Publications by authors named "Rainer Mittermayr"

46 Publications

Lugol's solution but not formaldehyde affects bone microstructure and bone mineral density parameters at the insertion site of the rotator cuff in rats.

J Orthop Surg Res 2021 Apr 13;16(1):254. Epub 2021 Apr 13.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria.

Background: This study aimed to investigate whether rodent shoulder specimens fixed in formaldehyde for histological and histomorphometric investigations and specimens stained using Lugol's solution for soft tissue visualization by micro-computed tomography (microCT) are still eligible to be used for bone architecture analysis by microCT.

Methods: In this controlled laboratory study, 11 male Sprague-Dawley rats were used. After sacrifice and exarticulation both shoulders of healthy rats were assigned into three groups: (A) control group (n = 2); (B) formaldehyde group (n = 4); (C) Lugol group (n = 5). Half of the specimens of groups B and C were placed in a 4% buffered formaldehyde or Lugol's solution for 24 h, whereas the contralateral sides and all specimens of group A were stored without any additives. MicroCT of both sides performed in all specimens focused on bone mineral density (BMD) and bone microstructure parameters.

Results: BMD measurements revealed higher values in specimens after placement in Lugol's solution (p < 0.05). Bone microstructure analyses showed increased BV/TV and Tb.Th values in group C (p < 0.05). Specimens of group C resulted in clearly decreased Tb.Sp values (p < 0.05) in comparison to the control group. Formaldehyde fixation showed minimally altered BMD and bone microstructure measurements without reaching any significance.

Conclusions: MicroCT scans of bone structures are recommended to be conducted natively and immediately after euthanizing rats. MicroCT scans of formaldehyde-fixed specimens must be performed with caution due to a possible slight shift of absolute values of BMD and bone microstructure. Bone analysis of specimens stained by Lugol's solution cannot be recommended.
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http://dx.doi.org/10.1186/s13018-021-02394-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045387PMC
April 2021

The role of shockwaves in the enhancement of bone repair - from basic principles to clinical application.

Injury 2021 Jun 2;52 Suppl 2:S84-S90. Epub 2021 Mar 2.

Ludwig Boltzmann Institute for experimental and clinical traumatology, Vienna, Austria; AUVA trauma research center, Vienna, Austria; Austrian Cluster for Tissue Engineering, Vienna, Austria; AUVA Medical Board, Vienna, Austria.

Extracorporeal shockwave therapy is a treatment modality, originally introduced into the clinic as lithotripsie, which has also been successfully used in the last two decades in the non-invasive treatment of delayed or non-healing fractures. Initially, the mechanism of action was attributed to microfracture-induced repair, but intensive basic research has now shown that the shockwave generates its effect in tissue via mechanotransduction. Numerous signal transduction pathways have already been demonstrated, which in their entirety trigger an endogenous regeneration process via cell proliferation, migration and differentiation. Clinically, these shockwave-conveyed biological signals support healing of acute, delayed and non-union fractures. The attainable outcome is comparable to surgery but avoiding an open approach with associated potential complications. These advantageous properties with a clearly positive cost-benefit ratio make shockwave therapy a first line treatment in delayed and non-union fractures.
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http://dx.doi.org/10.1016/j.injury.2021.02.081DOI Listing
June 2021

The secretome of stressed peripheral blood mononuclear cells increases tissue survival in a rodent epigastric flap model.

Bioeng Transl Med 2021 Jan 22;6(1):e10186. Epub 2020 Sep 22.

Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration Vienna Austria.

Reconstructive surgery transfers viable tissue to cover defects and to restore aesthetic and functional properties. Failure rates after free flap surgery range from 3 to 7%. Co-morbidities such as diabetes mellitus or peripheral vascular disease increase the risk of flap failure up to 4.5-fold. Experimental therapeutic concepts commonly use a monocausal approach by applying single growth factors. The secretome of γ-irradiated, stressed peripheral blood mononuclear cells (PBMCsec) resembles the physiological environment necessary for tissue regeneration. Its application led to improved wound healing rates and a two-fold increase in blood vessel counts in previous animal models. We hypothesized that PBMCsec has beneficial effects on the survival of compromised flap tissue by reducing the necrosis rate and increasing angiogenesis. Surgery was performed on 39 male Sprague-Dawley rats (control, = 13; fibrin sealant, = 14; PBMCsec, = 12). PBMCsec was produced according to good manufacturing practices (GMP) guidelines and 2 ml were administered intraoperatively at a concentration of 2.5 × 10 cells/ml using fibrin sealant as carrier substance. Flap perfusion and necrosis (as percentage of the total flap area) were analyzed using Laser Doppler Imaging and digital image planimetry on postoperative days 3 and 7. Immunohistochemical stainings for von Willebrand factor (vWF) and Vascular Endothelial Growth Factor-receptor-3 (Flt-4) were performed on postoperative day 7 to evaluate formation of blood vessels and lymphatic vessels. Seroma formation was quantified using a syringe and flap adhesion and tissue edema were evaluated clinically through a cranial incision by a blinded observer according to previously described criteria on postoperative day 7. We found a significantly reduced tissue necrosis rate (control: 27.8% ± 8.6; fibrin: 22.0% ± 6.2; 20.9% reduction, = .053 vs. control; PBMCsec: 19.1% ± 7.2; 31.1% reduction, = .012 vs. control; 12.9% reduction, 0.293 vs. fibrin) together with increased vWF+ vessel counts (control: 70.3 ± 16.3 vessels/4 fields at 200× magnification; fibrin: 67.8 ± 12.1; 3.6% reduction, = .651, vs. control; PBMCsec: 85.9 ± 20.4; 22.2% increase, = .045 vs. control; 26.7% increase, = .010 vs. fibrin) on postoperative day 7 after treatment with PBMCsec. Seroma formation was decreased after treatment with fibrin sealant with or without the addition of PBMCsec. (control: 11.9 ± 9.7 ml; fibrin: 1.7 ± 5.3, 86.0% reduction, 0.004 vs. control; PBMCsec: 0.6 ± 2.0; 94.8% reduction, = .001 vs. control; 62.8% reduction, = .523 vs. fibrin). We describe the beneficial effects of a secretome derived from γ-irradiated PBMCs on tissue survival, angiogenesis, and clinical parameters after flap surgery in a rodent epigastric flap model.
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http://dx.doi.org/10.1002/btm2.10186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823127PMC
January 2021

Implant-free iliac crest bone graft procedure shows anatomic remodelling without redislocation in recurrent anterior shoulder instability after short-term follow-up.

Arch Orthop Trauma Surg 2021 Jan 24. Epub 2021 Jan 24.

AUVA Trauma Centre Vienna Meidling, Kundratstraße 37, A-1120, Vienna, Austria.

Introduction: With the help of a J-shaped bicortical iliac crest bone graft, the morphology of the glenoid can be augmented without having to use screws to achieve glenohumeral stability. The aim of this retrospective clinical study was to evaluate the clinical stability and function of the shoulder joint as well as the radiological remodelling process and arthropathic outcomes following the J-bone graft technique.

Materials And Methods: 34 patients with recurrent shoulder dislocations and bony glenoid defects were treated with the J-bone graft technique between 2010 and 2018 at our level-I trauma centre. 15 patients (18 shoulders) could be recruited for the study. Pain levels, ASES, UCLA, SST, DASH, Rowe and WOSI Scores were collected using questionnaires. In 13 patients (16 shoulders) the Constant Score, ROM, CT with 3D reconstruction of the glenoid to assess the graft remodelling and X-rays were performed additionally.

Results: None of the patients suffered subluxations or recurrent dislocations during the follow-up period. The overall complication rate was 11%. The evaluation using objective and subjective shoulder function scores yielded good-to-excellent results. Radiological assessment at follow-up showed a low rate of moderate-to-severe arthritis (12%) and a high rate of shoulders without any signs of arthritic degeneration (53%). The CT scans all revealed an almost complete restoration of the glenoid with none of the grafts being resorbed. A rise in the average glenoid circumference and glenoid area could be demonstrated between preoperative measurements (81.6 and 82.4%, respectively) and follow-up measurements (104 and 102.5%, respectively).

Conclusion: The results of this study show a successful stabilisation of the shoulder joint and a low complication rate following the J-bone graft technique. Remodelling of the bone graft could be demonstrated, which in turn led to an almost perfect glenoid surface area of 100%.
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http://dx.doi.org/10.1007/s00402-021-03777-4DOI Listing
January 2021

Zoledronic Acid Substantially Improves Bone Microarchitecture and Biomechanical Properties After Rotator Cuff Repair in a Rodent Chronic Defect Model.

Am J Sports Med 2020 07 16;48(9):2151-2160. Epub 2020 Jun 16.

AUVA Trauma Center Vienna-Meidling, Department for Trauma Surgery, Vienna, Austria.

Background: Bone mineral density at the humeral head is reduced in patients with chronic rotator cuff tears. Bone loss in the humeral head is associated with repair failure after rotator cuff reconstruction. Bisphosphonates (eg, zoledronic acid) increase bone mineral density.

Hypothesis: Zoledronic acid improves bone mineral density of the humeral head and biomechanical properties of the enthesis after reconstruction of chronic rotator cuff tears in rats.

Study Design: Controlled laboratory study.

Methods: A total of 32 male Sprague-Dawley rats underwent unilateral (left) supraspinatus tenotomy with delayed transosseous rotator cuff reconstruction after 3 weeks. All rats were sacrificed 8 weeks after rotator cuff repair. Animals were randomly assigned to 1 of 2 groups. At 1 day after rotator cuff reconstruction, the intervention group was treated with a single subcutaneous dose of zoledronic acid at 100 µg/kg bodyweight, and the control group received 1 mL of subcutaneous saline solution. In 12 animals of each group, micro-computed tomography scans of both shoulders were performed as well as biomechanical testing of the supraspinatus enthesis of both sides. In 4 animals of each group, histological analyses were conducted.

Results: In the intervention group, bone volume fraction (bone volume/total volume [BV/TV]) of the operated side was higher at the lateral humeral head ( = .005) and the medial humeral head ( = .010) compared with the control group. Trabecular number on the operated side was higher at the lateral humeral head ( = .004) and the medial humeral head ( = .001) in the intervention group. Maximum load to failure rates on the operated side were higher in the intervention group ( < .001). Cortical thickness positively correlated with higher maximum load to failure rates in the intervention group ( = 0.69; = .026). Histological assessment revealed increased bone formation in the intervention group.

Conclusion: Single-dose therapy of zoledronic acid provided an improvement of bone microarchitecture at the humeral head as well as an increase of maximum load to failure rates after transosseous reconstruction of chronic rotator cuff lesions in rats.

Clinical Relevance: Zoledronic acid improves bone microarchitecture as well as biomechanical properties after reconstruction of chronic rotator cuff tears in rodents. These results need to be verified in clinical investigations.
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http://dx.doi.org/10.1177/0363546520926471DOI Listing
July 2020

Molecular Pattern and Density of Axons in the Long Head of the Biceps Tendon and the Superior Labrum.

J Clin Med 2019 Dec 3;8(12). Epub 2019 Dec 3.

AUVA Trauma Center Vienna Meidling, Kundratstraße 37, 1120 Vienna, Austria.

The type II superior labrum anterior to posterior (SLAP) repair is a viable option in young and demanding patients, although a prolonged period of pain after surgery is described in the literature. The reason for this fact remains unknown. Thus, the purpose of this study was to investigate the molecular pattern of the biceps tendon anchor, where the sutures for repair are placed. The long head of the biceps tendon (LHBT), including the superior labrum, was dissected in the setting of reverse total shoulder arthroplasty. Immunohistochemical staining was performed using neurofilament (NF) and protein gene product (PGP) 9.5 as general markers for axons and calcitonin gene-related peptide (CGRP) and substance P for nociceptive transmission. A quantitative assessment was performed according to the two regions of interest (ROIs), i.e., the anterosuperior (ROI I) and the posterosuperior labrum (ROI II). Eleven LHBTs with a mean age of 73 years (range: 66-87 years) were harvested intraoperatively. Six LHBTs were gained in osteoarthrosis and five in fractures. We found an inhomogeneous distribution of axons in the anterosuperior and posterosuperior parts of the labrum in all the specimens irrespective of the age, gender, and baseline situation. There was a significantly higher number ( < 0.01) as well as density ( < 0.001) of NF-positive axons in ROI I compared to ROI II. Nociceptive fibers were always found along the NF-positive axons. Thus, our results indicate that the biceps tendon anchor itself is a highly innervated region comprising different nerve qualities. The anterosuperior labrum contains a higher absolute number and density of axons compared to the posterosuperior parts. Furthermore, we were able to prove the presence of nociceptive fibers in the superior labrum. The results obtained in this study could contribute to the variability of pain after SLAP repair.
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http://dx.doi.org/10.3390/jcm8122129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947398PMC
December 2019

Structural and molecular characteristics of axons in the long head of the biceps tendon.

Cell Tissue Res 2020 Apr 7;380(1):43-57. Epub 2019 Dec 7.

AUVA Trauma Center Vienna Meidling, A-1120, Vienna, Austria.

The innervation of the long head of the biceps tendon (LHBT) is not sufficiently documented. This is a drawback since pathologies of the LHBT are a major source of shoulder pain. Thus, the study aimed to characterize structurally and molecularly nervous elements of the LHBT. The proximal part of 11 LHBTs was harvested intraoperatively. There were 8 female and 3 male specimens. Age ranged from 66 to 86 years. For structural analyses, nervous elements were viewed in the transmission electron microscope. For molecular characterization, we used general neuronal markers including antibodies against neurofilament and protein gene product 9.5 (PGP9.5) as well as specific neuronal markers including antibodies against myelin basic protein (MBP), calcitonin gene-related product (CGRP), substance P (SP), tyrosine hydroxylase (TH), and growth-associated protein 43 (GAP43). Anti-neurofilament and anti-PGP9.5 visualized the overall innervation. Anti-MBP visualized myelination, anti-CGRP and anti-SP nociceptive fibers, anti-TH sympathetic nerve fibers, and anti-GAP43 nerve fibers during development and regeneration. Immunolabeled sections were analyzed in the confocal laser scanning microscope. We show that the LHBT contains unmyelinated as well as myelinated nerve fibers which group in nerve fascicles and follow blood vessels. Manny myelinated and unmyelinated axons exhibit molecular features of nociceptive nerve fibers. Another subpopulation of unmyelinated axons exhibits molecular characteristics of sympathetic nerve fibers. Unmyelinated sympathetic fibers and unmyelinated nociceptive fibers express proteins that are found during development and regeneration. Present findings support the hypothesis that ingrowth of nociceptive fibers are the source of chronic tendon pain.
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http://dx.doi.org/10.1007/s00441-019-03141-4DOI Listing
April 2020

Bone Stress Injuries Are Associated With Differences in Bone Microarchitecture in Male Professional Soldiers.

J Orthop Res 2019 12 26;37(12):2516-2523. Epub 2019 Aug 26.

Medical Department II-VINFORCE Study Group, St. Vincent Hospital, Academic Teaching Hospital of the Medical University of Vienna, Stumpergasse 13, Vienna, A-1060, Austria.

Bone stress injuries are commonly due to repetitive loading, as often described in competitive athletes or military recruits. The underlying pathophysiology of bone stress injuries is multifactorial. The present cross-sectional study investigated (i) cortical and trabecular bone microstructure as well as volumetric bone mineral density in subjects with bone stress injuries at the tibial diaphysis, measured at the distal tibia and the distal radius by means of high-resolution peripheral quantitative computed tomography (CT), (ii) areal bone mineral density using dual-energy X-ray absorptiometry as well as calcaneal dual X-ray absorptiometry and laser, and (iii) the influence on bone turnover markers of formation and resorption at the early phase after injury. A total of 26 Caucasian male professional soldiers with post-training bone stress injury at the tibial diaphysis were included (case group). A total of 50 male, Caucasian professional soldiers from the same military institution served as controls (control group). High-resolution peripheral quantitative CT revealed a higher total area at the radius within the case group. Cortical bone mineral density was reduced at the radius and tibia within the case group. The trabecular number and trabecular thickness were reduced at the tibia in the case group. The trabecular network was more inhomogeneous at the radius and tibia within the case group. Calcaneal dual X-ray absorptiometry and laser was significantly reduced in the case group. This study quantified differences in bone microstructure among otherwise healthy individuals. Differences in bone microarchitecture may impair the biomechanical properties by increasing the susceptibility to sustain bone stress injuries. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2516-2523, 2019.
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http://dx.doi.org/10.1002/jor.24442DOI Listing
December 2019

miR-19a-3p containing exosomes improve function of ischaemic myocardium upon shock wave therapy.

Cardiovasc Res 2020 05;116(6):1226-1236

Department of Cardiac Surgery, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria.

Aims: As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia.

Methods And Results: Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography.

Conclusion: The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium.
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http://dx.doi.org/10.1093/cvr/cvz209DOI Listing
May 2020

Application of a new polyester patch in arthroscopic massive rotator cuff repair-a prospective cohort study.

J Shoulder Elbow Surg 2020 Jan 9;29(1):e11-e21. Epub 2019 Aug 9.

Etzel Clinic, Pfaeffikon, Switzerland.

Background: Massive rotator cuff (RC) tears still present a clinically challenging problem, with reported rerupture rates in up to 94%. The study objective was to determine the impact of synthetic patch augmentation for massive RC tears.

Methods: Between June 2012 and 2014, we performed 50 arthroscopic RC reconstructions augmented with a synthetic polyester patch. Pre- and postoperative imaging methods included arthrographic magnetic resonance imaging, arthrographic computed tomography, and ultrasound examination to determine tendon integrity or rerupture. Clinical outcome was evaluated using the Constant-Murley score and the subjective shoulder value. Mean clinical midterm and final follow-up was 22 months (9-35 months) and 52 months (25-74 months), respectively.

Results: The mean Constant-Murley score increased significantly from 36.5 (±16.4 standard deviation [SD]) preoperatively to a midterm value of 81.2 (±9.6 SD; P < .0001) and further improved to a mean of 83.4 (±10.8 SD) at final follow-up. The mean subjective shoulder value increased from 40.3 (±24.3 SD) to 89.2 (±12.9 SD; P < .0001) at midterm and to 89.6 (±15.2 SD) at final follow-up. We observed 7 complete reruptures (14%). However, reruptures did not correlate with revision surgery, which was performed in 8 patients. The main reason for revision was frozen shoulder or arthrofibrosis with an intact reconstruction and patch, which was performed in 6 cases.

Conclusions: The retear rate of 14% compared favorably with nonaugmented RC repairs in the literature. Therefore, we conclude that patch augmentation in massive RC tears is feasible to reduce retears and to improve clinical outcome.
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http://dx.doi.org/10.1016/j.jse.2019.05.015DOI Listing
January 2020

Substantial Biomechanical Improvement by Extracorporeal Shockwave Therapy After Surgical Repair of Rodent Chronic Rotator Cuff Tears.

Am J Sports Med 2019 07 17;47(9):2158-2166. Epub 2019 Jun 17.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria.

Background: Characteristics of chronic rotator cuff tears include continuous loss of tendon structure as well as tendon elasticity, followed by a high failure rate after surgical reconstruction. Several studies have already shown the beneficial effect of extracorporeal shockwave therapy (ESWT) on tissue regeneration in tendon pathologies.

Hypothesis: ESWT improves biomechanical tendon properties as well as functional shoulder outcomes in chronic rotator cuff reconstruction in rodents.

Study Design: Controlled laboratory study.

Methods: After tendon detachment and 3 weeks of degeneration, a subsequent transosseous reattachment of the supraspinatus tendon was performed in 48 adult male Sprague-Dawley rats (n = 16 per group). Rodents were randomly assigned to 3 study groups: no ESWT/control group, intraoperative ESWT (IntraESWT), and intra- and postoperative ESWT (IntraPostESWT). Shoulder joint function, as determined by gait analysis, was assessed repeatedly during the observation period. Eight weeks after tendon reconstruction, the rats were euthanized, and biomechanical and gene expression analyses were performed.

Results: Macroscopically, all repairs were intact at the time of euthanasia, with no ruptures detectable. Biomechanical analyses showed significantly improved load-to-failure testing results in both ESWT groups in comparison with the control group (control, 0.629; IntraESWT, 1.102; IntraPostESWT, 0.924; IntraESWT vs control, ≤ .001; IntraPostESWT vs control, ≤ .05). Furthermore, functional gait analyses showed a significant enhancement in intensity measurements for the IntraPostESWT group in comparison with the control group (≤ .05). Gene expression analysis revealed no significant differences among the 3 groups.

Conclusion: Clearly improved biomechanical results were shown in the single-application and repetitive ESWT groups. Furthermore, functional evaluation showed significantly improved intensity measurements for the repetitive ESWT group.

Clinical Relevance: This study underpins a new additional treatment possibility to prevent healing failure. Improved biomechanical stability and functionality may enable faster remobilization as well as an accelerated return to work and sports activities. Furthermore, as shockwave therapy is a noninvasive, easy-to-perform, cost-effective treatment tool with no undesired side effects, this study is of high clinical relevance in orthopaedic surgery. Based on these study results, a clinical study has already been initiated to clinically confirm the improved functionality by ESWT.
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http://dx.doi.org/10.1177/0363546519854760DOI Listing
July 2019

How to visualize the innervation pattern in tendons: A methodical guide.

Ann Anat 2019 Sep 11;225:21-27. Epub 2019 Jun 11.

AUVA Trauma Center Vienna Meidling, A-1120 Vienna, Austria.

Background: Tendon pathologies are common and several data suggests that the peripheral nervous system is involved in this disorder. Immunohistochemistry (IHC) is one of the pillars to characterize nervous structures and their implication in the pathogenesis of chronic tendon pain. Most commonly, formalin-fixed, paraffin-embedded (FFPE) tendons are used for immunohistochemical characterization of the innervation. However, FFPE specimens exhibit major disadvantages: First, antigens (proteins) are masked and antigen retrieval is necessary to restore antigenicity. Second, FFPE specimens involve immunolabeling with enzyme-conjugated antibodies but this approach has limitations when multiple antigens are of interest simultaneously. Consequently, there is a demand in the orthopedic community for an alternative immunohistochemical approach to visualize tendon innervations.

Results: Here, we present a guide how to visualize tendon innervation. This guide couples paraformaldehyde fixation, cryo-embedding, immunofluorescence, and confocal laser scanning microscopy. We demonstrate the utility of our approach in the long head of the biceps tendon. For nerve fiber characterization, we used different neuronal markers including antibodies against neurofilament, protein gene product 9.5, calcitonin gene related peptide, and substance P. We show that it is possible to collect high quality, multicolor images of the innervation pattern of tendons. To map immunolabeled structures and the anatomical structures of the tendon fluorescence images and bright field images were merged.

Conclusion: For the orthopedic community our approach might be a convenient research tool to simultaneously utilize multiple neuronal markers on the same tissue section and to define with greater accuracy the heterogeneity of tendon innervation.
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http://dx.doi.org/10.1016/j.aanat.2019.05.009DOI Listing
September 2019

Fracture patterns in patients with multiple fractures: the probability of multiple fractures and the most frequently associated regions.

Eur J Trauma Emerg Surg 2020 Oct 12;46(5):1151-1158. Epub 2019 Feb 12.

St. Vincent Hospital-Metabolic Bone Diseases Unit, The VINFORCE Study Group, Vienna, Austria.

Introduction: Multiple fractures are of high clinical relevance, as a significant increase in mortality rate has been described. The purpose of this study was to evaluate differences in age and gender distribution in multiple fractures dependent on severity of trauma. Furthermore, affected anatomic regions and frequently associated fracture regions were investigated.

Methods: Patients who had sustained multiple fractures between 2000 and 2012 were included in this study. At hospital admission, patients were divided according to trauma severity (high- vs low-traumatic), gender, and age for demographic analysis. Fractures were grouped in anatomical regions, and multiple fracture event probabilities as well as frequently associated regions were calculated.

Results: In total, 25,043 patients at an age range of 0-100 years (5.8% of all fracture patients; 14,769 male and 10,274 female patients) who sustained 57,862 multiple fractures were included. The lumbar/thoracic spine, cervical spine, femoral shaft, skull, and pelvis showed a probability of more than 40% of the presence of further fractures in each high-traumatic fracture event. In high-traumatic fracture events, male patients were more affected (p < 0.001). Considering low-traumatic fractures, female patients had a significantly higher proportion (p < 0.001) of multiple fractures among all fractures than male patients.

Conclusions: As a novelty, gender as well as age distributions in multiple fracture patients and a probability statement with the most affected anatomic regions, the risk of presence of further fractures for every region, and the frequently associated fracture regions including the percentage of occurrence are provided. These aspects yield new opportunities for clinical work and may reduce the high rate of overlooked fractures stated in the literature.
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http://dx.doi.org/10.1007/s00068-019-01087-4DOI Listing
October 2020

A Low Cost Implantation Model in the Rat That Allows a Spatial Assessment of Angiogenesis.

Front Bioeng Biotechnol 2018 5;6. Epub 2018 Feb 5.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria.

There is continual demand for animal models that allow a quantitative assessment of angiogenic properties of biomaterials, therapies, and pharmaceuticals. In its simplest form, this is done by subcutaneous material implantation and subsequent vessel counting which usually omits spatial data. We have refined an implantation model and paired it with a computational analytic routine which outputs not only vessel count but also vessel density, distribution, and vessel penetration depth, that relies on a centric vessel as a reference point. We have successfully validated our model by characterizing the angiogenic potential of a fibrin matrix in conjunction with recombinant human vascular endothelial growth factor (rhVEGF165). The inferior epigastric vascular pedicles of rats were sheathed with silicone tubes, which were subsequently filled with 0.2 ml of fibrin and different doses of rhVEGF165, centrically embedding the vessels. Over 4 weeks, tissue samples were harvested and subsequently immunohistologically stained and computationally analyzed. The model was able to detect variations over the angiogenic potentials of growth factor spiked fibrin matrices. Adding 20 ng of rhVEGF165 resulted in a significant increase in vasculature while 200 ng of rhVEGF165 did not improve vascular growth. Vascularized tissue volume increased during the first week and vascular density increased during the second week. Total vessel count increased significantly and exhibited a peak after 2 weeks which was followed by a resorption of vasculature by week 4. In summary, a simple implantation model to study vascularization with only a minimal workload attached was enhanced to include morphologic data of the emerging vascular tree.
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http://dx.doi.org/10.3389/fbioe.2018.00003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807912PMC
February 2018

Neurofilament distribution in the superior labrum and the long head of the biceps tendon.

J Orthop Surg Res 2017 Nov 22;12(1):181. Epub 2017 Nov 22.

AUVA Trauma Center Meidling, Kundratstraße 37, 1120, Vienna, Austria.

Background: The postoperative course after arthroscopic superior labrum anterior to posterior (SLAP) repair using suture anchors is accompanied by a prolonged period of pain, which might be caused by constriction of nerve fibres. The purpose was to histologically investigate the distribution of neurofilament in the superior labrum and the long head of the biceps tendon (LHBT), i.e. the location of type II SLAP lesions.

Methods: Ten LHBTs including the superior labrum were dissected from fresh human specimen and immunohistochemically stained against neurofilament (NF). All slides were scanned at high resolution and converted into tagged image file format, and regions of interest (ROIs) were defined as follows: ROI I-superior labrum anterior to the LHBT origin, ROI II-mid-portion of the superior labrum at the origin of the LHBT, ROI III-superior labrum posterior to the LHBT origin and ROI IV-the most proximal part of the LHBT before its attachment to the superior labrum. The entire images were automatically segmented according to the defined ROIs and measured using a programmed algorithm specifically created for this purpose. The NF-positive cells were counted, and their total size and the area of other tissue were measured separately for the different ROIs.

Results: Distribution of NF-positive cells in absolute numbers revealed a clear but insignificantly higher amount in favour of ROI I, representing the superior labrum anterior to the LHBT origin. Setting ROI I at 100%, a significant difference could be seen compared to ROI III, representing the superior labrum posterior to the LHBT origin (ROI I vs. ROI III with a p value < 0.05).

Conclusions: Summarizing, the density of neurofilament is inhomogeneously distributed throughout the superior labrum with the highest number of neurofilament in the anterior superior labrum. Thus, suture placement in type II SLAP repair could play an important role for the postoperative pain-related outcome.
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http://dx.doi.org/10.1186/s13018-017-0686-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700685PMC
November 2017

A Rodent Excision Model for Ischemia-Impaired Wound Healing.

Tissue Eng Part C Methods 2017 12;23(12):995-1002

1 Ludwig Boltzmann Institute for Clinical and Experimental Traumatology , AUVA Research Center, Austrian Cluster for Tissue Regeneration, Vienna, Austria .

Delayed wound healing and the potentially resulting chronic wounds are a challenging clinical problem. Available therapeutic strategies are limited in both number and efficacy. For developing and establishing novel treatment approaches appropriate clinically relevant animal models are essential. The aim of the study was to establish a reliable and reproducible delayed wound healing model, which simulates the clinical scenario of compromised vascular tissue perfusion (hypoxia/ischemia). Therefore a standard rodent ischemic flap model was modified by challenging the tissue with ascending degrees of ischemia using different surgical approaches (minimal, mild, moderate, and severe ischemic invasive approach). Then a full-thickness circular wound was excised in both the non-/hypoperfused flap area and in the normally perfused contralateral region serving as an internal control. Wound healing progress was compared. Superficial tissue perfusion was measured by Laser Doppler imaging technique, which showed persistent ischemia in the moderate and severe invasive surgical approaches 7 days after wounding. Wound closure assessed by planimetric analysis occurred significantly slower in the ischemic wounds compared to the contralateral nonischemic wounds in the moderate invasive approach. Histologic evaluations in this approach showed signs of tissue necrosis and impaired angiogenesis in the ischemic wounds. Therefore, it can be concluded that this clinically relevant animal model is suitable to study mechanism in ischemia-impaired wound healing. Furthermore, it allows evaluating the efficacy of therapeutic strategies for impaired wound healing and comparing the results with an internal control wound.
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http://dx.doi.org/10.1089/ten.TEC.2017.0212DOI Listing
December 2017

Extracorporeal shockwave therapy (ESWT) ameliorates healing of tibial fracture non-union unresponsive to conventional therapy.

Injury 2016 Jul 20;47(7):1506-13. Epub 2016 Apr 20.

Uniformed Services University for Health Sciences, Bethesda, MD, United States; Bon Secours Cancer Institute, Richmond, VA, United States.

Tibial non-unions are common cause of demanding revision surgeries and are associated with a significant impact on patients' quality of life and health care costs. Extracorporeal shockwave therapy (ESWT) has been shown to improve osseous healing in vitro and in vivo. The main objective of present study was to evaluate the efficacy of ESWT in healing of tibial non-unions unresponsive to previous surgical and non-surgical measures. A retrospective multivariant analysis of a prospective open, single-centre, clinical trial of tibia non-union was conducted. 56 patients with 58 eligible fractures who met the FDA criteria were included. All patients received 3000-4000 impulses of electrohydraulic shockwaves at an energy flux density of 0.4mJ/mm(2) (-6dB). On average patients underwent 1.9 times (±1.3SD) surgical interventions prior to ESWT displaying the rather negatively selected cohort and its limited therapy responsiveness. In 88.5% of patients receiving ESWT complete bone healing was observed after six months irrespective of underlying pathology. The multivariant analysis showed that time of application is important for therapy success. Patients achieving healing received ESWT earlier: mean number of days between last surgical intervention and ESWT (healed - 355.1 days±167.4SD vs. not healed - 836.7 days±383.0SD; p<0.0001). ESWT proved to be a safe, effective and non-invasive treatment modality in tibial non-unions recalcitrant to standard therapies. The procedure is well tolerated, time-saving, lacking side effects, with potential to significantly decrease health care costs. Thus, in our view, ESWT should be considered the treatment of first choice in established tibial non-unions.
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http://dx.doi.org/10.1016/j.injury.2016.04.010DOI Listing
July 2016

Paracrine Factors from Irradiated Peripheral Blood Mononuclear Cells Improve Skin Regeneration and Angiogenesis in a Porcine Burn Model.

Sci Rep 2016 04 29;6:25168. Epub 2016 Apr 29.

Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Waehringer Guertel 18-20, 1090 Vienna, Austria.

Burn wounds pose a serious threat to patients and often require surgical treatment. Skin grafting aims to achieve wound closure but requires a well-vascularized wound bed. The secretome of peripheral blood mononuclear cells (PBMCs) has been shown to improve wound healing and angiogenesis. We hypothesized that topical application of the PBMC secretome would improve the quality of regenerating skin, increase angiogenesis, and reduce scar formation after burn injury and skin grafting in a porcine model. Full-thickness burn injuries were created on the back of female pigs. Necrotic areas were excised and the wounds were covered with split-thickness mesh skin grafts. Wounds were treated repeatedly with either the secretome of cultured PBMCs (Sec(PBMC)), apoptotic PBMCs (Apo-Sec(PBMC)), or controls. The wounds treated with Apo-Sec(PBMC) had an increased epidermal thickness, higher number of rete ridges, and more advanced epidermal differentiation than controls. The samples treated with Apo-Sec(PBMC) had a two-fold increase in CD31+ cells, indicating more angiogenesis. These data suggest that the repeated application of Apo-Sec(PBMC) significantly improves epidermal thickness, angiogenesis, and skin quality in a porcine model of burn injury and skin grafting.
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http://dx.doi.org/10.1038/srep25168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850437PMC
April 2016

Effects of Shock Waves on Expression of IL-6, IL-8, MCP-1, and TNF-α Expression by Human Periodontal Ligament Fibroblasts: An In Vitro Study.

Med Sci Monit 2016 Mar 20;22:914-21. Epub 2016 Mar 20.

Competence Centre of Periodontal Research, Bernhard Gottlieb School of Dentistry, Medical University of Vienna, Vienna, Austria.

Background: Extracorporeal shock wave therapy (ESWT) can modulate cell behavior through mechanical information transduction. Human periodontal ligament fibroblasts (hPDLF) are sensible to mechanical stimulus and can express pro-inflammatory molecules in response. The aim of this study was to evaluate the impacts of shock waves on interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor-alpha (TNF-α) expression by hPDLF.

Material/methods: After being treated by shock waves with different parameters (100-500 times, 0.05-0.19 mJ/mm(2)), cell viability was tested using CCK-8. IL-6, IL-8, MCP-1, and TNF-α gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and IL-6 and IL-8 protein was measured by enzyme-linked immunosorbent assay (ELISA) at different time points.

Results: Shock waves with the parameters used in this study had no significant effects on the viability of hPDLF. A statistical inhibition of IL-6, IL-8, MCP-1, and TNF-α expression during the first few hours was observed (P<0.05). Expression of IL-8 was significantly elevated in the group receiving the most pulses of shock wave (500 times) after 4 h (P<0.05). At 8 h and 24 h, all treated groups demonstrated significantly enhanced IL-6 expression (P<0.05). TNF-α expression in the groups receiving more shock pulses (300, 500 times) or the highest energy shock treatment (0.19 mJ/mm(2)) was statistically decreased (P<0.05) at 24 h.

Conclusions: Under the condition of this study, a shock wave with energy density no higher than 0.19 mJ/mm(2) and pulses no more than 500 times elicited no negative effects on cell viability of hPDLF. After a uniform initial inhibition impact on expression of inflammatory mediators, a shock wave could cause dose-related up-regulation of IL-6 and IL-8 and down-regulation of TNF-α.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805137PMC
http://dx.doi.org/10.12659/msm.897507DOI Listing
March 2016

VEGF released from a fibrin biomatrix increases VEGFR-2 expression and improves early outcome after ischaemia-reperfusion injury.

J Tissue Eng Regen Med 2017 07 17;11(7):2153-2163. Epub 2016 Jan 17.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Centre, Austrian Cluster for Tissue Regeneration, Vienna, Austria.

Skeletal ischaemia-reperfusion (I-R) injury may influence patient outcome after severe vascular trauma or clamping of major vessels. The aim of this study was to observe whether locally applied vascular endothelial growth factor (VEGF) in fibrin could induce the expression of VEGF-receptor-2 (VEGFR-2) and improve the outcome after I-R injury. Transgenic mice expressing VEGFR-2 promoter-controlled luciferase were used for the assessment of VEGFR-2 expression. Ischaemia was induced for 2 h by a tension-controlled tourniquet to the hind limb, followed by 24 h of reperfusion. The animals were locally injected subcutaneously with fibrin sealant containing 20 or 200 ng VEGF; control animals received no treatment or fibrin sealant application. In vivo VEGFR-2 expression was quantified upon administration of luciferin at several observation times. For oedema and inflammation quantification, wet:dry ratio measurements and a myeloperoxidase assay of the muscle tissue were performed. Laser Doppler imaging showed that ischaemia was present and that the blood flow had returned to baseline levels after 24 h of reperfusion. VEGFR-2 expression levels in the fibrin + 200 ng VEGF were significantly higher than in all other groups. Granulocyte infiltration was reduced in both treatment groups, as well as reduced oedema formation. These results showed that VEGF released from fibrin had a positive effect on early I-R outcome in a mouse model, possibly via VEGFR-2. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.2114DOI Listing
July 2017

Controlled release of fibrin matrix-conjugated platelet derived growth factor improves ischemic tissue regeneration by functional angiogenesis.

Acta Biomater 2016 Jan 20;29:11-20. Epub 2015 Oct 20.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology - Austrian Cluster for Tissue Regeneration, AUVA Research Center, Vienna, Austria.

Unlabelled: Sustained, local, low dose growth factor stimulus of target tissues/cells is believed to be of imminent importance in tissue regeneration and engineering. Recently, a technology was developed to bind growth factors to a fibrin matrix using the transglutaminase (TG) activity of factor XIIIa, thus allowing prolonged release through enzymatic cleavage. In this study we aimed to determine whether TG-PDGF.AB in fibrin could improve tissue regeneration in a standard ischemic flap model. In vitro determination of binding and release kinetics of TG-PDGF.AB allowed proof of concept of the developed binding technology. A single spray application of TG-PDGF.AB in fibrin matrix at a concentration of 10 and 100ng/ml significantly reduced ischemia-induced flap tissue necrosis in vivo on day 7 after ischemic impact compared to controls. TG-PDGF.AB at a concentration of 100ng/ml fibrin induced distinct angiogenesis as reflected by significantly improved tissue perfusion assessed by laser Doppler imaging as well as enhanced von Willebrand factor (vWF) protein expression determined by immunohistochemical means. In addition, significantly more mature microvessels were observed with 100ng/ml TG-PDGF.AB in fibrin compared to control and vehicle groups as evidenced by an improved smooth muscle actin (sma)/vWF protein ratio. In conclusion, PDGF.AB in a conjugated fibrin matrix effectively reduced ischemia-induced tissue necrosis, increased tissue perfusion and induced the growth of a mature and functional neovasculature. The sealing properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by the TG-hook binding technology may present an innovative and suitable tool in tissue regeneration.

Statement Of Significance: In our experimental study we elucidated recombinant platelet derived growth factor (PDGF) as a potential candidate in inducing angiogenesis. To avoid preterm growth factor degradation in vivo PDGF.AB was covalently linked to a fibrin scaffold using a bi-domain functionalized peptide (FXIII substrate site and plasmin cleavage site). This allowed PDGF binding to fibrin during spray application to the donor site and subsequent prolonged release via endogenous plasmin. This resulted in a mature vascular network thus enhancing tissue perfusion and consequently improved clinical outcome. With our present work we could certainly provide researchers and clinicians with an innovative versatile and reproducible technology not only to induce functional vascularity but also to improve attempts in tissue engineering in general by e.g. using different growth factors. Hence, we believe that this approach studied in the present work may provide a valuable input in an effort to drive the aim forward bringing experimental work in tissue engineering to clinic by using a clinically well characterized and used fibrin scaffold in combination with a human recombinant growth factor (fibrin scaffold linked with the specific binding technology).
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http://dx.doi.org/10.1016/j.actbio.2015.10.028DOI Listing
January 2016

Extracorporeal shockwave therapy (ESWT)--First choice treatment of fracture non-unions?

Int J Surg 2015 Dec 9;24(Pt B):179-83. Epub 2015 Oct 9.

Center for Shockwave Medicine and Tissue Engineering, Department of Orthopedic Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.

Fracture non-unions are still a challenging problem in orthopedics. The treatment of non-unions remains highly individualized, complex, and demanding. In most countries the surgical approach with debridement of the non-union gap, anatomical reduction and appropriate osteosynthesis along with autologous bone grafting is considered as the standard of care. One of the very first non-urologic applications of extracorporeal shockwave treatment (ESWT) concerned non-healing fractures. Since the early 1990ties the knowledge of the working mechanism has increased enormously. The purpose of this review article is to demonstrate by peer-reviewed literature in conjunction with our own experiences that ESWT can be an efficient, non-invasive, almost complication-free and cost effective alternative to surgical treatment of non-healing fractures.
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http://dx.doi.org/10.1016/j.ijsu.2015.10.003DOI Listing
December 2015

Current evidence of extracorporeal shock wave therapy in chronic Achilles tendinopathy.

Int J Surg 2015 Dec 29;24(Pt B):154-9. Epub 2015 Aug 29.

Technical University of Munich, Dept. Orthopedics and Traumatology, Ismaninger Straße 22, D-81675 Munich, Germany.

Chronic Achilles tendinopathy has been described as the most common overuse injury in sports medicine. Several treatment modalities such as activity modification, heel lifts, arch supports, stretching exercises, nonsteroidal anti-inflammatories, and eccentric loading are known as standard treatment mostly without proven evidence. After failed conservative therapy, invasive treatment may be considered. Extracorporeal shock wave therapy (ESWT) has been successfully used in soft-tissue pathologies like lateral epicondylitis, plantar fasciitis, tendinopathy of the shoulder and also in bone and skin disorders. Conclusive evidence recommending ESWT as a treatment for Achilles tendinopathy is still lacking. In plantar fasciitis as well as in calcific shoulder tendinopathy shock wave therapy is recently the best evaluated treatment option. This article analysis the evidence based literature of ESWT in chronic Achilles tendinopathy. Recently published data have shown the efficacy of focused and radial extracorporeal shock wave therapy.
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http://dx.doi.org/10.1016/j.ijsu.2015.07.718DOI Listing
December 2015

Extracorporeal shockwave therapy in diabetic foot ulcers.

Int J Surg 2015 Dec 12;24(Pt B):207-9. Epub 2015 Jun 12.

AUVA-Trauma Center Meidling, Vienna, Austria; Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Austrian Cluster for Tissue Regeneration, Vienna, Austria.

Diabetic foot ulcers (DFUs) are among the most common foot disorders with ulceration, infection, and gangrene that may ultimately lead to lower extremity amputation. The goals of treatment include the control of diabetes and proper shoe wear. An effective therapy and appropriate foot care are important in wound healing in DFUs. Recently, extracorporeal shockwave therapy (ESWT) was reported to significantly promote and accelerate the healing of complex soft tissue wounds as compared to the standard methods of treatment in DFUs. ESWT showed positive results in short-term and long-term outcomes in diabetic patients suffering from foot ulcers. In this article, we review the clinical results of ESWT in DFUs.
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http://dx.doi.org/10.1016/j.ijsu.2015.06.024DOI Listing
December 2015

Low level light therapy by LED of different wavelength induces angiogenesis and improves ischemic wound healing.

Lasers Surg Med 2014 Dec 31;46(10):773-80. Epub 2014 Oct 31.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Austrian Cluster for Tissue Regeneration, Vienna, Austria.

Background And Objective: Low-level light therapy (LLLT) has been revealed as a potential means to improve wound healing. So far, most studies are being performed with irradiation in the red to near-infrared spectra. Recently, we showed that blue light (470 nm) can significantly influence biological systems such as nitric oxide (NO) metabolism and is able to release NO from nitrosyl-hemoglobin or mitochondrial protein complexes. Therefore, the aim of this study was to evaluate and compare the therapeutic value of blue or red light emitting diodes (LEDs) on wound healing in an ischemia disturbed rodent flap model.

Study Design/materials And Methods: An abdominal flap was rendered ischemic by ligation of one epigastric bundle and subjected to LED illumination with a wavelength of 470 nm (blue, n = 8) or 629 nm (red, n = 8) each at 50 mW/cm(2) and compared to a non-treated control group (n = 8). Illumination was performed for 10 minutes on five consecutive days.

Results: LED therapy with both wavelengths significantly increased angiogenesis in the sub-epidermal layer and intramuscularly (panniculus carnosus muscle) which was associated with significantly improved tissue perfusion 7 days after the ischemic insult. Accordingly, tissue necrosis was significantly reduced and shrinkage significantly less pronounced in the LED-treated groups of both wavelengths.

Conclusions: LED treatment of ischemia challenged tissue improved early wound healing by enhancing angiogenesis irrespective of the wavelength thus delineating this noninvasive means as a potential, cost effective tool in complicated wounds.
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http://dx.doi.org/10.1002/lsm.22299DOI Listing
December 2014

Thrombin as important factor for cutaneous wound healing: comparison of fibrin biomatrices in vitro and in a rat excisional wound healing model.

Wound Repair Regen 2014 Nov-Dec;22(6):740-8. Epub 2015 Jan 8.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria; Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria.

Fibrin biomatrices have been used for many years for hemostasis and sealing and are a well-established surgical tool. The objective of the present study was to compare two commercially available fibrin biomatrices regarding the effect of their thrombin concentration on keratinocytes and wound healing in vitro and in vivo. Keratinocytes showed significant differences in adhesion, viability, and morphology in the presence of the fibrin matrices in vitro. A high thrombin concentration (800-1,200 IU/mL) caused deteriorated cell compatibility. By using a thrombin inhibitor, those differences could be reversed. In a rat excisional wound healing model, we observed more rapid wound closure and less wound severity in wounds treated with a fibrin matrix containing a lower concentration of thrombin (4 IU/mL). Furthermore, fewer new functional vessels and a lower level of vascular endothelial growth factor were measured in wounds after 7 days treated with the matrix with higher thrombin concentration. These in vivo results may be partially explained by the in vitro biocompatibility data. Additionally, results show that low thrombin biomatrices were degraded faster than the high thrombin material. Hence, we conclude that the composition of fibrin biomatrices influences keratinocytes and therefore has an impact on wound healing.
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http://dx.doi.org/10.1111/wrr.12234DOI Listing
December 2015

In vitro extracorporeal shock wave treatment enhances stemness and preserves multipotency of rat and human adipose-derived stem cells.

Cytotherapy 2014 Dec 28;16(12):1666-78. Epub 2014 Aug 28.

Austrian Cluster for Tissue Regeneration, Vienna, Austria; University of Applied Sciences Technikum Wien-Department of Biochemical Engineering, Vienna, Austria.

Background Aims: Adipose-derived progenitor/stem cells (ASCs) are discussed as a promising candidate for various tissue engineering approaches. However, its applicability for the clinic is still difficult due to intra- and inter-donor heterogeneity and limited life span in vitro, influencing differentiation capacity as a consequence to decreased multipotency.

Methods: Extracorporeal shock wave treatment has been proven to be a suitable clinical tool to improve regeneration of a variety of tissues for several decades, whereas the mechanisms underlying these beneficial effects remain widely unknown.

Results: In this study we show that human and rat adipose derived stem cells respond strongly to repetitive shock wave treatment in vitro, resulting not only in maintenance and significant elevation of mesenchymal markers (CD73, CD90, CD105), but also in significantly increased differentiation capacity towards the osteogenic and adipogenic lineage as well as toward Schwann-cell like cells even after extended time in vitro, preserving multipotency of ASCs.

Conclusions: ESWT might be a promising tool to improve ASC quality for cell therapy in various tissue engineering and regenerative medicine applications.
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http://dx.doi.org/10.1016/j.jcyt.2014.07.005DOI Listing
December 2014

Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation.

J Biol Chem 2014 Sep 12;289(39):27090-27104. Epub 2014 Aug 12.

Department of Biochemical Engineering, University of Applied Sciences Technikum Wien, 1200 Vienna, Austria,; The Austrian Cluster for Tissue Regeneration, Vienna, Austria.

Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.
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http://dx.doi.org/10.1074/jbc.M114.580936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175346PMC
September 2014

Long-lasting fibrin matrices ensure stable and functional angiogenesis by highly tunable, sustained delivery of recombinant VEGF164.

Proc Natl Acad Sci U S A 2014 May 28;111(19):6952-7. Epub 2014 Apr 28.

Cell and Gene Therapy, Department of Biomedicine, University of Basel, and Department of Surgery, Basel University Hospital, CH-4031 Basel, Switzerland;

Clinical trials of therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery failed to show efficacy. Major challenges include the need to precisely control in vivo distribution of growth factor dose and duration of expression. Recombinant VEGF protein delivery could overcome these issues, but rapid in vivo clearance prevents the stabilization of induced angiogenesis. Here, we developed an optimized fibrin platform for controlled delivery of recombinant VEGF, to robustly induce normal, stable, and functional angiogenesis. Murine VEGF164 was fused to a sequence derived from α2-plasmin inhibitor (α2-PI1-8) that is a substrate for the coagulation factor fXIIIa, to allow its covalent cross-linking into fibrin hydrogels and release only by enzymatic cleavage. An α2-PI1-8-fused variant of the fibrinolysis inhibitor aprotinin was used to control the hydrogel degradation rate, which determines both the duration and effective dose of factor release. An optimized aprotinin-α2-PI1-8 concentration ensured ideal degradation over 4 wk. Under these conditions, fibrin-α2-PI1-8-VEGF164 allowed exquisitely dose-dependent angiogenesis: concentrations ≥25 μg/mL caused widespread aberrant vascular structures, but a 500-fold concentration range (0.01-5.0 μg/mL) induced exclusively normal, mature, nonleaky, and perfused capillaries, which were stable after 3 mo. Optimized delivery of fibrin-α2-PI1-8-VEGF164 was therapeutically effective both in ischemic hind limb and wound-healing models, significantly improving angiogenesis, tissue perfusion, and healing rate. In conclusion, this optimized platform ensured (i) controlled and highly tunable delivery of VEGF protein in ischemic tissue and (ii) stable and functional angiogenesis without introducing genetic material and with a limited and controllable duration of treatment. These findings suggest a strategy to improve safety and efficacy of therapeutic angiogenesis.
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http://dx.doi.org/10.1073/pnas.1404605111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4024904PMC
May 2014
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