scholarly journals An Improved Methodology to Evaluate Cell and Molecular Signals in the Reparative Callus During Fracture Healing

2020 ◽  
Vol 68 (3) ◽  
pp. 199-208
Author(s):  
Anuradha Valiya Kambrath ◽  
Justin N. Williams ◽  
Uma Sankar
Keyword(s):  
Fracture Healing ◽  
Bone Healing ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Structural Integrity ◽  
Bone Fractures ◽  
Early Stage ◽  
Antigen Retrieval ◽  
Sample Collection ◽  
Immunofluorescence Labeling

Approximately 5% to 10% of all bone fractures do not heal completely, contributing to significant patient suffering and medical costs. Even in healthy individuals, fracture healing is associated with significant downtime and loss of productivity. However, no pharmacological treatments are currently available to promote efficient bone healing. A better understanding of the underlying molecular mechanisms is crucial for developing novel therapies to hasten healing. The early reparative callus that forms around the site of bone injury is a fragile tissue consisting of shifting cell populations held together by loose connective tissue. The delicate callus is challenging to section and is vulnerable to disintegration during the harsh steps of immunostaining, namely, decalcification, deparaffinization, and antigen retrieval. Here, we describe an improved methodology for processing early-stage fracture calluses and immunofluorescence labeling of the sections to visualize the temporal (timing) and spatial (location) patterns of cellular and molecular events that regulate bone healing. This method has a short turnaround time from sample collection to microscopy as it does not require lengthy decalcification. It preserves the structural integrity of the fragile callus as the method does not entail deparaffinization or harsh methods of antigen retrieval. Our method can be adapted for high-throughput screening of drugs that promote efficacious bone healing:

scholarly journals Effects of Local Opioid Antagonist on Diabetic Fracture Rat Model

2018 ◽  
Vol 3 (3) ◽  
pp. 2473011418S0017
Author(s):  
Jarrett D. Cain ◽  
Michelle Titunick ◽  
Patricia McLaughlin ◽  
Ian Zagon
Keyword(s):  
Growth Factor ◽  
Fracture Healing ◽  
Bone Healing ◽  
Calcium Sulfate ◽  
Bone Fractures ◽  
Diabetic Rats ◽  
Receptor Expression ◽  
Opioid Antagonist ◽  
Diabetic Patients ◽  
Opioid Growth Factor

Category: Diabetes Introduction/Purpose: Complications associated with the diabetes include increased incidence of fracture healing, delayed fracture healing, delayed osteoblasts cell replication, decreased angiogenesis, migration and/or osteoblast cell differentiation. The cellular events involved in bone healing are adversely affected by diabetes; however, can be modulated by the Opioid Growth Factor (OGF)–OGF receptor (OGFr) is an inhibitory peptide that downregulates DNA synthesis in a tissue nonspecific manner. Diabetes is associated with elevated serum levels of OGF and dysregulation of the OGFr leading to multiple complications related to healing, sensitivity, and regeneration. This study explores the presence and function of the OGF-OGFr axis in bone tissue from type 1 diabetic rats examining intact and fractured femurs during early phases of the repair process Methods: Seven-week-old Sprague Dawley rats were injected with streptozotocin (40mg/kg i.p.) to induce T1D; other rats received buffer only and served as controls. After one month, hyperglycemia rats underwent surgery to produce a fracture at the distal third of the femur. Four diabetic rats received opioid antagoinist (naltrexone) and calcium sulfate and all remaining rats received calcium sulfate with water only. X-rays were taken immediately after surgery and after rats were euthanized on post-surgery; femur and tibia were collected for protein isolation, western blot analysis along with frozen or paraffin-embedded for histological analysis Results: Immunofluorescence indicated approximately 90% increase in opioid growth factor receptor expression in diabetic femurs compared to age-matched normal femurs. Western Blotting also suggested an increase in the receptor protein in diabetic bones relative to normal bone. TRAP staining for osteoclasts was greater in control and opioid antagonist-treated diabetic fractures when compared to the number of osteoclasts in vehicle-treated diabetic fractured femurs. Safranin O stained sections revealed approximately more bone in opioid growth receptor antagonist-treated diabetic bone fractures than in vehicle-treated bone fractures Conclusion: These data support our hypothesis that expression levels of OGFr are dysregulated in the bone of diabetic patients leading to complications in bone healing. Moreover, modulation of the OGF-OGFr pathway with receptor antagonists restored some aspects of bone healing. With further study, these preliminary results support the role of the OGF-OGFr axis in treatment of diabetic bone healing. New therapies to target dysregulation of the OGF-OGFr regulatory pathway in diabetes would provide a safe and effective disease-modifying treatment for delayed bone healing.

scholarly journals Virtual Structural Analysis of Tibial Fracture Healing from Low-Dose Clinical CT Scans

2018 ◽  
Author(s):  
Peter Schwarzenberg ◽  
Hannah L. Dailey
Keyword(s):  
Structural Analysis ◽  
Fracture Healing ◽  
Bone Healing ◽  
Structural Integrity ◽  
Torsional Rigidity ◽  
Torsion Test ◽  
Ct Scans ◽  
Orthopaedic Trauma ◽  
Element Analysis ◽  
Trauma Research

Quantitative assessment of bone fracture healing remains a significant challenge in orthopaedic trauma research. Accordingly, we developed a new technique for assessing bone healing using virtual mechano-structural analysis of computed tomography (CT) scans. CT scans from 19 fractured human tibiae at 12 weeks after surgery were segmented and prepared for finite element analysis (FEA). Boundary conditions were applied to the models to simulate a torsion test that is commonly used to access the structural integrity of long bones in animal models of fracture healing. The output of each model was the virtual torsional rigidity (VTR) of the healing zone, normalized to the torsional rigidity of each patient’s virtually reconstructed tibia. This provided a structural measure to track the percentage of healing each patient had undergone. Callus morphometric measurements were also collected from the CT scans. Results showed that at 12 weeks post-op, more than 75% of patients achieved a normalized VTR (torsional rigidity relative to uninjured bone) of 85% or above. The predicted intact torsional rigidities compared well with published cadaveric data. Across all patients, callus volume and density were weakly and non-significantly correlated with normalized VTR and time to clinical union. Conversely, normalized VTR was significantly correlated with time to union (R2 = 0.383, p = 0.005). This suggests that fracture scoring methods based on the visual appearance of callus may not accurately predict mechanical integrity. The image-based structural analysis presented here may be a useful technique for assessment of bone healing in orthopaedic trauma research.

scholarly journals Akkermansia muciniphila promotes type H vessel formation and bone fracture healing by reducing gut permeability and inflammation

2020 ◽  
Vol 13 (11) ◽  
pp. dmm043620 ◽  
Author(s):  
Jiang-Hua Liu ◽  
Tao Yue ◽  
Zhong-Wei Luo ◽  
Jia Cao ◽  
Zi-Qi Yan ◽  
...  
Keyword(s):  
Fracture Healing ◽  
Systemic Inflammation ◽  
Bone Healing ◽  
Bone Fracture ◽  
Early Stage ◽  
Gut Permeability ◽  
Bone Fracture Healing ◽  
Gut Barrier ◽  
Vessel Formation ◽  
Akkermansia Muciniphila

ABSTRACTImproving revascularization is one of the major measures in fracture treatment. Moderate local inflammation triggers angiogenesis, whereas systemic inflammation hampers angiogenesis. Previous studies showed that Akkermansia muciniphila, a gut probiotic, ameliorates systemic inflammation by tightening the intestinal barrier. In this study, fractured mice intragastrically administrated with A. muciniphila were found to display better fracture healing than mice treated with vehicle. Notably, more preosteclasts positive for platelet-derived growth factor-BB (PDGF-BB) were induced by A. muciniphila at 2 weeks post fracture, coinciding with increased formation of type H vessels, a specific vessel subtype that couples angiogenesis and osteogenesis, and can be stimulated by PDGF-BB. Moreover, A. muciniphila treatment significantly reduced gut permeability and inflammation at the early stage. Dextran sulfate sodium (DSS) was used to disrupt the gut barrier to determine its role in fracture healing and whether A. muciniphila still can stimulate bone fracture healing. As expected, A. muciniphila evidently improved gut barrier, reduced inflammation and restored the impaired bone healing and angiogenesis in DSS-treated mice. Our results suggest that A. muciniphila reduces intestinal permeability and alleviates inflammation, which probably induces more PDGF-BB+ preosteoclasts and type H vessel formation in callus, thereby promoting fracture healing. This study provides the evidence for the involvement of type H vessels in fracture healing and suggests the potential of A. muciniphila as a promising strategy for bone healing.This article has an associated First Person interview with the first author of the paper.

scholarly journals Bone turnover markers for early detection of fracture healing disturbances: A review of the scientific literature

2015 ◽  
Vol 87 (2) ◽  
pp. 1049-1061 ◽  
Author(s):  
Cristina P. Sousa ◽  
Isabel R. Dias ◽  
Mónica Lopez-peña ◽  
José A. Camassa ◽  
Paulo J. Lourenço ◽  
...  
Keyword(s):  
Early Detection ◽  
Bone Turnover ◽  
Fracture Healing ◽  
Bone Turnover Markers ◽  
New Technologies ◽  
Bone Fractures ◽  
Early Stage ◽  
Imaging Techniques ◽  
Non Union ◽  
Turnover Markers

Imaging techniques are the standard method for assessment of fracture healing processes. However, these methods are perhaps not entirely reliable for early detection of complications, the most frequent of these being delayed union and non-union. A prompt diagnosis of such disorders could prevent prolonged patient distress and disability. Efforts should be directed towards the development of new technologies for improving accuracy in diagnosing complications following bone fractures. The variation in the levels of bone turnover markers (BTMs) have been assessed with regard to there ability to predict impaired fracture healing at an early stage, nevertheless the conclusions of some studies are not consensual. In this article the authors have revised the potential of BTMs as early predictors of prognosis in adult patients presenting traumatic bone fractures but who did not suffer from osteopenia or postmenopausal osteoporosis. The available information from the different studies performed in this field was systematized in order to highlight the most promising BTMs for the assessment of fracture healing outcome.

scholarly journals Biomechanical duality of fracture healing captured using virtual modeling and validated in ovine bones

2021 ◽  
Author(s):  
Brendan Inglis ◽  
Peter Schwarzenberg ◽  
Karina Klein ◽  
Brigitte von Rechenberg ◽  
Salim Darwiche ◽  
...  
Keyword(s):  
Soft Tissue ◽  
Fracture Healing ◽  
Mechanical Testing ◽  
Large Scale ◽  
Structural Integrity ◽  
Bone Fractures ◽  
Diagnostic Testing ◽  
Material Model ◽  
Mechanical Tests ◽  
Constitutive Material Model

Bone fractures commonly repair by forming a bridging structure called callus, which begins as soft tissue and gradually ossifies to restore the rigidity of the bone. In large animals and humans, this process typically requires only a few months to complete. Occasionally bones can fail to heal, resulting in nonunions that are difficult to diagnose and treat, partly because early detection of poor healing remains challenging. We present a translational technique for measuring what really matters in fracture healing – the structural integrity of the bone – using image-based virtual mechanical testing. This work shows that physical mechanical tests of osteotomized ovine tibiae can be reliably recapitulated in a simulation environment only when the soft-hard mechanical duality of the healing zone is accurately captured by the constitutive material model. A large-scale optimization analysis was performed on thousands of 3D finite element models derived from computed tomography (CT) scans of 33 osteotomized sheep. This identified a piecewise material model that successfully replicated the postmortem torsion testing data by differentiating between soft and hard callus based on material density alone. The results suggest that most of the structural integrity of a healing bone is conferred by an internal architecture of mineralized hard callus that is supported by interstitial soft tissue. We conclude that with appropriate material modeling, virtual mechanical testing is a reliable surrogate for physical testing and that this technique has high translational potential for diagnostic testing of nonunion.

Molecular Processes Involved in Pancreatic Cancer and Therapeutics

2020 ◽  
Vol 14 ◽  
Author(s):  
Subhajit Makar ◽  
Abhrajyoti Ghosh ◽  
Divya ◽  
Shalini Shivhare ◽  
Ashok Kumar ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Locally Advanced ◽  
Therapeutic Strategies ◽  
Screening Methods ◽  
Pancreatic Cancer Cells ◽  
Systemic Treatments ◽  
Cancer Initiation ◽  
Novel Therapeutic Strategies

: Despite advances in the development of cytotoxic and targeted therapies, pancreatic adenocarcinoma (PAC) remains a significant cause of cancer mortality worldwide. It is also difficult to detect it at an early stage due to numbers of factors. Most of the patients are present with locally advanced or metastatic disease, which precludes curative resection. In the absence of effective screening methods, considerable efforts have been made to identify better systemic treatments during the past decade. This review describes the recent advances in molecular mechanisms involved in pancreatic cancer initiation, progression, and metastasis. Additionally, the importance of deregulated cellular signalling pathways and various cellular proteins as potential targets for developing novel therapeutic strategies against incurable forms of pancreatic cancer is reported. The emphasis is on the critical functions associated with growth factors and their receptors viz. c-MET/HGF, CTHRC1, TGF-β, JAK-STAT, cyclooxygenase pathway, WNT, CCK, MAPK-RAS-RAF, PI3K-AKT, Notch, src, IGF-1R, CDK2NA and chromatin regulation for the sustained growth, survival, and metastasis of pancreatic cancer cells. It also includes various therapeutic strategies viz. immunotherapy, surgical therapy, radiation therapy and chemotherapy.

scholarly journals Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

2021 ◽  
Vol 18 (7) ◽  
pp. 3332
Author(s):  
Olga V. Naidenko ◽  
David Q. Andrews ◽  
Alexis M. Temkin ◽  
Tasha Stoiber ◽  
Uloma Igara Uche ◽  
...  
Keyword(s):  
Immune System ◽  
High Throughput ◽  
Environmental Protection Agency ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Specific Activity ◽  
Laboratory Animals ◽  
In Vitro Assays ◽  
Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

scholarly journals Enzyme-Loaded Flower-Shaped Nanomaterials: A Versatile Platform with Biosensing, Biocatalytic, and Environmental Promise

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1460
Author(s):  
Khadega A. Al-Maqdi ◽  
Muhammad Bilal ◽  
Ahmed Alzamly ◽  
Hafiz M. N. Iqbal ◽  
Iltaf Shah ◽  
...  
Keyword(s):  
Organic Matter ◽  
Metal Ions ◽  
Critical Points ◽  
Structural Integrity ◽  
Early Stage ◽  
Surface Orientation ◽  
Critical Factors ◽  
Inorganic Hybrid ◽  
Iron Based ◽  
Significant Attention

As a result of their unique structural and multifunctional characteristics, organic–inorganic hybrid nanoflowers (hNFs), a newly developed class of flower-like, well-structured and well-oriented materials has gained significant attention. The structural attributes along with the surface-engineered functional entities of hNFs, e.g., their size, shape, surface orientation, structural integrity, stability under reactive environments, enzyme stabilizing capability, and organic–inorganic ratio, all significantly contribute to and determine their applications. Although hNFs are still in their infancy and in the early stage of robust development, the recent hike in biotechnology at large and nanotechnology in particular is making hNFs a versatile platform for constructing enzyme-loaded/immobilized structures for different applications. For instance, detection- and sensing-based applications, environmental- and sustainability-based applications, and biocatalytic and biotransformation applications are of supreme interest. Considering the above points, herein we reviewed current advances in multifunctional hNFs, with particular emphasis on (1) critical factors, (2) different metal/non-metal-based synthesizing processes (i.e., (i) copper-based hNFs, (ii) calcium-based hNFs, (iii) manganese-based hNFs, (iv) zinc-based hNFs, (v) cobalt-based hNFs, (vi) iron-based hNFs, (vii) multi-metal-based hNFs, and (viii) non-metal-based hNFs), and (3) their applications. Moreover, the interfacial mechanism involved in hNF development is also discussed considering the following three critical points: (1) the combination of metal ions and organic matter, (2) petal formation, and (3) the generation of hNFs. In summary, the literature given herein could be used to engineer hNFs for multipurpose applications in the biosensing, biocatalysis, and other environmental sectors.

scholarly journals OP0244 A PRECLINICAL TESTING TOOL: THE IN VITRO 3D FRACTURE GAP MODEL

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 154.1-154
Author(s):  
M. Pfeiffenberger ◽  
A. Damerau ◽  
P. Hoff ◽  
A. Lang ◽  
F. Buttgereit ◽  
...  
Keyword(s):  
Fracture Healing ◽  
Bone Healing ◽  
Inflammatory Markers ◽  
Initial Phase ◽  
Gap Model ◽  
Osteogenic Markers ◽  
Immunosuppressed Patients ◽  
The Impact

Background:Approximately 10% of fractures lead to significant fracture healing disorders, with a tendency to further increase due to the aging population. Of note, especially immunosuppressed patients with ongoing inflammation show difficulties in the correct course of fracture healing leading to fracture healing disorders. Most notably, invading immune cells and secreted cytokines are considered to provide an inflammatory microenvironment within the fracture gap, primarily during the initial phase of fracture healing. Current research has the focus on small animal models, facing the problem of translation towards the human system. In order to improve the therapy of fracture healing disorders, we have developed a human cell-basedin vitromodel to mimic the initial phase of fracture healing adequately. This model will be used for the development of new therapeutic strategies.Objectives:Our aim is to develop anin vitro3D fracture gap model (FG model) which mimics thein vivosituation in order to provide a reliable preclinical test system for fracture healing disorders.Methods:To assemble our FG model, we co-cultivated coagulated peripheral blood and primary human mesenchymal stromal cells (MSCs) mimicking the fracture hematoma (FH model) together with a scaffold-free bone-like construct mimicking the bony part of the fracture gap for 48 h under hypoxic conditions (n=3), in order to reflect thein vivosituation after fracture most adequately. To analyze the impact of the bone-like construct on thein vitroFH model with regard to its osteogenic induction capacity, we cultivated the fracture gap models in either medium with or without osteogenic supplements. To analyze the impact of Deferoxamine (DFO, known to foster fracture healing) on the FG model, we further treated our FG models with either 250 µmol DFO or left them untreated. After incubation and subsequent preparation of the fracture hematomas, we evaluated gene expression of osteogenic (RUNX2,SPP1), angiogenic (VEGF,IL8), inflammatory markers (IL6,IL8) and markers for the adaptation towards hypoxia (LDHA,PGK1) as well as secretion of cytokines/chemokines using quantitative PCR and multiplex suspension assay, respectively.Results:We found via histology that both the fracture hematoma model and the bone-like construct had close contact during the incubation, allowing the cells to interact with each other through direct cell-cell contact, signal molecules or metabolites. Additionally, we could show that the bone-like constructs induced the upregulation of osteogenic markers (RUNX2, SPP1) within the FH models irrespective of the supplementation of osteogenic supplements. Furthermore, we observed an upregulation of hypoxia-related, angiogenic and osteogenic markers (RUNX2,SPP1) under the influence of DFO, and the downregulation of inflammatory markers (IL6,IL8) as compared to the untreated control. The latter was also confirmed on protein level (e.g. IL-6 and IL-8). Within the bone-like constructs, we observed an upregulation of angiogenic markers (RNA-expression ofVEGF,IL8), even more pronounced under the treatment of DFO.Conclusion:In summary, our findings demonstrate that our establishedin vitroFG model provides all osteogenic cues to induce the initial bone healing process, which could be enhanced by the fracture-healing promoting substance DFO. Therefore, we conclude that our model is indeed able to mimic correctly the human fracture gap situation and is therefore suitable to study the influence and efficacy of potential therapeutics for the treatment of bone healing disorders in immunosuppressed patients with ongoing inflammation.Disclosure of Interests:Moritz Pfeiffenberger: None declared, Alexandra Damerau: None declared, Paula Hoff: None declared, Annemarie Lang: None declared, Frank Buttgereit Grant/research support from: Amgen, BMS, Celgene, Generic Assays, GSK, Hexal, Horizon, Lilly, medac, Mundipharma, Novartis, Pfizer, Roche, and Sanofi., Timo Gaber: None declared

scholarly journals Can Optimizing the Mechanical Environment Deliver a Clinically Significant Reduction in Fracture Healing Time?

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 691
Author(s):  
Jan Barcik ◽  
Devakara R. Epari
Keyword(s):  
Fracture Healing ◽  
Bone Healing ◽  
Mechanical Stimulation ◽  
Callus Formation ◽  
Weight Bearing ◽  
Healing Process ◽  
Healing Time ◽  
Mechanical Environment ◽  
Clinically Significant ◽  
The Impact

The impact of the local mechanical environment in the fracture gap on the bone healing process has been extensively investigated. Whilst it is widely accepted that mechanical stimulation is integral to callus formation and secondary bone healing, treatment strategies that aim to harness that potential are rare. In fact, the current clinical practice with an initially partial or non-weight-bearing approach appears to contradict the findings from animal experiments that early mechanical stimulation is critical. Therefore, we posed the question as to whether optimizing the mechanical environment over the course of healing can deliver a clinically significant reduction in fracture healing time. In reviewing the evidence from pre-clinical studies that investigate the influence of mechanics on bone healing, we formulate a hypothesis for the stimulation protocol which has the potential to shorten healing time. The protocol involves confining stimulation predominantly to the proliferative phase of healing and including adequate rest periods between applications of stimulation.

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