Wavelength-Dependent Effects of Photobiomodulation for Wound Care in Diabetic Wounds.

Photobiomodulation, showing positive effects on wound healing processes, has been performed mainly with lasers in the red/infrared spectrum. Light of shorter wavelengths can significantly influence biological systems. This study aimed to evaluate and compare the therapeutic effects of pulsed LED light of different wavelengths on wound healing in a diabetic (db/db) mouse excision wound model. LED therapy by Repuls was applied at either 470 nm (blue), 540 nm (green) or 635 nm (red), at 40 mW/cm2 each. Wound size and wound perfusion were assessed and correlated to wound temperature and light absorption in the tissue. Red and trend-wise green light positively stimulated wound healing, while blue light was ineffective. Light absorption was wavelength-dependent and was associated with significantly increased wound perfusion as measured by laser Doppler imaging. Shorter wavelengths ranging from green to blue significantly increased wound surface temperature, while red light, which penetrates deeper into tissue, led to a significant increase in core body temperature. In summary, wound treatment with pulsed red or green light resulted in improved wound healing in diabetic mice. Since impeded wound healing in diabetic patients poses an ever-increasing socio-economic problem, LED therapy may be an effective, easily applied and cost-efficient supportive treatment for diabetic wound therapy.

ESWT Diminishes Axonal Regeneration following Repair of the Rat Median Nerve with Muscle-In-Vein Conduits but Not after Autologous Nerve Grafting.

Investigations reporting positive effects of extracorporeal shockwave therapy (ESWT) on nerve regeneration are limited to the rat sciatic nerve model. The effects of ESWT on muscle-in-vein conduits (MVCs) have also not been investigated yet. This study aimed to evaluate the effects of ESWT after repair of the rat median nerve with either autografts (ANGs) or MVCs. In male Lewis rats, a 7 mm segment of the right median nerve was reconstructed either with an ANG or an MVC. For each reconstructive technique, one group of animals received one application of ESWT while the other rats served as controls. The animals were observed for 12 weeks, and nerve regeneration was assessed using computerized gait analysis, the grasping test, electrophysiological evaluations and histological quantification of axons, blood vessels and lymphatic vasculature. Here, we provide for the first time a comprehensive analysis of ESWT effects on nerve regeneration in a rat model of median nerve injury. Furthermore, this study is among the first reporting the quantification of lymphatic vessels following peripheral nerve injury and reconstruction in vivo. While we found no significant direct positive effects of ESWT on peripheral nerve regeneration, results following nerve repair with MVCs were significantly inferior to those after ANG repair.

Effects of Extracorporeal Shockwave Therapy on Functional Recovery and Circulating miR-375 and miR-382-5p after Subacute and Chronic Spinal Cord Contusion Injury in Rats.

Extracorporeal shockwave therapy (ESWT) can stimulate processes to promote regeneration, including cell proliferation and modulation of inflammation. Specific miRNA expression panels have been established to define correlations with regulatory targets within these pathways. This study aims to investigate the influence of low-energy ESWT-applied within the subacute and chronic phase of SCI (spinal cord injury) on recovery in a rat spinal cord contusion model. Outcomes were evaluated by gait analysis, µCT and histological analysis of spinal cords. A panel of serum-derived miRNAs after SCI and after ESWT was investigated to identify injury-, regeneration- and treatment-associated expression patterns. Rats receiving ESWT showed significant improvement in motor function in both a subacute and a chronic experimental setting. This effect was not reflected in changes in morphology, µCT-parameters or histological markers after ESWT. Expression analysis of various miRNAs, however, revealed changes after SCI and ESWT, with increased miR-375, indicating a neuroprotective effect, and decreased miR-382-5p potentially improving neuroplasticity via its regulatory involvement with BDNF. We were able to demonstrate a functional improvement of ESWT-treated animals after SCI in a subacute and chronic setting. Furthermore, the identification of miR-375 and miR-382-5p could potentially provide new targets for therapeutic intervention in future studies.

Improved biomechanics in experimental chronic rotator cuff repair after shockwaves is not reflected by bone microarchitecture.

PURPOSE: The aim of this study was to investigate the effect of extracorporeal shockwave therapy (ESWT) on bone microstructure as well as the bone-tendon-interface and the musculo-tendinous transition zone to explain the previously shown improved biomechanics in a degenerative rotator cuff tear animal model. This study hypothesized that biomechanical improvements related to ESWT are a result of improved bone microstructure and muscle tendon properties. METHODS: In this controlled laboratory study unilateral supraspinatus (SSP) tendon detachment was performed in 48 male Sprague-Dawley rats. After a degeneration period of three weeks, SSP tendon was reconstructed transosseously. Rats were randomly assigned into three groups (n = 16 per group): control (noSW); intraoperative shockwave treatment (IntraSW); intra- and postoperative shockwave treatment (IntraPostSW). Eight weeks after SSP repair, all rats were sacrificed and underwent bone microstructure analysis as well as histological and immunohistochemical analyses. RESULTS: With exception of cortical porosity at the tendon area, bone microstructure analyses revealed no significant differences between the three study groups regarding cortical and trabecular bone parameters. Cortical Porosity at the Tendon Area was lowest in the IntraPostSW (p≤0.05) group. Histological analyses showed well-regenerated muscle and tendon structures in all groups. Immunohistochemistry detected augmented angiogenesis at the musculo-tendinous transition zone in both shockwave groups indicated by CD31 positive stained blood vessels. CONCLUSION: In conclusion, bone microarchitecture changes are not responsible for previously described improved biomechanical results after shockwave treatment in rotator cuff repair in rodents. Immunohistochemical analysis showed neovascularization at the musculo-tendinous transition zone within ESWT-treated animals. Further studies focusing on neovascularization at the musculo-tendinous transition zone are necessary to explain the enhanced biomechanical and functional properties observed previously. CLINICAL RELEVANCE: In patients treated with a double-row SSP tendon repair, an improvement in healing through ESWT, especially in this area, could prevent a failure of the medial row, which is considered a constantly observed tear pattern.

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

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.

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

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, N = 13; fibrin sealant, N = 14; PBMCsec, N = 12). PBMCsec was produced according to good manufacturing practices (GMP) guidelines and 2 ml were administered intraoperatively at a concentration of 2.5 × 107 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, p = .053 vs. control; PBMCsec: 19.1% ± 7.2; 31.1% reduction, p = .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, p = .651, vs. control; PBMCsec: 85.9 ± 20.4; 22.2% increase, p = .045 vs. control; 26.7% increase, p = .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, p = .001 vs. control; 62.8% reduction, p = .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.

A Comparative Efficacy Evaluation of Recombinant Topical Thrombin (RECOTHROM®) With A Gelatin Sponge Carrier Versus Topical Oxidized Regenerated Cellulose (TABOTAMP®/SURGICEL®) In A Porcine Liver Bleeding Model.

PURPOSE: Topical hemostatic agents can be classified as active or passive. This study compared the hemostatic efficacy of an active agent, recombinant thrombin (RECOTHROM® [rT]) plus gelatin sponge carrier versus a passive agent, oxidized regenerated cellulose (TABOTAMP®/SURGICEL® [ORC]), in a porcine liver abrasion model. MATERIALS AND METHODS: Eight pigs were used, four of them were heparinized. A total of 80 liver lesions were created, 40 of them in heparinized pigs. Lesions were treated with rT plus gelatin sponge or ORC. Bleeding rate was quantified before treatment by applying pre-weighed gauze. Time to hemostasis was assessed visually for 10 minutes. RESULTS: Seven of the 80 lesions were excluded for having initial bleeding rates exceeding the target of 10 g/min. Sixteen and 20 lesions were treated with rT plus gelatin sponge and 19 and 18 lesions were treated with ORC, in non-heparinized and heparinized animals, respectively. Time to hemostasis (median [IQR]) was significantly shorter with rT plus gelatin sponge (30 [30,30] seconds) in heparinized and non-heparinized animals versus ORC in non-heparinized (180 [120,210] seconds) and heparinized animals (215 [135,345] seconds); P < 0.0001 for both comparisons. In heparinized animals, ORC took longer to achieve hemostasis, with treatment failure in 2/18 lesions. Time to hemostasis with ORC was longer for lesions in heparinized animals with initial bleeding rates of >5-10 g/min (285 [225,394] seconds) versus ≤5 g/min (175 [108,290] seconds). CONCLUSIONS: In this model, rT plus gelatin sponge carrier (active) was a more effective hemostat than ORC (passive) in both heparinized and non-heparinized animals.

Circulating miRNAs Associated With ER Stress and Organ Damage in a Preclinical Model of Trauma Hemorrhagic Shock.

Circulating microRNAs (miRNA) alterations have been reported in severe trauma patients but the pathophysiological relevance of these changes is still unclear. miRNAs are critical biologic regulators of pathological events such as hypoxia and inflammation, which are known to induce endoplasmic reticulum (ER) stress. ER stress is emerging as an important process contributing to the development of single and/or multiple organ dysfunction after trauma hemorrhagic shock (THS) accompanied by impaired tissue microcirculation and inflammation. Here, we aim to bring new insights into the involvement of miRNAs associated with ER stress in THS. THS was induced in rats by a median laparotomy and blood withdrawal until mean arterial pressure (MAP) dropped to 30-35 mmHg followed by a restrictive (40 min) and full reperfusion (60 min) with Ringer's solution. Tunicamycin was used to induce ER stress. Blood samples were collected 24 h after THS for the determination of pathological changes in the blood (PCB) and circulating miRNAs. Plasma levels of circulating miRNAs were compared between THS, tunicamycin, and sham groups and correlated to biomarkers of PCB. MiRNA profile of THS animals showed that 40 out of 91 (44%) miRNAs were significantly upregulated compared to sham (p < 0.01). The data showed a very strong correlation between liver injury and miR-122-5p (r = 0.91, p < 0.00001). MiR-638, miR-135a-5p, miR-135b-5p, miR-668-3p, miR-204-5p, miR-146a-5p, miR-200a-3p, miR-17-5p, miR-30a-5p, and miR-214-3p were found positively correlated with lactate (r > 0.7, p < 0.05), and negatively with base excess (r ≤ 0.8, p < 0.05) and bicarbonate (r ≤ 0.8, p < 0.05), which are clinical parameters that reflected the shock severity. Tunicamycin significantly modified the microRNA profile of the animals, 33 out of 91 miRNAs were found differentially expressed. In addition, principal component analysis revealed that THS and tunicamycin induced similar changes in plasma miRNA patterns. Strikingly, the data showed that 15 (25.9%) miRNAs were regulated by both THS and tunicamycin (p < 0.01). This included miR-122-5p, a liver-specific microRNA, but also miR-17-5p and miR-125b-5p which are miRNAs remarkably involved in unfolded protein response (UPR)-mediating pro-survival signaling (IRE1α). Since miRNAs associated with ER stress are clearly correlated with THS, our data strongly suggest that interaction between miRNAs and ER stress is an important pathologic event occurring during THS. Overall, we consider that the miRNA profile developed in this study can provide a rationale for the development of bench-to-bedside strategies that target miRNAs in critical care diseases or be used as biomarkers in the prognosis of trauma patients.

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

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 (P = .005) and the medial humeral head (P = .010) compared with the control group. Trabecular number on the operated side was higher at the lateral humeral head (P = .004) and the medial humeral head (P = .001) in the intervention group. Maximum load to failure rates on the operated side were higher in the intervention group (P < .001). Cortical thickness positively correlated with higher maximum load to failure rates in the intervention group (r = 0.69; P = .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.

Evaluation of Functional Recovery in Rats After Median Nerve Resection and Autograft Repair Using Computerized Gait Analysis.

Computerized gait analysis is a common evaluation method in rat models of hind limb nerve injuries, but its use remains unpublished in models of segmental nerve injury of the forelimb. It was the aim of this work to investigate if computerized gait analysis is a feasible evaluation method in a rat model of segmental median nerve injury and autograft repair. Ten male Lewis rats underwent 7-mm resection of the right median nerve with immediate autograft repair. The left median nerve was resected without repair and served as an internal control. Animals were assessed for 12 weeks after surgery via CatWalk (CW) gait analysis every 2 weeks. Evaluation of motor recovery by means of the grasping test was performed weekly while electrophysiological measurements were performed at the end of the observation period. CW data were correlated with grasping strength at each post-operative time point. CW data were also correlated with electrophysiology using linear regression analysis. Principal component analysis was performed to identify clusters of outcome metrics. Recovery of motor function was observable 4 weeks after surgery, but grasping strength was significantly reduced (p < 0.01) compared to baseline values until post-operative week 6. In terms of sensory recovery, the pain-related parameter Duty Cycle showed significant (p < 0.05) recovery starting from post-operative week 8. The Print Area of the right paw was significantly (p < 0.05) increased compared to the left side starting from post-operative week 10. Various parameters of gait correlated significantly (p < 0.05) with mean and maximum grasping strength. However, only Stand Index showed a significant correlation with compound muscle action potential (CMAP) amplitude (p < 0.05). With this work, we prove that computerized gait analysis is a valid and feasible method to evaluate functional recovery after autograft repair of the rat median nerve. We were able to identify parameters such as Print Area, Duty Cycle, and Stand Index, which allow assessment of nerve regeneration. The course of these parameters following nerve resection without repair was also assessed. Additionally, external paw rotation was identified as a valid parameter to evaluate motor reinnervation. In summary, computerized gait analysis is a valuable additional tool to study nerve regeneration in rats with median nerve injury.

An Efficacy Comparison of Two Hemostatic Agents in a Porcine Liver Bleeding Model: Gelatin/Thrombin Flowable Matrix versus Collagen/Thrombin Powder.

Purpose: Management of bleeding during surgery can be aided by the application of topical hemostatic agents. This study compared the hemostatic efficacy of a new powder agent containing collagen, chondroitin sulfate, and thrombin (PCCT) with a flowable gelatin-thrombin matrix with smooth particles (SmGM) in a porcine liver bleeding model. Materials and Methods: Lesions 4-6 mm deep and ∼10 mm in diameter were created in porcine livers and treated with either SmGM or PCCT. Bleeding rate and grade were quantified before and 3, 7, and 11 minutes after treatment. Results: Thirty-two lesions each were treated with SmGM or PCCT; the median (Q1, Q3) initial bleeding rate was comparable between the two groups (8.43 [6.18, 10.68] g/min and 7.15 [5.16, 9.63] g/min, respectively). The residual bleeding rate was significantly lower at all time-points post treatment for SmGM compared with PCCT (3 minutes: 0.14 [0.07, 0.21] versus 0.46 [0.20, 1.20] g/min, p < 0.0001; 7 minutes: 0.07 [0.04, 0.11] versus 0.12 [0.08, 0.39] g/min, p = 0.001; 11 minutes: 0.05 [0.03, 0.08] versus 0.07 [0.05, 0.12] g/min, p = 0.043). Bleeding grade at 3 minutes was also significantly lower for SmGM compared with PCCT (median [Q1, Q3] 0.0 [0.0, 0.0] versus 1.0 [1.0, 2.0], p < 0.0001). PCCT required reapplication in approximately one-third of applications due to insufficient hemostasis 4 minutes after initial application and showed a tendency to stick to the wet gauze during approximation. Conclusions: In this bleeding model, treatment with SmGM resulted in reduced blood loss, no need for reapplication and was easier to apply compared with PCCT.

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

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, P≤ .001; IntraPostESWT vs control, P≤ .05). Furthermore, functional gait analyses showed a significant enhancement in intensity measurements for the IntraPostESWT group in comparison with the control group (P≤ .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.

Efficacy of Topical Hemostatic Agents: A Comparative Evaluation of Two Gelatin/Thrombin-Based Hemostatic Matrices in a Porcine Kidney Surgical Model.

Purpose: Topical hemostatic agents are an important means of controlling or preventing bleeding. This study was performed to compare gelatin-thrombin matrix with smooth particles (SmGM) versus gelatin-thrombin matrix with stellate particles (StGM) in a porcine kidney bleeding model. Materials and methods: In male pigs, reproducible lesions (diameter and depth ∼10 mm) were created in the renal cortex. Each lesion was treated topically using either SmGM or StGM. Blood loss was quantified before and 2, 5 and 10 minutes after treatment. Dry mass, ultrastructural and histologic analyses were also performed. Results: Thirty-two lesions were treated with SmGM and 32 with StGM; median initial bleeding rates were 27.6 and 29.1 mL/min, respectively. Two minutes post-application, SmGM was associated with significantly less bleeding than StGM (0.574 vs 0.920 mL/min; p < .0001). This difference stemmed principally from lesions with initial blood loss >29 mL/min, where bleeding rates at 2 minutes were ∼3-fold higher with StGM (1.636 vs 0.567 mL/min; p ≥ 0.040). Dry mass per unit volume of hemostatic agent was significantly higher with SmGM versus StGM. SmGM formed discrete, smooth particles, while StGM particles were stellate and tended to coalesce. Histologic analysis showed more solid mass, larger particles and less intervening space with SmGM versus StGM. Conclusions: In a severe, high-volume bleeding model, residual bleeding at 2 minutes was significantly lower with SmGM versus StGM, and SmGM showed greater consistency across bleeding intensities. These findings may be attributable to dry mass per unit volume and/or ultrastructural differences between the two agents.

Effect of Coagulation Factor Concentrates on Markers of Endothelial Cell Damage in Experimental Hemorrhagic Shock.

BACKGROUND: Plasma-based resuscitation showed protective effects on the endothelial glycocalyx compared with crystalloid resuscitation. There is paucity of data regarding the effect of coagulation factor concentrates (CFC) on the glycocalyx in hemorrhagic shock (HS). We hypothesized that colloid-based resuscitation supplemented with CFCs offers a therapeutic value to treat endothelial damage following HS. METHODS: Eighty-four rats were subjected to pressure-controlled (mean arterial pressure (MAP) 30-35 mm Hg) and lab-guided (targeted cutoff: lactate >2.2. mmol/L and base deficit > 5.5 mmol/L) HS. Animals were resuscitated with fresh frozen plasma (FFP), human albumin (HA) or Ringer's lactate (RL) and RL or HA supplemented with fibrinogen concentrate (FC) or prothrombin complex concentrate (PCC). Serum epinephrine and the following markers of endothelial damage were assessed at baseline and at the end-of-observation (120 min after shock was terminated): syndecan-1, heparan sulfate, and soluble vascular endothelial growth factor receptor 1 (sVEGFR 1). RESULTS: Resuscitation with FFP had no effect on sVEGFR1 compared with crystalloid-based resuscitation (FFP: 19.3 ng/mL vs. RL: 15.9 ng/mL; RL+FC: 19.7 ng/mL; RL+PCC: 18.9 ng/mL; n.s.). At the end-of-observation, syndecan-1 was similar among all groups. Interestingly, HA+FC treated animals displayed the highest syndecan-1 concentration (12.07 ng/mL). Resuscitation with FFP restored heparan sulfate back to baseline (baseline: 36 ng/mL vs. end-of-observation: 36 ng/mL). CONCLUSION: The current study revealed that plasma-based resuscitation normalized circulating heparan sulfate but not syndecan-1. Co-administration of CFC had no further effect on glycocalyx shedding suggesting a lack of its therapeutic potential. LEVEL OF EVIDENCE: VExperimental in vivo study.

Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions.

Secretomes from various cell sources exert strong regenerative activities on numerous organs, including the skin. Although secretomes consist of many diverse components, a growing body of evidence suggests that small extracellular vesicles (EVs) account for their regenerative capacity. We previously demonstrated that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs) exhibits wound healing capacity. Therefore, we sought to dissect the molecular composition of EVs present in the secretome and compared wound healing-related activities of these EVs to other subfractions of the secretome and the fully supplemented secretome (MNCaposec). Compared to EVs derived from non-irradiated PBMCs, γ-irradiation significantly increased the size and number and changed the composition of released EVs. Detailed characterization of the molecular components of EVs, i.e. miRNA, proteins, and lipids, derived from irradiated PBMCs revealed a strong association with regenerative processes. Reporter gene assays and aortic ring sprouting assays revealed diminished activity of the subfractions compared to MNCaposec. In addition, we showed that MNCaposec accelerated wound closure in a diabetic mouse model. Taken together, our results suggest that secretome-based wound healing represents a promising new therapeutic avenue, and strongly recommend using the complete secretome instead of purified subfractions, such as EVs, to exploit its full regenerative capacity.

Experimental Models of Endotheliopathy: Impact of Shock Severity.

BACKGROUND: Hemorrhagic shock (HS) followed by resuscitation is often associated with sympathoadrenal activation (SAA) and endothelial damage (ED). OBJECTIVE: We aimed to evaluate the impact of HS alone on the magnitude of SAA and consecutive ED, and to characterize potential targets for a standardized and reproducible model of HS-induced endotheliopathy in rats. METHODS: Rats were subjected either to a volume-controlled HS (40% of total blood volume: v-HS group) or to a laboratory-guided HS (l-HS) targeting base deficit (BD) more than 5.5 mmol/L and/or lactate more than 2.2 mmol/L using a pressure-controlled volume loss. RESULTS: At the end of shock, mean arterial pressure was significantly higher in the v-HS than the l-HS group (36 ±â€Š5.6 vs. 30 ±â€Š3.0 mmHg; P < 0.01). Base deficit and lactate were higher in l-HS than the v-HS group (BD: 9.5 ±â€Š2.5 vs. 3.0 ±â€Š1.0 mmol/L; P < 0.001; lactate: 4.1 ±â€Š1.3 vs. 1.6 ±â€Š0.6 mmol/L; P < 0.001). sVEGFR-1 and syndecan-1 were approximately 50% higher in the l-HS than the v-HS group (% changes vs. baseline: 160 ±â€Š10 vs. 116 ±â€Š36; P < 0.01; 170 ±â€Š37 vs. 113 ±â€Š27; P < 0.001). Adrenaline was 2-fold higher in l-HS than the v-HS group (1964 ±â€Š961% vs. 855 ±â€Š451%; P < 0.02, respectively). Moreover, linear regression analysis revealed an independent association of shock severity BD with syndecan-1 (rho = 0.55, P = 0.0005), sVEGFR1 (rho = 0.25, P < 0.05), and adrenaline (rho = 0.31, P = 0.021). CONCLUSIONS: Our findings indicate that ED has already occurred during HS without reperfusion; intensity is strongly related to the severity of HS and consecutive SAA; and severity may appropriately be targeted and standardized in a HS model controlled by biological endpoints such as BD and/or lactate.

Comparison of nanoparticular hydroxyapatite pastes of different particle content and size in a novel scapula defect model.

Nanocrystalline hydroxyapatite (HA) has good biocompatibility and the potential to support bone formation. It represents a promising alternative to autologous bone grafting, which is considered the current gold standard for the treatment of low weight bearing bone defects. The purpose of this study was to compare three bone substitute pastes of different HA content and particle size with autologous bone and empty defects, at two time points (6 and 12 months) in an ovine scapula drillhole model using micro-CT, histology and histomorphometry evaluation. The nHA-LC (38% HA content) paste supported bone formation with a high defect bridging-rate. Compared to nHA-LC, Ostim® (35% HA content) showed less and smaller particle agglomerates but also a reduced defect bridging-rate due to its fast degradation The highly concentrated nHA-HC paste (48% HA content) formed oversized particle agglomerates which supported the defect bridging but left little space for bone formation in the defect site. Interestingly, the gold standard treatment of the defect site with autologous bone tissue did not improve bone formation or defect bridging compared to the empty control. We concluded that the material resorption and bone formation was highly impacted by the particle-specific agglomeration behaviour in this study.

Induced Hypothermia Preserves the Functional Enterocyte Mass in A Porcine Multiple Trauma Model: Retracted.

INTRODUCTION: Multiple organ dysfunction syndrome (MODS) and the resulting multiple organ failure (MOF) following severe trauma are associated with increased morbidity and mortality. Due to intestinal mucosal lesions and gut barrier disorders, the intestine contributes decisively to how post-traumatic MOF develops. As mild therapeutic hypothermia has been found to have protective effects on post-traumatic organ injuries, we analysed its effects on the intestine. METHODS: In a porcine model, Forty pigs were assigned to four groups: sham or trauma groups each with two sub-groups receiving either hypothermia or normothermia. The trauma was a combined trauma of blunt chest trauma, liver laceration and haemorrhagic shock. Functional enterocyte mass and enterocyte necrosis were evaluated by measuring plasma citrulline and iFABP. Mucosal lesions were assessed using a semi-quantitative histological scoring system. RESULTS: In normothermic trauma animals, citrulline decreased significantly compared to both sham groups and to the hypothermic trauma group. However, citrulline levels did not differ significantly between the hypothermic trauma group and the hypothermic sham group. Although histological analysis demonstrated subepithelial lifting and mucosal oedema in the ileal mucosa of all trauma animals, the semi-quantitative score of the group treated with hypothermia was comparable to that of the hypothermic sham group. However, the score was significantly elevated in normothermic trauma animals in comparison to sham and hypothermic trauma animals. CONCLUSION: Induced hypothermia preserves the functional enterocyte mass after severe trauma. Therefore induced hypothermia might represent a therapeutic strategy to avoid posttraumatic organ dysfunction, although further studies regarding the safety and long-term effects are required. LEVEL OF EVIDENCE: Level III; therapeutic study.

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

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.

Preferential effects of low volume versus high volume replacement with crystalloid fluid in a hemorrhagic shock model in pigs.

BACKGROUND: Fluid resuscitation is a core stone of hemorrhagic shock therapy, and crystalloid fluids seem to be associated with lower mortality compared to colloids. However, as redistribution starts within minutes, it has been suggested to replace blood loss with a minimum of a three-fold amount of crystalloids. The hypothesis was that in comparison to high volume (HV), a lower crystalloid volume (LV) achieves a favorable coagulation profile and exerts sufficient haemodynamics in the acute phase of resuscitation. METHODS: In 24 anaesthetized pigs, controlled arterial blood loss of 50 % of the estimated blood volume was either (n = 12) replaced with a LV (one-fold) or a HV (three-fold) volume of a balanced, acetated crystalloid solution at room temperature. Hemodynamic parameters, dilution effects and coagulation profile by standard coagulation tests and thromboelastometry at baseline and after resuscitation were determined in both groups. RESULTS: LV resuscitation increased MAP significantly less compared to the HV, 61 ± 7 vs. 82 ± 14 mmHg (p < 0.001) respectively, with no difference between lactate and base excess between groups. Haematocrit after fluid replacement was 0.20 vs. 0.16 (LV vs. HV, p < 0.001), suggesting a grade of blood dilution of 32 vs. 42 % (p < 0.001) compared to baseline values. Compared to LV, HV resulted in decreased core temperature (37.5 ± 0.2 vs. 36.0 ± 0.6 °C, p < 0.001), lower platelet count (318 ± 77 vs. 231 ± 53 K/µL, p < 0.01) and lower plasma fibrinogen levels (205 ± 19 vs. 168 ± 24 mg/dL, p < 0.001). Thromboelastometric measurements showed a significant impairment on viscoelastic clot properties following HV group. While prothrombin time index decreased significantly more in the HV group, activated partial thromboplastin time did not differ between both groups. HV did not result in hyperchloraemic acidosis. DISCUSSION: Coagulation parameters represented by plasma fibrinogen and ROTEM parameters were also less impaired with LV. With regrad to hematocrit, 60 % of LV remained intracascular , while in HV only 30 % remained in circulation within the first hour of administration. In the acute setting of 50 % controlled blood loss, a one fold LV crystalloid replacement strategy is sufficient to adequately raise blood pressure up to a mean arterial pressure >50 mm Hg. The concept of damage control resuscitation (DCR) with permissive hypotension may be better met by using LV as compared to a three fold HV resuscitation strategy. CONCLUSION: High volume administration of an acetated balanced crystalloid does not lead to hyperchloraemic acidosis, but may negatively influence clinical parameters, such as higher blood pressure, lower body temperature and impaired coagulation parameters, which could potentially increase bleeding after trauma. Replacement of acute blood loss with just an equal amount of an acetated balanced crystalloid appears to be the preferential treatment strategy in the acute phase after controlled bleeding.