Comparison of extraction sites versus artificial defects with xenogenic bone substitute in minipigs

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

Regulatory Mechanisms of Somatostatin Expression

Somatostatin is a peptide hormone, which most commonly is produced by endocrine cells and the central nervous system. In mammals, somatostatin originates from pre-prosomatostatin and is processed to a shorter form, i.e., somatostatin-14, and a longer form, i.e., somatostatin-28. The two peptides repress growth hormone secretion and are involved in the regulation of glucagon and insulin synthesis in the pancreas. In recent years, the processing and secretion of somatostatin have been studied intensively. However, little attention has been paid to the regulatory mechanisms that control its expression. This review provides an up-to-date overview of these mechanisms. In particular, it focuses on the role of enhancers and silencers within the promoter region as well as on the binding of modulatory transcription factors to these elements. Moreover, it addresses extracellular factors, which trigger key signaling pathways, leading to an enhanced somatostatin expression in health and disease

Tissue-Protective Mechanisms of Bioactive Phytochemicals in Flap Surgery

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

Targeting sphingosine kinase-1 with the low MW inhibitor SKI-5C suppresses the development of endometriotic lesions in mice

Background and Purpose Limited evidence suggests that the sphingosine-1-phosphate/sphingosine kinase 1 (S1P/SPHK1) signalling pathway is involved in the pathogenesis of endometriosis. Therefore, we analyzed in this study whether the inhibition of SPHK1 and, consequently, decreased levels of S1P affected the vascularization and growth of endometriotic lesions. Experimental Approach Endometriotic lesions were surgically induced in the peritoneal cavity and the dorsal skinfold chamber of female BALB/c mice. The animals received a daily dose of the SPHK1 inhibitor SKI-5C or vehicle (control). Analyses involved the determination of lesion growth, cyst formation, microvessel density and cell proliferation within peritoneal endometriotic lesions by means of high-resolution ultrasound imaging, caliper measurement, histology and immunohistochemistry. In the dorsal skinfold chamber model the development of newly formed microvascular networks and their microhemodynamic parameters within endometriotic lesions were investigated by means of intravital fluorescence microscopy. Key Results SKI-5C significantly inhibited the development and vascularization of peritoneal endometriotic lesions, as indicated by a reduced growth and cyst formation, a lower microvessel density and a suppressed cell proliferation, when compared to vehicle-treated controls. Endometriotic lesions in dorsal skinfold chambers of SKI-5C-treated animals exhibited a significantly smaller lesion size, lower functional microvessel density, smaller microvessel diameters and a reduced blood perfusion of the newly developing microvascular networks. Conclusions and Implications SPHK1/S1P signalling promotes the establishment and progression of endometriotic lesions. The inhibition of this pathway suppresses the development of endometriotic lesions, suggesting SPHK1 as a potential novel target for future endometriosis therapy

Maslinic acid alleviates ischemia/reperfusion-induced inflammation by downregulation of NFκB-mediated adhesion molecule expression

Ischemia/reperfusion (I/R)-induced inflammation is associated with enhanced leukocyte rolling, adhesion and transmigration within the microcirculation. These steps are mediated by hypoxia-triggered signaling pathways, which upregulate adhesion molecule expression on endothelial cells and pericytes. We analyzed whether these cellular events are affected by maslinic acid (MA). Mitochondrial activity and viability of MA-exposed endothelial cells and pericytes were assessed by water-soluble tetrazolium (WST)-1 and lactate dehydrogenase (LDH) assays as well as Annexin V/propidium iodide (PI) stainings. Effects of MA on hypoxia and reoxygenation-induced expression of E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were determined by flow cytometry. The subcellular localization of the NFκB subunit p65 was analyzed by immunofluorescence and Western blot. I/R-induced leukocytic inflammation was studied in MA- and vehicle-treated mouse dorsal skinfold chambers by intravital fluorescence microscopy and immunohistochemistry. MA did not affect viability, but suppressed the mitochondrial activity of endothelial cells. Furthermore, MA reduced adhesion molecule expression on endothelial cells and pericytes due to an inhibitory action on NFκB signaling. Numbers of adherent and transmigrated leukocytes were lower in post-ischemic tissue of MA-treated mice when compared to vehicle-treated controls. In addition, MA affected reactive oxygen species (ROS) formation, resulting in a diminished oxidative DNA damage. Hence, MA represents an attractive compound for the establishment of novel therapeutic approaches against I/R-induced inflammation

Cold atmospheric plasma does not affect the regenerative potential of the pulp in rats

The aim of this study was to investigate the effect of cold atmospheric plasma (CAP) treatment combined with adhesive filling therapy on rat dental pulps. Cavities were prepared in the first maxillary molars of 20 Sprague Dawley rats. The first molar and the unprepared second molar of one randomly selected maxillary quadrant were treated with CAP. The prepared cavities were filled with composite. After 24 h and 28 days, 10 rats each were killed. Teeth were demineralized and embedded in paraffin and histological sections were stained with hematoxylin–eosin and chloracetatesterase. None of the pulps displayed necrosis. Plasma treatment caused no additional alteration to the dental pulp in combination with adhesive filling therapy. These findings indicate that plasma treatment is compatible with the regenerative potential of the pulp

The regulatory mechanisms of NG2/CSPG4 expression

Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), is a surface type I transmembrane core proteoglycan that is crucially involved in cell survival, migration and angiogenesis. NG2 is frequently used as a marker for the identification and characterization of certain cell types, but little is known about the mechanisms regulating its expression. In this review, we provide evidence that the regulation of NG2 expression underlies inflammation and hypoxia and is mediated by methyltransferases, transcription factors, including Sp1, paired box (Pax) 3 and Egr-1, and the microRNA miR129-2. These regulatory factors crucially determine NG2-mediated cellular processes such as glial scar formation in the central nervous system (CNS) or tumor growth and metastasis. Therefore, they are potential targets for the establishment of novel NG2-based therapeutic strategies in the treatment of CNS injuries, cancer and other conditions of these types

Bromelain Protects Critically Perfused Musculocutaneous Flap Tissue from Necrosis

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 devel oped 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

The Marine-Derived Triterpenoid Frondoside A Inhibits Thrombus Formation

Background: The marine-derived triterpenoid frondoside A inhibits the phosphatidylinositol-3-kinase (PI3K) pathway in cancer cells. Because this pathway is also crucially involved in platelet activation, we studied the effect of frondoside A on thrombus formation. Methods: Frondoside A effects on platelet viability, surface adhesion molecule expression, and intracellular signaling were analyzed by flow cytometry and Western blot. The effect of frondoside A was analyzed by photochemically induced thrombus formation in the mouse dorsal skinfold chamber model and by tail vein bleeding. Results: Concentrations of up to 15 µM frondoside A did not affect the viability of platelets, but reduced their surface expression of P-selectin (CD62P) and the activation of glycoprotein (GP)IIb/IIIa after agonist stimulation. Additional mechanistic analyses revealed that this was mediated by downregulation of PI3K-dependent Akt and extracellular-stimuli-responsive kinase (ERK) phosphorylation. Frondoside A significantly prolonged the complete vessel occlusion time in the mouse dorsal skinfold chamber model of photochemically induced thrombus formation and also the tail vein bleeding time when compared to vehicle-treated controls. Conclusion: Our findings demonstrated that frondoside A inhibits agonist-induced CD62P expression and activation of GPIIb/IIIa. Moreover, frondoside A suppresses thrombus formation. Therefore, this marine-derived triterpenoid may serve as a lead compound for the development of novel antithrombotic drugs

Microvascular Fragments Protect Ischemic Musculocutaneous Flap Tissue from Necrosis by Improving Nutritive Tissue Perfusion and Suppressing Apoptosis

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