this study was to compare gene expression differences between AC with diabetes mellitus and AC without diabetes mellitus. METHODS: Shoulder capsule samples were prospectively obtained from diabetic or nondiabetic patients who presented with shoulder dysfunction and underwent arthroscopy (N = 16). Shoulder samples of AC with and without diabetes (n = 8) were compared with normal shoulder samples with and without diabetes as the control group (n = 8). Shoulder capsule samples were subjected to whole-transcriptome RNA sequencing, and differential expression was analyzed with EdgeR. Only genes with a false discovery rate < 5% were included for further functional enrichment analysis. RESULTS: The sample population had a mean age of 47 years (range, 24-62 years), and the mean hemoglobin A1c level for nondiabetic and diabetic patients was 5.18% and 8.71%, respectively. RNA-sequencing analysis revealed that 66 genes were differentially expressed between diabetic patients and nondiabetic patients with AC whereas only 3 genes were differentially expressed when control patients with and without diabetes were compared. Furthermore, 286 genes were differentially expressed in idiopathic AC patients, and 61 genes were differentially expressed in diabetic AC patients. On gene clustering analysis, idiopathic AC was enriched with multiple structural and muscle-related pathways, such as muscle filament sliding, whereas diabetic AC included a greater number of hormonal and inflammatory signaling pathways, such as cellular response to corticotropin-releasing factor. CONCLUSIONS: Whole-transcriptome expression profiles demonstrate a fundamentally different underlying pathophysiology when comparing diabetic AC with idiopathic AC, suggesting that these conditions are distinct clinical entities. The new genes expressed explain the differences in the disease course and suggest new therapeutic targets that may lead to different treatment paradigms in these 2 subsets.

Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 Isolates.

We report the genome of a B.1.1.7+E484K severe acute respiratory syndrome coronavirus 2 from Southeastern Pennsylvania and compare it with all high-coverage B.1.1.7+E484K genomes (n = 235) available. Analyses showed the existence of at least 4 distinct clades of this variant circulating in the United States and the possibility of at least 59 independent acquisitions of the E484K mutation.

Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 isolates from Pennsylvania.

Rapid whole genome sequencing of SARS-CoV-2 has presented the ability to detect new emerging variants of concern in near real time. Here we report the genome of a virus isolated in Pennsylvania in March 2021 that was identified as lineage B.1.1.7 (VOC-202012/01) that also harbors the E484K spike mutation, which has been shown to promote "escape" from neutralizing antibodies in vitro. We compare this sequence to the only 5 other B.1.1.7+E484K genomes from Pennsylvania, all of which were isolated in mid March. Beginning in February 2021, only a small number (n=60) of isolates with this profile have been detected in the US, and only a total of 253 have been reported globally (first in the UK in December 2020). Comparative genomics of all currently available high coverage B.1.1.7+E484K genomes (n=235) available on GISAID suggested the existence of 7 distinct groups or clonal complexes (CC; as defined by GNUVID) bearing the E484K mutation raising the possibility of 7 independent acquisitions of the E484K spike mutation in each background. Phylogenetic analysis suggested the presence of at least 3 distinct clades of B.1.1.7+E484K circulating in the US, with the Pennsylvanian isolates belonging to two distinct clades. Increased genomic surveillance will be crucial for detection of emerging variants of concern that can escape natural and vaccine induced immunity.

Characterization of Injury Induced by Routine Surgical Manipulations of Nasal Septal Cartilage.

IMPORTANCE: This study characterizes and compares common surgical manipulations' effects on septal cartilage to understand their implications for rhinoplasty outcomes based on cell viability and cartilage health. OBJECTIVE: To illustrate distinct differences in the impact of various surgical manipulations on septal cartilage in an in vitro septal cartilage model. A secondary objective is to better understand the chondrocyte's response to injury as well as how alterations in the extracellular matrix correspond to chondrocyte viability. DESIGN, SETTING, AND PARTICIPANTS: In this bench-top in vitro porcine model using juvenile bovine septal cartilage from bovine snouts, easily obtainable septal cartilage was used to generate large numbers of homogenous cartilage specimens. Quantitative outcomes at early and late time points were cell viability, cell stress, matrix loss, and qualitative assessment through histologic examination. The study was performed at a single academic tertiary care research hospital. INTERVENTIONS: Four common surgical manipulations were contrasted with a control group: crushed cartilage, scored cartilage, diced cartilage, and shaved cartilage. MAIN OUTCOMES AND MEASURES: Following the manipulation of the cartilage, the quantitative outcomes were glycosaminoglycan release to the media, lactate dehydrogenase release to the media, and cell death analysis through apoptosis staining. The qualitative outcomes were histologic staining of the manipulated cartilage with safranin-O/fast green stain to identify proteoglycan loss. RESULTS: The crushing followed by shaving manipulations were the most damaging as indicated by increased levels of lactate dehydrogenase release, glycosaminoglycans loss, and cell death. Matrix loss did not increase until after 48 hours postinjury. Furthermore, chondrocyte death was seen early after injury and accelerated to the late time point, day 9, in all manipulations. Conversely, cell stress was found to be greater at 48 hours postinjury, which then declined to the late time point, day 9. CONCLUSIONS AND RELEVANCE: The crushing manipulation followed by shaving and then dicing were the most destructive methods of cartilage manipulation relative to control specimens. Collectively, these outcomes demonstrate the range of injury which occurs with all septal cartilage manipulations and can inform rhinoplasty practice to use the least damaging effective surgical manipulation to obtain the desired outcome. LEVEL OF EVIDENCE: NA.