Cicero Predicts cis-Regulatory DNA Interactions from Single-Cell Chromatin Accessibility Data.

Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of "chromatin hubs"-they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.

Enhanced fear memory after social defeat in mice is dependent on interleukin-1 receptor signaling in glutamatergic neurons.

Chronic stress is associated with increased anxiety, cognitive deficits, and post-traumatic stress disorder. Repeated social defeat (RSD) in mice causes long-term stress-sensitization associated with increased microglia activation, monocyte accumulation, and enhanced interleukin (IL)-1 signaling in endothelia and neurons. With stress-sensitization, mice have amplified neuronal, immune, and behavioral responses to acute stress 24 days later. This is clinically relevant as it shares key aspects with post-traumatic stress disorder. The mechanisms underlying stress-sensitization are unclear, but enhanced fear memory may be critical. The purpose of this study was to determine the influence of microglia and IL-1R1 signaling in neurons in the development of sensitization and increased fear memory after RSD. Here, RSD accelerated fear acquisition, delayed fear extinction, and increased cued-based freezing at 0.5 day. The enhancement in contextual fear memory after RSD persisted 24 days later. Next, microglia were depleted with a CSF1R antagonist prior to RSD and several parameters were assessed. Microglia depletion blocked monocyte recruitment to the brain. Nonetheless, neuronal reactivity (pCREB) and IL-1ß RNA expression in the hippocampus and enhanced fear memory after RSD were microglial-independent. Because IL-1ß RNA was prominent in the hippocampus after RSD even with microglia depletion, IL-1R1 mediated signaling in glutamatergic neurons was assessed using neuronal Vglut2+/IL-1R1-/- mice. RSD-induced neuronal reactivity (pCREB) in the hippocampus and enhancement in fear memory were dependent on neuronal IL-1R1 signaling. Furthermore, single-nuclei RNA sequencing (snRNAseq) showed that RSD influenced transcription in specific hippocampal neurons (DG neurons, CA2/3, CA1 neurons) associated with glutamate signaling, inflammation and synaptic plasticity, which were neuronal IL-1R1-dependent. Furthermore, snRNAseq data provided evidence that RSD increased CREB, BDNF, and calcium signaling in DG neurons in an IL-1R1-dependent manner. Collectively, increased IL-1R1-mediated signaling (monocytes/microglia independent) in glutamatergic neurons after RSD enhanced neuronal reactivity and fear memory.

Impaired cortical neuronal homeostasis and cognition after diffuse traumatic brain injury are dependent on microglia and type I interferon responses.

Neuropsychiatric complications including depression and cognitive decline develop in the years after traumatic brain injury (TBI), negatively affecting quality of life. Microglial and type 1 interferon (IFN-I) responses are associated with the transition from acute to chronic neuroinflammation after diffuse TBI in mice. Thus, the purpose of this study was to determine if impaired neuronal homeostasis and increased IFN-I responses intersected after TBI to cause cognitive impairment. Here, the RNA profile of neurons and microglia after TBI (single nucleus RNA-sequencing) with or without microglia depletion (CSF1R antagonist) was assessed 7 dpi. There was a TBI-dependent suppression of cortical neuronal homeostasis with reductions in CREB signaling, synaptogenesis, and synaptic migration and increases in RhoGDI and PTEN signaling (Ingenuity Pathway Analysis). Microglial depletion reversed 50% of TBI-induced gene changes in cortical neurons depending on subtype. Moreover, the microglial RNA signature 7 dpi was associated with increased stimulator of interferon genes (STING) activation and IFN-I responses. Therefore, we sought to reduce IFN-I signaling after TBI using STING knockout mice and a STING antagonist, chloroquine (CQ). TBI-associated cognitive deficits in novel object location and recognition (NOL/NOR) tasks at 7 and 30 dpi were STING dependent. In addition, TBI-induced STING expression, microglial morphological restructuring, inflammatory (Tnf, Cd68, Ccl2) and IFN-related (Irf3, Irf7, Ifi27) gene expression in the cortex were attenuated in STINGKO mice. CQ also reversed TBI-induced cognitive deficits and reduced TBI-induced inflammatory (Tnf, Cd68, Ccl2) and IFN (Irf7, Sting) cortical gene expression. Collectively, reducing IFN-I signaling after TBI with STING-dependent interventions attenuated the prolonged microglial activation and cognitive impairment.

Amplified Gliosis and Interferon-Associated Inflammation in the Aging Brain following Diffuse Traumatic Brain Injury.

Traumatic brain injury (TBI) is associated with chronic psychiatric complications and increased risk for development of neurodegenerative pathology. Aged individuals account for most TBI-related hospitalizations and deaths. Nonetheless, neurobiological mechanisms that underlie worsened functional outcomes after TBI in the elderly remain unclear. Therefore, this study aimed to identify pathways that govern differential responses to TBI with age. Here, adult (2 months of age) and aged (16-18 months of age) male C57BL/6 mice were subjected to diffuse brain injury (midline fluid percussion), and cognition, gliosis, and neuroinflammation were determined 7 or 30 d postinjury (dpi). Cognitive impairment was evident 7 dpi, independent of age. There was enhanced morphologic restructuring of microglia and astrocytes 7 dpi in the cortex and hippocampus of aged mice compared with adults. Transcriptional analysis revealed robust age-dependent amplification of cytokine/chemokine, complement, innate immune, and interferon-associated inflammatory gene expression in the cortex 7 dpi. Ingenuity pathway analysis of the transcriptional data showed that type I interferon (IFN) signaling was significantly enhanced in the aged brain after TBI compared with adults. Age prolonged inflammatory signaling and microgliosis 30 dpi with an increased presence of rod microglia. Based on these results, a STING (stimulator of interferon genes) agonist, DMXAA, was used to determine whether augmenting IFN signaling worsened cortical inflammation and gliosis after TBI. DMXAA-treated Adult-TBI mice showed comparable expression of myriad genes that were overexpressed in the cortex of Aged-TBI mice, including Irf7, Clec7a, Cxcl10, and Ccl5 Overall, diffuse TBI promoted amplified IFN signaling in aged mice, resulting in extended inflammation and gliosis.SIGNIFICANCE STATEMENT Elderly individuals are at higher risk of complications following traumatic brain injury (TBI). Individuals >70 years old have the highest rates of TBI-related hospitalization, neurodegenerative pathology, and death. Although inflammation has been linked with poor outcomes in aging, the specific biological pathways driving worsened outcomes after TBI in aging remain undefined. In this study, we identify amplified interferon-associated inflammation and gliosis in aged mice following TBI that was associated with persistent inflammatory gene expression and microglial morphologic diversity 30 dpi. STING (stimulator of interferon genes) agonist DMXAA was used to demonstrate a causal link between augmented interferon signaling and worsened neuroinflammation after TBI. Therefore, interferon signaling may represent a therapeutic target to reduce inflammation-associated complications following TBI.

Chronic Cortical Inflammation, Cognitive Impairment, and Immune Reactivity Associated with Diffuse Brain Injury Are Ameliorated by Forced Turnover of Microglia.

Traumatic brain injury (TBI) is associated with an increased risk of cognitive, psychiatric, and neurodegenerative complications that may develop after injury. Increased microglial reactivity following TBI may underlie chronic neuroinflammation, neuropathology, and exaggerated responses to immune challenges. Therefore, the goal of this study was to force turnover of trauma-associated microglia that develop after diffuse TBI and determine whether this alleviated chronic inflammation, improved functional recovery and attenuated reduced immune reactivity to lipopolysaccharide (LPS) challenge. Male mice received a midline fluid percussion injury (mFPI) and 7 d later were subjected to a forced microglia turnover paradigm using CSF1R antagonism (PLX5622). At 30 d postinjury (dpi), cortical gene expression, dendritic complexity, myelin content, neuronal connectivity, cognition, and immune reactivity were assessed. Myriad neuropathology-related genes were increased 30 dpi in the cortex, and 90% of these gene changes were reversed by microglial turnover. Reduced neuronal connectivity was evident 30 dpi and these deficits were attenuated by microglial turnover. TBI-associated dendritic remodeling and myelin alterations, however, remained 30 dpi independent of microglial turnover. In assessments of functional recovery, increased depressive-like behavior, and cognitive impairment 30 dpi were ameliorated by microglia turnover. To investigate microglial priming and reactivity 30 dpi, mice were injected intraperitoneally with LPS. This immune challenge caused prolonged lethargy, sickness behavior, and microglial reactivity in the TBI mice. These extended complications with LPS in TBI mice were prevented by microglia turnover. Collectively, microglial turnover 7 dpi alleviated behavioral and cognitive impairments associated with microglial priming and immune reactivity 30 dpi.SIGNIFICANCE STATEMENT A striking feature of traumatic brain injury (TBI), even mild injuries, is that over 70% of individuals have long-term neuropsychiatric complications. Chronic inflammatory processes are implicated in the pathology of these complications and these issues can be exaggerated by immune challenge. Therefore, our goal was to force the turnover of microglia 7 d after TBI. This subacute 7 d postinjury (dpi) time point is a critical transitional period in the shift toward chronic inflammatory processes and microglia priming. This forced microglia turnover intervention in mice attenuated the deficits in behavior and cognition 30 dpi. Moreover, microglia priming and immune reactivity after TBI were also reduced with microglia turnover. Therefore, microglia represent therapeutic targets after TBI to reduce persistent neuroinflammation and improve recovery.

Single-Cell Multi-omics: An Engine for New Quantitative Models of Gene Regulation.

Cells in a multicellular organism fulfill specific functions by enacting cell-type-specific programs of gene regulation. Single-cell RNA sequencing technologies have provided a transformative view of cell-type-specific gene expression, the output of cell-type-specific gene regulatory programs. This review discusses new single-cell genomic technologies that complement single-cell RNA sequencing by providing additional readouts of cellular state beyond the transcriptome. We highlight regression models as a simple yet powerful approach to relate gene expression to other aspects of cellular state, and in doing so, gain insights into the biochemical mechanisms that are necessary to produce a given gene expression output.

A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease.

BACKGROUND: Elucidation of the genetic factors underlying chronic liver disease may reveal new therapeutic targets. METHODS: We used exome sequence data and electronic health records from 46,544 participants in the DiscovEHR human genetics study to identify genetic variants associated with serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Variants that were replicated in three additional cohorts (12,527 persons) were evaluated for association with clinical diagnoses of chronic liver disease in DiscovEHR study participants and two independent cohorts (total of 37,173 persons) and with histopathological severity of liver disease in 2391 human liver samples. RESULTS: A splice variant (rs72613567:TA) in HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13, was associated with reduced levels of ALT (P=4.2×10-12) and AST (P=6.2×10-10). Among DiscovEHR study participants, this variant was associated with a reduced risk of alcoholic liver disease (by 42% [95% confidence interval {CI}, 20 to 58] among heterozygotes and by 53% [95% CI, 3 to 77] among homozygotes), nonalcoholic liver disease (by 17% [95% CI, 8 to 25] among heterozygotes and by 30% [95% CI, 13 to 43] among homozygotes), alcoholic cirrhosis (by 42% [95% CI, 14 to 61] among heterozygotes and by 73% [95% CI, 15 to 91] among homozygotes), and nonalcoholic cirrhosis (by 26% [95% CI, 7 to 40] among heterozygotes and by 49% [95% CI, 15 to 69] among homozygotes). Associations were confirmed in two independent cohorts. The rs72613567:TA variant was associated with a reduced risk of nonalcoholic steatohepatitis, but not steatosis, in human liver samples. The rs72613567:TA variant mitigated liver injury associated with the risk-increasing PNPLA3 p.I148M allele and resulted in an unstable and truncated protein with reduced enzymatic activity. CONCLUSIONS: A loss-of-function variant in HSD17B13 was associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.).

On the design of CRISPR-based single-cell molecular screens.

Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to ∼50% swapping of guide RNA-barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.

An accurate method of measuring shoulder sling compliance: a validation study.

BACKGROUND: The effect of postoperative shoulder sling compliance on surgical outcomes is unknown. The goal was to determine an accurate method to measure sling compliance. We compared volunteer recorded sling wear time with temperature-based sensors to monitor sling compliance. METHODS: Data loggers sutured at three locations measured heat generated in 15-minute intervals. Slings wearers logged sling wear to accurately cross-reference with temperature sensors. Secondary experiments analyzed whether surrounding ambient temperature can be discerned from actual sling wear. We created an algorithm to describe actual sling wear time as a function of heat recorded and calculated percent wear accuracy. RESULTS: The modified sling was worn for 172 h. The algorithm modeled sling on/off times by analyzing cutoff temperatures. Diagnostic accuracy was >99 % for the three locations, with no statistically significant differences among them. Compared with sling wear, ambient temperature took longer to reach critical temperature values determined by the algorithm, helping distinguish compliance from false positives. CONCLUSIONS: The described algorithm can effectively quantify shoulder sling wear time based on heat-generated sensor readings. False positives from ambient temperature are minimal. This measurement method could be used to study the relationship between postoperative sling use and functional outcomes after shoulder surgery.

Impact of pre-operative recreational marijuana use on outcomes two years after orthopaedic surgery.

PURPOSE: The purpose of this study was to investigate the relationship between recreational marijuana use and patient-reported outcomes two years after orthopaedic surgery. We hypothesized that pre-operative recreational marijuana use would be associated with less pain, better function, and better mental health measures two years after orthopaedic surgery. METHODS: Patients were retrospectively analyzed from a prospective orthopaedic registry at a single urban institution. A total of 1710 patients completed the pre-operative assessment and 1103 patients (64.5%) completed the two-year follow-up questionnaires. The cohort was then divided into two groups based on reported preoperative recreational marijuana usage, and statistical analysis was performed to determine if marijuana use was associated with two-year outcomes. Multivariable analysis was used to control for confounding variables. RESULTS: Marijuana use was reported by 47 (4.3%) patients. Significantly worse scores for two-year PROMIS Anxiety (53.2 vs. 49.2, p = 0.005), PROMIS Depression (51.1 vs. 46.5, p = 0.001), Met Expectations (63.1 vs. 74.4, p = 0.024), Surgical Satisfaction Questionnaire-8 (71.7 vs. 80.4, p = 0.005), and Numeric Satisfaction Scale (75.6 vs. 83.1, p = 0.041) were associated with marijuana use. Marijuana users also had less improvement of Numeric Pain Scores at the operative site (- 1.8 vs. - 2.7, p = 0.037) and greater decrease in Marx activity scores for lower extremities (- 12.3 vs. - 3.9, p = 0.024). Marijuana use was not an independent predictor of any outcome measure in the multivariable analysis. CONCLUSION: Marijuana use was associated with worse mental health scores, lower activity level, less pain relief, and worse satisfaction two years after orthopaedic surgery. However, after controlling for confounding variables, marijuana use was not predictive of any two-year outcome measure. STUDY DESIGN: Cross-sectional study.

Single-cell mRNA quantification and differential analysis with Census.

Single-cell gene expression studies promise to reveal rare cell types and cryptic states, but the high variability of single-cell RNA-seq measurements frustrates efforts to assay transcriptional differences between cells. We introduce the Census algorithm to convert relative RNA-seq expression levels into relative transcript counts without the need for experimental spike-in controls. Analyzing changes in relative transcript counts led to dramatic improvements in accuracy compared to normalized read counts and enabled new statistical tests for identifying developmentally regulated genes. Census counts can be analyzed with widely used regression techniques to reveal changes in cell-fate-dependent gene expression, splicing patterns and allelic imbalances. We reanalyzed single-cell data from several developmental and disease studies, and demonstrate that Census enabled robust analysis at multiple layers of gene regulation. Census is freely available through our updated single-cell analysis toolkit, Monocle 2.

Outcomes of Anterior Cruciate Ligament Reconstruction in Obese and Overweight Patients: A Systematic Review.

OBJECTIVE: To evaluate the mechanism of injury, outcomes, and complications of anterior cruciate ligament (ACL) reconstruction in overweight and obese patients. DATA SOURCES: MEDLINE, EMBASE, and OVID electronic libraries were systematically searched from inception to December, 2017 for any eligible articles using a combination of the phrases "anterior cruciate ligament," "ACL," "overweight," "obese," and "BMI." RESULTS: Studies that evaluated patients with primary ACL reconstruction, classified patients as overweight or obese, and reported a minimum of 1-year follow-up data were included. Eight cohorts from 9 studies fulfilled the inclusion criteria. There were no significant differences for mechanism of injury, Lysholm scores, Knee injury and Osteoarthritis Outcome Scores values, or return to sports with a body mass index (BMI) above or below 25 kg/m. A significant difference was described in International Knee Documentation Committee (IKDC) scores when comparing obese patients (BMI >30 kg/m) to patients with BMI <25 kg/m (P <0.01). In patients with BMI >25 kg/m, the risk for arthritis was significantly higher but the risk for revision surgery or contralateral ACL tear was lower (P <0.05). There was no significant difference in complication rates (P = 0.77). CONCLUSION: Patient-reported outcome measures were similar for patients with BMI above and below 25 kg/m, but there is evidence that obese patients have lower IKDC scores. There is a consistent association between overweight status and developing arthritis among patients having an ACL reconstruction. Overweight and obese patients have a lower risk of revision ACL reconstruction and contralateral ACL tear. There is insufficient data to make any conclusions regarding mechanism of injury or complications. More research is needed to better understand what is the appropriate counsel and treatment for overweight or obese patients with ACL tears. PROSPERO REGISTRATION NUMBER: CRD42017055594.

Orthopaedic surgery patients who use recreational marijuana have less pre-operative pain.

AIMS: To determine the baseline clinical characteristics of recreational marijuana users undergoing outpatient orthopaedic surgery. We hypothesized that patients who report marijuana use would have worse pain, function, and general health status. PATIENTS AND METHODS: Nine-hundred and thirty-seven patients undergoing outpatient orthopaedic surgery were asked to fill out patient-reported outcome (PRO) tools. These PROs included the Patient-Reported Outcomes Measurement Information Systems (PROMIS) computer adaptive tests and legacy PROs unique to each patients' surgical site. RESULTS: Forty patients (4.2%) reported marijuana use. Marijuana use was associated with younger age (33 vs. 43 years, p < 0.001), having a history of fewer operations (1.8 vs. 3.2, p < 0.05), single marital status (68 vs. 38%, p < 0.01), and having a history of smoking cigarettes (63 vs. 31%, p < 0.0001). Marijuana use was found to be significantly associated with greater Marx lower extremity activity rating scale scores (8.5 points vs. 6.1 points, p < 0.05) and decreased pain intensity in the operative site (3.7 points vs. 5.0 points, p < 0.05). Multivariable analysis found that marijuana use was an independent factor associated with less pain intensity in the operative site (p < 0.05). CONCLUSION: Our studies support other national studies that report increased marijuana use among younger patients and those who smoke cigarettes. The results do not support our hypothesis, as marijuana use was associated with less pain and better lower extremity activity rating scale scores when compared to non-users. Further research is warranted to analyze the effects of marijuana use on orthopaedic surgery patients. STUDY DESIGN: Cross-sectional study.

Ibuprofen impairs capsulolabral healing in a rat model of anterior glenohumeral instability.

BACKGROUND: Failure of glenoid labrum and capsular healing after glenohumeral dislocation can lead to persistent shoulder instability. The purpose of this study was to determine the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the healing glenoid labrum and capsule after glenohumeral dislocation in a rat model. METHODS: Sixty-six rats had surgically induced anterior-inferior labral tears and anterior glenohumeral dislocation. Postoperatively, the animals were assigned to either normal (n = 32) or ibuprofen drinking water (n = 31). Animals were euthanized at 2 and 4 weeks postoperatively for biomechanical testing and histologic analysis. RESULTS: The maximum load increased from 2 to 4 weeks after injury in the NSAID groups but not in the control groups. At 2 weeks, the maximum load was lower in the NSAID group compared with the control group. In a matched comparison between injured and uninjured limbs, the maximum load was significantly decreased in the injured limb of the 2-week NSAID group. At 4 weeks, the NSAID group had decreased stiffness compared with the 4-week control group. CONCLUSIONS: In a new rat model of glenohumeral instability, the postinjury administration of ibuprofen resulted in decreased capsulolabral healing. A matched pair analysis of injured to uninjured limbs supported the findings of impaired healing in the NSAID-treated animals. These findings demonstrate that the use of NSAIDs after glenohumeral dislocation may impair capsulolabral healing and should be limited or avoided to optimize glenohumeral stability.

End-of-life Decision-making for People in a Minimally Conscious State: A Review of the Application of the Mental Capacity Act 2005.

The last 15 years has seen clarification of the terminology used to describe prolonged disorders of consciousness within the United Kingdom leading to the emergence of a new diagnosis - minimally conscious state (MCS) in 2002. MCS is distinct from vegetative states, in that a person demonstrates wakefulness with some degree of minimal awareness. The Mental Capacity Act (MCA) 2005 in England and Wales provides a legal framework for assessing an individuals' capacity to make decisions for themselves. The Act also authorizes others to make decisions on behalf of an individual who is assessed as lacking capacity in their best interests. The Act has an accompanying Code of Practice which provides guidance and a best interests "test" to be applied when assessing best interests. Since the advent of the Act, approximately two cases each year go to the Court of Protection for final decisions regarding end-of-life care in people in an MCS. Currently, any decision involving the withdrawal of clinically assisted nutrition and hydration (CANH) for people in an MCS must be referred to the court. In each case, the courts analyze the application of the Act which has become central in the court's decision-making process, particularly when assessing best interests. This article provides an overview of key MCA sections applied in such end-of-life MCS cases and reviews seminal cases elucidating how the Act has been applied. It further describes the evolution of how courts have interpreted the doctrine of best interests when considering withholding or withdrawing CANH and other life-sustaining treatments.

CLAMMS: a scalable algorithm for calling common and rare copy number variants from exome sequencing data.

MOTIVATION: Several algorithms exist for detecting copy number variants (CNVs) from human exome sequencing read depth, but previous tools have not been well suited for large population studies on the order of tens or hundreds of thousands of exomes. Their limitations include being difficult to integrate into automated variant-calling pipelines and being ill-suited for detecting common variants. To address these issues, we developed a new algorithm--Copy number estimation using Lattice-Aligned Mixture Models (CLAMMS)--which is highly scalable and suitable for detecting CNVs across the whole allele frequency spectrum. RESULTS: In this note, we summarize the methods and intended use-case of CLAMMS, compare it to previous algorithms and briefly describe results of validation experiments. We evaluate the adherence of CNV calls from CLAMMS and four other algorithms to Mendelian inheritance patterns on a pedigree; we compare calls from CLAMMS and other algorithms to calls from SNP genotyping arrays for a set of 3164 samples; and we use TaqMan quantitative polymerase chain reaction to validate CNVs predicted by CLAMMS at 39 loci (95% of rare variants validate; across 19 common variant loci, the mean precision and recall are 99% and 94%, respectively). In the Supplementary Materials (available at the CLAMMS Github repository), we present our methods and validation results in greater detail. AVAILABILITY AND IMPLEMENTATION: https://github.com/rgcgithub/clamms (implemented in C). CONTACT: jeffrey.reid@regeneron.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Designing Biomimetic 3D-Printed Osteochondral Scaffolds for Enhanced Load-Bearing Capacity.

Osteoarthritis is a debilitating chronic joint disorder that affects millions of people worldwide. Since palliative and surgical treatments cannot completely regenerate hyaline cartilage within the articulating joint, osteochondral (OC) tissue engineering has been explored to heal OC defects. Utilizing computational simulations and three-dimensional (3D) printing, we aimed to build rationale around fabricating OC scaffolds with enhanced biomechanics. First, computational simulations revealed that interfacial fibrils within a bilayer alter OC scaffold deformation patterns by redirecting load-induced stresses toward the top of the cartilage layer. Principal component analysis revealed that scaffolds with 800 µm long fibrils (scaffolds 8A-8H) possessed optimal biomechanical properties to withstand compression and shear forces. While compression testing indicated that OC scaffolds with 800 µm fibrils did not have greater compressive moduli than other scaffolds, interfacial shear tests indicated that scaffold 8H possessed the greatest shear strength. Lastly, failure analysis demonstrated that yielding or buckling models describe interfacial fibril failure depending on fibril slenderness S. Specifically for scaffolds with packing density n = 6 and n = 8, the yielding failure model fits experimental loads with S < 10, while the buckling model fitted scaffolds with S < 10 slenderness. The research presented provides critical insights into designing 3D printed interfacial scaffolds with refined biomechanics toward improving OC tissue engineering outcomes.

Strategies for the Co-Delivery of Osteoclasts and MSCs in 3D-Printable Osteochondral Scaffolds.

Osteochondral defects, characterized by structural compromises to articular cartilage and subchondral bone, can cause pain and lead to progressive cartilage damage and eventual osteoarthritis. Unfortunately, repairing these defects remains difficult due to the poor regenerative properties of cartilage and complex mechanical demands of the joint. As such, the field of tissue engineering aims to develop multiphasic implants that replace pathological cartilage and bone tissue and restore mechanical functionality to the joint. Recent bone physiology investigations have demonstrated that osteoclast lineage cells are inextricably involved in osteoblastic bone formation through an extensive network of anabolic signaling pathways, and so the co-delivery osteoclast and osteoblast lineage cells within scaffolds is being actively explored for bone tissue engineering purposes. However, it remains unclear how these cells can be incorporated into the design of multiphasic osteochondral scaffolds to potentially enhance subchondral bone formation and subsequent implant osseointegration. To explore this question, we examined direct surface seeding and hydrogel encapsulation as potential scaffold cellularization strategies. First, we examined how osteoclast precursor cells (OCps) and peripheral blood monocytes (PBMCs) influence early-stage bone matrix development and osteogenesis in 2D co-culture. Then, we evaluated the osteogenic potential of mesenchymal stem cells (MSCs) and PBMCs co-cultures encapsulated within a gelatin methacrylate (GelMA) hydrogel system. Our findings demonstrate that co-culturing PBMCs with MSCs in 2D cultures significantly enhanced cell proliferation, early bone matrix deposition, and the formation of cell clusters by Day 28. However, we observed no significant difference in Type I collagen (COLI) deposition between GelMA hydrogel scaffolds cultured in basal and OC conditions during the same period. Additionally, we found that the GelMA hydrogel system with MSC/PBMC co-cultures in OC conditions exhibited decreased osteogenic activity by Day 28. Collectively, our findings support the osteogenic potential of osteoclast-lineage cells in 2D culture conditions, and the potential benefits of surface-seeding for the co-delivery of osteoclast-lineage cells and MSCs in osteo-scaffolds for enhanced osteochondral regeneration and broader bone tissue engineering purposes.

Biomechanical Comparison of Four- versus Six-Strand Transosseous Suture Repair for Patellar Tendon Rupture.

BACKGROUND: Surgical repair is indicated for patellar tendon ruptures that result in loss of knee extensor mechanism function. However, biomechanical studies report conflicting results when comparing transosseous suture versus suture anchor repair techniques. This discrepancy may be due to inconsistencies in experimental design as these studies use various numbers of suture strands. Therefore, the main objective of this study is to compare the ultimate load of four- versus six-strand transosseous suture repair. Secondary objectives are to compare gap formation after cyclical loading and mode of failure. METHODS: Six pairs of fresh-frozen cadaveric specimen were randomly allocated to either four- or six-strand transosseous suture repair. Specimen underwent preconditioning cyclical loading and then load to failure. RESULTS: The six-strand repair had a significantly higher maximum load to failure compared with the four-strand repair (mean difference = 319.3 N [57.9%], p = 0.03). There was no significant difference in gap length after cyclical loading or at max load. There were no significant differences in mode of failure. CONCLUSION: Utilizing a six-stand transosseous patella tendon repair construct with one additional suture increases overall construct strength by over 50% compared with a four-strand construct.

Patient-Reported Outcome Measurement Information System Depression and Anxiety in Elective Knee Surgery Patients.

Mental health has been shown to play an important role in patient-reported outcomes (PRO); however, there is a general lack of literature describing patient-reported outcome measurement information system (PROMIS) depression and anxiety computer adaptive tests in elective knee surgery patients. The purpose of our study was to assess the prevalence of depression and anxiety symptoms before and after elective knee surgery and to determine whether these symptoms influence postoperative functional outcomes. An institutional review board-approved prospective orthopaedic registry was retrospectively queried for patients undergoing elective knee surgery from June 2015 to November 2018. Electronic surveys collecting patient demographic information and PROs were administered pre- and postoperatively. Of the 663 patients that completed baseline questionnaires, 466 completed 2-year follow-up (70.3%). PROs included PROMIS depression, PROMIS anxiety, International Knee Documentation Committee Subjective Knee Form (IKDC), and PROMIS physical function (PF). Wilcoxon rank sum and Spearman's rank order correlation were utilized to determine associations between variables. Multivariable analysis was used to control for confounding variables. Average PROMIS depression and anxiety scores significantly improved 2 years after surgery. PROMIS depression and anxiety scores significantly correlated with each other. PROMIS depression and anxiety scores significantly correlated with PROMIS PF and IKDC scores. After controlling for confounders on multivariable analysis, worse 2-year PROMIS anxiety was predictive of less functional improvement and worse 2-year PF and IKDC, while worse 2-year PROMIS depression was predictive of less improvement in IKDC. This study confirms the important relationship between mental health and functional outcomes. Given that psychiatric comorbidities are potentially modifiable with treatment, proper recognition could potentially lead to better orthopaedic outcomes. In addition, the prevalence of depression and anxiety symptoms postoperatively, as documented by PROMIS computer adaptive tests, may act as a barrier to achieving optimal functional outcomes after elective knee surgery. LEVEL OF EVIDENCE: Level III.