MAP3K1 regulates female reproductive tract development.

Mitogen-activated protein 3 kinase 1 (MAP3K1) has a plethora of cell type-specific functions not yet fully understood. Herein, we describe a role for MAP3K1 in female reproductive tract (FRT) development. MAP3K1 kinase domain-deficient female mice exhibited an imperforate vagina, labor failure and infertility. These defects corresponded with shunted Müllerian ducts (MDs), the embryonic precursors of FRT, that manifested as a contorted caudal vagina and abrogated vaginal-urogenital sinus fusion in neonates. The MAP3K1 kinase domain is required for optimal activation of the Jun-N-terminal kinase (JNK) and cell polarity in the MD epithelium, and for upregulation of WNT signaling in the mesenchyme surrounding the caudal MD. The MAP3K1-deficient epithelial cells and MD epithelium had reduced expression of WNT7B ligands. Correspondingly, conditioned media derived from MAP3K1-competent, but not -deficient, epithelial cells activated a TCF/Lef-luciferase reporter in fibroblasts. These observations indicate that MAP3K1 regulates MD caudal elongation and FRT development, in part through the induction of paracrine factors in the epithelium that trans-activate WNT signaling in the mesenchyme.

Pathologists' perspective on the study design, analysis, and interpretation of proliferative lesions in a lifetime rodent carcinogenicity bioassay of sucralose.

Sucralose, a sugar substitute first approved for use in 1991, is a non-caloric sweetener regulated globally as a food additive. Based on numerous experimental animal studies (dating to the 1980s) and human epidemiology studies, international health agencies have determined that sucralose is safe when consumed as intended. A single lifetime rodent carcinogenicity bioassay conducted by the Ramazzini Institute (RI) reported that mice fed diets containing sucralose develop hematopoietic neoplasia, but controversy continues regarding the validity and relevance of these data for predicting health effects in humans. The present paper addresses the controversy by providing the perspective of experienced pathologists on sucralose-related animal toxicity and carcinogenicity data generally, and the RI carcinogenicity bioassay findings specifically, using results from publicly available papers and international regulatory authority decisions. In the authors' view, flaws in the design, methodology, data evaluation, and reporting of the RI carcinogenicity bioassay for sucralose diminish the value of the data as evidence that this agent represents a carcinogenic hazard to humans. This limitation will remain until the RI bioassay is repeated under Good Laboratory Practices and the design, data, and accuracy of the pathology diagnoses and interpretations are reviewed by qualified pathologists with experience in evaluating potential chemically-induced carcinogenic hazards.

Scientific and Regulatory Policy Committee Technical Review: Biology and Pathology of Ganglia in Animal Species Used for Nonclinical Safety Testing.

Dorsal root ganglia (DRG), trigeminal ganglia (TG), other sensory ganglia, and autonomic ganglia may be injured by some test article classes, including anti-neoplastic chemotherapeutics, adeno-associated virus-based gene therapies, antisense oligonucleotides, nerve growth factor inhibitors, and aminoglycoside antibiotics. This article reviews ganglion anatomy, cytology, and pathology (emphasizing sensory ganglia) among common nonclinical species used in assessing product safety for such test articles (TAs). Principal histopathologic findings associated with sensory ganglion injury include neuron degeneration, necrosis, and/or loss; increased satellite glial cell and/or Schwann cell numbers; and leukocyte infiltration and/or inflammation. Secondary nerve fiber degeneration and/or glial reactions may occur in nerves, dorsal spinal nerve roots, spinal cord (dorsal and occasionally lateral funiculi), and sometimes the brainstem. Ganglion findings related to TA administration may result from TA exposure and/or trauma related to direct TA delivery into the central nervous system or ganglia. In some cases, TA-related effects may need to be differentiated from a spectrum of artifactual and/or spontaneous background changes.

Novel MECP2 gene therapy is effective in a multicenter study using two mouse models of Rett syndrome and is safe in non-human primates.

The AAV9 gene therapy vector presented in this study is safe in mice and non-human primates and highly efficacious without causing overexpression toxicity, a major challenge for clinical translation of Rett syndrome gene therapy vectors to date. Our team designed a new truncated methyl-CpG-binding protein 2 (MECP2) promoter allowing widespread expression of MECP2 in mice and non-human primates after a single injection into the cerebrospinal fluid without causing overexpression symptoms up to 18 months after injection. Additionally, this new vector is highly efficacious at lower doses compared with previous constructs as demonstrated in extensive efficacy studies performed by two independent laboratories in two different Rett syndrome mouse models carrying either a knockout or one of the most frequent human mutations of Mecp2. Overall, data from this multicenter study highlight the efficacy and safety of this gene therapy construct, making it a promising candidate for first-in-human studies to treat Rett syndrome.

Scientific and Regulatory Policy Committee Points to Consider: Sampling, Processing, Evaluation, Interpretation, and Reporting of Test Article-Related Ganglion Pathology for Nonclinical Toxicity Studies.

Certain biopharmaceutical products consistently affect dorsal root ganglia, trigeminal ganglia, and/or autonomic ganglia. Product classes targeting ganglia include antineoplastic chemotherapeutics, adeno-associated virus-based gene therapies, antisense oligonucleotides, and anti-nerve growth factor agents. This article outlines "points to consider" for sample collection, processing, evaluation, interpretation, and reporting of ganglion findings; these points are consistent with published best practices for peripheral nervous system evaluation in nonclinical toxicity studies. Ganglion findings often occur as a combination of neuronal injury (e.g., degeneration, necrosis, and/or loss) and/or glial effects (e.g., increased satellite glial cell cellularity) with leukocyte accumulation (e.g., mononuclear cell infiltration or inflammation). Nerve fiber degeneration and/or glial reactions may be seen in nerves, dorsal spinal nerve roots, spinal cord, and occasionally brainstem. Interpretation of test article (TA)-associated effects may be confounded by incidental background changes or experimental procedure-related changes and limited historical control data. Reports should describe findings at these sites, any TA relationship, and the criteria used for assigning severity grades. Contextualizing adversity of ganglia findings can require a weight-of-evidence approach because morphologic changes of variable severity occur in ganglia but often are not accompanied by observable overt in-life functional alterations detectable by conventional behavioral and neurological testing techniques.

Toxicologic Pathology Forum Opinion: Interpretation of Gliosis in the Brain and Spinal Cord Observed During Nonclinical Safety Studies.

Gliosis, defined as a nonneoplastic reaction (hypertrophy and/or proliferation) of astrocytes and/or microglial cells, is a frequent finding in the central nervous system (CNS [brain and/or spinal cord]) in nonclinical safety studies. Gliosis in rodents and nonrodents occurs at low incidence as a spontaneous finding and is induced by various test articles (e.g., biomolecules, cell and gene therapies, small molecules) delivered centrally (i.e., by injection or infusion into cerebrospinal fluid or neural tissue) or systemically. Several CNS gliosis patterns occur in nonclinical species. First, gliosis may accompany degeneration and/or necrosis of cells (mainly neurons) or neural parenchyma (neuron processes and myelin). Second, gliosis often follows inflammation (i.e., leukocyte accumulation causing parenchymal damage) or neoplasm formation. Third, gliosis may appear as variably sized, randomly scattered foci of reactive glial cells in the absence of visible parenchymal damage or inflammation. In interpreting test article-related CNS gliosis, adversity is indicated by parenchymal injury (e.g., degeneration, necrosis, or inflammation) and not the mere existence of a glial reaction. In the absence of clear structural damage to the parenchyma, gliosis as a standalone CNS finding should be interpreted as a nonadverse reaction to regional alterations in microenvironmental conditions rather than as evidence of a glial reaction associated with neurotoxicity.

Oral chronic toxicity and carcinogenicity study of alpha-glycosyl isoquercitrin (AGIQ) in Sprague Dawley rats.

alpha-Glycosyl isoquercitrin (AGIQ) is a flavonoid that possesses antioxidant and tumor suppressive capabilities and is marketed as a food additive in Japan. The aim of this study was to assess the potential for oral chronic toxicity and carcinogenicity of AGIQ in male and female Sprague Dawley rats following up to 5.0% dietary exposure. In the chronic toxicity study, rats were exposed to AGIQ or vehicle for one year with a 6-month interim termination point; for the carcinogenicity study, rats were treated for 24 months. No signs of AGIQ-related toxicity clinically or histologically were observed for up to one year except for yellow discoloration of bone. In the carcinogenicity study, a statistically significant increase in the incidence of malignant glioma of the brain or spinal cord was observed in female rats exposed to 5.0% AGIQ compared to those exposed to control feed. A Scientific Advisory Panel of experienced neuropathologists reviewed the gliomas (routine stains and glial cell markers) and concluded that the gliomas were a rare, spontaneous, rat-specific neoplasm: malignant microglial tumor. The lesions could not definitively be attributed to AGIQ exposure and have limited implications with respect to predicting human cancer risk.

Pathologists' perspective on the study design, analysis, and interpretation of proliferative lesions in lifetime and prenatal rodent carcinogenicity bioassays of aspartame.

Aspartame, an artificial sweetener commonly used as a sugar substitute, is currently authorized for use in more than 100 countries. Hundreds of studies, conducted in various countries dating back to the 1970s, have shown that aspartame is safe at real-world exposure levels. Furthermore, multiple human epidemiology studies have provided no indication that consumption of aspartame induces cancer. Given the continued controversy surrounding the Ramazzini Institute's (RI) studies suggesting that aspartame is a carcinogenic hazard in rodents and evaluation by the International Agency for Research on Cancer, this report aims to provide the perspective of experienced pathologists on publicly available pathology data regarding purported proliferative lesions in liver, lung, lymphoid organs, and mammary gland as well as their implications for human risk assessment as reported for three lifetime rodent carcinogenicity bioassays of aspartame conducted at the RI. In the authors' view, flaws in the design, methodology and reporting of the RI aspartame studies limit the utility of the data sets as evidence that this agent represents a carcinogenic hazard. Therefore, all three RI studies, and particularly the accuracy of their pathology diagnoses and interpretations, should be rigorously reviewed by qualified and experienced veterinary toxicologic pathologists in assessing aspartame's carcinogenic risk.

Preclinical safety assessment of MV-s-NAP, a novel oncolytic measles virus strain armed with an H . pylori immunostimulatory bacterial transgene.

Despite recent therapeutic advances, metastatic breast cancer (MBC) remains incurable. Engineered measles virus (MV) constructs based on the attenuated MV Edmonston vaccine platform have demonstrated significant oncolytic activity against solid tumors. The Helicobacter pylori neutrophil-activating protein (NAP) is responsible for the robust inflammatory reaction in gastroduodenal mucosa during bacterial infection. NAP attracts and activates immune cells at the site of infection, inducing expression of pro-inflammatory mediators. We engineered an MV strain to express the secretory form of NAP (MV-s-NAP) and showed that it exhibits anti-tumor and immunostimulatory activity in human breast cancer xenograft models. In this study, we utilized a measles-infection-permissive mouse model (transgenic IFNAR KO-CD46Ge) to evaluate the biodistribution and safety of MV-s-NAP. The primary objective was to identify potential toxic side effects and confirm the safety of the proposed clinical doses of MV-s-NAP prior to a phase I clinical trial of intratumoral administration of MV-s-NAP in patients with MBC. Both subcutaneous delivery (corresponding to the clinical trial intratumoral administration route) and intravenous (worst case scenario) delivery of MV-s-NAP were well tolerated: no significant clinical, laboratory or histologic toxicity was observed. This outcome supports the safety of MV-s-NAP for oncolytic virotherapy of MBC. The first-in-human clinical trial of MV-s-NAP in patients with MBC (ClinicalTrials.gov: NCT04521764) was subsequently activated.

Scientific and Regulatory Policy Committee Points to Consider: Nonclinical Research and Development of In Vivo Gene Therapy Products, Emphasizing Adeno-Associated Virus Vectors.

Sequencing of the human genome and numerous advances in molecular techniques have launched the era of genetic medicine. Increasingly precise technologies for genetic modification, manufacturing, and administration of pharmaceutical-grade biologics have proved the viability of in vivo gene therapy (GTx) as a therapeutic modality as shown in several thousand clinical trials and recent approval of several GTx products for treating rare diseases and cancers. In recognition of the rapidly advancing knowledge in this field, the regulatory landscape has evolved considerably to maintain appropriate monitoring of safety concerns associated with this modality. Nonetheless, GTx safety assessment remains complex and is designed on a case-by-case basis that is determined by the disease indication and product attributes. This article describes our current understanding of fundamental biological principles and possible procedures (emphasizing those related to toxicology and toxicologic pathology) needed to support research and development of in vivo GTx products. This article is not intended to provide comprehensive guidance on all GTx modalities but instead provides an overview relevant to in vivo GTx generally by utilizing recombinant adeno-associated virus-based GTx-the most common in vivo GTx platform-to exemplify the main points to be considered in nonclinical research and development of GTx products.

Comparative Analysis of Chemotherapy-Induced Peripheral Neuropathy in Bioengineered Sensory Nerve Tissue Distinguishes Mechanistic Differences in Early-Stage Vincristine-, Cisplatin-, and Paclitaxel-Induced Nerve Damage.

Chemotherapy-induced peripheral neuropathy (CIPN) is a well-known, potentially permanent side effect of widely used antineoplastic agents. The mechanisms of neuropathic progression are poorly understood, and the need to test efficacy of novel interventions to treat CIPN continues to grow. Bioengineered microphysiological nerve tissue ("nerve on a chip") has been suggested as an in vitro platform for modeling the structure and physiology of in situ peripheral nerve tissue. Here, we find that length-dependent nerve conduction and histopathologic changes induced by cisplatin, paclitaxel, or vincristine in rat dorsal root ganglion-derived microphysiological sensory nerve tissue recapitulate published descriptions of clinical electrophysiological changes and neuropathologic biopsy findings in test animals and human patients with CIPN. We additionally confirm the postulated link between vincristine-induced axoplasmic transport failure and functional impairment of nerve conduction, the postulated paclitaxel-induced somal toxicity, and identify a potential central role of gliotoxicity in cisplatin-induced sensory neuropathy. Microphysiological CIPN combines the tight experimental control afforded by in vitro experimentation with clinically relevant functional and structural outputs that conventionally require in vivo models. Microphysiological nerve tissue provides a low-cost, high-throughput alternative to conventional nonclinical models for efficiently and effectively investigating lesions, mechanisms, and treatments of CIPN. Neural microphysiological systems are capable of modeling complex neurological disease at the tissue level offering unique advantages over conventional methodology for both testing and generating hypotheses in neurological disease modeling. Impact Statement Recapitulation of distinct hallmarks of clinical CIPN in microphysiological sensory nerve validates a novel peripheral neurotoxicity model with unique advantages over conventional model systems.

Impact of Preanalytical Factors During Histology Processing on Section Suitability for Digital Image Analysis.

Digital image analysis (DIA) is impacted by the quality of tissue staining. This study examined the influence of preanalytical variables-staining protocol design, reagent quality, section attributes, and instrumentation-on the performance of automated DIA software. Our hypotheses were that (1) staining intensity is impacted by subtle differences in protocol design, reagent quality, and section composition and that (2) identically programmed and loaded stainers will produce equivalent immunohistochemical (IHC) staining. We tested these propositions by using 1 hematoxylin and eosin stainer to process 13 formalin-fixed, paraffin-embedded (FFPE) mouse tissues and by using 3 identically programmed and loaded immunostainers to process 5 FFPE mouse tissues for 4 cell biomarkers. Digital images of stained sections acquired with a commercial whole slide scanner were analyzed by customizable algorithms incorporated into commercially available DIA software. Staining intensity as viewed qualitatively by an observer and/or quantitatively by DIA was affected by staining conditions and tissue attributes. Intrarun and inter-run IHC staining intensities were equivalent for each tissue when processed on a given stainer but varied measurably across stainers. Our data indicate that staining quality must be monitored for each method and stainer to ensure that preanalytical factors do not impact digital pathology data quality.

Comparative Pathology of the Peripheral Nervous System.

The peripheral nervous system (PNS) relays messages between the central nervous system (brain and spinal cord) and the body. Despite this critical role and widespread distribution, the PNS is often overlooked when investigating disease in diagnostic and experimental pathology. This review highlights key features of neuroanatomy and physiology of the somatic and autonomic PNS, and appropriate PNS sampling and processing techniques. The review considers major classes of PNS lesions including neuronopathy, axonopathy, and myelinopathy, and major categories of PNS disease including toxic, metabolic, and paraneoplastic neuropathies; infectious and inflammatory diseases; and neoplasms. This review describes a broad range of common PNS lesions and their diagnostic criteria and provides many useful references for pathologists who perform PNS evaluations as a regular or occasional task in their comparative pathology practice.

Anti-fibrotic effects of the antihistamine ketotifen in a rabbit model of arthrofibrosis.

AIMS: Arthrofibrosis is a relatively common complication after joint injuries and surgery, particularly in the knee. The present study used a previously described and validated rabbit model to assess the biomechanical, histopathological, and molecular effects of the mast cell stabilizer ketotifen on surgically induced knee joint contractures in female rabbits. METHODS: A group of 12 skeletally mature rabbits were randomly divided into two groups. One group received subcutaneous (SQ) saline, and a second group received SQ ketotifen injections. Biomechanical data were collected at eight, ten, 16, and 24 weeks. At the time of necropsy, posterior capsule tissue was collected for histopathological and gene expression analyses (messenger RNA (mRNA) and protein). RESULTS: At the 24-week timepoint, there was a statistically significant increase in passive extension among rabbits treated with ketotifen compared to those treated with saline (p = 0.03). However, no difference in capsular stiffness was detected. Histopathological data failed to demonstrate a decrease in the density of fibrous tissue or a decrease in α-smooth muscle actin (α-SMA) staining with ketotifen treatment. In contrast, tryptase and α-SMA protein expression in the ketotifen group were decreased when compared to saline controls (p = 0.007 and p = 0.01, respectively). Furthermore, there was a significant decrease in α-SMA (ACTA2) gene expression in the ketotifen group compared to the control group (p < 0.001). CONCLUSION: Collectively, these data suggest that ketotifen mitigates the severity of contracture formation in a rabbit model of arthrofibrosis.

Reduction of arthrofibrosis utilizing a collagen membrane drug-eluting scaffold with celecoxib and subcutaneous injections with ketotifen.

The dense formation of abnormal scar tissue after total knee arthroplasty results in arthrofibrosis, an unfortunate sequela of inflammation. The purpose of this study was to use a validated rabbit model to assess the effects on surgically-induced knee joint contractures of two combined pharmacological interventions: celecoxib (CXB) loaded on an implanted collagen membrane, and subcutaneously (SQ) injected ketotifen. Thirty rabbits were randomly divided into five groups. The first group received no intervention after the index surgery. The remaining four groups underwent intra-articular implantation of collagen membranes loaded with or without CXB at the time of the index surgery; two of which were also treated with SQ ketotifen. Biomechanical joint contracture data were collected at 8, 10, 16, and 24 weeks. At the time of necropsy (24 weeks), posterior capsule tissue was collected for messenger RNA and histopathologic analyses. At 24 weeks, there was a statistically significant increase in passive extension among rabbits in all groups treated with CXB and/or ketotifen compared to those in the contracture control group. There was a statistically significant decrease in COL3A1, COL6A1, and ACTA2 gene expression in the treatment groups compared to the contracture control group (P < .001). Histopathologic data also demonstrated a trend towards decreased fibrous tissue density in the CXB membrane group compared to the vehicle membrane group. The present data suggest that intra-articular placement of a treated collagen membrane blunts the severity of contracture development in a rabbit model of arthrofibrosis, and that ketotifen and CXB may independently contribute to the prevention of arthrofibrosis. Statement of clinical significance: Current literature has demonstrated that arthrofibrosis may affect up to 5% of primary total knee arthroplasty patients. For that reason, novel pharmacologic prophylaxis and treatment modalities are critical to mitigating reoperations and revisions while improving the quality of life for patients with this debilitating condition.

Optical density-based image analysis method for the evaluation of hematoxylin and eosin staining precision.

Staining quality and reproducibility are essential factors to monitor laboratory quality assurance. In the last decade, there has been an increase in the use of digital pathology and image analysis. While the adoption of these tools provides a potential means to track staining precision by optical density (OD), it also presents challenges. Results from image analysis are more sensitive to variations in staining than microscopic evaluation by a pathologist. There are two goals with this study. The first was to track the precision of hematoxylin and eosin (H&E) staining, in both nuclear and cytoplasmic components by OD. The second was to determine the impact of different pre-analytical and analytical variables on the OD results. Specifically, the endpoints investigated were quality parameters including impacts of section thickness, protocol manipulation, expired hematoxylin on staining precision and reproducibility of staining over time. Our results show that image analysis of H&E-stained tissue sections is a viable tool for assessing and verifying staining quality. We also show that OD analysis results for H&E-stained sections are affected by changing pre-analytical and/or reagent variables. These authors chose a graphical rather than fully statistical analysis of the results to highlight the utility of visual aids in demonstrating H&E staining reproducibility.

An Image Analysis Solution For Quantification and Determination of Immunohistochemistry Staining Reproducibility.

With immunohistochemical (IHC) staining increasingly being used to guide clinical decisions, variability in staining quality and reproducibility are becoming essential factors in generating diagnoses using IHC tissue preparations. The current study tested a method to track and quantify the interrun, intrarun, and intersite variability of IHC staining intensity. Our hypothesis was that staining precision between laboratory sites, staining runs, and individual slides may be verified quantitatively, efficiently and effectively utilizing algorithm-based, automated image analysis. To investigate this premise, we tested the consistency of IHC staining in 40 routinely processed (formalin-fixed, paraffin-embedded) human tissues using 10 common antibiomarker antibodies on 2 Dako Omnis instruments at 2 locations (Carpinteria, CA: 30 m above sea level and Longmont, CO: 1500 m above sea level) programmed with identical, default settings and sample pretreatments. Digital images of IHC-labeled sections produced by a whole slide scanner were analyzed by a simple commercially available algorithm and compared with a board-certified veterinary pathologist's semiquantitative scoring of staining intensity. The image analysis output correlated well with pathology scores but had increased sensitivity for discriminating subtle variations and providing reproducible digital quantification across sites as well as within and among staining runs at the same site. Taken together, our data indicate that digital image analysis offers an objective and quantifiable means of verifying IHC staining parameters as a part of laboratory quality assurance systems.

A conditional mouse model of complex II deficiency manifesting as Leigh-like syndrome.

Leigh syndrome embodies degenerative disorders with a collection of symptoms secondary to inborn errors of metabolism. Combinations of hypomorphic and loss-of-function alleles in many genes have been shown to result in Leigh syndrome. Interestingly, deficiency for the tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH) can lead to Leigh-like syndrome in some circumstances and to cancer (paraganglioma, renal cell carcinoma, gastrointestinal stromal tumor) in others. In our experiments originally intended to create an inducible whole-body SDH-loss mouse model of tumorigenesis, we generated a condition reminiscent of Leigh-like syndrome that is lethal to mice within 4 wk. Remarkably, as has been shown for other mitochondrial diseases, chronic hypoxia offers substantial protection to mice from this condition after systemic SDH loss, allowing survival in the context of profoundly impaired oxidative metabolism.-Al Khazal, F., Holte, M. N., Bolon, B., White, T. A., LeBrasseur, N., Maher, L. J. III. A conditional mouse model of complex II deficiency manifesting as Leigh-like syndrome.

Inhibition of COX-2 Pathway as a Potential Prophylaxis Against Arthrofibrogenesis in a Rabbit Model of Joint Contracture.

Arthrofibrosis is a common complication following total knee arthroplasty caused by pathologic fibroblast activation and excessive collagen deposition around a synovial joint leading to debilitating loss of motion. Treatment options are limited because the pathologic mechanisms remain to be characterized. Dysregulation of the inflammatory cascade may lead to communication between myofibroblasts and immune cells triggering tissue metaplasia, and excessive collagen deposition described clinically as arthrofibrosis. We explored the novel use of celecoxib (selective cyclooxygenase-2 [COX-2] inhibitor) to disrupt the downstream effects of the post-traumatic inflammatory cascade and inhibit scar tissue formation in a validated rabbit model of arthrofibrosis combined with new parameters for quantifying the stiffness of the posterior capsule. Biomechanical and molecular analyses, of contracted rabbit knee posterior capsule tissue after COX-2 inhibition revealed increased maximal passive extension and down-regulation of collagen messenger RNA compared with controls. Histopathologic examination suggested a trend of decreased quantities of dense fibrous connective tissue with COX-2 inhibition. These data may suggest that inhibiting the inflammatory cascade could potentially reduce pathologic myofibroblast activation, thereby reducing scar tissue formation and increasing the range of motion in arthrofibrotic joints. Implementing a multi-modal pharmacologic approach may simultaneously target numerous cellular components contributing to the complex process of arthrofibrogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2609-2620, 2019.