Cellular and molecular analysis of fracture healing in a neurofibromatosis type 1 conditional knockout mice model

NF1 ist eine autosomal dominante Erbkrankheit, die durch inaktivierende Mutationen im Neurofibromin-Gen verursacht wird. NF1 manifestiert sich durch eine erhöhte Tumor-Inzidenz des neuralen Gewebes in der Haut (Neurofibroma). Neben diesen häufigeren klinischen Manifestationen haben rund 50% der NF1-Patienten Skelett-Anomalien. Häufiger sind Röhrenknochen betroffen, die klinischen Symptome reichen von Tibia-Krümmung über Spontanfrakturen bis hin zu Nonunions. Diese Studie analysiert den Heilungsverlauf von Femurfrakturen in Nf1Prx1- Mäusen. Der Frakturkallus von Mäusen wurde an den Tagen 7, 10, 14 und 21 durch µCT, Histologie und molekulare Analysen evaluiert. µCT und histologische Analysen haben eine beeinträchtigte Knochenheilung in Nf1Prx1-Mäusen gezeigt. Eine erhöhte periostale Knochenbildung in den frühen Stadien der Heilung war zu beobachten, sowie eine reduzierte, aber anhaltende Knorpelbildung und Bindegewebs-Akkumulation innerhalb der Fraktur. Wir konnten zeigen, dass der normalen Heilungsprozess durch dieses Bindegewebe behindert wird, welches durch alpha smooth muscle actin-positive Myofibroblasten gebildet wird, die ihrerseits aus einer bisher noch nicht identifizierten Muskelfaszie abgeleitet sind. Dieser Zusammenhang wird durch eine Microarray-Analyse der Kallus-Gewebe bestätigt, die ergab, dass durch den Knock-Out Gene reguliert wurden, die in Physiologie, Proliferation und Differenzierung von Muskelzellen involviert sind. Darüber hinaus waren extrazelluläre-Matrix-Gene in den Mutanten hoch regeuliert. Zusammenfassend konnten wir zeigen, dass eine Ähnlichkeit des Heilungsverlauf zwischen dem Nf1Prx1-Mausmodell und NF1-Patienten besteht. Folglich kann an diesem Mausmodell untersucht werden, durch welche Mechanismen die Mutationen im NF1 zu Knochenheilungsstörungen führen. Außerdem konnte in einer Pilotstudie der Effekt des Neurofibromin-Mangels auf die Knochenheilung durch Behandlung mit MEK-Inhibitoren in vitro und in vivo weitestgehend behoben werdenNeurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease resulting from inactivating mutations in the gene encoding the protein neurofibromin. NF1 patients – around 50% – have abnormalities of the skeleton. Long bones are often affected, and the clinical signs range from tibial bowing to spontaneous fractures and even non-unions. Moreover, NF1 mice models could provide the understanding of the cell types involved in the resulting non-union and their behavior. This study analyzed the healing progress of femur fractures in a model of NF1 long bone dysplasia. Fracture callus was assessed at days 7, 10, 14, and 21 by µCT, histology, biomechanics, and molecular analyses. Bone healing was impaired in Nf1Prx1 mice femoral fracture. Results revealed increased periosteal bone deposition at the early stages of healing, decreased but persistent cartilage formation concomitant with fibrous tissue accumulation within the fracture site, decreased torsional stiffness, decreased bone mineral density, and increased fibrous tissue infiltration in the callus of mutant mice. This fibrous tissue accumulation hindered bone fracture healing, and was deposited by alpha smooth muscle actin-positive myofibroblasts, which were derived from a yet unidentified muscle fascia. This is further supported by the microarray analysis of callus tissues showing that genes crucial to muscle cells physiology, proliferation and differentiation were affected. In addition, extracellular matrix related genes were up-regulated in the mutants. In summary, this study shows a resemblance in the healing progression to the Nf1Prx1 mice model and NF1 patients, thereby, confirming the suitability of this mice model to explore the mechanism by which mutations in NF1 lead to non-unions. Moreover, in vitro and in vivo pilot assessments of MEK inhibitor treatment demonstrated a potential remedy for the lack of neurofibromin in bone healing

A tissue-based approach to selection of reference genes for quantitative real-time PCR in a sheep osteoporosis model

Background: In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability. Methods: Twelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes. Results: We identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples. Conclusion: This study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs

Osteocytes Influence on Bone Matrix Integrity Affects Biomechanical Competence at Bone-Implant Interface of Bioactive-Coated Titanium Implants in Rat Tibiae

Osseointegration is a prerequisite for the long-term success of implants. Titanium implants are preferred for their biocompatibility and mechanical properties. Nonetheless, the need for early and immediate loading requires enhancing these properties by adding bioactive coatings. In this preclinical study, extracellular matrix properties and cellular balance at the implant/bone interface was examined. Polyelectrolyte multilayers of chitosan and gelatin or with chitosan and Hyaluronic acid fabricated on titanium alloy using a layer-by-layer self-assembly process were compared with native titanium alloy. The study aimed to histologically evaluate bone parameters that correlate to the biomechanical anchorage enhancement resulted from bioactive coatings of titanium implants in a rat animal model. Superior collagen fiber arrangements and an increased number of active osteocytes reflected a significant improvement of bone matrix quality at the bone interface of the chitosan/gelatin-coated titan implants over chitosan/hyaluronic acid-coated and native implants. Furthermore, the numbers and localization of osteoblasts and osteoclasts in the reparative and remodeling phases suggested a better cellular balance in the chitosan/Gel-coated group over the other two groups. Investigating the micro-mechanical properties of bone tissue at the interface can elucidate detailed discrepancies between different promising bioactive coatings of titanium alloys to maximize their benefit in future medical applications

Awareness of predatory journals and open access publishing among orthopaedic and trauma surgeons – results from an online survey in Germany

Background Along with emerging open access journals (OAJ) predatory journals increasingly appear. As they harm accurate and good scientific research, we aimed to examine the awareness of predatory journals and open access publishing among orthopaedic and trauma surgeons. Methods In an online survey between August and December 2019 the knowledge on predatory journals and OAJ was tested with a hyperlink made available to the participants via the German Society for Orthopaedics and Trauma Surgery (DGOU) email distributor. Results Three hundred fifty orthopaedic and trauma surgeons participated, of which 291 complete responses (231 males (79.4%), 54 females (18.6%) and 5 N/A (2.0%)) were obtained. 39.9% were aware of predatory journals. However, 21.0% knew about the “Directory of Open Access Journals” (DOAJ) as a register for non-predatory open access journals. The level of profession (e.g. clinic director, consultant) (p = 0.018) influenced the awareness of predatory journals. Interestingly, participants aware of predatory journals had more often been listed as corresponding authors (p < 0.001) and were well published as first or last author (p < 0.001). Awareness of OAJ was masked when journal selection options did not to provide any information on the editorial board, the peer review process or the publication costs. Conclusion The impending hazard of predatory journals is unknown to many orthopaedic and trauma surgeons. Early stage clinical researchers must be trained to differentiate between predatory and scientifically accurate journals

Influence of Thoracic Trauma on Fracture Healing in Long Bones—A Retrospective Analysis

Purpose: Pre-clinical studies indicate that concomitant thoracic trauma impairs fracture healing of long bones and reduces callus formation. The aim of this study was to investigate whether patients with accompanying chest trauma suffer from delayed fracture healing of long bones in comparison with patients with fractures of two long bones or isolated fractures. Patients and Methods: This is a clinical retrospective study from a level I trauma center. The patients were divided into three groups: (1) thoracic trauma and fracture of a long bone, (2) fractures of two long bones, (3) isolated fracture of a long bone. The fracture consolidation was defined using the radiographic union scale in tibial fractures (RUST). A RUST value of ≥10 six-to-eight months after definitive operative intervention represented complete fracture healing. Results: In the first group 19 (43.2%) fractures did not show full consolidation, in the second group 14 (45.2%) and 13 (41.9%) and in the third group 14 (36.8%). The analysis revealed no statistically significant differences between the groups regarding consolidation of the fractures six-to-eight months after definitive operative intervention (p = 0.84). Conclusions: Unlike previously reported pre-clinical data, this study did not demonstrate a negative effect on fracture consolidation in long bones when accompanied by thoracic trauma. Furthermore, the results demonstrated that concomitant fractures of two long bones does not have a negative effect on fracture consolidation

T Lymphocytes Influence the Mineralization Process of Bone

Bone is a unique organ able to regenerate itself after injuries. This regeneration requires the local interplay between different biological systems such as inflammation and matrix formation. Structural reconstitution is initiated by an inflammatory response orchestrated by the host immune system. However, the individual role of T cells and B cells in regeneration and their relationship to bone tissue reconstitution remain unknown. Comparing bone and fracture healing in animals with and without mature T and B cells revealed the essential role of these immune cells in determining the tissue mineralization and thus the bone quality. Bone without mature T and B cells is stiffer when compared to wild-type bone thus lacking the elasticity that helps to absorb forces, thus preventing fractures. In-depth analysis showed dysregulations in collagen deposition and osteoblast distribution upon lack of mature T and B cells. These changes in matrix deposition have been correlated with T cells rather than B cells within this study. This work presents, for the first time, a direct link between immune cells and matrix formation during bone healing after fracture. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells

2018 Proceedings of the International Conference on Trauma Surgery Technology in Giessen

The overarching goal of the gathering was to define a concept for technology design research in Giessen to improve patient outcomes through the design of assistive technology that assists both patients and surgeons. The emerging field of regenerative rehabilitation where trauma patients are treated by methods of regenerative medicine promises great improvements for our field, but clinical success is yet to be realised. The symposium was specifically organised to acknowledge and address these issues

An Optimized Approach to Perform Bone Histomorphometry

Bone histomorphometry allows quantitative evaluation of bone micro-architecture, bone formation, and bone remodeling by providing an insight to cellular changes. Histomorphometry plays an important role in monitoring changes in bone properties because of systemic skeletal diseases like osteoporosis and osteomalacia. Besides, quantitative evaluation plays an important role in fracture healing studies to explore the effect of biomaterial or drug treatment. However, until today, to our knowledge, bone histomorphometry remain time-consuming and expensive. This incited us to set up an open-source freely available semi-automated solution to measure parameters like trabecular area, osteoid area, trabecular thickness, and osteoclast activity. Here in this study, the authors present the adaptation of Trainable Weka Segmentation plugin of ImageJ to allow fast evaluation of bone parameters (trabecular area, osteoid area) to diagnose bone related diseases. Also, ImageJ toolbox and plugins (BoneJ) were adapted to measure osteoclast activity, trabecular thickness, and trabecular separation. The optimized two different scripts are based on ImageJ, by providing simple user-interface and easy accessibility for biologists and clinicians. The scripts developed for bone histomorphometry can be optimized globally for other histological samples. The showed scripts will benefit the scientific community in histological evaluation

Structural Analysis of Mitochondrial Dynamics—From Cardiomyocytes to Osteoblasts: A Critical Review

Mitochondria play a crucial role in cell physiology and pathophysiology. In this context, mitochondrial dynamics and, subsequently, mitochondrial ultrastructure have increasingly become hot topics in modern research, with a focus on mitochondrial fission and fusion. Thus, the dynamics of mitochondria in several diseases have been intensively investigated, especially with a view to developing new promising treatment options. However, the majority of recent studies are performed in highly energy-dependent tissues, such as cardiac, hepatic, and neuronal tissues. In contrast, publications on mitochondrial dynamics from the orthopedic or trauma fields are quite rare, even if there are common cellular mechanisms in cardiovascular and bone tissue, especially regarding bone infection. The present report summarizes the spectrum of mitochondrial alterations in the cardiovascular system and compares it to the state of knowledge in the musculoskeletal system. The present paper summarizes recent knowledge regarding mitochondrial dynamics and gives a short, but not exhaustive, overview of its regulation via fission and fusion. Furthermore, the article highlights hypoxia and its accompanying increased mitochondrial fission as a possible link between cardiac ischemia and inflammatory diseases of the bone, such as osteomyelitis. This opens new innovative perspectives not only for the understanding of cellular pathomechanisms in osteomyelitis but also for potential new treatment options

Retrospective Analysis of Bone Substitute Material for Traumatic Long Bone Fractures: Sex-Specific Outcomes

Male patients often experience increased bone and muscle loss after traumatic fractures. This study aims to compare the treatment outcomes of male and female patients with large bone defects. A total of 345 trauma patients underwent surgery, with participants divided into two groups: one receiving bone substitute material (BSM) for augmented defects (n = 192) and the other without augmentation (empty defects = ED, n = 153). Outcome parameters were assessed among female (n = 184) and male (n = 161) patients. Descriptive statistics revealed no significant differences between male and female patients. Approximately one-half of the fractures resulted from high-energy trauma (n = 187). The BSM group experienced fewer complications (p = 0.004), including pseudarthrosis (BSM: n = 1, ED: n = 7; p = 0.02). Among female patients over 65, the incidence of pseudarthrosis was lower in the BSM group (p = 0.01), while younger females showed no significant differences (p = 0.4). Radiologically, we observed premature bone healing with subsequent harmonization. Post hoc power analysis demonstrated a power of 0.99. Augmenting bone defects, especially with bone substitute material, may reduce complications, including pseudarthrosis, in female patients. Additionally, this material accelerates bone healing. Further prospective studies are necessary for confirmation