Electrical stimulation in bone tissue engineering treatments Leppik, Liudmila; Oliveira, Karla Mychellyne Costa; Bhavsar, Mit Balvantray ...
European journal of trauma and emergency surgery (Munich : 2007),
04/2020, Letnik:
46, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Electrical stimulation (EStim) has been shown to promote bone healing and regeneration both in animal experiments and clinical treatments. Therefore, incorporating EStim into promising new bone ...tissue engineering (BTE) therapies is a logical next step. The goal of current BTE research is to develop combinations of cells, scaffolds, and chemical and physical stimuli that optimize treatment outcomes. Recent studies demonstrating EStim’s positive osteogenic effects at the cellular and molecular level provide intriguing clues to the underlying mechanisms by which it promotes bone healing. In this review, we discuss results of recent in vitro and in vivo research focused on using EStim to promote bone healing and regeneration and consider possible strategies for its application to improve outcomes in BTE treatments. Technical aspects of exposing cells and tissues to EStim in in vitro and in vivo model systems are also discussed.
Sepsis as a severe systemic inflammation leads oftentimes to organ dysfunction and subsequently to death. In polytrauma patients, septic complications represent with 45% the predominant cause of late ...death and are responsible for extremely high costs in the healthcare system. Therefore, clinicians have to detect as early as possible the begin of sepsis to improve the patient's outcome. One new promising diagnostic tool to diagnose septic complications in polytraumatized patients are exosomes.
Plasma samples from polytraumatized patients (Injury Severity Score (ISS) ≥16) which developed sepsis (n = 10) and without sepsis (n = 10), were collected at emergency room (ER), 24h and 5 days after trauma. The EVs subpopulations were investigated by a bead-based multiplex flow cytometry measurement of surface epitopes and were compared with plasma EVs from healthy controls (n = 10). Moreover, exosomal cytokine concentrations were measured via high-sensitive ELISA and were correlated with systemic concentrations. For miRNA cargo analysis, we analysed the miRNAs miR-1298-5p, miR-1262, miR-125b-5p, miR-92a-3p, miR-93-5p, miR-155-5p and miR-21-5p and compared their exosomal concentrations by means of RT-qPCR.
CD62p + exosomes were significantly increased in septic polytrauma-patients (p ≤ 0.05), while CD40+exosomes, as well as CD49e + exosomes were diminished (p ≤ 0.05). Furthermore, we observed that the exosomal IL-6 concentration reflects the systemic IL-6 concentration (r2 = 0.63) and did not significantly alter between patients with and without sepsis. The exosomal IL-10 concentration seemed to be constant in all patients and healthy controls. We observed that a decrease of miR-21-5p in exosomes was associated with the development of sepsis (p ≤ 0.05), while exosomal miR-93-5p, miR-155-5p and miR-92a-3p were not specifically altered in septic patients.
Taken together, the present study in polytraumatized patients demonstrated that the development of sepsis is associated with an increase of CD62p + exosomes. Furthermore, the exosomal cargo was changed in septic patients: miR-21-5p was diminished.
•CD62p + exosomes were significantly increased in septic polytrauma-patients, while CD40+, as well as CD49e + exosomes were diminished.•Exosomal IL-6 concentration in septic patients reflects the systemic IL-6.•Exosomal IL-10 concentration seemed to be constant in patients and healthy controls.•Decrease of miR-21 in exosomes was associated with the development of sepsis, while exosomal miR-93, miR-155 and miR-92a were not specifically altered.
A macrophage shift from the M1 to the M2 phenotype is relevant for promoting tissue repair and regeneration. In a previous in vivo study, we found that direct current (DC) electrical stimulation ...(EStim) increased the proportion of M2 macrophages in healing tissues and directed the balance of the injury response away from healing/scarring towards regeneration. These observations led us to hypothesize that DC EStim regulates macrophage polarization towards an M2 phenotype. THP-1-derived M0, M1 (IFN-γ and LPS), and M2 (IL-4 and IL-13) macrophages were exposed (or not: control group) to 100 mV/mm of DC EStim, 1 h/day for three days. Macrophage polarization was assessed through gene and surface marker expressions and cytokine secretion profiles. Following DC EStim treatment, M0 cells exhibited an upregulation of M2 marker genes IL10, CD163, and PPARG. In M1 cells, DC EStim upregulated the gene expressions of M2 markers IL10, TGM2, and CD206 and downregulated M1 marker gene CD86. EStim treatment also reduced the surface expression of CD86 and secretion of pro-inflammatory cytokines IL-1β and IL-6. Our results suggest that DC EStim differentially exerts pro-M2 effects depending on the macrophage phenotype: it upregulates typical M2 genes in M0 and M1 cells while inhibiting M1 marker CD86 at the nuclear and protein levels and the secretion of pro-inflammatory interleukins in M1 cells. Conversely, M2 cells appear to be less responsive to the EStim treatment employed in this study.
Background
Hemorrhagic shock (HS) is responsible for approximately 2 million deaths per year worldwide and is caused in 80% by polytrauma. These patients need a precise and quick diagnostic, which ...should be based on a combination of laboratory markers and radiological data. Extracellular vesicles (EVs) were described as potential new markers and mediators in trauma. The aim of the present study was to analyze, whether the surface epitopes of plasma-EVs reflect HS in polytraumatized patients and whether cell-specific EV subpopulations are useful diagnostic tools.
Material and methods
Plasma samples from polytraumatized patients (ISS ≥16) with HS (n=10) and without (n=15), were collected at emergency room (ER) and 24h after trauma. Plasma-EVs were isolated via size exclusion chromatography and EV-concentrations were detected by Coomassie Plus (Bradford) Assay. The EVs subpopulations were investigated by a bead-based multiplex flow cytometry measurement of surface epitopes and were compared with healthy controls (n=10). To investigate the diagnostic and prognostic potential of EVs subpopulations, results were correlated with clinical outcome parameters documented in the electronical patients’ record.
Results
We observed a significant reduction of the total amount of plasma EVs in polytrauma patients with HS, as compared to polytrauma patients without HS and healthy controls. We found significant reduction of CD42a+ and CD41b+ (platelet-derived) EVs in all polytrauma patients, as well as a reduction of CD29+ EVs compared to healthy volunteers (*p<0.05). CD44+ and CD31+ EVs were specifically altered in patients with HS (*p<0.05). Both EV populations showed a moderate correlation (r² = 0.42) with the transfusion of erythrocyte concentrate, were associated with non-survival and the need for catecholamines (*p<0.05).
Conclusion
Our data reveal that polytrauma patients with a hemorrhagic shock are characterized by a reduction of CD44+ and CD31+ plasma-EVs. Both EV populations showed a moderate correlation with the need of erythrocyte transfusion, were associated with non-survival and the need for catecholamines.
This study was designed to characterize morphologic stages during neuroma development post amputation with an eye toward developing better treatment strategies that intervene before neuromas are ...fully formed. Right forelimbs of 30 Sprague Dawley rats were amputated and limb stumps were collected at 3, 7, 28, 60 and 90 Days Post Amputation (DPA). Morphology of newly formed nerves and neuromas were assessed via general histology and neurofilament protein antibody staining. Analysis revealed six morphological characteristics during nerve and neuroma development; 1) normal nerve, 2) degenerating axons, 3) axonal sprouts, 4) unorganized bundles of axons, 5) unorganized axon growth into muscles, and 6) unorganized axon growth into fibrotic tissue (neuroma). At early stages (3 & 7 DPA) after amputation, normal nerves could be identified throughout the limb stump and small areas of axonal sprouts were present near the site of injury. Signs of degenerating axons were evident from 7 to 90 DPA. From day 28 on, variability of nerve characteristics with signs of unorganized axon growth into muscle and fibrotic tissue and neuroma formation became visible in multiple areas of stump tissue. These pathological features became more evident on days 60 and 90. At 90 DPA frank neuroma formation was present in all stump tissue. By following nerve regrowth and neuroma formation after amputation we were able to identify 6 separate histological stages of nerve regrowth and neuroma development. Axonal regrowth was observed as early as 3 DPA and signs of unorganized axonal growth and neuroma formation were evident by 28 DPA. Based on these observations we speculate that neuroma treatment and or prevention strategies might be more successful if targeted at the initial stages of development and not after 28 DPA.
Trauma remains a leading cause of morbidity and mortality. Polytraumatized patients need a precise, early diagnosis to avoid complications such as multiorgan failure or sepsis. Inflammatory ...cytokines, commonly used for diagnosis, have a short half-life, which limits their efficacy as a diagnostic or prognostic marker. In this study, we hypothesized that cytokines in exosomes could have a longer half-life, and therefore could be used as diagnostic and prognostic markers in polytrauma patients. Plasma samples from polytraumatized patients (ISS ≥ 16,
= 18) were collected in the emergency room (ER) 1, 2, 3 and 5 days after trauma. Plasma-exosomes were isolated via size exclusion chromatography from polytraumatized patients and healthy volunteers (
= 10). The systemic and exosomal concentrations of interleukin (IL)-6, IL-10, IL-1β and TNF were measured using high-sensitive ELISAs. To investigate the diagnostic and prognostic potential of exosomal cytokines, data were correlated with clinical outcome parameters (injury severity, ventilation time, time in ICU and survival) documented in the patients' electronic records. Despite the use of high-sensitive ELISAs, IL-1β and TNF alpha were not detected in exosomes. IL-6 and IL-10 were detectable in polytraumatized patient exosomes at all time points. A decrease over time of both systemic and exosomal IL-6 concentrations was observed. Furthermore, exosomal and systemic IL-6 concentrations moderately correlated (r = 0.63). Exosomal IL-6 in the ER moderately correlated with the Injury Severity Score (ISS) (mean 35.5 ± 11.5) (r = 0.45) and was associated with non-survival in polytrauma patients (
< 0.05). In contrast to IL-6, no correlation between systemic and exosomal IL-10 concentrations was found. Exosomal IL-10 concentrations remained unchanged throughout the observation time, whereas systemic IL-10 concentrations peaked in the ER and were significantly reduced after 24 h. Data from this study support our hypothesis that some cytokines (IL-10), but not all (IL-6), are detectable in exosomes significantly longer than they are in plasma. This might indicate that they are protected from degradation. Although we did not find a correlation between IL-10 exosomal concentration and patient outcome, our data confirm that exosomal cytokines are of interest as potential diagnostic and prognostic markers in polytrauma patients, and require further detailed research.
Electrical stimulation (ES) has been successfully used to treat bone defects clinically. Recently, both cellular and molecular approaches have demonstrated that ES can change cell behavior such as ...migration, proliferation and differentiation.
In the present study we exposed rat bone marrow- (BM-) and adipose tissue- (AT-) derived mesenchymal stem cells (MSCs) to direct current electrical stimulation (DC ES) and assessed temporal changes in osteogenic differentiation. We applied 100 mV/mm of DC ES for 1 h per day for three, seven and 14 days to cells cultivated in osteogenic differentiation medium and assessed viability and calcium deposition at the different time points. In addition, expression of osteogenic genes, Runx2, Osteopontin, and Col1A2 was assessed in BM- and AT-derived MSCs at the different time points.
Results showed that ES changed osteogenic gene expression patterns in both BM- and AT-MSCs, and these changes differed between the two groups. In BM-MSCs, ES caused a significant increase in mRNA levels of Runx2, Osteopontin and Col1A2 at day 7, while in AT-MSCs, the increase in Runx2 and Osteopontin expression were observed after 14 days of ES.
This study shows that rat bone marrow- and adipose tissue-derived stem cells react differently to electrical stimuli, an observation that could be important for application of electrical stimulation in tissue engineering.
Limb loss is a devastating disability and while current treatments provide aesthetic and functional restoration, they are associated with complications and risks. The optimal solution would be to ...harness the body's regenerative capabilities to regrow new limbs. Several methods have been tried to regrow limbs in mammals, but none have succeeded. One such attempt, in the early 1970s, used electrical stimulation and demonstrated partial limb regeneration. Several researchers reproduced these findings, applying low voltage DC electrical stimulation to the stumps of amputated rat forelimbs reporting "blastema, and new bone, bone marrow, cartilage, nerve, skin, muscle and epiphyseal plate formation". In spite of these encouraging results this research was discontinued. Recently there has been renewed interest in studying electrical stimulation, primarily at a cellular and subcellular level, and studies have demonstrated changes in stem cell behavior with increased proliferation, differentiation, matrix formation and migration, all important in tissue regeneration. We applied electrical stimulation, in vivo, to the stumps of amputated rat limbs and observed significant new bone, cartilage and vessel formation and prevention of neuroma formation. These findings demonstrate that electricity stimulates tissue regeneration and form the basis for further research leading to possible new treatments for regenerating limbs.
Trauma is still a leading cause of morbidity and mortality, especially in the younger population. Trauma patients need a precise, early diagnostic to avoid complications like multiorgan failure and ...sepsis. Exosomes were described as markers and mediators in trauma. The aim of the present study was to analyze, whether the surface epitopes of plasma-exosomes can reflect the injury pattern in polytrauma.
Polytraumatized patients (Injury Severity Score = ISS ≥16, n = 38) were subdivided according to the predominant injury in either abdominal trauma, chest trauma or traumatic brain injury (TBI). Plasma exosomes were isolated via size exclusion chromatography. The concentration and size distribution of the plasma exosomes from emergency room samples were measured by nanoparticle tracking analysis. The exosomal surface antigens were investigated by bead-based multiplex flow cytometry and compared with healthy controls (n=10).
In contrast to other studies, we did not observe an increase in the total amount of plasma exosomes in polytrauma patients (1,15x109 vs. 1,13x109 particles/ml), but found changes in the exosomal surface epitopes. We found a significant reduction of CD42a+ (platelet-derived) exosomes in polytrauma patients, CD209+ (dendritic cell-derived) exosomes in the patients with predominant abdominal trauma, and CD11+ (monocyte-derived) exosomes in the patients with chest trauma. The group of patients with TBI was characterized in contrast by an increase of CD62p+ (endothelial/platelet-derived) exosomes (*p<0.05).
Our data showed that the polytrauma injury pattern might be reflected by the cellular origin/surface epitopes of plasma-released exosomes immediately after trauma. The observed reduction of CD42+ exosomes in polytrauma patients was not associated with a reduction of total platelets in polytrauma patients.
The goal of this study was to identify changes in extracellular vesicles (EV) surface proteins specific to traumatic brain injury (TBI), which could be used as a diagnostic and prognostic tool in ...polytrauma patients.
Known serum TBI-specific biomarkers (S100B, NSE, and GFAP), which can predict the severity and outcome of isolated TBI, lose their predictive value in the presence of additional extracranial injuries. Extracellular vesicles (EVs) are released from cells in response to various stimuli and carry specific cargo/surface molecules that could be used for tracking injury-responding cells.
EVs were isolated using size exclusion chromatography (SEC) from the plasma of two groups of patients (with isolated TBI, ISS≥16, AIShead≥4, n=10; and polytraumatized patients without TBI ISS≥16, AIShead=0, n=10) collected in the emergency room and 48 h after trauma. EVs' surface epitope expression was investigated using a neurospecific multiplex flow cytometry assay and compared with healthy controls (n=10). Three enrichments of EV epitopes found to be specific to TBI were validated by western blot.
The expression of 10 EV epitopes differed significantly among the patient and control groups, and five of these epitopes (CD13, CD196, MOG, CD133, and MBP) were TBI-specific. The increased expression of CD196, CD13, and MOG-positive EVs was validated by western blot.
Our data showed that TBI is characterized by a significant increase of CD13, CD196, MOG, CD133, and MBP-positive EVs in patients' plasma. A high level of MOG-positive EVs negatively correlated with the Glasgow Coma Scale score at admission and could be an indicator of poor neurological status.
PDF
