Refinement of Animal Experiments: Replacing Traumatic Methods of Laboratory Animal Marking with Non-Invasive Alternatives.

Reliable methods for identifying rodents play an important role in ensuring the success of preclinical studies. However, animal identification remains a trivial laboratory routine that is not often discussed, despite the fact that more than 6 million rodents are used in animal studies each year. Currently, there are extensive regulations in place to ensure adequate anesthesia and to reduce animal suffering during experiments. At the same time, not enough attention is paid to the comfort of rodents during routine identification procedures, which can be painful and cause some complications. In order to achieve the highest ethical standards in laboratory research, we must minimize animal discomfort during the identification phase. In this article, we discuss traumatic methods of identification and describe several painless methods for marking in long-term experimental studies. The use of non-traumatic and non-invasive methods requires the renewal of marks as they fade and additional handling of the rodents. Laboratory personnel must be trained in stress-minimizing handling techniques to make mark renewal less stressful.

Gene-Activated Materials in Regenerative Dentistry: Narrative Review of Technology and Study Results.

Treatment of a wide variety of defects in the oral and maxillofacial regions requires the use of innovative approaches to achieve best outcomes. One of the promising directions is the use of gene-activated materials (GAMs) that represent a combination of tissue engineering and gene therapy. This approach implies that biocompatible materials will be enriched with gene-carrying vectors and implanted into the defect site resulting in transfection of the recipient's cells and secretion of encoded therapeutic protein in situ. GAMs may be presented in various designs depending on the type of material, encoded protein, vector, and way of connecting the vector and the material. Thus, it is possible to choose the most suitable GAM design for the treatment of a particular pathology. The use of plasmids for delivery of therapeutic genes is of particular interest. In the present review, we aimed to delineate the principle of work and various designs of plasmid-based GAMs and to highlight results of experimental and clinical studies devoted to the treatment of periodontitis, jaw bone defects, teeth avulsion, and other pathologies in the oral and maxillofacial regions.

World's First Experience of the Low-Dose Radionuclide Inhalation Therapy in the Treatment of COVID-19-Associated Viral Pneumonia: Phase 1/2 Clinical Trial.

OBJECTIVE: Previously, low-dose radiation therapy was used for pneumonia treatment. We aimed to investigate the safety and effectiveness of carbon nanoparticles labeled with Technetium isotope (99mTc) in a form of ultradispersed aerosol in combination with standard COVID-19 therapy. The study was a randomized phase 1 and phase 2 clinical trial of low-dose radionuclide inhalation therapy for patients with COVID-19 related pneumonia. METHODS: We enrolled 47 patients with confirmed COVID-19 infection and early laboratory signs of cytokine storm and randomized them into the Treatment and Control groups. We analyzed blood parameters reflecting the COVID-19 severity and inflammatory response. RESULTS: Low-dose 99mTc-labeled inhalation showed a minimal accumulation of radionuclide in lungs in healthy volunteers. We observed no significant differences between the groups before treatment in WBC-count, D-dimer, CRP, Ferritin or LDH levels. We found that Ferritin and LDH levels significantly raised after the 7th day follow-up only in the Control group (p < 0.0001 and p = 0.0005, respectively), while mean values of the same indicators did not change in patients in the Treatment group after the radionuclide treatment. D-dimer values also lowered in the radionuclide treated group, however, this effect was not statistically significant. Furthermore, we observed a significant decrease in CD19+ cell counts in patients of the radionuclide-treated group. CONCLUSION: Inhalation low-dose radionuclide therapy of 99mTc aerosol affects the major prognostic indicators of COVID-19- related pneumonia restraining inflammatory response. Overall, we identified no evidence of major adverse events in the group receiving radionuclide.

Biomechanical Behaviors and Degradation Properties of Multilayered Polymer Scaffolds: The Phase Space Method for Bile Duct Design and Bioengineering.

This article reports the electrospinning technique for the manufacturing of multilayered scaffolds for bile duct tissue engineering based on an inner layer of polycaprolactone (PCL) and an outer layer either of a copolymer of D,L-lactide and glycolide (PLGA) or a copolymer of L-lactide and ε-caprolactone (PLCL). A study of the degradation properties of separate polymers showed that flat PCL samples exhibited the highest resistance to hydrolysis in comparison with PLGA and PLCL. Irrespective of the liquid-phase nature, no significant mass loss of PCL samples was found in 140 days of incubation. The PLCL- and PLGA-based flat samples were more prone to hydrolysis within the same period of time, which was confirmed by the increased loss of mass and a significant reduction of weight-average molecular mass. The study of the mechanical properties of developed multi-layered tubular scaffolds revealed that their strength in the longitudinal and transverse directions was comparable with the values measured for a decellularized bile duct. The strength of three-layered scaffolds declined significantly because of the active degradation of the outer layer made of PLGA. The strength of scaffolds with the PLCL outer layer deteriorated much less with time, both in the axial (p-value = 0.0016) and radial (p-value = 0.0022) directions. A novel method for assessment of the physiological relevance of synthetic scaffolds was developed and named the phase space approach for assessment of physiological relevance. Two-dimensional phase space (elongation modulus and tensile strength) was used for the assessment and visualization of the physiological relevance of scaffolds for bile duct bioengineering. In conclusion, the design of scaffolds for the creation of physiologically relevant tissue-engineered bile ducts should be based not only on biodegradation properties but also on the biomechanical time-related behavior of various compositions of polymers and copolymers.

Angiogenic Modification of Microfibrous Polycaprolactone by pCMV-VEGF165 Plasmid Promotes Local Vascular Growth after Implantation in Rats.

Insufficient vascular growth in the area of artificial-material implantation contributes to ischemia, fibrosis, the development of bacterial infections, and tissue necrosis around the graft. The purpose of this study was to evaluate angiogenesis after implantation of polycaprolactone microfiber scaffolds modified by a pCMV-VEGF165-plasmid in rats. Influence of vascularization on scaffold degradation was also examined. We investigated flat microfibrous scaffolds obtained by electrospinning polycaprolactone with incorporation of the pCMV-VEGF-165 plasmid into the microfibers at concentrations of 0.005 ng of plasmid per 1 mg of polycaprolactone (0.005 ng/mg) (LCGroup) and 0.05 ng/mg (HCGroup). The samples were subcutaneously implanted in the interscapular area of rats. On days 7, 16, 33, 46, and 64, the scaffolds were removed, and a histological study with a morphometric evaluation of the density and diameter of the vessels and microfiber diameter was performed. The number of vessels was increased in all groups, as well as the resorption of the scaffold. On day 33, the vascular density in the HCGroup was 42% higher compared to the control group (p = 0.0344). The dose-dependent effect of the pCMV-VEGF165-plasmid was confirmed by enhanced angiogenesis in the HCGroup compared to the LCGroup on day 33 (p-value = 0.0259). We did not find a statistically significant correlation between scaffold degradation rate and vessel growth (the Pearson correlation coefficient was ρ = 0.20, p-value = 0.6134). Functionalization of polycaprolactone by incorporation of the pCMV-VEGF165 plasmid provided improved vascularization within 33 days after implantation, however, vessel growth did not seem to correlate with scaffold degradation rate.

Intraoperative Creation of Tissue-Engineered Grafts with Minimally Manipulated Cells: New Concept of Bone Tissue Engineering In Situ.

Transfer of regenerative approaches into clinical practice is limited by strict legal regulation of in vitro expanded cells and risks associated with substantial manipulations. Isolation of cells for the enrichment of bone grafts directly in the Operating Room appears to be a promising solution for the translation of biomedical technologies into clinical practice. These intraoperative approaches could be generally characterized as a joint concept of tissue engineering in situ. Our review covers techniques of intraoperative cell isolation and seeding for the creation of tissue-engineered grafts in situ, that is, directly in the Operating Room. Up-to-date, the clinical use of tissue-engineered grafts created in vitro remains a highly inaccessible option. Fortunately, intraoperative tissue engineering in situ is already available for patients who need advanced treatment modalities.

Insulin Diminishes Superoxide Increase in Cytosol and Mitochondria of Cultured Cortical Neurons Treated with Toxic Glutamate.

Glutamate excitotoxicity is involved in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer's disease, for which central insulin resistance is a comorbid condition. Neurotoxicity of glutamate (Glu) is primarily associated with hyperactivation of the ionotropic N-methyl-D-aspartate receptors (NMDARs), causing a sustained increase in intracellular free calcium concentration ([Ca2+]i) and synchronous mitochondrial depolarization and an increase in intracellular superoxide anion radical (O2-•) production. Recently, we found that insulin protects neurons against excitotoxicity by decreasing the delayed calcium deregulation (DCD). However, the role of insulin in O2-• production in excitotoxicity still needs to be clarified. The present study aims to investigate insulin's effects on glutamate-evoked O2-• generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in cortical neurons. We found a linear correlation between [Ca2+]i and [O2-•] in primary cultures of the rat neuron exposed to Glu, with insulin significantly reducing the production of intracellular and mitochondrial O2-• in the primary cultures of the rat neuron. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments in sister cultures, insulin diminished neuronal death and O2 consumption rate (OCR).

Engineering of Tracheal Grafts Based on Recellularization of Laser-Engraved Human Airway Cartilage Substrates.

OBJECTIVE: Implantation of tissue-engineered tracheal grafts represents a visionary strategy for the reconstruction of tracheal wall defects after resections and may develop into a last chance for a number of patients with severe cicatricial stenosis. The use of a decellularized tracheal substrate would offer an ideally stiff graft, but the matrix density would challenge efficient remodeling into a living cartilage. In this study, we hypothesized that the pores of decellularized laser-perforated tracheal cartilage (LPTC) tissues can be colonized by adult nasal chondrocytes (NCs) to produce new cartilage tissue suitable for the repair of tracheal defects. DESIGN: Human, native tracheal specimens, isolated from cadaveric donors, were exposed to decellularized and laser engraving-controlled superficial perforation (300 µm depth). Human or rabbit NCs were cultured on the LPTCs for 1 week. The resulting revitalized tissues were implanted ectopically in nude mice or orthotopically in tracheal wall defects in rabbits. Tissues were assayed histologically and by microtomography analyses before and after implantation. RESULTS: NCs were able to efficiently colonize the pores of the LPTCs. The extent of colonization (i.e., percentage of viable cells spanning >300 µm of tissue depth), cell morphology, and cartilage matrix deposition improved once the revitalized constructs were implanted ectopically in nude mice. LPTCs could be successfully grafted onto the tracheal wall of rabbits without any evidence of dislocation or tracheal stenosis, 8 weeks after implantation. Rabbit NCs, within the LPTCs, actively produced new cartilage matrix. CONCLUSION: Implantation of NC-revitalized LPTCs represents a feasible strategy for the repair of tracheal wall defects.

Mesenchymal stromal cell therapy alone does not lead to complete restoration of skin parameters in diabetic foot patients within a 3-year follow-up period.

Introduction: Mesenchymal stromal cells (MSCs) administration is an effective option for the treatment of diabetic foot ulcers (DFUs). However, to date, studies assessing long-term outcomes and evaluating skin parameters after cell-based therapy are lacking. We presented the clinical outcomes of 3 patients, treated for DFUs with the bone marrow MSCs 3 years earlier. Methods: Ultrasound examination was used to compare collagen density and epidermal thickness in areas of healed ulcers in comparison with non-affected skin used as a control. Ultrasound and dermatoscopy were used to exclude neoplasm formation, to assess scar contracture and wound recurrence. Results: In all patients, no ulcer recurrence was detected, which was lower than the expected 60% rate of re-ulceration in diabetic patients in a 3-year period (OD [odds ratio] = 0.095, P = 0.12). No neoplasm formation, no contracture of hypertrophic scar, and adjacent tissue were registered. Collagen ultrasound density was decreased by 57% (P = 0.053) and epidermal thickness was increased by 72% (P = 0.01) in the area of healed ulcers in all patients. Conclusion: MSCs therapy alone did not result in the complete restoration of the skin parameters within a 3-year period. MSCs may represent important adjuvant to the therapy, however, other novel approaches are required to achieve better results.

Mesenchymal Stromal Cells Enhance Vascularization and Epithelialization within 7 Days after Gingival Augmentation with Collagen Matrices in Rabbits.

Soft gingival tissue deficiency remains a severe problem leading to postoperative recession, peri-implantitis, and bone resorption. The use of collagen matrices does not always lead to complete rebuilding of the gingiva volume. The application of mesenchymal stromal cells (MSCs) simultaneously with collagen materials represents a promising approach for the restoration of soft gingival tissues. However, short-term effects of MSCs-enriched collagen grafts after gingival augmentation have not yet been studied properly. Mucograft and Mucoderm matrices were implanted in rabbits (n = 12) simultaneously with the intraoperative injection of rabbit bone marrow-derived mesenchymal stromal cells (BM-MSCs) or without cells. Collagen matrices were implanted under the flap or by the surface technique without intentional primary closure. The samples were harvested seven days after implantation, histological staining with hematoxylin and eosin, and immunohistochemical staining for VEGF, IGF1, and TGF were performed. The use of Mucoderm led to better augmentation outcomes on day 7 compared with Mucograft (p < 0.0001). Gingival augmentation in combination with the local administration of BM-MSCs led to better regeneration of the soft gingival tissues independently of the type of implanted collagen matrices (p < 0.0001). Furthermore, injection of BM-MSCs significantly enhanced gingival vascularization and epithelization with a clear positive correlation between vascular growth and epithelial response. Administration of BM-MSCs in combination with various collagen materials may potentially improve gingiva regeneration.

Defense Advanced Research Projects Agency: Comment on 'Engineering biology and the grand challenges: Do we need a new R&D&I model?'

This comment paper refers to the article published by Gauvreau et al. in Engineering Biology in 2018. Gauvreau et al. discussed various civilian models in the private and public sectors that could boost R&D plus Innovation (R&D&I) progress in the environmental sphere. The authors concluded that there is no tried-and-tested R&D&I model for engineering biology that is suited to help solve the problems of many grand and interacting challenges, and further, that the required model will not work without the private sector, so governments must collaborate to develop such a model. However, the authors have not mentioned one of the most famous classic Research & Development (R&D) models used in the creation of disruptive technologies-a unique model of the Defense Advanced Research Projects Agency (DARPA) of the US Department of Defense. The present commentary poses questions about the models currently used in innovative research, known as DARPA-like R&D agencies, and the innovative ecosystems around them.

Innovative approach to aesthetical medicine services quality assessment.

Recently, both doctors and patients pay particular attention to aesthetic medical services quality and efficacy of aesthetic adjustment implemented. It is essential to ensure research, analysis, and improving of botulinum toxin therapy quality and provide patients' orientation on the adjustment schedule, which includes long-term observation period, in order to accomplish the expected goals and increase degree of satisfaction. Injections of medication such as botulinum A toxin are highly demanded in many types of aesthetic treatment. However, practically, sufficient cosmetological effect may not always be achieved, and moreover, complications are possible. This article presents an attempt to evaluate botulinum toxin therapy efficacy using computer program, with calculation of integral quality factor (IQF) and plotting injection modeling visual diagram in long-term observation period. In 3 selected clinical examples, aggregate IQF figure makes: in case of static forehead wrinkles 91.67%, for dynamic forehead wrinkles 76.67%; in case of static eyebrow wrinkles 96.67%, for dynamic eyebrow wrinkles 85%; as applied to static wrinkles of the outer corned of the eye 91.67%, whereas applied to dynamic wrinkles of the outer corner of the eye 73.33%. Although the benchmark is thought to be 100% of remodeling result, the procedures outcome still meets patients' expectations. At the same time, present approach demands further discussion and collection for the purpose of development of procedural basis for aesthetic services quality assessment and interpretation of findings.

Prolonged prothrombin time as an early prognostic indicator of severe acute respiratory distress syndrome in patients with COVID-19 related pneumonia.

BACKGROUND: Clinical observations demonstrated that COVID-19 related pneumonia is often accompanied by hematological and coagulation abnormalities including lymphopenia, thrombocytopenia, and prolonged prothrombin time. The evaluation of laboratory findings including coagulation and inflammation parameters may represent a promising approach for early determination of COVID-19 severity. METHODS AND MATERIALS: In the present study, we aimed to identify laboratory parameters present upon admission in patients with COVID-19 related viral pneumonia and associated with an early in-hospital development of refractory respiratory failure or severe acute respiratory distress syndrome requiring treatment in an intensive care unit. We investigated differences in the C-reactive protein (CRP) and fibrinogen levels, prothrombin time (PT) and international normalized ratio (INR) between COVID-19 patients who had been transferred to an ICU within two weeks after admission (n = 82) and COVID-19 patients with stable course of the disease (n = 74). RESULTS: Multiple comparisons showed statistically significantly prolonged PT on admission in ICU-transferred COVID-19 patients (14.15 sec, median, CI 95% 13.4 ÷ 14.9) compared to the stable COVID-19 patients (13.25 sec, median, CI 95% 12.9 ÷ 13.6) (p-value = .0005). CRP levels upon admission were statistically significantly higher in ICU-transferred COVID-19 patients (132 mg/L, median, CI95% 113 ÷ 159) compared to the stable COVID-19 patients (51 mg/L, median, CI95% 33 ÷ 72) (p-value < .0001). On-admission fibrinogen and INR levels did not statistically significantly differ between ICU-transferred COVID-19 patients and stable COVID-19 patients. CONCLUSION: We suggest that CRP and PT levels present on admission in COVID-19 patients may be used as early prognostic markers of severe pneumonia requiring transfer to ICU.