Single-cell RNAseq provides insight into altered immune cell populations in human fracture nonunions.

Nonunion describes bone fractures that fail to heal, resulting in the fracture callus failing to fully ossify or, in atrophic cases, not forming altogether. Fracture healing is regulated, in part, by the balance of proinflammatory and anti-inflammatory processes occurring within the bone marrow and surface cell populations. We sought to further understand the role of osteoimmunology (i.e., study of the close relationship between the immune system and bone) by examining immune cell gene expression via single-cell RNA sequencing of intramedullary canal tissue obtained from human patients with femoral nonunions. Intramedullary canal tissue samples obtained by reaming were collected at the time of surgical repair for femur fracture nonunion (n = 5) or from native bone controls when harvesting autologous bone graft (n = 4). Cells within the samples were isolated and analyzed using the Chromium Single-Cell System (10x Genomics Inc.) and Illumina sequencers. Twenty-three distinct cell clusters were identified, with higher cell proportions in the nonunion samples for monocytes and CD14 + dendritic cells (DCs), and lower proportions of T cells, myelocytes, and promyelocytes in nonunion samples. Gene expression differences were identified in each of the cell clusters from cell types associated with osteoimmunology, including CD14 + DC, monocytes, T cells, promyelocytes, and myelocytes. These results provide human-derived gene profiles that can further our understanding of pathways that may be a cause or a consequence of nonunion, providing the clinical rationale to focus on specific components of osteoimmunology. Clinical significance: The novel single-cell approach may lead to clinically relevant diagnostic biomarkers during earlier stages of nonunion development and/or investigation into therapeutic options.

Utility of Plasma Protein Biomarkers and Mid-infrared Spectroscopy for Diagnosing Fracture-related Infections: A Pilot Study.

OBJECTIVES: To compare a large panel of plasma protein inflammatory biomarkers and mid-infrared (MIR) spectral patterns in patients with confirmed fracture-related infections (FRIs) with those in controls without infection. DESIGN: Prospective case-control study. SETTING: Academic, Level 1 trauma center. PATIENTS: Thirteen patients meeting confirmatory FRI criteria were matched to 13 controls based on age, time after surgery, and fracture region. INTERVENTION: Plasma levels of 49 proteins were measured using enzyme-linked immunosorbent assay techniques. Fourier transform infrared spectroscopy of dried films was used to obtain MIR spectra of plasma samples. MAIN OUTCOME MEASUREMENTS: The main outcome measurements included plasma protein levels and MIR spectra of samples. RESULTS: Multivariate analysis-based predictive model developed using enzyme-linked immunosorbent assay-based biomarkers had sensitivity, specificity, and accuracy of 69.2% ± 0.0%, 99.9% ± 1.0%, and 84.5% ± 0.6%, respectively, with platelet-derived growth factor-AB/BB, C-reactive protein, and MIG selected as the minimum number of variables explaining group differences ( P < 0.05). Sensitivity, specificity, and accuracy of the predictive model based on MIR spectra were 69.9% ± 6.2%, 71.9% ± 5.9%, and 70.9% ± 4.8%, respectively, with 6 wavenumbers as explanatory variables ( P < 0.05). CONCLUSIONS: This pilot study demonstrates the feasibility of using a select panel of plasma proteins and Fourier transform infrared spectroscopy to diagnose FRIs. Preliminary data suggest that the measurement of these select proteins and MIR spectra may be potential clinical tools to detect FRIs. Further investigation of these biomarkers in a larger cohort of patients is warranted. LEVEL OF EVIDENCE: Diagnostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Patient-Specific Precision Injury Signatures to Optimize Orthopaedic Interventions in Multiply Injured Patients (PRECISE STUDY).

SUMMARY: Optimal timing and procedure selection that define staged treatment strategies can affect outcomes dramatically and remain an area of major debate in the treatment of multiply injured orthopaedic trauma patients. Decisions regarding timing and choice of orthopaedic procedure(s) are currently based on the physiologic condition of the patient, resource availability, and the expected magnitude of the intervention. Surgical decision-making algorithms rarely rely on precision-type data that account for demographics, magnitude of injury, and the physiologic/immunologic response to injury on a patient-specific basis. This study is a multicenter prospective investigation that will work toward developing a precision medicine approach to managing multiply injured patients by incorporating patient-specific indices that quantify (1) mechanical tissue damage volume; (2) cumulative hypoperfusion; (3) immunologic response; and (4) demographics. These indices will formulate a precision injury signature, unique to each patient, which will be explored for correspondence to outcomes and response to surgical interventions. The impact of the timing and magnitude of initial and staged surgical interventions on patient-specific physiologic and immunologic responses will be evaluated and described. The primary goal of the study will be the development of data-driven models that will inform clinical decision-making tools that can be used to predict outcomes and guide intervention decisions.

Single-cell RNA sequencing of intramedullary canal tissue to improve methods for studying fracture repair biology.

The ability to study the bone microenvironment of failed fracture healing may lead to biomarkers for fracture nonunion. Herein the authors describe a technique for isolating individual cells suitable for single-cell RNA sequencing analyses from intramedullary canal tissue collected by reaming during surgery. The purpose was to detail challenges and solutions inherent to the collection and processing of intramedullary canal tissue samples. The authors then examined single-cell RNA sequencing data from fresh and reanimated samples to demonstrate the feasibility of this approach for prospective studies.

Fixation using Alternative Implants for the Treatment of Hip Fractures (FAITH-2): The Exploratory Health-Related Quality of Life and Patient-Reported Functional Outcomes of a Multi-Centre 2 × 2 Factorial Randomized Controlled Pilot Trial in Young Femoral Neck Fracture Patients.

PURPOSE: Femoral neck fractures in young patients are typically managed with internal fixation using either cancellous screws or a sliding hip screw (SHS). Although fixation preserves the hip joint, patients are still at risk of complications and poor clinical outcomes which lead to diminished function and health related quality of life (HRQL). The Fixation using Alternative Implants for the Treatment of Hip Fractures (FAITH-2) pilot randomized controlled factorial trial evaluated the effect of surgical fixation (cancellous screws vs. SHS) and vitamin D supplementation vs. placebo on patient-reported function and HRQL. METHODS: Patients between the ages of 18-60 years with a femoral neck fracture requiring surgical fixation were eligible. Eligible patients were randomized to receive either a sliding hip screw or cancellous screws for fracture fixation AND vitamin D3 4,000 IU or placebo daily for 6 months. Patient-reported function (Hip Outcome Score) and HRQL (Short Form-12) were assessed at standardized time points in the 12 months following their fixation surgery. Patient-reported function and HRQL were summarized using means, SD, and 95% confidence intervals (CIs), or percentages and counts. Longitudinal data analysis with mixed models was used to explore the effect of treatment group and time on the patient-reported function and HRQL. RESULTS: 86 of the 91 patients randomized into the FAITH-2 pilot study were deemed eligible. There were no significant differences in patient-reported function or HRQL between the treatment groups at 12 months post-fracture. At the 6- and 9-month assessments, a potential benefit in hip function was seen in the cancellous screw group. In all treatment groups, participants reported lower function and HRQL at 12 months post-fracture as compared to their pre-injury assessment. CONCLUSIONS: Few differences were found in function and HRQL among the treatment groups in the FAITH-2 pilot study. Despite modern implants and vitamin D supplementation, neither function nor HRQL returns to baseline in this population. Additional efforts to improve the outcomes of these challenging injuries are still needed. LEVEL OF EVIDENCE: Therapeutic Level II.

Fixation Using Alternative Implants for the Treatment of Hip Fractures (FAITH-2): The Clinical Outcomes of a Multicenter 2 × 2 Factorial Randomized Controlled Pilot Trial in Young Femoral Neck Fracture Patients.

OBJECTIVE: To assess whether the fixation method and vitamin D supplementation affect the risk of patient-important outcomes within 12 months of injury in nongeriatric femoral neck fracture patients. DESIGN: A pilot factorial randomized controlled trial. SETTING: Fifteen North American clinical sites. PARTICIPANTS: Ninety-one adults 18-60 years of age with a femoral neck fracture requiring surgical fixation. INTERVENTION: Participants were randomized to a surgical intervention (sliding hip screw or cancellous screws) and a vitamin D intervention (vitamin D3 4000 IU daily vs. placebo for 6 months). MAIN OUTCOME MEASUREMENTS: The primary clinical outcome was a composite of patient-important complications (reoperation, femoral head osteonecrosis, severe femoral neck malunion, and nonunion). Secondary outcomes included fracture-healing complications and radiographic fracture healing. RESULTS: Eighty-six participants with a mean age of 41 years were included. We found no statistically significant difference in the risk of patient-important outcomes between the surgical treatment arms (hazard ratio 0.90, 95% confidence interval 0.40-2.02, P = 0.80) and vitamin D supplementation treatment arms (hazard ratio 0.96, 95% confidence interval 0.42-2.18, P = 0.92). CONCLUSIONS: These pilot trial results continue to describe the results of current fixation implants, inform the challenges of improving outcomes in this fracture population, and may guide future vitamin D trials to improve healing outcomes in young fracture populations. Although the pilot trial was not adequately powered to detect treatment effects, publishing these results may facilitate future meta-analyses on this topic. LEVEL OF EVIDENCE: Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Fixation Using Alternative Implants for the Treatment of Hip Fractures: The feasibility of a multicenter 2 × 2 factorial randomized controlled trial evaluating surgical treatment and vitamin D supplementation in young femoral neck fracture patients.

OBJECTIVES: To conduct a pilot trial for the Fixation using Alternative Implants for the Treatment of Hip Fractures (FAITH-2) protocol to assess feasibility of a definitive trial. DESIGN: Pilot trial. SETTING: Twenty-five clinical sites across North America and Australia were initiated, but enrolment occurred in only 15 North American sites. PATIENTS/PARTICIPANTS: Ninety-one randomized adults aged 18 to 60 years with a femoral neck fracture requiring surgical fixation. INTERVENTION: Eligible patients were randomized to receive surgical treatment (sliding hip screw or cancellous screws) AND nutritional supplementation (4000 IU of vitamin D or placebo) for 6 months postfracture. MAIN OUTCOME MEASUREMENTS: Feasibility outcomes included: clinical site initiation, participant enrolment rate, proportion of participants with complete 12-month follow-up, level of data quality, and rate of protocol adherence (number of randomization errors, crossovers between treatment groups, and daily supplementation adherence). RESULTS: Eighty-six of 91 participants randomized into the pilot trial from 15 North American hospitals were deemed eligible. Four of five primary feasibility criteria were not achieved as we were unable to initiate clinical sites outside of North America and Australia due to feasibility constraints, slow participant enrolment (60 participants recruited over 36 mo), low adherence with daily nutritional supplementation at the 6-week (72.1%), 3-month (60.5%), and 6-month (54.7%) follow-up visits, and a high loss to follow-up rate of 22.1% at 12 months. CONCLUSIONS: Despite not meeting key feasibility criteria, we increased our knowledge on the logistics and anticipated barriers when conducting vitamin D supplementation trials in this trauma population, which can be used to inform the design and conduct of future trials on this topic.

Correction: Type V Collagen Induced Tolerance Suppresses Collagen Deposition, TGF-ß and Associated Transcripts in Pulmonary Fibrosis.

[This corrects the article DOI: 10.1371/journal.pone.0076451.].

Type V collagen induced tolerance suppresses collagen deposition, TGF-ß and associated transcripts in pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by progressive scarring and matrix deposition. Recent reports highlight an autoimmune component in IPF pathogenesis. We have reported anti-col(V) immunity in IPF patients. The objective of our study was to determine the specificity of col(V) expression profile and anti-col(V) immunity relative to col(I) in clinical IPF and the efficacy of nebulized col(V) in pre-clinical IPF models. METHODS: Col(V) and col(I) expression profile was analyzed in normal human and IPF tissues. C57-BL6 mice were intratracheally instilled with bleomycin (0.025 U) followed by col(V) nebulization at pre-/post-fibrotic stage and analyzed for systemic and local responses. RESULTS: Compared to normal lungs, IPF lungs had higher protein and transcript expression of the alpha 1 chain of col(V) and col(I). Systemic anti-col(V) antibody concentrations, but not of anti-col(I), were higher in IPF patients. Nebulized col(V), but not col(I), prevented bleomycin-induced fibrosis, collagen deposition, and myofibroblast differentiation. Col(V) treatment suppressed systemic levels of anti-col(V) antibodies, IL-6 and TNF-α; and local Il-17a transcripts. Compared to controls, nebulized col(V)-induced tolerance abrogated antigen-specific proliferation in mediastinal lymphocytes and production of IL-17A, IL-6, TNF-α and IFN-γ. In a clinically relevant established fibrosis model, nebulized col(V) decreased collagen deposition. mRNA array revealed downregulation of genes specific to fibrosis (Tgf-ß, Il-1ß, Pdgfb), matrix (Acta2, Col1a2, Col3a1, Lox, Itgb1/6, Itga2/3) and members of the TGF-ß superfamily (Tgfbr1/2, Smad2/3, Ltbp1, Serpine1, Nfkb/Sp1/Cebpb). CONCLUSIONS: Anti-col(V) immunity is pathogenic in IPF, and col(V)-induced tolerance abrogates bleomycin-induced fibrogenesis and down regulates TGF- ß-related signaling pathways.

Role of complement activation in obliterative bronchiolitis post-lung transplantation.

Obliterative bronchiolitis (OB) post-lung transplantation involves IL-17-regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB is unknown. The current study examines the role of complement activation in OB. Complement-regulatory protein (CRP) (CD55, CD46, complement receptor 1-related protein y/CD46) expression was downregulated in human and murine OB; and C3a, a marker of complement activation, was upregulated locally. IL-17 differentially suppressed complement receptor 1-related protein y expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen- or autoantigen (type V collagen)-reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17-mediated downregulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed-forward loop that may enhance CRP downregulation, suggesting that complement blockade could be a therapeutic strategy for OB.