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Non-thermal plasma, a partially ionized gas, holds significant potential for clinical applications, including wound-healing support, oral therapies, and anti-tumour treatments. While its applications showed promising outcomes, the underlying molecular mechanisms remain incompletely understood. We thus apply non-thermal plasma to mouse auricular skin and conducted non-coding RNA sequencing, as well as single-cell blood sequencing. In a time-series analysis (five timepoints spanning 2 hours), we compare the expression of microRNAs in the plasma-treated left ears to the unexposed right ears of the same mice as well as to the ears of unexposed control mice. Our findings indicate specific effects in the treated ears for a set of five miRNAs: mmu-miR-144-5p, mmu-miR-144-3p, mmu-miR-142a-5p, mmu-miR-223-3p, and mmu-miR-451a. Interestingly, mmu-miR-223-3p also exhibits an increase over time in the right non-treated ear of the exposed mice, suggesting systemic effects. Notably, this miRNA, along with mmu-miR-142a-5p and mmu-miR-144-3p, regulates genes and pathways associated with wound healing and tissue regeneration (namely ErbB, FoxO, Hippo, and PI3K-Akt signalling). This co-regulation is particularly remarkable considering the significant seed dissimilarities among the miRNAs. Finally, single-cell sequencing of PBMCs reveals the downregulation of 12 from 15 target genes in B-cells, Cd4+ and Cd8+ T-cells. Collectively, our data provide evidence for a systemic effect of non-thermal plasma.

Abstract Electrochemotherapy (ECT) combines the reversible electroporation (rEP) with intravenous (i.v.) or intratumoral (i.t.) administration of chemotherapeutic drugs. We conducted this study to compare the efficacy of i.v., i.t., and i.v. + i.t. injection of bleomycin (BLM) in ECT treatment of colorectal hepatic metastases in a rat model. WAG/Rij rats were randomized into three groups and underwent ECT with i.v., i.t., or i.v. + i.t. injection of BLM. Tumor volumes and oxygenation were measured by means of ultrasound and photoacoustic imaging. Moreover, liver and tumor tissue were analyzed by histology and immunohistochemistry. The i.v. and i.v. + i.t. groups exhibited a 44.0% and 46.6% reduction in oxygen saturation of the tumor tissue when compared to pretreatment values, whereas the i.t. group only showed a reduction of 35.2%. The extent of tumor tissue necrosis did not statistically differ between the groups. However, the i.t. group showed a tendency towards a lower necrosis rate. Cell proliferation, apoptotic cell death, vascularization, and immune cell infiltration were comparable in the treated tumors of the three groups. ECT with i.v. administration of BLM should be preferred in clinical practice, as the combined i.v. + i.t. therapy did not show superior oncological outcomes in the present study.

IntroductionSpheroids are promising building blocks for scaffold-free bone tissue engineering. Their rapid vascularization is of major importance to guarantee their survival after transplantation. To achieve this, we herein introduce the biofabrication of prevascularized spheroids by fusion of adipose tissue-derived microvascular fragments (MVF) with osteoblasts (OB).MethodsFor this purpose, 200 MVF from donor mice and 5,000, 10,000 or 20,000 murine OB (MC3T3-E1) were co-cultured in a liquid overlay system for 3 days to generate OB + MVF spheroids. OB mono-culture spheroids served as controls.Results and discussionDuring the generation process, the diameters of all spheroids progressively decreased, resulting in compact, viable spheroids of homogeneous sizes. MVF promoted the maturation of spheroids containing 5,000 OB, as shown by an accelerated decline of cell proliferation due to contact inhibition. Moreover, MVF most effectively reassembled into new microvascular networks within these small spheroids when compared to the other spheroid types, indicating the most beneficial MVF to OB ratio. Accordingly, these spheroids also showed a high angiogenic sprouting activity in vitro. In contrast to OB spheroids, they further rapidly vascularized in vivo after transplantation into dorsal skinfold chambers. This was caused by the interconnection of incorporated MVF with surrounding blood vessels. These findings indicate that OB + MVF spheroids may be suitable for bone tissue engineering, which should be next tested in appropriate in vivo bone defect models.

Nanofat is an autologous fat derivative with high regenerative activity, which is usually administered immediately after its generation by mechanical emulsification of adipose tissue. For its potential repeated use over longer time, we herein tested whether cryopreservation of nanofat is feasible. For this purpose, the inguinal fat pads of donor mice were processed to nanofat, which was i) frozen and stored in a freezer at −20°C, ii) shock frozen in liquid nitrogen with subsequent storage at −80°C or iii) gradually frozen and stored at −80°C. After 7 days, the cryopreserved nanofat samples were thawed and immunohistochemically compared with freshly generated nanofat (control). Nanofat frozen and stored at −20°C exhibited the lowest apoptotic rate and highest densities of blood and lymph vessels, which were comparable to those of control. Accordingly, nanofat cryopreserved at −20°C or control nanofat were subsequently fixed with platelet-rich plasma in full-thickness skin defects within dorsal skinfold chambers of recipient mice to assess vascularization, formation of granulation tissue and wound closure by means of stereomicroscopy, intravital fluorescence microscopy, histology and immunohistochemistry over 14 days. These analyses revealed no marked differences between the healing capacity of wounds filled with cryopreserved or control nanofat. Therefore, it can be concluded that cryopreservation of nanofat is simply feasible without affecting its viability and regenerative potential. This may broaden the range of future nanofat applications, which would particularly benefit from repeated administration of this autologous biological product.

Previous work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of RNAs. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory RNAs (sncRNAs) in two mouse plasma fractions at five time points across the lifespan from 2–18 months: (1) sncRNAs that are free-circulating (fc-RNA) and (2) sncRNAs bound outside or inside EVs (EV-RNA). Different sncRNA classes exhibit unique ageing patterns that vary between the fcRNA and EV-RNA fractions. While tRNAs showed the highest correlation with ageing in both fractions, rRNAs exhibited inverse correlation trajectories between the EV- and fc-fractions. For miRNAs, the EV-RNA fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncRNAs and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR.

Abstract Background Non-union formation still represents a major burden in trauma and orthopedic surgery. Moreover, aged patients are at an increased risk for bone healing failure. Parathyroid hormone (PTH) has been shown to accelerate fracture healing in young adult animals. However, there is no information whether PTH also stimulates bone regeneration in atrophic non-unions in the aged. Therefore, the aim of the present study was to analyze the effect of PTH on bone regeneration in an atrophic non-union model in aged CD-1 mice. Methods After creation of a 1.8 mm segmental defect, mice femora were stabilized by pin-clip fixation. The animals were treated daily with either 200 μg/kg body weight PTH 1–34 (n = 17) or saline (control; n = 17) subcutaneously. Bone regeneration was analyzed by means of X-ray, biomechanics, micro-computed tomography (µCT) imaging as well as histological, immunohistochemical and Western blot analyses. Results In PTH-treated animals bone formation was markedly improved when compared to controls. This was associated with an increased bending stiffness as well as a higher number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and CD31-positive microvessels within the callus tissue. Furthermore, PTH-treated aged animals showed a decreased inflammatory response, characterized by a lower number of MPO-positive granulocytes and CD68-positive macrophages within the bone defects when compared to controls. Additional Western blot analyses demonstrated a significantly higher expression of cyclooxygenase (COX)-2 and phosphoinositide 3-kinase (PI3K) in PTH-treated mice. Conclusion Taken together, these findings indicate that PTH is an effective pharmacological compound for the treatment of non-union formation in aged animals.

(1) Objective: This study aims to lay a foundation for noncontact intensive care monitoring of premature babies. (2) Methods: Arterial oxygen saturation and heart rate were measured using a monochrome camera and time-division multiplex controlled lighting at three different wavelengths (660 nm, 810 nm and 940 nm) on a piglet model. (3) Results: Using this camera system and our newly designed algorithm for further analysis, the detection of a heartbeat and the calculation of oxygen saturation were evaluated. In motionless individuals, heartbeat and respiration were separated clearly during light breathing and with only minor intervention. In this case, the mean difference between noncontact and contact saturation measurements was 0.7% (RMSE = 3.8%, MAE = 2.93%). (4) Conclusions: The new sensor was proven effective under ideal animal experimental conditions. The results allow a systematic improvement for the further development of contactless vital sign monitoring systems. The results presented here are a major step towards the development of an incubator with noncontact sensor systems for use in the neonatal intensive care unit.

The elderly exhibit a reduced healing capacity after fracture, which is often associated with delayed or failed bone healing. This is due to a plethora of factors, such as an impaired bone vascular system and delayed angiogenesis. The phosphodiesterase-5 (PDE-5) inhibitor sildenafil exerts pro-angiogenic and pro-osteogenic effects. Hence, we herein investigated in aged mice whether sildenafil can improve fracture healing. For this purpose, 40 aged CD-1 mice (16–18 months) were daily treated with 5 mg/kg body weight sildenafil (n = 20) or vehicle (control, n = 20) by oral gavage. The callus tissue of their femora was analyzed at 2 and 5 weeks after fracture by X-ray, biomechanics, micro-computed tomography (µCT), histology, immunohistochemistry as well as Western blotting. These analyses revealed a significantly increased bone volume and higher ratio of callus to femoral bone diameter in sildenafil-treated mice at 5 weeks after fracture when compared to controls. This was associated with a reduced number and activity of osteoclasts at 2 weeks after fracture, most likely caused by an increased expression of osteoprotegerin (OPG). Taken together, these findings indicate that sildenafil does not improve fracture healing in the elderly but delays the process of bone remodeling most likely by reducing the number and activity of osteoclasts within the callus tissue.

Nerve/glial antigen (NG)2 is highly expressed in glioblastoma multiforme (GBM). However, the underlying mechanisms of its upregulated expression are largely unknown. In silico analyses reveal that the tumor-suppressive miR-29b targets NG2. We used GBM-based data from The Cancer Genome Atlas databases to analyze the expression pattern of miR-29b and different target genes, including NG2. Moreover, we investigated the regulatory function of miR-29b on NG2 expression and NG2-related signaling pathways. We further studied upstream mechanisms affecting miR-29b-dependent NG2 expression. We found that miR-29b downregulates NG2 expression directly and indirectly via the transcription factor Sp1. Furthermore, we identified the NG2 coreceptor platelet-derived growth factor receptor (PDGFR)α as an additional miR-29b target. As shown by a panel of functional cell assays, a reduced miR-29b-dependent NG2 expression suppresses tumor cell proliferation and migration. Signaling pathway analyses revealed that this is associated with a decreased ERK1/2 activity. In addition, we found that the long noncoding RNA H19 and c-Myc act as upstream repressors of miR-29b in GBM cells, resulting in an increased NG2 expression. These findings indicate that the c-Myc/H19/miR-29b axis crucially regulates NG2 expression in GBM and, thus, represents a target for the development of future GBM therapies.

Abstract The hierarchical design of the toe pad surface in geckos and its reversible adhesiveness have inspired material scientists for many years. Micro‐ and nano‐patterned surfaces with impressive adhesive performance have been developed to mimic gecko's properties. While the adhesive performance achieved in some examples has surpassed living counterparts, the durability of the fabricated surfaces is limited and the capability to self‐renew and restore function—inherent to biological systems—is unimaginable. Here the morphogenesis of gecko setae using skin samples from the Bibron´s gecko (Chondrodactylus bibronii) is studied. Gecko setae develop as specialized apical differentiation structures at a distinct cell–cell layer interface within the skin epidermis. A primary role for F‐actin and microtubules as templating structural elements is necessary for the development of setae's hierarchical morphology, and a stabilization role of keratins and corneus beta proteins is identified. Setae grow from single cells in a bottom layer protruding into four neighboring cells in the upper layer. The resulting multicellular junction can play a role during shedding by facilitating fracture of the cell–cell interface and release of the high aspect ratio setae. The results contribute to the understanding of setae regeneration and may inspire future concepts to bioengineer self‐renewable patterned adhesive surfaces.

Abstract Non-union formation represents a major complication in trauma and orthopedic surgery. The phosphodiesterase-5 (PDE-5) inhibitor sildenafil has been shown to exert pro-angiogenic and pro-osteogenic effects in vitro and in vivo. Therefore, the aim of the present study was to analyze the impact of sildenafil in an atrophic non-union model in mice. After creation of a 1.8 mm segmental defect, mice femora were stabilized by pin-clip fixation. Bone regeneration was analyzed by means of X-ray, biomechanics, photoacoustic and micro-computed tomography (µCT) imaging as well as histological, immunohistochemical and Western blot analyses at 2, 5 and 10 weeks after surgery. The animals were treated daily with either 5 mg/kg body weight sildenafil (n = 35) or saline (control; n = 35) per os. Bone formation was markedly improved in defects of sildenafil-treated mice when compared to controls. This was associated with a higher bending stiffness as well as an increased number of CD31-positive microvessels and a higher oxygen saturation within the callus tissue. Moreover, the bone defects of sildenafil-treated animals contained more tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and CD68-positive macrophages and exhibited a higher expression of the pro-angiogenic and pro-osteogenic markers cysteine rich protein (CYR)61 and vascular endothelial growth factor (VEGF) when compared to controls. These findings demonstrate that sildenafil acts as a potent stimulator of angiogenesis and bone regeneration in atrophic non-unions.

Non-unions represent a major complication in trauma and orthopedic surgery. Many factors contribute to bone regeneration, out of which an adequate vascularization has been recognized as crucial. The phosphodiesterase-3 (PDE-3) inhibitor cilostazol has been shown to exert pro-angiogenic and pro-osteogenic effects in a variety of preclinical studies. Hence, we herein investigated the effects of cilostazol on bone regeneration in an atrophic non-union model in mice. For this purpose, a 1.8 mm femoral segmental defect was stabilized by pin-clip fixation and the animals were treated daily with 30 mg/kg body weight cilostazol or saline (control) per os. At 2, 5 and 10 weeks after surgery the healing of femora was analyzed by X-ray, biomechanics, photoacoustic imaging, and micro-computed tomography (µCT). To investigate the cellular composition and the growth factor expression of the callus tissue additional histological, immunohistochemical and Western blot analyses were performed. Cilostazol-treated animals showed increased bone formation within the callus, resulting in an enhanced bending stiffness when compared to controls. This was associated with a more pronounced expression of vascular endothelial growth factor (VEGF), a higher number of CD31-positive microvessels and an increased oxygen saturation within the callus tissue. Furthermore, cilostazol induced higher numbers of tartrate-resistant acidic phosphate (TRAP)-positive osteoclasts and CD68-positive macrophages. Taken together, these findings demonstrate that cilostazol is a promising drug candidate for the adjuvant treatment of atrophic non-unions in clinical practice.

The tumor microenvironment stimulates the angiogenic activity of endothelial cells (ECs) to facilitate tumor vascularization, growth, and metastasis. The involvement of microRNA-186-5p (miR-186) in regulating the aberrant activity of tumor-associated ECs has so far not been clarified. In the present study, we demonstrated that miR-186 is significantly downregulated in ECs microdissected from human non-small cell lung cancer (NSCLC) tissues compared with matched non-malignant lung tissues. In vitro analyses of primary human dermal microvascular ECs (HDMECs) exposed to different stimuli indicated that this miR-186 downregulation is triggered by hypoxia via activation of hypoxia-inducible factor 1 alpha (HIF1α). Transfection of HDMECs with miR-186 mimic (miR-186m) significantly inhibited their proliferation, migration, tube formation, and spheroid sprouting. In contrast, miR-186 inhibitor (miR-186i) exerted pro-angiogenic effects. In vivo, endothelial miR-186 overexpression inhibited the vascularization of Matrigel plugs and the initial growth of tumors composed of NSCLC cells (NCI-H460) and HDMECs. Mechanistic analyses revealed that the gene encoding for protein kinase C alpha (PKCα) is a bona fide target of miR-186. Activation of this kinase significantly reversed the miR-186m-repressed angiogenic activity of HDMECs. These findings indicate that downregulation of miR-186 in ECs mediates hypoxia-stimulated NSCLC angiogenesis by upregulating PKCα.

Periodic fasting (PF) as a form of dietary restriction has been shown to induce tissue-protective effects against ischemic injury in several different tissues. Accordingly, in this study we analyzed whether a short-term 24 h fast is suitable to prevent necrosis of musculocutaneous flap tissue undergoing acute persistent ischemia. C57BL/6N mice were randomly divided into a PF group (n = 8) and a control group that was given unrestricted access to standard chow (n = 8). The PF animals underwent a 24 h fast immediately before flap elevation and had unrestricted access to food for the rest of the 10 day observation period. Musculocutaneous flaps with a random pattern design were dissected on the animals’ backs and mounted into dorsal skinfold chambers. On days 1, 3, 5, 7 and 10 after surgery, nutritive tissue perfusion, angiogenesis and flap necrosis were evaluated using intravital fluorescence microscopy. Thereafter, the flap tissue was excised and fixed for histological and immunohistochemical analyses. The flaps of PF-treated animals exhibited a higher functional capillary density and more newly formed microvessels, resulting in a significantly increased flap survival rate. Moreover, they contained a lower number of myeloperoxidase (MPO)-positive neutrophilic granulocytes and cleaved caspase-3-positive apoptotic cells in the transition zone between vital and necrotic flap tissue. These findings indicate that short-term PF improves tissue survival in ischemically challenged musculocutaneous flaps by maintaining nutritive blood perfusion and dampening ischemia-induced inflammation.

Electrochemical disinfection in dentistry using boron-doped diamond (BDD) electrodes bears the potential risk of disturbing vital functions. Applying different arrays of BDD electrodes and an electrotome as reference, it was the goal of this animal study to compare their effects on an electrocorticogram (ECoG) and electrocardiogram (ECG). Following the trepanation of teeth in rats, the electrodes and electrotome were applied in a randomized manner while recording ECoG and ECG. The recordings were classified according to an electrophysiological significance score based on involvement, extent of disruption and duration. The scores obtained were compared by means of ANOVA followed by Dunn’s multiple comparisons test (α = 0.05). Voltage type and electrode design had a significant influence on the detectable electrophysiological effects. The results seen with BDD electrodes ranged from no detectable electrophysiological effects to a pronounced effect. The application of the electrotome induced the most pronounced effects. Given that electrotomes are safe medical devices, despite evoking greater disturbance compared to BDD electrodes, regardless of their design, electrochemical disinfection may be considered a safe procedure.

Abstract Insufficient revascularization of pancreatic islets is one of the major obstacles impairing the success of islet transplantation. To overcome this problem, we introduce in the present study a straightforward strategy to accelerate the engraftment of isolated islets. For this purpose, we co-transplanted 250 islets and 20,000 adipose tissue-derived microvascular fragments (MVF) from donor mice under the kidney capsule as well as 500 or 1000 islets with 40,000 MVF into the subcutaneous space of diabetic mice. We found that the co-transplantation of islets and MVF markedly accelerates the restoration of normoglycemia in diabetic recipients compared with the transplantation of islets alone. In fact, the transplantation of 250 islets with 20,000 MVF under the kidney capsule reversed diabetes in 88% of mice and the subcutaneous transplantation of 500 or 1000 islets with 40,000 MVF restored normoglycemia in 100% of mice. Moreover, diabetic mice receiving islets and MVF exhibited plasma insulin levels similar to nondiabetic control animals. Additional immunohistochemical analyses of the grafts revealed a significantly higher number of islet cells and microvessels in the co-transplantation groups. These findings demonstrate that the co-transplantation of islets and MVF is a promising strategy to improve the success rates of islet transplantation, which could be easily implemented into future clinical practice.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, belong to the most prescribed analgesic medication after traumatic injuries. However, there is accumulating evidence that NSAIDs impair fracture healing. Because bone regeneration in aged patients is subject to significant changes in cell differentiation and proliferation as well as a markedly altered pharmacological action of drugs, we herein analyzed the effects of diclofenac on bone healing in aged mice using a stable closed femoral facture model. Thirty-three mice (male n = 14, female n = 19) received a daily intraperitoneal injection of diclofenac (5 mg/kg body weight). Vehicle-treated mice (n = 29; male n = 13, female n = 16) served as controls. Fractured mice femora were analyzed by means of X-ray, biomechanics, micro computed tomography (μCT), histology and Western blotting. Biomechanical analyses revealed a significantly reduced bending stiffness in diclofenac-treated animals at 5 weeks after fracture when compared to vehicle-treated controls. Moreover, the callus tissue in diclofenac-treated aged animals exhibited a significantly reduced amount of bone tissue and higher amounts of fibrous tissue. Further histological analyses demonstrated less lamellar bone after diclofenac treatment, indicating a delay in callus remodeling. This was associated with a decreased number of osteoclasts and an increased expression of osteoprotegerin (OPG) during the early phase of fracture healing. These findings indicate that diclofenac delays fracture healing in aged mice by affecting osteogenic growth factor expression and bone formation as well as osteoclast activity and callus remodeling.

Fracture healing in the aged is associated with a reduced healing capacity, which often results in delayed healing or non-union formation. Many factors may contribute to this deterioration of bone regeneration, including a reduced ‘angiogenic trauma response’. The phosphodiesterase-3 (PDE-3) inhibitor cilostazol has been shown to exert pro-angiogenic and pro-osteogenic effects in preclinical studies. Therefore, we herein analyzed in a stable closed femoral fracture model whether this compound also promotes fracture healing in aged mice. Forty-two aged CD-1 mice (age: 16–18 months) were daily treated with 30 mg/kg body weight cilostazol (n = 21) or vehicle (control, n = 21) by oral gavage. At 2 and 5 weeks after fracture, the femora were analyzed by X-ray, biomechanics, micro-computed tomography (µCT), histology, immunohistochemistry, and Western blotting. These analyses revealed a significantly increased bending stiffness at 2 weeks (2.2 ± 0.4 vs. 4.3 ± 0.7 N/mm) and an enhanced bone formation at 5 weeks (4.4 ± 0.7 vs. 9.1 ± 0.7 mm3) in cilostazol-treated mice when compared to controls. This was associated with a higher number of newly formed CD31-positive microvessels (3.3 ± 0.9 vs. 5.5 ± 0.7 microvessels/HPF) as well as an elevated expression of phosphoinositide-3-kinase (PI3K) (3.6 ± 0.8 vs. 17.4 ± 5.5-pixel intensity × 104) and runt-related transcription factor (RUNX)2 (6.4 ± 1.2 vs. 18.2 ± 2.7-pixel intensity × 104) within the callus tissue. These findings indicate that cilostazol accelerates fracture healing in aged mice by stimulating angiogenesis and the expression of PI3K and RUNX2. Hence, cilostazol may represent a promising compound to promote bone regeneration in geriatric patients.

The treatment of wounds using the body’s own resources is a promising approach to support the physiological regenerative process. To advance this concept, we evaluated the effect of nanofat (NF) on wound healing. For this purpose, full-thickness skin defects were created in dorsal skinfold chambers of wild-type mice. These defects were filled with NF generated from the inguinal subcutaneous adipose tissue of green fluorescent protein (GFP)+ donor mice, which was stabilized using platelet-rich plasma (PRP). Empty wounds and wounds solely filled with PRP served as controls. Wound closure, vascularization and formation of granulation tissue were repeatedly analyzed using stereomicroscopy, intravital fluorescence microscopy, histology and immunohistochemistry over an observation period of 14 days. PRP + NF-treated wounds exhibited accelerated vascularization and wound closure when compared to controls. This was primarily due to the fact that the grafted NF contained a substantial fraction of viable GFP+ vascular and lymph vessel fragments, which interconnected with the GFP− vessels of the host tissue. Moreover, the switch from inflammatory M1- to regenerative M2-polarized macrophages was promoted in PRP + NF-treated wounds. These findings indicate that NF markedly accelerates and improves the wound healing process and, thus, represents a promising autologous product for future wound management.

Clinical islet transplantation is limited by ischemia-induced islet cell death. Recently, it has been reported that the absent in melanoma (AIM)2 inflammasome is upregulated by ischemic cell death due to recognition of aberrant cytoplasmic self-dsDNA. However, it is unknown whether AIM2 determines the outcome of islet transplantation. To investigate this, isolated wild type (WT) and AIM2-deficient (AIM2−/−) islets were exposed to oxygen-glucose deprivation to mimic ischemia, and their viability, endocrine function, and interferon (IFN) signaling were assessed. Moreover, the revascularization and endocrine function of grafted WT and AIM2−/− islets were analyzed in the mouse dorsal skinfold chamber model and the diabetic kidney capsule model. Ischemic WT and AIM2−/− islets did not differ in their viability. However, AIM2−/− islets exhibited a higher protein level of p202, a transcriptional regulator of IFN-β and IFN-γ gene expression. Accordingly, these cytokines were upregulated in AIM2−/− islets, resulting in a suppressed gene expression and secretion of insulin. Moreover, the revascularization of AIM2−/− islet grafts was deteriorated when compared to WT controls. Furthermore, transplantation of AIM2−/− islets in diabetic mice failed to restore physiological blood glucose levels. These findings indicate that AIM2 crucially determines the engraftment and endocrine function of transplanted islets by repressing IFN signaling.

Abstract Endometriosis develop from shed endometrial fragments via retrograde menstruation. This affects the survival, proliferation and vascularization of the tissue and its final ability to form endometriotic lesions. Within this study, uterine tissue samples from donor mice were precultivated for 24 h or 72 h to simulate avascular periods. Their morphology, microvessel density, apoptotic activity and expression of angiogenesis-related proteins were analyzed in vitro. The formation of endometriotic lesions in vivo was assessed after transplantation of precultivated uterine tissue samples to the abdominal wall and dorsal skinfold chambers by means of high-resolution ultrasound, intravital fluorescence microscopy, histology and immunohistochemistry. In vitro, 72-h-precultivated uterine tissue samples exhibit extensive areas of tissue necrosis and high numbers of apoptotic cells as well as a significantly reduced cell and microvessel density. These samples failed to develop into endometriotic lesions. In contrast, the 24-h-precultivated samples showed, that their early vascularization and growth in vivo was improved when compared to controls. This indicates that avascular periods have a strong impact on the survival of ectopic endometrial tissue and the chance for the development of endometriosis.

Endometriosis is a common gynecological disease which is characterized by endometriotic lesions outside the uterine cavity. In this study, we investigated whether the presence of pre-existing endometriotic lesions promotes the development of new lesions due to the exchange of cells and an altered peritoneal environment. For this purpose, uterine tissue samples from FVB/N wild-type donor mice were transplanted simultaneously or time-delayed with samples from transgenic FVB-Tg(CAG-luc-GFP)L2G85Chco/J donor mice into the abdominal cavity of FVB/N wild-type recipient mice. The formation of endometriotic lesions was analyzed by means of high-resolution ultrasound, bioluminescence imaging, histology and immunohistochemistry. Moreover, immune cells and inflammatory factors in the peritoneal fluid were assessed by flow cytometry and a cytokine array. These analyses revealed that the growth of newly developing endometriotic lesions is promoted by the presence of pre-existing ones. This is not due to an exchange of cells between both lesion types but rather caused by peritoneal inflammation induced by already established lesions. These findings indicate that, among other pathogenic mechanisms, the chronic nature of endometriosis may be driven by a lesion-induced inflammatory milieu in the peritoneal cavity, which creates favorable conditions for the development of new lesions.

Caloric restriction (CR) is a cost-effective and easy-to-perform approach to counteracting surgical stress. The present study therefore evaluates the tissue-protective effects of a 30% CR in musculocutaneous flaps undergoing ischemia. For this purpose, a well-established murine dorsal skinfold chamber model, in combination with random pattern musculocutaneous flaps, was used. C57BL/6N mice were divided at random into a CR group (n = 8) and a control group with unrestricted access to standard chow (n = 8). The CR animals were subjected to a 30% reduction in caloric intake for 10 days before flap elevation. Intravital fluorescence microscopy was carried out on days 1, 3, 5, 7 and 10 after flap elevation to assess the nutritive blood perfusion, angiogenesis and flap necrosis. Subsequently, the flap tissue was harvested for additional histological and immunohistochemical analyses. The CR-treated animals exhibited a significantly higher functional capillary density and more newly formed microvessels within the flap tissue when compared to the controls; this was associated with a significantly higher flap survival rate. Immunohistochemical analyses showed a decreased invasion of myeloperoxidase-positive neutrophilic granulocytes into the flap tissue of the CR-treated mice. Moreover, the detection of cleaved caspase-3 revealed fewer cells undergoing apoptosis in the transition zone between the vital and necrotic tissue in the flaps of the CR-treated mice. These results demonstrate that a CR of 30% effectively prevents flap necrosis by maintaining microperfusion on a capillary level and inhibiting inflammation under ischemic stress. Hence, CR represents a promising novel conditioning strategy for improving the survival of musculocutaneous flaps with random pattern perfusion.

MicroRNAs (miRNAs) expressed in endothelial cells (ECs) are powerful regulators of angiogenesis, which is essential for tumor growth and metastasis. Here, we demonstrated that miR-22 is preferentially and highly expressed in ECs, while its endothelial level is significantly downregulated in human non-small cell lung cancer (NSCLC) tissues when compared to matched nontumor lung tissues. This reduction of endothelial miR-22 is possibly induced by NSCLC cell-secreted interleukin-1β and subsequently activated transcription factor nuclear factor-κB. Endothelial miR-22 functions as a potent angiogenesis inhibitor that inhibits all of the key angiogenic activities of ECs and consequently NSCLC growth through directly targeting sirtuin 1 and fibroblast growth factor receptor 1 in ECs, leading to inactivation of AKT/mammalian target of rapamycin signaling. These findings provide insight into the molecular mechanisms of NSCLC angiogenesis and indicate that endothelial miR-22 represents a potential target for the future antiangiogenic treatment of NSCLC.

Non-union formation represents a major complication in trauma surgery. Adequate vascularization has been recognized as vital for bone healing. However, the role of vascularization in the pathophysiology of non-union formation remains elusive. This is due to difficulties in studying bone microcirculation in vivo. Therefore, we herein studied in a murine osteotomy model whether photoacoustic imaging may be used to analyze vascularization in bone healing and non-union formation. We found that oxygen saturation within the callus tissue is significantly lower in non-unions compared to unions and further declines over time. Moreover, the amount of total hemoglobin (HbT) within the callus tissue was markedly reduced in non-unions. Correlation analyses showed a strong positive correlation between microvessel density and HbT, indicating that photoacoustically determined HbT is a valid parameter to assess vascularization during bone healing. In summary, photoacoustic imaging is a promising approach to study vascular function and tissue oxygenation in bone regeneration.

Abstract Objectives The preclinical evaluation of bone substitutes is frequently performed in artificially created defects. However, such defects do not reflect the predominant clinical application of bone substitutes for socket preservation. Hence, the goal of this animal study was to compare the performance of a xenogenic bone substitute in extraction sites versus artificial defects. Material and Methods Four study sites each were created in the mandibles of four minipigs in the region of the third premolars and first molars, respectively. On one side, fresh extraction sockets were established while contralaterally trephine defects were created in healed alveolar bone. All sites were augmented using a particulate xenogenic bone substitute, covered by resorbable membranes and allowed to heal for 12 weeks. The amounts of new bone, non‐bone tissue and remaining bone substitute granules were quantified through histological and micro‐CT analysis. Comparative statistics were based on t‐tests for two samples and ANOVA with the level of significance set at α = 0.05. Results Histomorphometric data from only two animals could be quantitatively analyzed due to difficulty with identifying the surgical sites. The percentage of newly formed bone ranged between 53.2% ± 5.6% for artificial defects and 54.9% ± 12.4% for extraction sites. With the exception of ANOVA indicating a greater amount of non‐bone tissue in extraction sites as compared to artificial sites (p = 0.047), no statistically significant differences were observed. Micro‐CT scans showed patterns similar to the ones observed in histomorphometry. As extraction sites could be identified only in two micro‐CT reconstructions, quantitative assessment was not undertaken. Conclusions Despite the comparable performance of bone substitute material in artificial defects and extraction sites found here, the data gathered with this experiment was insufficient for showing equivalence of both approaches.

Islet transplantation is a promising treatment strategy for type 1 diabetes mellitus (T1DM) patients. However, oxidative stress-induced graft failure due to an insufficient revascularization is a major problem of this therapeutic approach. NADPH oxidase (NOX)2 is an important producer of reactive oxygen species (ROS) and several studies have already reported that this enzyme plays a crucial role in the endocrine function and viability of β-cells. Therefore, we hypothesized that targeting islet NOX2 improves the outcome of islet transplantation. To test this, we analyzed the cellular composition and viability of isolated wild-type (WT) and Nox2−/− islets by immunohistochemistry as well as different viability assays. Ex vivo, the effect of Nox2 deficiency on superoxide production, endocrine function and anti-oxidant protein expression was studied under hypoxic conditions. In vivo, we transplanted WT and Nox2−/− islets into mouse dorsal skinfold chambers and under the kidney capsule of diabetic mice to assess their revascularization and endocrine function, respectively. We found that the loss of NOX2 does not affect the cellular composition and viability of isolated islets. However, decreased superoxide production, higher glucose-stimulated insulin secretion as well as expression of nuclear factor erythroid 2-related factor (Nrf)2, heme oxygenase (HO)-1 and superoxide dismutase 1 (SOD1) was detected in hypoxic Nox2−/− islets when compared to WT islets. Moreover, we detected an early revascularization, a higher take rate and restoration of normoglycemia in diabetic mice transplanted with Nox2−/− islets. These findings indicate that the suppression of NOX2 activity represents a promising therapeutic strategy to improve engraftment and function of isolated islets.

Angiogenesis, the development of new blood vessels from pre-existing ones, is an essential process determining numerous physiological and pathological conditions. Accordingly, there is a high demand for research approaches allowing the investigation of angiogenic mechanisms and the assessment of pro- and anti-angiogenic therapeutics. The present review provides a selective overview and critical discussion of such approaches, which, in line with the 3R principle, all share the common feature that they are not based on animal experiments. They include in vitro assays to study the viability, proliferation, migration, tube formation and sprouting activity of endothelial cells in two- and three-dimensional environments, the degradation of extracellular matrix compounds as well as the impact of hemodynamic forces on blood vessel formation. These assays can be complemented by in vivo analyses of microvascular network formation in the chorioallantoic membrane assay and early stages of zebrafish larvae. In addition, the combination of experimental data and physical laws enables the mathematical modeling of tissue-specific vascularization, blood flow patterns, interstitial fluid flow as well as oxygen, nutrient and drug distribution. All these animal-free approaches markedly contribute to an improved understanding of fundamental biological mechanisms underlying angiogenesis. Hence, they do not only represent essential tools in basic science but also in early stages of drug development. Moreover, their advancement bears the great potential to analyze angiogenesis in all its complexity and, thus, to make animal experiments superfluous in the future.

Tumor endothelial cells (TECs) are key stromal components of the tumor microenvironment, and are essential for tumor angiogenesis, growth and metastasis. Accumulating evidence has shown that small single-stranded non-coding microRNAs (miRNAs) act as powerful endogenous regulators of TEC function and blood vessel formation. This systematic review provides an up-to-date overview of these endothelial miRNAs. Their expression is mainly regulated by hypoxia, pro-angiogenic factors, gap junctions and extracellular vesicles, as well as long non-coding RNAs and circular RNAs. In preclinical studies, they have been shown to modulate diverse fundamental angiogenesis-related signaling pathways and proteins, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway; the rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; the phosphoinositide 3-kinase (PI3K)/AKT pathway; and the transforming growth factor (TGF)-β/TGF-β receptor (TGFBR) pathway, as well as krüppel-like factors (KLFs), suppressor of cytokine signaling (SOCS) and metalloproteinases (MMPs). Accordingly, endothelial miRNAs represent promising targets for future anti-angiogenic cancer therapy. To achieve this, it will be necessary to further unravel the regulatory and functional networks of endothelial miRNAs and to develop safe and efficient TEC-specific miRNA delivery technologies.

Blood flow in the microcirculatory system is crucially affected by intrinsic red blood cell (RBC) properties, such as their deformability. In the smallest vessels of this network, RBCs adapt their shapes to the flow conditions. Although it is known that the age of RBCs modifies their physical properties, such as increased cytosol viscosity and altered viscoelastic membrane properties, the evolution of their shape-adapting abilities during senescence remains unclear. In this study, we investigated the effect of RBC properties on the microcapillary in vitro flow behavior and their characteristic shapes in microfluidic channels. For this, we fractioned RBCs from healthy donors according to their age. Moreover, the membranes of fresh RBCs were chemically rigidified using diamide to study the effect of isolated graded-membrane rigidity. Our results show that a fraction of stable, asymmetric, off-centered slipper-like cells at high velocities decreases with increasing age or diamide concentration. However, while old cells form an enhanced number of stable symmetric croissants at the channel centerline, this shape class is suppressed for purely rigidified cells with diamide. Our study provides further knowledge about the distinct effects of age-related changes of intrinsic cell properties on the single-cell flow behavior of RBCs in confined flows due to inter-cellular age-related cell heterogeneity.

Microvascular fragments (MVF) derived from enzymatically digested adipose tissue are functional vessel segments that have been shown to increase the survival rate of surgical flaps. However, the underlying mechanisms have not been clarified so far. To achieve this, we raised random-pattern musculocutaneous flaps on the back of wild-type mice and mounted them into dorsal skinfold chambers. The flaps were injected with MVF that were freshly isolated from green fluorescent protein-positive (GFP+) donor mice or saline solution (control). On days 1, 3, 5, 7, and 10 after surgery, intravital fluorescence microscopy was performed for the quantitative assessment of angiogenesis, nutritive blood perfusion, and flap necrosis. Subsequently, the flaps were analyzed by histology and immunohistochemistry. The injection of MVF reduced necrosis of the ischemic flap tissue by ~20%. When compared to controls, MVF-injected flaps also displayed a significantly higher functional capillary density and number of newly formed microvessels in the transition zone, where vital tissue bordered on necrotic tissue. Immunohistochemical analyses revealed a markedly lower number of cleaved caspase-3+ apoptotic cells in the transition zone of MVF-injected flaps and a significantly increased number of CD31+ microvessels in both the flaps’ base and transition zone. Up to ~10% of these microvessels were GFP+, proving their origin from injected MVF. These findings demonstrate that MVF reduce flap necrosis by increasing angiogenesis, improving nutritive tissue perfusion, and suppressing apoptosis. Hence, the injection of MVF may represent a promising strategy to reduce ischemia-induced flap necrosis in future clinical practice.

Abstract Background There is increasing evidence that Parkinson’s disease (PD) might start in the gut, thus involving and compromising also the enteric nervous system (ENS). At the clinical onset of the disease the majority of dopaminergic neurons in the midbrain is already destroyed, so that the lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use a transgenic A30P-α-synuclein-overexpressing PD mouse model to identify appropriate candidate markers in the gut before hallmark symptoms begin to manifest. Methods Based on a gait analysis and striatal dopamine levels, we defined 2-month-old A30P mice as pre-symptomatic (psA30P), since they are not showing any motoric impairments of the skeletal neuromuscular system and no reduced dopamine levels, but an intestinal α-synuclein pathology. Mice at this particular age were further used to analyze functional and molecular alterations in both, the gastrointestinal tract and the ENS, to identify early pathological changes. We examined the gastrointestinal motility, the molecular composition of the ENS, as well as the expression of regulating miRNAs. Moreover, we applied A30P-α-synuclein challenges in vitro to simulate PD in the ENS. Results A retarded gut motility and early molecular dysregulations were found in the myenteric plexus of psA30P mice. We found that i.e. neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are significantly altered in the psA30P, thus representing potential biomarkers for early PD. Many of the dysregulated miRNAs found in the psA30P mice are reported to be changed in PD patients as well, either in blood, cerebrospinal fluid or brain tissue. Interestingly, the in vitro approaches delivered similar changes in the ENS cultures as seen in the transgenic animals, thus confirming the data from the mouse model. Conclusions These findings provide an interesting and novel approach for the identification of appropriate biomarkers in men.

Abstract Peritonitis is a common complication of peritoneal dialysis (PD). Our root cause analysis allowed to attribute some cases to leakage of the PD catheter. Accordingly, a clinically based stress test study on potential material damage issues of PD catheters was performed, focusing on material damage caused by cleaning, de- and attachment procedures during dialysate changes and on the individual storage methods of PD catheters between dialysate changes. PD catheters were exposed to both chemical stress by repeating dialysate-flow and physical stress simulating de- and connecting, fixation, pressure, flexing, folding etc.—simulating standard clinical daily routine of 8–10 years PD catheter usage. Potentially by normal usage caused damages should be then detected by intraluminal pressure, light- and electron microscopy. The multi-step visual control showed no obvious damages on PD catheters nor any leakage or barrier indulgence. Our tests simulating daily routine usage of PD catheters for several years could not detect any material defects under chemical or physical stress. Hence, we presume that most PD catheter damages, as identified cause for peritonitis in some of our patients, may be due to accidental, unnoticed external damage (e.g. through scissors, while changing dressings) or neglecting PD catheter handling specifications.

With a gradually increasing elderly population, the treatment of geriatric patients represents a major challenge for trauma and reconstructive surgery. Although, it is well established that aging affects bone metabolism, it is still controversial if aging impairs bone healing. Accordingly, we investigated fracture healing in young adult (3–4 months) and aged (16–18 months) CD-1 mice using a stable closed femoral fracture model. Bone healing was analyzed by radiographic, biomechanical and histological analysis at 1, 2, 3, 4 and 5 weeks after fracture. Our results demonstrated an increased callus diameter to femoral diameter ratio in aged animals at later time points of fracture healing when compared to young adult mice. Moreover, our biomechanical analysis revealed a significantly decreased bending stiffness at 3 and 4 weeks after fracture in aged animals. In contrast, at 5 weeks after fracture, the analysis showed no significant difference in bending stiffness between the two study groups. Additional histological analysis showed a delayed endochondral ossification in aged animals as well as a higher amounts of fibrous tissue at early healing time points. These findings indicate a delayed process of callus remodeling in aged CD-1 mice, resulting in a delayed fracture healing when compared to young adult animals. However, the overall healing capacity of the fractured femora was not affected by aging.

Despite careful preoperative planning, surgical flaps are prone to ischemic tissue damage and ischemia–reperfusion injury. The resulting wound breakdown and flap necrosis increase both treatment costs and patient morbidity. Hence, there is a need for strategies to promote flap survival and prevent ischemia-induced tissue damage. Phytochemicals, defined as non-essential, bioactive, and plant-derived molecules, are attractive candidates for perioperative treatment as they have little to no side effects and are well tolerated by most patients. Furthermore, they have been shown to exert beneficial combinations of pro-angiogenic, anti-inflammatory, anti-oxidant, and anti-apoptotic effects. This review provides an overview of bioactive phytochemicals that have been used to increase flap survival in preclinical animal models and discusses the underlying molecular and cellular mechanisms.

Nanofat is increasingly applied in plastic surgery for the improvement of scar quality and skin rejuvenation. However, little is known about the underlying regenerative mechanisms. Therefore, we herein investigated nanofat grafts in a murine dorsal skinfold chamber model. Nanofat generated from subcutaneous, inguinal adipose tissue of green fluorescent protein (GFP)+ C57BL/6 male and female donor mice was injected intracutaneously into dorsal skinfold chambers of gender-matched GFP− wild-type mice. The vascularization and tissue composition of the grafted nanofat were analyzed by means of intravital fluorescence microscopy, histology and immunohistochemistry over an observation period of 14 days. The freshly generated nanofat consisted of small fragments of perilipin+ adipocytes surrounded by Sirius red+ collagen fibers and still contained intact CD31+/GFP+ vessel segments. After transplantation into the dorsal skinfold chamber, these vessel segments survived and developed interconnections to the surrounding CD31+/GFP− host microvasculature. Accordingly, the grafted nanofat rapidly vascularized and formed new microvascular networks with a high functional microvessel density on day 14 without marked differences between male and female mice. Even though further research is needed to confirm these findings, the present study suggests that nanofat boosts tissue vascularization. Thus, nanofat may represent a versatile resource for many applications in tissue engineering and regenerative medicine.

Bone healing is a multifarious process involving mesenchymal stem cells, osteoprogenitor cells, macrophages, osteoblasts and -clasts, and chondrocytes to restore the osseous tissue. Particularly in long bones including the tibia, clavicle, humerus and femur, this process fails in 2–10% of all fractures, with devastating effects for the patient and the healthcare system. Underlying reasons for this failure are manifold, from lack of biomechanical stability to impaired biological host conditions and wound-immanent intricacies. In this review, we describe the cellular components involved in impaired bone healing and how they interfere with the delicately orchestrated processes of bone repair and formation. We subsequently outline and weigh the risk factors for the development of non-unions that have been established in the literature. Therapeutic prospects are illustrated and put into clinical perspective, before the applicability of biomarkers is finally discussed.

Endometriosis represents an estrogen-dependent disorder with a complex pathophysiology. Phytochemicals are promising candidates for endometriosis therapy, because they simultaneously target different cellular processes involved in the pathogenesis of endometriosis. Herein, we analyzed whether indole-3-carbinol (I3C) suppresses the development of endometriotic lesions, which were surgically induced by fixation of uterine tissue samples (diameter: 2 mm) from female BALB/c donor mice to the peritoneum of recipient animals. The mice received either I3C or vehicle (control) by peroral administration once per day. Growth, cyst formation, cell proliferation, microvascularization and protein expression of the lesions were assessed by high-resolution ultrasound imaging, caliper measurements, histology, immunohistochemistry and Western blotting. I3C inhibited the vascularization and growth of endometriotic lesions without inducing anti-angiogenic and anti-proliferative side effects on reproductive organs. This was associated with a significantly reduced number of proliferating stromal and endothelial cells and a lower expression of the pro-angiogenic signaling molecules vascular endothelial growth factor receptor-2 (VEGFR2), phosphoinositide 3-kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (pERK) within I3C-treated lesions when compared to controls. These findings indicate that I3C effectively inhibits endometriotic lesion formation in mice. Thus, further studies should clarify whether I3C may be also beneficial for the prevention and therapy of the human disease.

Adipose tissue-derived microvascular fragments (MVF) represent effective vascularization units for tissue engineering. Most experimental studies exclusively use epididymal fat tissue of male donor mice as a source for MVF isolation. However, in future clinical practice, MVF-based approaches may be applied in both male and female patients. Therefore, we herein compared the vascularization capacity of MVF isolated from the epididymal and peri-ovarian fat tissue of male and female donor mice. Freshly isolated MVF from male and female donors did not differ in their number, length distribution, viability and cellular composition. After their assembly into spheroids, they also exhibited a comparable in vitro sprouting activity. Moreover, they could be seeded onto collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects within mouse dorsal skinfold chambers. Repetitive intravital fluorescence microscopy as well as histological and immunohistochemical analyses revealed a comparable vascularization and incorporation of implants seeded with MVF of male and female origin. Taken together, these findings demonstrate that the vascularization capacity of MVF is not gender-specific.

Thrombus formation is dependent on the interaction of platelets, leukocytes and endothelial cells as well as proteins of the coagulation cascade. This interaction is tightly controlled by phospho-regulated pathways involving protein kinase CK2. A growing number of studies have demonstrated an important role of this kinase in the regulation of primary and secondary hemostasis. Inhibition of CK2 downregulates the expression of important adhesion molecules on platelets and endothelial cells, such as glycoprotein (GP)IIb/IIIa, P-selectin, von Willebrand factor and vascular cell adhesion molecule. Moreover, the reduced CK2-dependent phosphorylation of different coagulation factors prevents the conversion of fibrinogen to fibrin. Targeting these mechanisms may open the door for the development of novel anti-thrombotic therapies.

Bromelain has previously been shown to prevent ischemia-induced necrosis in different types of tissues. In the present study, we, therefore, evaluated for the first time, the tissue-protective effects of bromelain in musculocutaneous flaps in mice. Adult C57BL/6N mice were randomly assigned to a bromelain treatment group and a control group. The animals were treated daily with intraperitoneal injections of 20 mg/kg bromelain or saline (control), starting 1 h before the flap elevation throughout a 10-day observation period. The random-pattern musculocutaneous flaps were raised on the backs of the animals and mounted into a dorsal skinfold chamber. Angiogenesis, nutritive blood perfusion and flap necrosis were quantitatively analyzed by means of repeated intravital fluorescence microscopy over 10 days after surgery. After the last microscopy, the flaps were harvested for additional histological and immunohistochemical analyses. Bromelain reduced necrosis of the critically perfused flap tissue by ~25%. The bromelain-treated flaps also exhibited a significantly higher functional microvessel density and an elevated formation of newly developed microvessels in the transition zone between the vital and necrotic tissues when compared to the controls. Immunohistochemical analyses demonstrated a markedly lower invasion of the myeloperoxidase-positive neutrophilic granulocytes and a significantly reduced number of cleaved caspase 3-positive apoptotic cells in the transition zone of bromelain-treated musculocutaneous flaps. These findings indicate that bromelain prevents flap necrosis by maintaining nutritive tissue perfusion and by suppressing ischemia-induced inflammation and apoptosis. Hence, bromelain may represent a promising compound to prevent ischemia-induced flap necrosis in clinical practice.

Abstract Background The seeding of scaffolds with the stromal vascular fraction (SVF) of adipose tissue is a common prevascularization strategy in tissue engineering. Alternatively, adipose tissue-derived microvascular fragments (ad-MVF) may serve as vascularization units. In contrast to SVF single cells, they represent a mixture of intact arteriolar, capillary and venular vessel segments. Therefore, we herein hypothesized that the ad-MVF-based prevascularization of scaffolds is superior to the conventional SVF single cells-based approach. Results SVF single cells and ad-MVF were enzymatically isolated from epididymal fat pads of green fluorescent protein (GFP)+ donor mice to assess their viability and cellular composition using fluorescence microscopy and flow cytometry. Moreover, collagen-glycosaminoglycan matrices (Integra®) were seeded with identical amounts of the isolates and implanted into full-thickness skin defects within dorsal skinfold chambers of GFP− recipient mice for the intravital fluorescent microscopic, histological and immunohistochemical analysis of implant vascularization and incorporation throughout an observation period of 2 weeks. Non-seeded matrices served as controls. While both isolates contained a comparable fraction of endothelial cells, perivascular cells, adipocytes and stem cells, ad-MVF exhibited a significantly higher viability. After in vivo implantation, the vascularization of ad-MVF-seeded scaffolds was improved when compared to SVF-seeded ones, as indicated by a significantly higher functional microvessel density. This was associated with an enhanced cellular infiltration, collagen content and density of CD31+/GFP+ microvessels particularly in the center of the implants, demonstrating a better incorporation into the surrounding host tissue. In contrast, non-seeded matrices exhibited a poor vascularization, incorporation and epithelialization over time. Conclusions The present study demonstrates that ad-MVF are highly potent vascularization units that markedly accelerate and improve scaffold vascularization when compared to the SVF.

Juvenile angiofibroma (JA) is a rare fibrovascular neoplasm predominately found within the posterior nasal cavity of adolescent males. JA expresses the proteoglycan nerve–glial antigen (NG)2, which crucially determines the migratory capacity of distinct cancer cells. Moreover, it is known that the protein kinase CK2 regulates NG2 gene expression. Therefore, in the present study, we analyzed whether the inhibition of CK2 suppresses NG2-dependent JA cell proliferation and migration. For this purpose, we assessed the expression of NG2 and CK2 in patient-derived JA tissue samples, as well as in patient-derived JA cell cultures by Western blot, immunohistochemistry, flow cytometry and quantitative real-time PCR. The mitochondrial activity, proliferation and migratory capacity of the JA cells were determined by water-soluble tetrazolium (WST)-1, 5-bromo-2′-deoxyuridine (BrdU) and collagen sprouting assays. We found that NG2 and CK2 were expressed in both the JA tissue samples and cell cultures. The treatment of the JA cells with the two CK2 inhibitors, CX-4945 and SGC-CK2-1, significantly reduced NG2 gene and protein expression when compared to the vehicle-treated cells. In addition, the loss of CK2 activity suppressed the JA cell proliferation and migration. These findings indicate that the inhibition of CK2 may represent a promising therapeutic approach for the treatment of NG2-expressing JA.

VS-5584 is a highly selective dual kinase inhibitor which suppresses phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) activity. Because these kinases are crucially involved in primary hemostasis, we herein investigated the effect of this compound on thrombus formation in vitro and in vivo. Pretreatment of washed platelets (WP) or platelet-rich plasma (PRP) with VS-5584 inhibited the agonist-induced activation of surface glycoprotein complex (GP)IIb/IIIa and the upregulation of P-selectin. This was associated with a significantly reduced formation of platelet-leukocyte aggregates (PLA). VS-5584 further attenuated platelet aggregation and adhesion after agonist stimulation. In contrast, endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cellular adhesion molecule (VCAM)-1 and secretion of von Willebrand Factor (vWF) were not affected by the dual kinase inhibitor. In vivo, VS-5584 inhibited photochemically induced thrombus formation as shown by a significantly prolonged time to complete vessel occlusion when compared to vehicle-treated controls. This was associated with an elevated tail vein bleeding time, indicating a potential hemorrhagic risk in VS-5584-treated mice. Taken together, these novel findings demonstrate that VS-5584 is a potent inhibitor of primary hemostasis targeting multiple platelet functions.

Summary: Voltage-gated calcium channels (Cavs) are major Ca2+ entry pathways in excitable cells. Their β subunits facilitate membrane trafficking of the channel’s ion-conducting α1 pore and modulate its gating properties. We report that one β subunit, β3, reduces Ca2+ release following stimulation of phospholipase C-coupled receptors and inositol 1,4,5-trisphosphate (IP3) formation. This effect requires the SH3-HOOK domain of Cavβ3, includes physical β3/IP3 receptor interaction, and prevails when agonist-induced IP3 formation is bypassed by photolysis of caged IP3. In agreement with β3 acting as a brake on Ca2+ release, fibroblast migration is enhanced in vitro, and in vivo, closure of skin wounds is accelerated in the absence of β3. To mediate specific physiological responses and to prevent Ca2+ toxicity, cytoplasmic Ca2+ signals must be tightly controlled. The described function of β3, unrelated to its function as a Cav subunit, adds to this tight control. : Belkacemi et al. show that the calcium channel subunit Cavβ3 binds to the IP3R and desensitizes cells to low IP3 levels, influencing fibroblast migration and collagen secretion. Removal of the Cavβ3 protein in mice results in faster skin wound healing. Keywords: Ca2+ signaling, Cavβ3, cell migration, IP3 receptor, wound healing, Cavβ2, Cavβ3 KO, IP3 binding, voltage-gated Ca2+ channel, Ca2+ release

Fat grafting is a frequently applied procedure in plastic surgery for volume reconstruction. Moreover, the transplantation of white adipose tissue (WAT) and brown adipose tissue (BAT) increasingly gains interest in preclinical research for the treatment of obesity-related metabolic defects. Therefore, we herein directly compared the vascularization capacity and survival of WAT and BAT grafts. For this purpose, size-matched grafts isolated from the inguinal WAT pad and the interscapular BAT depot of C57BL/6N donor mice were syngeneically transplanted into the dorsal skinfold chamber of recipient animals. The vascularization and survival of the grafts were analyzed by means of intravital fluorescence microscopy, histology, and immunohistochemistry over an observation period of 14 days. WAT grafts showed an identical microvascular architecture and functional microvessel density as native WAT. In contrast, BAT grafts developed an erratic microvasculature with a significantly lower functional microvessel density when compared to native BAT. Accordingly, they also contained a markedly lower number of CD31-positive microvessels, which was associated with a massive loss of perilipin-positive adipocytes. These findings indicate that in contrast to WAT grafts, BAT grafts exhibit an impaired vascularization capacity and survival, which may be due to their higher metabolic demand. Hence, future studies should focus on the establishment of strategies to improve the engraftment of transplanted BAT.

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2–12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival.