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Dermal fibroblasts (DF) constitute one of key cells involved in wound healing. However, the functions they perform in wound conditions remain poorly understood. This study involved exposing DF to low nutrition and to low nutrition + LPS for 5 days as conditions representing the wound. Although DF exhibited increasing metabolic activity in time under all conditions including control, the proliferation did not change in both low nutrition and low nutrition + LPS. Only the low nutrition + LPS was found to potentiate the migration and pro-inflammatory phenotype (IL6 release) of DF. The potential of DF to contract collagen hydrogel declined only under the low nutrition as a consequence of low cell number. The expression of α-SMA was reduced under the both conditions independently of the cell number. The remodeling capability of DF was affected under the both conditions as documented by the enhanced MMP2 activity. Finally, the production of collagen type I was not affected by either condition. The study shows that low nutrition as the single factor is able to delay the healing process. Moreover, the addition of the mild pro-inflammatory stimulus represented by LPS may amplify the cell response in case of decreased α-SMA expression or excite DF to produce IL6 impairing the healing process.

Only a fraction of specimens under study are usually selected for quantification in histology. Multilevel sampling or tissue probes, slides and fields of view (FOVs) in the regions of interest (ROIs) are required. In general, all parts of the organs under study should be given the same probability to be taken into account; that is, the sampling should be unbiased on all levels. The objective of our study was to provide an overview of the use of virtual microscopy in the context of developing sampling strategies of FOVs for stereological quantification. We elaborated this idea on 18 examples from multiple fields of histology, including quantification of extracellular matrix and muscle tissue, quantification of organ and tumour microvessels and tumour-infiltrating lymphocytes, assessing osseointegration of bone implants, healing of intestine anastomoses and osteochondral defects, counting brain neurons, counting nuclei in vitro cell cultures and others. We provided practical implications for the most common situations, such as exhaustive sampling of ROIs, sampling ROIs of different sizes, sampling the same ROIs for multiple histological methods, sampling more ROIs with variable intensities or using various objectives, multistage sampling and virtual sampling. Recommendations were provided for pilot studies on systematic uniform random sampling of FOVs as a part of optimizing the efficiency of histological quantification to prevent over- or undersampling. We critically discussed the pros and cons of using virtual sections for sampling FOVs from whole scanned sections. Our review demonstrated that whole slide scans of histological sections facilitate the design of sampling strategies for quantitative histology.

Background In patients with colorectal liver metastases, the possibility for radical liver resection can be limited by oxaliplatin-induced sinusoidal obstruction syndrome (SOS). This study investigates the potential of mesenchymal stem cells (MSC) to improve the outcome of liver resections in pigs with SOS. Materials and methods SOS was induced in all animals (n=20) on day 0. Animals in the experimental group (n=8) received allogeneic MSC on day 7. Liver resection was performed in all animals on day 14 and the animals were observed until day 28. Ultrasound volumetry, biochemical analysis and histological examination of liver parenchyma was performed during the follow-up period. Results Six animals from the control group died prematurely, while all animals survived in the experimental group. According to histology, biochemical analysis and ultrasound volumetry, there were no significant differences between the groups documenting the effect of MSC. Conclusion Single dose allogeneic MSC administration improved survival of animals with SOS undergoing partial liver resection. Further experiments with different timing of liver resection and MSC administration should be performed to investigate the effect of MSC in more detail.

Background/Aim: Patients with unresectable liver colorectal cancer metastases are treated with neoadjuvant chemotherapy often accompanied by biological therapy aimed at reducing the mass of metastases and thus increasing the chances of resectability. Bevacizumab comprises an anti-VEGF (vascular endothelial growth factor) humanized IgG monoclonal antibody that is used for biological therapy purposes. It acts to inhibit angiogenesis, thereby slowing down the growth of metastases. Due to its being administered systematically, bevacizumab also exerts an effect on the surrounding healthy liver parenchyma and potentially limits the process of neovascularization and thus regeneration of the liver. Since the remnant liver volume forms an important factor in postoperative morbidity and mortality following a major hepatectomy, we decided to study the effect of bevacizumab on vascular and biliary microarchitecture in healthy liver parenchyma and its ability to regenerate following major hepatectomy. Materials and Methods: We performed an experiment employing a large animal model where a total of 16 piglets were divided into two groups (8 piglets in the control group and 8 piglets in the experimental group with bevacizumab). All the animals were subjected to major hepatectomy and the experimental group was given bevacizumab prior to hepatectomy. All the animals were sacrificed after 4 weeks. We performed biochemical analyses at regular time intervals during the follow-up period. Histological examination of the liver tissue was performed following sacrifice of the animals. Results: No statistical difference was shown between groups in terms of the biochemical and immunohistochemical parameters. The histological examination of the regenerating liver tissue revealed the higher length density of sinusoids in the experimental group. Conclusion: Bevacizumab does not act to impair liver regeneration following hepatectomy.

The theme of this paper is the analysis of mechanical and structural properties of nanofibrous COL under simulated body conditions and in the presence of osteoblasts and dermal fibroblasts. COL were prepared by electrostatic spinning of 8wt% collagen type I dispersion with 8wt% (to COL) of PEG in phosphate buffer/ethanol solution (1/1vol). The stability of COL was enhanced by means of cross-linking with EDC and NHS at a molar ratio of 4:1. COL were exposed in culture medium for 21 days and human SAOS-2 human dermal fibroblasts and osteoblasts were cultured therein for 21 days as well. The cell culture on COL was assessed by fluorescence microscopy and metabolic activity. Then the metabolic activity of both cell types grown on COL and PS were measured after 1, 7, 14 and 21 days using the Alamar Blue assay method. Mechanical properties were determined using an tensile test. The influence of the cell activity on secondary structure of COL was verified by IR spectroscopy. Furthermore, the influence of cells on COL was evaluated by SEM.

In a biological system, nanoparticles (NPs) may interact with biomolecules. Specifically, the adsorption of proteins on the nanoparticle surface may influence both the nanoparticles' and proteins' overall bio-reactivity. Nevertheless, our knowledge of the biocompatibility and risk of exposure to nanomaterials is limited. Here, in vitro and ex ovo biocompatibility of naturally based crosslinked freeze-dried 3D porous collagen/chitosan scaffolds, modified with thermostable fibroblast growth factor 2 (FGF2-STAB®), to enhance healing and selenium nanoparticles (SeNPs) to provide antibacterial activity, were evaluated. Biocompatibility and cytotoxicity were tested in vitro using normal human dermal fibroblasts (NHDF) with scaffolds and SeNPs and FGF2-STAB® solutions. Metabolic activity assays indicated an antagonistic effect of SeNPs and FGF2-STAB® at high concentrations of SeNPs. The half-maximal inhibitory concentration (IC50) of SeNPs for NHDF was 18.9 µg/ml and IC80 was 5.6 µg/ml. The angiogenic properties of the scaffolds were monitored ex ovo using a chick chorioallantoic membrane (CAM) assay and the cytotoxicity of SeNPs over IC80 value was confirmed. Furthermore, the positive effect of FGF2-STAB® at very low concentrations (0.01 µg/ml) on NHDF metabolic activity was observed. Based on detailed in vitro testing, the optimal concentrations of additives in the scaffolds were determined, specifically 1 µg/ml of FGF2-STAB® and 1 µg/ml of SeNPs. The scaffolds were further subjected to antimicrobial tests, where an increase in selenium concentration in the collagen/chitosan scaffolds increased the antibacterial activity. This work highlights the antimicrobial ability and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® and SeNPs. Moreover, we suggest that these sponges could be used as scaffolds for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration. Due to their antimicrobial properties, these scaffolds are also highly promising for tissue replacement requiring the prevention of infection.

Introduction: Nonalcoholic fatty liver disease (NAFLD) is the most widespread chronic liver disease in the world. It can evolve into nonalcoholic steatohepatitis (NASH) where inflammation and hepat...

Introduction: Maternal sepsis is a leading cause of maternal and neonatal mortality. Despite the availability of management protocols, there is disparity in case fatality rates for pregnancy-related sepsis compared to other maternity-related complications. The main aim of this systematic review was to assess concordance between international evidence-based guidelines for the prevention and management of childbirth-related bacterial infections. Material and methods: The PRISMA statement was followed during the conduct and reporting of this review. PubMed and G-I-N were searched electronically from 2009 to November 2019 for clinical guidelines covering the topic of childbirth-related infections and specific searches for relevant guidelines on the websites of the top 5 international professional bodies most commonly identified by our searches. We did not apply any language restrictions. Guidelines were included if they provided any information about the prevention or management of childbirth-related bacterial infections irrespective of whether the guideline stated a recommendation or not. Two independent reviewers undertook study selection, decisions about inclusion of selected guidelines and data extraction. Extracted information was synthesized under the following topics: Asymptomatic bacteriuria; group B streptococcal infection (GBS); preterm premature rupture of membranes (P-PROM); Intrauterine infection; Interventions and procedures; maternal sepsis; miscellaneous. Concordance was defined as absence of contradictory information between the different guidelines with regards to a specific topic, subtopic or recommendation. Quality of included guidelines was assessed against the AGREE II guideline reporting domains.Results: A total of 43 guidelines were selected of which 11 were excluded leaving 32 guidelines that fulfilled our inclusion criteria. None of the guidelines fulfilled all the quality assessment domains and 11 (34%) of the guidelines satisfied 1 - 2 of domains only. Two guidelines covered the topic of asymptomatic bacteriuria, nine for GBS, five for P-PROM and three covered each of intra-amniotic infections maternal sepsis, obstetric procedures and interventions topics. The remaining guidelines covered miscellaneous topics.Conclusions: There was concordance between guidelines with regards to several aspects in the prophylaxis and treatment of bacteriological infections in pregnancy. Nevertheless, there were several areas of discordance, some of which reached the extent of contradictory information as in the case of antenatal screening for GBS.

Maternal sepsis is a leading cause of maternal and neonatal mortality. Despite the availability of management protocols, there is disparity in case fatality rates for pregnancy-related sepsis compared to other maternity-related complications. The main aim of this systematic review was to assess concordance between international evidence-based guidelines for the prevention and management of childbirth-related bacterial infections. The PRISMA statement was followed during the conduct and reporting of this review. PubMed was searched electronically from 2009 to November 2019 for clinical guidelines covering the topic of childbirth-related infections and specific searches for relevant guidelines on the websites of the top five international professional bodies most commonly identified by our searches. We did not apply any language restrictions. Guidelines were included if they provided any information about the prevention or management of childbirth-related bacterial infections irrespective of whether the guideline stated a recommendation or not. Two independent reviewers undertook study selection, decisions about inclusion of selected guidelines and data extraction. Extracted information was synthesized under the following topics: Asymptomatic bacteriuria; group B streptococcal infection (GBS); preterm premature rupture of membranes (P-PROM); intrauterine infection; procedures; maternal sepsis; miscellaneous. Concordance was defined as absence of contradictory information between the different guidelines with regards to a specific topic, subtopic or recommendation. Quality of included guidelines was assessed against the AGREE II guideline reporting domains. A total of 43 guidelines were selected of which 11 were excluded leaving 32 guidelines that fulfilled our inclusion criteria. None of the guidelines fulfilled all the quality assessment domains and 11 (34%) of the guidelines satisfied 1−2 of domains only. Two guidelines covered the topic of asymptomatic bacteriuria, nine for GBS, five for P-PROM and three covered each of intra-amniotic infections maternal sepsis, obstetric procedures and interventions topics. The remaining guidelines covered miscellaneous topics. There was concordance between guidelines with regards to several aspects in the prophylaxis and treatment of bacteriological infections in pregnancy. Nevertheless, there were several areas of discordance, some of which reached the extent of contradictory information as in the case of antenatal screening for GBS.

Background: Treatment with mesenchymal stem cells (MSCs) has elicited considerable interest as an adjunctive therapy in sepsis. However, the encouraging effects of experiments with MSC in rodents have not been adequately studied in large-animal models with better relevance to human sepsis. Objectives: Here, we aimed to assess safety and efficacy of bone marrow-derived MSCs in a clinically relevant porcine model of progressive peritonitis-induced sepsis. Methods: Thirty-two anesthetized, mechanically ventilated, and instrumented pigs were randomly assigned into four groups (n = 8 per group): (1) sham-operated group (CONTROL); (2) sham-operated group treated with MSCs (MSC-CONTROL); (3) sepsis group with standard supportive care (SEPSIS); and (4) sepsis group treated with MSCs (MSC-SEPSIS). Peritoneal sepsis was induced by inoculating cultivated autologous feces. MSCs (1 × 106/kg) were administered intravenously at 6 h after sepsis induction. Results: Before, 12, 18, and 24 h after the induction of peritonitis, we measured systemic, regional, and microvascular hemodynamics, multiple-organ functions, mitochondrial energy metabolism, systemic immune-inflammatory response, and oxidative stress. Administration of MSCs in the MSC-CONTROL group did not elicit any measurable acute effects. Treatment of septic animals with MSCs failed to mitigate sepsis-induced hemodynamic alterations or the gradual rise in Sepsis-related organ failure assessment scores. MSCs did not confer any protection against sepsis-mediated cellular myocardial depression and mitochondrial dysfunction. MSCs also failed to modulate the deregulated immune-inflammatory response. Conclusion: Intravenous administration of bone marrow-derived MSCs to healthy animals was well-tolerated. However, in this large-animal, clinically relevant peritonitis-induced sepsis model, MSCs were not capable of reversing any of the sepsis-induced disturbances in multiple biological, organ, and cellular systems.

To investigate the potential benefits of vagus nerve stimulation in a clinically-relevant large animal model of progressive sepsis.Prospective, controlled, randomized trial.University animal research laboratory.Twenty-five domestic pigs were divided into three groups: 1) sepsis group (eight pigs), 2) sepsis + vagus nerve stimulation group (nine pigs), and 3) control sham group (eight pigs).Sepsis was induced by cultivated autologous feces inoculation in anesthetized, mechanically ventilated, and surgically instrumented pigs and followed for 24 hours. Electrical stimulation of the cervical vagus nerve was initiated 6 hours after the induction of peritonitis and maintained throughout the experiment.Measurements of hemodynamics, electrocardiography, biochemistry, blood gases, cytokines, and blood cells were collected at baseline (just before peritonitis induction) and at the end of the in vivo experiment (24 hr after peritonitis induction). Subsequent in vitro analyses addressed cardiac contractility and calcium handling in isolated tissues and myocytes and analyzed mitochondrial function by ultrasensitive oxygraphy. Vagus nerve stimulation partially or completely prevented the development of hyperlactatemia, hyperdynamic circulation, cellular myocardial depression, shift in sympathovagal balance toward sympathetic dominance, and cardiac mitochondrial dysfunction, and reduced the number of activated monocytes. Sequential Organ Failure Assessment scores and vasopressor requirements significantly decreased after vagus nerve stimulation.In a clinically-relevant large animal model of progressive sepsis, vagus nerve stimulation was associated with a number of beneficial effects that resulted in significantly attenuated multiple organ dysfunction and reduced vasopressor and fluid resuscitation requirements. This suggests that vagus nerve stimulation might provide a significant therapeutic potential that warrants further thorough investigation.

Titanium, the gold standard for dental implant materials, is distinguished by its exceptional biocompatibility; however, from a long-term perspective, titanium still lacks sufficient loading strength. Commercially pure titanium with grain size 30 μm was processed by equal channel angular pressing, and two novel mechanically improved types of titanium material with grain size 4.6 and ≤1 µm were obtained. The surfaces of these ultra-fine grained titanium samples were further mechanically treated by grinding and polishing, and their surfaces were characterized by atomic force microscopy and contact angle measurement. Osteoblast-like cells and human mesenchymal stem cells were used to evaluate the cytocompatibility of these titanium samples. The cell metabolic activity, cell number, cell area, morphology, and cell adhesion quality during the early stage (2 h) and prolonged cultivation (48 h) were determined. Both cell types displayed increased initial attachment to all tested titanium materials in comparison to reference tissue culture plastic. Importantly, results revealed that the novel titanium materials with improved strength were equivalent to the original commercially pure titanium, thus confirming their suitability for osteoblast and mesenchymal stem cell growth and proliferation. The present study proved the cytocompatibility of the novel forms of titanium with superior mechanical properties and revealed their potential for manufacturing of long-term dental implants.

Dermal fibroblasts, which make up the major cell type in the dermis, have, historically, been considered to be relatively 'passive' cells which are responsible for the synthesis and remodeling of extracellular matrix proteins. However, the dermal fibroblast population is composed of heterogeneous and distinct cell types, and it has been established that, under the stress conditions of healing wound environments, dermal fibroblasts participate in the regulation of ongoing inflammation and cell proliferation by secreting a variety of signaling molecules that modulate the functions of immune cells, keratinocyte, endothelial cells and mast cells via both direct cell to cell communication and autocrine and paracrine interactions. This review describes the capacity of dermal fibroblasts to sense and respond to signals from the micro-environment and to communicate with surrounding cells during cutaneous wound healing. The review further emphasizes the, to date, poorly understood roles of heterogeneous dermal fibroblast populations in the wound healing process.

Physical and chemical structure of paclitaxel (PTX) was studied after its incorporation into polymeric micelles made of hyaluronic acid (HA) (Mw=15 kDa) grafted with C6 or C18:1 acyl chains. PTX was physically incorporated into the micellar core by solvent evaporation technique. Maximum loading capacity for HAC6 and HAC18:1 was determined to be 2 and 14 wt.%, respectively. The loading efficiency was higher for HAC18:1 and reached 70%. Independently of the derivative, loaded HA micelles had spherical size of approximately 60-80 nm and demonstrated slow and sustained release of PTX in vitro. PTX largely changed its form from crystalline to amorphous after its incorporation into the micelle's interior. This transformation increased PTX sensitivity towards stressing conditions, mainly to UV light exposure, during which the structure of amorphous PTX isomerized and formed C3C11 bond within its structure. In vitro cytotoxicity assay revealed that polymeric micelles loaded with PTX isomer had higher cytotoxic effect to normal human dermal fibroblasts (NHDF) and human colon carcinoma cells (HCT-116) than the same micelles loaded with non-isomerized PTX. Further observation indicated that PTX isomer influenced in different ways cell morphology and markers of cell cycle. Taken together, PTX isomer loaded in nanocarrier systems may have improved anticancer activity in vivo than pure PTX.

The generation of degradation byproducts from hyaluronan during its acid hydrolysis was studied. The degradation byproducts were removed from reaction mixture by ultrafiltration, followed by ionex separation and purification on a reversed-phase HPLC column. Isolated fractions were structurally analyzed by NMR and mass spectrometry. Results indicated that when acid hydrolysis is performed at 100 °C for 24 h, about 0.7% of high molar mass hyaluronan (1.2 × 106 g mol−1) was converted into furan-like derivatives. Other major degradation byproducts present in 0.2 wt% amounts were identified as derivatives of cyclopentanone. Cell viability tests indicated that most of the degradation byproducts have negative effect on cell growth. These tests were performed using mouse fibroblast cell line 3T3 and human keranocytes cell line HaCaT. For this reason, it is important to evaluate the amount of degradation byproducts in low molar mass hyaluronan produced by acid hydrolysis, mainly when these products are used in pharmaceutical and cosmetic applications. During this study it was found out that the unwanted degradation byproducts can be completely removed when an ultrafiltration step is introduced after acid hydrolysis prior hyaluronan isolation.

Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3 kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.

Diabetes mellitus (DM) is associated with many health complications and foot ulcers are one of the escalating, which brings uncomfortable and painful lifestyle for DM patients. In severe cases, foot ulcers can lead to a lower extremity amputation or even death. The treatment of such affected patients results in a high economic load [1]. Immune system of DM patients shows improper function leading to chronic wound of foot ulcers. In a physiological wound healing, wound is cleared from bacteria by immune cells, keratinocytes perform re-epithelization, fibroblasts restore dermis matrix and endothelial cells ensure angiogenesis. All is perfectly driven by growth factors, cytokines and chemokines which are dozen in a sensitive balance. In the foot ulcers of DM patients, immune cells over-express pro-inflammatory cytokines and chemokines (IL-1, IL-6, TNF-α, CCL2, CCL3, CCL4, CXCL1, CXCL5, CXCL8) and are not able to clear wound bed from infection. Fibroblasts and keratinocytes have reduced proliferative, locomotive and secreting functions and are not able to renovate epidermis and dermis followed by lacking angiogenesis [2]. High levels of pro-inflammatory cytokines, chemokines and proteases produced by persisting bacteria and activated immune cells represent main factors supporting the improper healing of foot ulcers. The standard treatment of diabetic ulcers includes optimization of glycemic control, extensive debridement, infection elimination, use of moisture dressings, and offloading high pressure. New approaches such as autologous skins transplantation, mesenchymal stem cell application or dressings containing growth factors started to be utilized [1]. The development of new treatments requires a multidisciplinary cooperation and a deep molecular-biological research. The identification of microbiological contamination is a common approach and serves for the development and the evaluation of efficient antibacterial preparations. The protein analysis of wound debris and wound exudates utilizing multiplex immunological methods represent a quite new method and can provide an overview of chronic wound protein content, which can bring information for the development of curing preparations focused on cytokine, chemokine or proteases function. The determination of protein composition of chronic wound further enables the monitoring of healing process and provides the data about efficacy of healing management. Although the foot ulcers care is at high level and not all patients are judged to extremity amputation, the healing of foot ulcers in DM patients is a constant challenge. The field for development of an effective, economically and patient’s friendly medical preparation with doctor’s favorable application form is still open.

Determination of cell numbers is a crucial step in studies focused on cytokinetics and cell toxicity. The impedance-based analysis employing electronic sensor array system xCELLigence System allowing label-free dynamic monitoring of relative viable adherent cell amounts was compared with the most utilized methods for relative quantification of viable cell numbers based on a determination of cellular metabolism.Colorimetric assay based on reduction of tetrazolium salt (MTT) by mitochondrial enzymes and chemiluminiscent assay based on intracellular adenosine triphosphate (ATP) determination were compared with the impedance-based system. Cell morphology was compared by microscopic evaluation. Normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF), together with 3T3 mouse fibroblast and HaCaT keratinocyte cell lines were employed.The progress of cell growth curves obtained by different methods during 72 hours reflected cell type and cell seeding densities. The impedance-based method was found to be applicable for the determination of the cell proliferation of 3T3 fibroblasts, HaCaT and NHDF, since the comparison of this method with ATP and MTT determinations showed a comparable results. In contrast, the proliferation of NHEK measured by the impedance-based method did not correlate with other methodological approaches. This could be accounted to the specific morphological appearance of these cells.The study shows the impedance-based detection of viable adherent cells is a valuable approach for cytokinetics and pharmacological studies. However, the specific morphological characteristics of cell lines have to be considered employing this method for determination of cell proliferation without using other reference methods.