Viable Tissue Research Articles

Hydrogels in cardiac tissue engineering: application and challenges.

Cardiovascular disease remains the leading cause of global mortality. Current stem cell therapy and heart transplant therapy have limited long-term stability in cardiac function. Cardiac tissue engineering may be one of the key methods for regenerating damaged myocardial tissue. As an ideal scaffold material, hydrogel has become a viable tissue engineering therapy for the heart. Hydrogel can not only provide mechanical support for infarcted myocardium but also serve as a carrier for various drugs, bioactive factors, and cells to increase myocardial contractility and improve the cell microenvironment in the infarcted area, thereby improving cardiac function. This paper reviews the applications of hydrogels and biomedical mechanisms in cardiac tissue engineering and discusses the challenge of clinical transformation of hydrogel in cardiac tissue engineering, providing new strategies for treating cardiovascular diseases.

Viability and Diagnostic Potential of Tissues Obtained Through Cryobiopsy

BackgroundTransbronchial lung cryobiopsy is primarily used for diagnosing interstitial lung diseases and tumors, providing larger tissue samples with reduced tissue crushing than traditional biopsies. However, freezing during cryobiopsy may damage cells, potentially affecting diagnostic methods that require live cells, such as flow cytometry (FCM). We aimed to determine the extent of freezing-related cell damage in cryobiopsies using cells cultured in vitro. MethodsTo investigate the relationship between freezing duration and sample volume, Jurkat cells underwent freezing for durations ranging from 2 to 6 s, with 1-s intervals, using either 1-mm- or 1.7-mm cryoprobes. FCM was conducted to assess both cell viability (2, 4, and 6 s) and cell-surface molecule expression (3 and 6 s) over varying freezing times. Additionally, we describe a clinical case involving a 70-year-old man suspected of malignant lymphoma, in which tissue samples were obtained via both forceps biopsy and cryobiopsy methods to compare the pathological and cytological features between the methods. ResultsHarvested cell count increased with freezing duration, with a notable increase in viable cell percentage. Moreover, cells distant from the cryoprobe exhibited higher survival rates under milder freezing conditions. FCM revealed significantly higher marker expression levels in viable cryobiopsy samples than in non-viable samples. The clinical case demonstrated that cryobiopsy yields a significant proportion of live cells (>90%), with cytological findings consistent with those of non-frozen samples. ConclusionsCryobiopsy may be beneficial for histopathological diagnosis, providing sufficient viable cells for FCM, and can be used for diagnosing malignant lymphomas and other pulmonary conditions.

The DAIR-procedure in fracture-related infection–When and how

This narrative review discusses treatment strategies and key considerations guiding decisions in the surgical management of fracture-related infections (FRI). Treatment options primarily revolve around two approaches: debridement antibiotics and implant retention (DAIR), or implant exchange, either in a one or multiple-stage strategy. Several considerations, including time since infection onset, implant type, stability, causative pathogens, host physiology, and soft tissue conditions, inform the choice of surgical intervention for FRI. Current literature supports the preference for a DAIR approach in cases with a short duration of ongoing symptoms, a stable implant with satisfactory fracture reduction, and a viable soft tissue envelope. Conversely, one- or multiple-stage implant exchange is deemed beneficial in instances of compromised local and systemic host physiology, mature biofilm, difficult-to-treat pathogens, intramedullary implants, and cases involving reinfections or failed DAIR procedures. Notably, these recommendations draw parallels from periprosthetic joint infection treatment strategies, constrained by the limited availability of randomized controlled trials comparing these options specifically in acute FRI. In conclusion, future perspectives call for extensive investigations into biofilm maturation and the impact of time on treatment outcomes. Additionally, there is a need for a standardized classification system for FRI to enhance the comparability of treatment outcomes in FRI management.

Quantification of myocardial oxygen extraction fraction on noncontrast MRI enabled by deep learning

Abstract Purpose To develop a new deep learning enabled cardiovascular magnetic resonance (CMR) approach for noncontrast quantification of myocardial oxygen extraction fraction (mOEF) and myocardial blood volume (MBV) in vivo. Materials and Methods An asymmetric-spin-echo prepared CMR sequence was created in a 3 T MRI clinical system. A UNet-based fully connected neural network was developed based on a theoretical model of CMR signals to calculate mOEF and MBV. Twenty healthy volunteers (20–30 years old, 11 females) underwent CMR scans at three short-axial slices (16 myocardial segments) on two different days. The reproducibility was assessed by the coefficient of variation (CV). Ten patients with chronic myocardial infarction were examined to evaluate the feasibility of this CMR method to detect abnormality of mOEF and MBV. Results Among the volunteers, the average global mOEF and MBV on both days was 0.58 ± 0.07 and 9.5% ± 1.5%, respectively, which agreed well with data measured by other imaging modalities. The CV of mOEF was 8.4%, 4.5%, and 2.6%, on a basis of segment, slice, and participant, respectively. No significant difference in mOEF was shown among three slices or among different myocardial segments. Female participants showed significantly higher segmental mOEF than male participants (p < 0.001). Regional mOEF decrease 40% in CMR confirmed myocardial infarction core, compared to normal myocardial regions. Conclusion The new deep learning enabled CMR approach allows noncontrast quantification of mOEF and MBV with good to excellent reproducibility. This technique could provide an objective contrast-free means to assess and serially measure hypoxia-relief effects of therapeutic interventional strategies to save viable myocardial tissues.

Procurement and Preservation of Neonatal Porcine Cardiac Tissue.

The porcine and human heart are remarkably similar in cardiac physiology and biochemistry. Translational research involving the porcine biomedical model is becoming increasingly applicable for the study of human cardiac function in health and disease. Presently, few protocols exist for collecting experimentally viable cardiac tissue from large animal models, particularly during neonatal maturation. To address this deficiency, we have developed a technique to procure and preserve ventricular tissue from neonatal piglets at day 3 (n=4) and day 30 (n=6) post-partum. Piglets were subjected to a strict sedation, anesthesia, and analgesia regimen. During surgery, cardiopulmonary indices of electrocardiogram, heart rate, systolic and diastolic blood pressure, respiration rate, peripheral O2 saturation, and end-tidal CO2 were monitored continuously to ensure normal cardiac function. Prior to cardiectomy, each heart was perfused with an intravenous administration of heparin (10 ml/kg) and ice-cold Custodiol HTK cardioplegia solution (10 ml/kg). After cardiac explantation, myocardial samples (dimensions: 1 x 1 x 1 cm) were dissected from the left and right ventricles and snap-frozen in liquid nitrogen. Analysis via SDS-PAGE and densitometry demonstrated that myofibrillar proteins are stable and undegraded. Western Blots showed full expression of protein. These results suggest that the detailed cardiac tissue procurement technique preserves the experimental viability of the cardiac tissue and prevents the degradation of myofibrillar proteins.

Pangolin scales as adaptations for innate immunity against pathogens

BackgroundPangolins are the only mammals that have overlapping scales covering most of their bodies, and they play a crucial role in the ecosystem, biological research, and human health and disease. Previous studies indicated pangolin scale might provide an important mechanical defense to themselves. The origin and exact functions of this unique trait remain a mystery. Using a multi-omics analysis approach, we report a novel functional explanation for how mammalian scales can provide host–pathogen defense.ResultsOur data suggest that pangolin scales have a sophisticated structure that could potentially trap pathogens. We identified numerous proteins and metabolites exhibiting antimicrobial activity, which could suggest a role for scales in pathogen defense. Notably, we found evidence suggesting the presence of exosomes derived from diverse cellular origins, including mesenchymal stem cells, immune cells, and keratinocytes. This observation suggests a complex interplay where various cell types may contribute to the release of exosomes and antimicrobial compounds at the interface between scales and viable tissue. These findings indicate that pangolin scales may serve as a multifaceted defense system, potentially contributing to innate immunity. Comparisons with human nail and hair revealed pangolin-specific proteins that were enriched in functions relating to sensing, immune responses, neutrophil degranulation, and stress responses. We demonstrated the antimicrobial activity of key pangolin scale components on pathogenic bacteria by antimicrobial assays.ConclusionsThis study identifies a potential role of pangolin scales and implicates scales, as possible determinants of pathogen defense due to their structure and contents. We indicate for the first time the presence of exosomes in pangolin scales and propose the new functions of scales and their mechanisms. This new mechanism could have implications for multiple fields, including providing interesting new research directions and important insights that can be useful for synthesizing and implementing new biomimetic antimicrobial approaches.

Effect of Hyperbaric Oxygen Therapy and Adipocyte Stem Cell on the Viability of Degloving Injury: A Murine Model.

Degloving soft tissue injuries (DSTIs) involve skin and tissue detachment from muscle or fascia. Surgical treatments exist, but they cannot prevent necrosis. Our aim was to investigate the effects of hyperbaric oxygen therapy (HOT) and adipocyte stem cell (ASC) treatment on tissue viability in degloving injuries in a murine model. 32 animals were submitted to a degloving flap surgery in the dorsal region and were allocated in four groups (n=8/group): Control: suture only; HOT: 2-hour daily therapy in 100% oxygen at 2.0 ATA for 7 days; ASC: injected with 1x106 stem cells; ASC+HOT: stem cells injection plus HOT therapy. We performed macroscopic measurements, blood flow, histology, and expression of inflammation genes. After 7days, HOT, ASC, and ASC+HOT groups had significantly more viable tissue compared to Control (97%, 90%, 81% vs. 6%). Viable area ratios were higher in HOT and ASC than Control. Blood flow in the injury's distal region was higher in HOT, ASC, and ASC+HOT compared to Control. Vascular density was higher in HOT and ASC+HOT than Control. Inflammatory cells decreased by 40% in HOT, 50% in ASC+HOT, and 75% in ASC. Gene Cd68 expression was lower in HOT than Control. Il10 expression was lower in HOT but higher in ASC and ASC+HOT than Control. This study suggests that the HOT can benefit the degloving injury flap model in the early phase of wound healing, and the association of ASC with HOT could benefit the wound healing in a later phase. This journal requires that authors assign a level of evidence to each submission to which evidence-based medicine rankings are applicable. This excludes review articles, book reviews, and manuscripts that concern basic science, animal studies, cadaver studies, and experimental studies. For a full description of these evidence-based medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Acute Ischemic Stroke during Extracorporeal Membrane Oxygenation (ECMO): A Narrative Review of the Literature

Ischemic stroke (IS) is a severe complication and leading cause of mortality in patients under extracorporeal membrane oxygenation (ECMO). The aim of our narrative review is to summarize the existing evidence and provide a deep examination of the diagnosis and treatment of acute ischemic stroke patients undergoing ECMO support. The incidence rate of ISs is estimated to be between 1 and 8%, while the mortality rate ranges from 44 to 76%, depending on several factors, including ECMO type, duration of support and patient characteristics. Several mechanisms leading to ISs during ECMO have been identified, with thromboembolic events and cerebral hypoperfusion being the most common causes. However, considering that most of the ECMO patients are severely ill or under sedation, stroke symptoms are often underdiagnosed. Multimodal monitoring and daily clinical assessment could be useful preventive techniques. Early recognition of neurological deficits is of paramount importance for prompt therapeutic interventions. All ECMO patients with suspected strokes should immediately receive brain computed tomography (CT) and CT angiography (CTA) for the identification of large vessel occlusion (LVO) and assessment of collateral blood flow. CT perfusion (CTP) can further assist in the detection of viable tissue (penumbra), especially in cases of strokes of unknown onset. Catheter angiography is required to confirm LVO detected on CTA. Intravenous thrombolytic therapy is usually contraindicated in ECMO as most patients are on active anticoagulation treatment. Therefore, mechanical thrombectomy is the preferred treatment option in cases where there is evidence of LVO. The choice of the arterial vascular access used to perform mechanical thrombectomy should be discussed between interventional radiologists and an ECMO team. Anticoagulation management during the acute phase of IS should be individualized after the thromboembolic risk has been carefully balanced against hemorrhagic risk. A multidisciplinary approach is essential for the optimal management of ISs in patients treated with ECMO.

Plasma-Functionalized Liquids for Decontamination of Viable Tissues: A Comparative Approach

Plasma-functionalized liquids (PFLs) are rich in chemical species, such as ozone, hydrogen peroxide, singlet oxygen, hydroxyl radical and nitrogen oxides, commonly referred to as reactive oxygen and nitrogen species (RONS). Therefore, manifold applications are being investigated for their use in medicine, agriculture, and the environment. Depending on the goal, a suitable plasma source concept for the generation of PFLs has to be determined because the plasma generation setup determines the composition of reactive species. This study investigates three PFL-generating plasma sources—two spark discharges and a flow dielectric barrier discharge (DBD) system—for their efficacy in eliminating microbial contaminants from tissue samples aiming to replace antibiotics in the rinsing process. The final goal is to use these tissues as a cell source for cell-based meat production in bioreactors and thereby completely avoid antibiotics. Initially, a physicochemical characterization was conducted to better understand the decontamination capabilities of PFLs and their potential impact on tissue viability. The results indicate that the flow DBD system demonstrated the highest antimicrobial efficacy due to its elevated reactive species output and the possibility of direct treatment of tissues while tissue integrity remained. Achieving a balance between effective large-scale decontamination and the biocompatibility of PFLs remains a critical challenge.

6797 Pituitary Apoplexy as initial Presentation of IgG4-Related hypophysitis

Abstract Disclosure: M. Alameri: None. A. Alnuaimi: None. Introduction: IgG4-related hypophysitis is a rare disease due to IgG4 lymphoplasmacytic infiltration of the pituitary gland. It can either be isolated or part of a multisystemic disease. We report a rare case of pituitary apoplexy as first manifestation of IgG4-RD. Case report: A 27-year-old previously healthy male presented to emergency department with severe headache, nausea, vomiting and visual impairment. CT brain showed a large sellar suprasellar mass of 3cm with thick rim enhancement. Blood test showed evidence of central adrenal insufficiency, central hypothyroidism and secondary hypogonadism. Patient was started on hormone replacement therapy for central adrenal insufficiency and central hypothyroidism. Pituitary magnetic resonance imaging (MRI) demonstrated a 3.1 x 2.4 x 2.4 cm well-defined large sellar suprasellar mass with thick rim enhancement with intrinsic heterogeneous T2 signal with hypointense T1 signal on fat-suppressed post contrast imaging consistent with necrotic haemorrhagic suggestive of pituitary apoplexy and background of hypophysitis. Upward displacement of optic chiasm was noted without involvement of cavernous sinus. The patient was initially treated with high-dose steroids aiming for reduction of the pituitary mass. However, eventually, he underwent transsphenoidal surgery due to alternated level of consciousness. Pituitary histopathology showed that vast majority of the tissue is necrotic. A small fragment of viable anterior pituitary tissue is present, which measures up to 5.5 mm in greatest dimension which exhibited marked chronic inflammation, predominantly comprised of small lymphocytes. Plasma cells were present and staining with IgG/IgG4 revealed two high-power fields with clusters of approximately 25 and 50 IgG4 positive plasma cells, respectively. Blood test showed elevated blood IgG4 levels of >2.60 g/L (normal reference range 0.03-2.01 g/L). Case was discussed at Endocrine MDT meeting which agreed that blood tests result along with histopathology findings were in keeping with IgG4-Related hypophysitis as per the Mayo Clinic, Leporati and Japan-Korea proposed criteria. Because blood IgG4 levels were elevated, a systemic evaluation was performed. No evidence of other systemic disease found. High dose steroid therapy was initiated, which reduced remnant inflammatory sellar changes. Discussion and conclusion: IgG4-related pituitary disease should be considered as differential diagnosis in unusual presentations of pituitary apoplexy in the context of sellar suprasellar hypophysitis. Presentation: 6/2/2024

Long-term outcomes of ovary preserving procedure for neonatal ovarian cysts

Objective: We aim to evaluate the long-term outcomes of ovarian preserving surgery for neonatal ovarian cysts. Method: The clinical data of neonatal ovarian cysts treated in our hospital from January 2015 to December 2022 were retrospectively analyzed. Results: Our study included 22 patients, of which 21 were unilateral and 1 was bilateral. There were 13 cases of simple cysts and 9 cases of complex cysts, and one cyst changed from simple to complex. The mean largest diameter of simple cysts was 5.7±2.1cm, and that of complex cysts was 4.6±2.0cm. There was no significant difference between the two groups (P=0.2264). Among the 22 patients, 21 underwent laparoscopic cystectomy. The only patient with bilateral cysts underwent percutaneous drainage of the right simple cyst. Twenty-one histological specimens were reviewed, of which 18(85.7%) contained viable ovarian tissues. Five cases lost follow-up after surgery, and the remaining 17 cases had a follow-up period of 6 months to 5 years. The last ultrasound follow-up showed that 9 cases had bilateral normal ovaries, and 8 cases lost the ipsilateral ovary. The ovarian preservation rate of simple cyst was 90% (9/10), which was significantly higher than that of complex cyst (12.5%, 1/8) (P=0.003). Conclusions: Simple cysts (≥ 4cm) had an excellent prognosis, with a long-term ovarian preservation rate of 90%. The long-term ovarian preservation rate of complex cysts after surgery was relatively low. Our study suggested that surgical treatment did not increase the chances of preserving the ovaries of patients with complex cysts.

Use of Radioguided Surgery for Small and Difficult-to-Locate Relapsed MIBG (+) High-Risk Neuroblastoma Lesions.

High-risk neuroblastoma, particularly in the relapse/refractory (R/R) setting, poses unique challenges to obtaining the representative-quality tissue that is mostly required for molecular analysis. This study explores the use of 123I-MIBG radioguided surgery to access complex locations of MIBG-positive neuroblastoma as a tool to overcome the difficulties associated with repeated surgeries in these patients. This study is a retrospective review of all patients with R/R neuroblastoma and MIBG-uptaking lesions who underwent radioguided surgery between February 2020 and 2023 at SJD Barcelona Children's Hospital. The Europrobe 3.2 gamma probe was used to identify neuroblastoma tissue in the operating room. Ten patients were identified. Radioguided surgery was useful in all patients. One patient with previous multiple operations developed an entero-cutaneous fistula with posterior full recovery. Mean surgical time was 111.7 min. The gamma probe identified 100% of neuroblastoma lesions which were all completely removed (123I-MIBG-SPECT/CT negative post-surgery). Pathology and molecular studies could be successfully performed in all samples. 123I-MIBG radioguided surgery proved effective in obtaining viable tissue from difficult-to-access sites in high-risk relapsed neuroblastoma.

Bridging the Gap: Advances and Challenges in Heart Regeneration from In Vitro to In Vivo Applications.

Cardiovascular diseases, particularly ischemic heart disease, area leading cause of morbidity and mortality worldwide. Myocardial infarction (MI) results in extensive cardiomyocyte loss, inflammation, extracellular matrix (ECM) degradation, fibrosis, and ultimately, adverse ventricular remodeling associated with impaired heart function. While heart transplantation is the only definitive treatment for end-stage heart failure, donor organ scarcity necessitates the development of alternative therapies. In such cases, methods to promote endogenous tissue regeneration by stimulating growth factor secretion and vascular formation alone are insufficient. Techniques for the creation and transplantation of viable tissues are therefore highly sought after. Approaches to cardiac regeneration range from stem cell injections to epicardial patches and interposition grafts. While numerous preclinical trials have demonstrated the positive effects of tissue transplantation on vasculogenesis and functional recovery, long-term graft survival in large animal models is rare. Adequate vascularization is essential for the survival of transplanted tissues, yet pre-formed microvasculature often fails to achieve sufficient engraftment. Recent studies report success in enhancing cell survival rates in vitro via tissue perfusion. However, the transition of these techniques to in vivo models remains challenging, especially in large animals. This review aims to highlight the evolution of cardiac patch and stem cell therapies for the treatment of cardiovascular disease, identify discrepancies between in vitro and in vivo studies, and discuss critical factors for establishing effective myocardial tissue regeneration in vivo.

Clinical outcomes of hydrofiber foam dressing in the treatment of abrasion wounds: A retrospective study.

Mopeds are a popular mode of transportation in Taiwan, yet collision accidents often lead to abrasion requiring medical attention. While hydrofiber foam dressing (HFD) have proven to help control exudates, reduce the frequency of dressing changes, and preserve more viable tissue, there is currently no established clinical guideline for the treatment of abrasion wounds with dressings. Therefore, the aim of this study was to investigate the clinical outcomes of abrasions treated with hydrofiber foam dressings. A total of 127 patients treated with HFDs between June 2018 and January 2021 were included in this study. Abrasions were categorized into 3 grades: grade I (n = 0) involving the epidermal layer, grade II (n = 89) involving the dermal layer, and grade III (n = 38) involving subcutaneous layer, bone or tendon. Statistical analysis showed that there was a significant difference in healing time (P < 0.001) and the number of outpatient department (OPD) visits (P = 0.013) between grade II and grade III. However, there was no significant difference in the number of dressing pieces used. This study offers a clinical reference for the use of hydrofiber foam dressings in treating different grades of abrasion wounds caused by moped accidents.

Decalcification-free technique on the analysis of dental pulp tissue: histological and immunohistochemical analysis.

Histological techniques are essential for the microscopic study and investigation of the human dental pulp. The aim of this study was to evaluate the impact of decalcification-free technique by examining dental pulp morphology by histological staining with haematoxylin and eosin and immunohistochemistry. The sample consisted of 30 healthy third molars extracted for orthodontic indication, the pulp tissue was obtained by removing the mineralized tissues, separating the enamel and dentine and by marking with a flexible diamond disc on the coronal surface and longitudinal axis of the root. These guides made it possible to separate the fragments and obtain the pulp tissue for fixation and staining with H&E and subsequent immunohistochemistry with CD34 and S-100 antibodies. The technique showed preservation of pulp morphology with adequate preservation of microscopic structures. No alterations in tissue viability were observed. The staining allowed an accurate assessment of vascular and nervous components by means of CD34 and S-100 markers, respectively. This technique allows preservation of pulp tissue, maintaining viable tissue for histological analysis and immunohistochemistry tests, as well as reducing sample processing time.

Long-Term Functional Outcomes Following Enzymatic Debridement of Deep Hand Burns Using Nexobrid®: A Retrospective Analysis.

Background: For years, surgical debridement with autografting has been considered the standard of care in the treatment of severe burns of the hand. However, in recent years, enzymatic debridement has increasingly been reported as a good alternative, especially for burns of the hand, as it selectively preserves viable tissue. In this study, we aim to evaluate the long-term function of the hand after enzymatic debridement in deep dermal burns. Methods: A retrospective chart review was conducted as well as measurements of subjective and objective outcome measures through physical examination and Disabilities of the Arm, Shoulder, and Hand (DASH), Patient and Observer Scar Assessment Scale (POSAS), and Vancouver Scar Scale (VSS) scores. Results: A total of 32 enzymatically debrided hands of 24 patients were included with a mean age of 42.4 ± 16.8 years and a mean follow-up of 31 months. Postoperatively, 19 of these could be managed conservatively using skin substitutes such as "Suprathel", 13 had to undergo subsequent autografting. The mean DASH score for the entire study population was eight with a mean value of four in the conservatively managed group and fourteen in the autografted group. The mean Patient, Observer POSAS, and VSS values were nineteen, thirteen, and two. A total of 30 cases showed an effortless complete fist closure, and, also in 30 cases, patients attested to be satisfied with the esthetic appearance of the hand on being asked. Conclusions: The descriptive analysis of these results in our study population suggests that the enzymatic debridement of deep burns of the hand, especially combined with subsequent conservative management with skin substitutes, was associated with low long-term hand disability scores at a follow-up of two years.

Chemical and sensory analyses of cultivated pork fat tissue as a flavor enhancer for meat alternatives

The emerging field of cellular agriculture has accelerated the development of cell-cultivated adipose tissue as an additive to enhance the flavor of alternative meat products. However, there has been limited research to evaluate the sensory profile of in vitro-grown tissues compared to conventionally obtained animal fat. This study aimed to investigate the aromatic characteristics of cell-cultivated fat tissue as a flavor enhancer for meat alternatives. Porcine dedifferentiated fat (PDFAT) cells were clonally isolated and differentiated into adipocytes. This cultured adipose tissue was then analyzed alongside native porcine fat using gas chromatography-mass spectrometry (GC/MS) coupled with descriptive sensory analysis by human consumers. This evaluation enabled quantitative and qualitative assessments of volatile compounds released during cooking for both in vitro and in vivo porcine fats. The volatile profiles generated during the cooking process and fatty aroma characteristics reported by sensory consumers were largely similar between the two fat sources, with some differences in select compounds and aroma attributes. Ultimately, the consumers found comparable overall liking scores reported between the conventional and cultured porcine fats. These findings provide valuable sensory evidence supporting the viability of cell-cultivated adipose tissue as a flavor component of meat alternatives, substituting for conventional animal fat.

A history of repeated antibiotic usage leads to microbiota-dependent mucus defects

ABSTRACT Recent evidence indicates that repeated antibiotic usage lowers microbial diversity and ultimately changes the gut microbiota community. However, the physiological effects of repeated – but not recent – antibiotic usage on microbiota-mediated mucosal barrier function are largely unknown. By selecting human individuals from the deeply phenotyped Estonian Microbiome Cohort (EstMB), we here utilized human-to-mouse fecal microbiota transplantation to explore long-term impacts of repeated antibiotic use on intestinal mucus function. While a healthy mucus layer protects the intestinal epithelium against infection and inflammation, using ex vivo mucus function analyses of viable colonic tissue explants, we show that microbiota from humans with a history of repeated antibiotic use causes reduced mucus growth rate and increased mucus penetrability compared to healthy controls in the transplanted mice. Moreover, shotgun metagenomic sequencing identified a significantly altered microbiota composition in the antibiotic-shaped microbial community, with known mucus-utilizing bacteria, including Akkermansia muciniphila and Bacteroides fragilis, dominating in the gut. The altered microbiota composition was further characterized by a distinct metabolite profile, which may be caused by differential mucus degradation capacity. Consequently, our proof-of-concept study suggests that long-term antibiotic use in humans can result in an altered microbial community that has reduced capacity to maintain proper mucus function in the gut.

Endometriotic tissue fragments are viable after cryopreservation in an ex vivo tissue model recapitulating the fibrotic microenvironment.

Is it possible to establish an ex vivo endometriosis model using cryopreserved endometriotic tissue fragments? Cryopreserved endometriotic tissue fragments remain viable after thawing and during at least 3days of culture and can therefore be used to establish an ex vivo endometriosis model to efficiently test potential therapeutic agents. Endometriosis is the most prevalent benign gynecologic disease with an enormous societal burden; however, curative therapies are still lacking. To efficiently test potential new therapies, an ex vivo model based on previously cryopreserved endometriotic tissue that recapitulates the different endometriosis subtypes and their microenvironment is highly desirable. Endometriotic tissue fragments of three different subtypes were obtained from 28 patients by surgical resection. After cryopreservation and thawing, viability and metabolic activity of these tissue fragments were assessed. Viability was compared with fresh fragments from 11 patients directly after surgical removal. Experimental intervention studies were performed in cryopreserved and thawed tissue fragments from two patients to confirm the usability of these tissues for ex vivo intervention studies. Endometriotic tissue fragments (n = 45) were cryopreserved according to three different protocols. After thawing, fragments were cultured for 24 h. A resazurin-based assay was performed to assess the metabolic activity of the tissue fragments. In addition, cell type-specific viability was analyzed by VivaFix, Hoechst 33342, and α-smooth muscle actin immunofluorescence staining and confocal microscopy. The presence of endometriosis was histologically confirmed based on hematoxylin-eosin staining. Cryopreserved and thawed tissue fragments were treated for 72 h with pirfenidone or metformin and COL1A1 and CEMIP gene expressions were assessed using RT-PCR and RT-qPCR, either in the whole tissue fragments or in myofibroblasts isolated by laser capture microdissection. Metabolic activity of endometriotic tissue fragments obtained from peritoneal (PER), ovarian (OMA), and deep (DE) endometriotic lesions was well preserved after cryopreservation in a dimethyl sulfoxide-based medium and was comparable with fresh tissue fragments. Relative metabolic activity compared to fresh tissue was 70% (CI: 92-47%) in PER, 43% (CI: 53-15%) in OMA and 94% (CI: 186-3%) in DE lesions. In fragments from PE lesions 92% (CI: 87-96%), from OMA lesions 95% (CI: 91-98%), and from DE lesions 88% (CI: 78-98%) of cells were viable after cryopreservation and thawing followed by a 24-h culture period. Differences in gene expression of fibrotic markers COL1A1 and CEMIP after 72-h treatment with pirfenidone or metformin could be detected in whole tissue fragments and in isolated myofibroblasts, indicating that cryopreserved and thawed endometriotic tissue fragments are suitable for testing anti-fibrotic interventions. N/A. Viability and metabolic activity of the endometriotic tissue fragments may have been partially compromised by damage sustained during the surgical procedure, contributing to inter-sample variance. The storage of viable endometriotic tissue fragments for later usage in an ex vivo model creates the possibility to efficiently test potential new therapeutic strategies and facilitates the exchange of viable endometriotic tissue between different research laboratories. This study was not financially supported by external funding. The authors declare no competing interest. N/A.

Debridement efficacy of serine protease and formulated cream by In Vitro assessment against artificial wound eschar

Chronic wounds typically comprise of necrotic tissue and dried secretions, often culminating in the formation of a thick and tough layer of dead skin known as eschar. Removal of eschar is imperative to facilitate wound healing. Conventional approach for eschar removal involves surgical excision and grafting, which can be traumatic and frequently leads to viable tissue damage. There has been growing interest in the use of enzymatic agents for a gentler approach to debridement, utilizing proteolytic enzymes. In this study, a purified intracellular recombinant serine protease from Bacillus sp. (SPB) and its cream formulation were employed to evaluate their ability to degrade artificial wound eschar; composed of collagen, fibrin, and elastin. Degradation was assessed based on percentage weight reduction of eschar biomass, analysis via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and scanning electron microscopy (SEM). Both SPB and its cream formulation were able to degrade up to 50 % artificial wound eschar, with the SPB cream maintaining its degradation efficiency for up to 24 hours. Additionally, the SPB-based cream demonstrated the ability to hydrolyze proteinaceous components of eschars individually (fibrin and collagen) as determined through qualitative assessment. These findings suggest that SPB holds promise for the debridement of wound eschar.