Connective Tissue Constituents Research Articles

Surfactin attenuates particulate matter-induced COX-2-dependent PGE2 production in human gingival fibroblasts by inhibiting TLR2 and TLR4/MyD88/NADPH oxidase/ROS/PI3K/Akt/NF-κB signaling pathway.

To evaluate the anti-inflammatory effects of surfactin and underlying mechanisms against particulate matter (PM)-induced inflammatory responses in human gingival fibroblasts (HGFs). PM, a major air pollutant, may associate with certain oral diseases possibly by inducing inflammation and oxidative stress. Surfactin, a potent biosurfactant, possesses various biological properties including anti-inflammatory activity. However, the underlying mechanisms are unclear. Also, there is no study investigating the effects of surfactin on PM-induced oral inflammatory responses. As an essential constituent of human periodontal connective tissues which involves immune-inflammatory responses, HGFs serve as useful study models. HGFs were pretreated with surfactin prior to PM incubation. The PGE2 production was determined by ELISA, while the protein expression and mRNA levels of COX-2 and upstream regulators were measured using Western blot and real-time PCR, respectively. The transcriptional activity of COX-2 and NF-κB were determined using promoter assay. ROS generation and NADPH oxidase activity were identified by specific assays. Co-immunoprecipitation assay, pharmacologic inhibitors, and siRNA transfection were applied to explore the interplay of molecules. Mice were given one dose of surfactin or different pharmacologic inhibitors, then PM was delivered into the gingiva for three consecutive days. Gingival tissues were obtained for analyzing COX-2 expression. PM-treated HGFs released significantly higher COX-2-dependent PGE2 , which were regulated by TLR2 and TLR4/MyD88/NADPH oxidase/ROS/PI3K/Akt/NF-κB pathway. PM-induced COX-2/PGE2 increase was effectively reversed by surfactin through the disruption of regulatory pathway. Similar inhibitory effects of surfactin was observed in mice. Surfactin may elicit anti-inflammatory effects against PM-induced oral inflammatory responses.

Histomorphological and Histochemical Studies on Proventriculus in Guinea Fowl (Numida meleagris)

Background: The proventriculus, component of digestive system of bird secretes hydrochloric acid and pepsinogen which helps the churning of food material that takes place in ventriculus. Pepsinogen produces pepsin and it smashes the peptide bonds of amino acid of food material. Methods: The twelve proventriculus samples of Guinea fowl birds were fixed in 10% neutral buffered formalin and Bouin’s fluid for histological procedure while chilled acetone and chilled ethanol were used as the fixatives for histochemical studies. The fresh unfixed samples were used on cryostat for studies of succinic dehydrogenase enzyme.Result: Histomorphologically the proventriculus of Guinea fowl was observed as the typical tubular component with four tunics from inside to outside as tunica mucosa, tunica sub mucosa, tunica muscularis and tunica serosa. The submucosal layer comprised with proventricular submucosal gland (PVSMG) which was encapsulated by elastic fibers. Tunica muscularis was inner longitudinal and outer circular layer, while the tunica serosa was observed with surrounding adipose tissue as the connective tissue constituent to endow the access of nutritional supply in the form of blood vessels. The luminal surface of proventricular submucosal glands has shown the significant activity of glycogen while intercalated, intralobular and interlobular ducts have exhibited the weak activity towards the same. The glandular cell rod of PVSMG, serosal layer and adjoining connective tissue with blood vessels has exhibited very weak activity of alkaline phosphatase. Mucosal folds and lumen of PVSMG has shown the weak activity of acidic phosphatase but the serosal limiting membrane has shown very weak activity. The succinic dehydrogenase activity was seen in the connective tissue septa of submucosal glandular cellular rods and the intense activity was observed at tunica muscularis.

Evaluation of metalloproteinases-2, -9, and -13 post photobiomodulation in mice talocrural joint.

The extracellular matrix (ECM) is the main constituent of connective tissue with structural and regulatory functions, stimulating cell differentiation and proliferation. Moreover, ECM is a dynamic structure in the constant remodeling process, which is controlled by a balance between metalloproteinases (MMPs) and their inhibitors (TIMPs). Photobiomodulation (PBM) is widely described in the literature and applied in clinical practices, although its effects on ECM have not yet been elucidated. Therefore, it was evaluated if PBM could alter ECM components, such as MMP-2, -9, -13, and TIMP-2 from mice talocrural joints. Mice were divided into 3 groups (n = 6): control, PBM 3Jcm-2, and PBM 30Jcm-2. A low-level laser (830nm, 10mW, 0.05 irradiated area, energy densities 3Jcm-2 and 30Jcm-2, the irradiation time of 15 and 150s, respectively, continuous wave) was applied on the joint for 4 consecutive days. mRNA levels of metalloproteinases genes (MMP-2, MMP-9, and MMP-13), their regulator (TIMP-2), and protein expressions of MMP-13 and TIMP-2 were quantified. PBM can alter only mRNA relative levels of MMP-2 at 30Jcm-2 (p < 0.05), while MMP-9, MMP-13, and TIMP-2 mRNA relative levels did not demonstrate statistical differences for any of the groups (p > 0.05). Regarding protein expressions, MMP-13 demonstrated positive-labeled cells, only in articular cartilage, although the cell quantification did not demonstrate statistical differences when compared with the control group (p > 0.05). TIMP-2 did not present positive-labeled cells for any tissues evaluated. Our results indicate that PBM can alter MMP-2 mRNA relative level but cannot alter MMP-9, MMP-13, and TIMP mRNA relative levels. Moreover, both MMP-13 and TIMP-2 proteins were also unaltered after PBM.

An RNA-seq screen of P. gingivalis LPS treated human gingival fibroblasts

BackgroundIn gingival tissues, lipopolysaccharide (LPS) from Porphyromonas gingivalis (P. gingivalis) is the most critical stimulator for inducing inflammatory response. Human gingival fibroblasts (HGFs) are the major constituents of gingival connective tissues. The aim of this study was to investigate P. gingivalis LPS induced whole transcriptional profile in HGFs and the potential crosstalk between microRNAs (miRNAs) and inflammatory cytokines. MethodsRNA-seq was performed on HGFs with and without P. gingivalis LPS treatment. The gene expression of selected inflammatory cytokines and miRNAs induced by LPS at different time points was evaluated by quantitative RT-PCR. The protein expression of chemokines was further confirmed by ELISA. ResultsInterestingly, most of the significantly changed genes (198/204) were up-regulated at 4 h after 10 μg/ml LPS stimulation, including inflammatory cytokines and miRNAs. Confirmed by quantitative RT-PCR, the mRNA levels of IL-1β, IL-6 and IL-8 showed single up-regulation peak (4 h/6 h) after 1 μg/ml and 10 μg/ml LPS treatment. Similarly, 1 μg/ml LPS induced single up-regulation peak (8 h) of miRNA-146a, −146b and −155 expression. However, 10 μg/ml LPS induced the increased expression of miRNA-146a and −155 at both early stage (2 h/4 h) and late stage (24 h). ConclusionTaken together, we investigated P. gingivalis LPS induced whole transcriptional profile, and the different behaviors of miRNA expression induced by different doses of LPS in HGFs.

Study the effect of heat processing on the vitamin C of some fruits

Ascorbic Acid also known as Vitamin C. Vitamin C is very essential for growth and maintenance of the human body. It is necessary for the normal formation of the protein collagen, which is an important constituent of skin and connective tissue. The deficiency of vitamin C causes will be known as disease “Scurvy”. Vitamin C is present in all citrus fruits, gooseberry (Aonala), tomato, apple, pine, pineapple, grapes and other foods. The vitamin C is very sensitive to heat light air and strong alkali. In this paper, we study the effect of heat processing on the vitamin C of Citrus Limon, Ananas Cosmosus, Psidium Guajana, Vitis Vinifera.

Biomimetic heterogenous elastic tissue development

There is an unmet need for artificial tissue to address current limitations with donor organs and problems with donor site morbidity. Despite the success with sophisticated tissue engineering endeavours, which employ cells as building blocks, they are limited to dedicated labs suitable for cell culture, with associated high costs and long tissue maturation times before available for clinical use. Direct 3D printing presents rapid, bespoke, acellular solutions for skull and bone repair or replacement, and can potentially address the need for elastic tissue, which is a major constituent of smooth muscle, cartilage, ligaments and connective tissue that support organs. Thermoplastic polyurethanes are one of the most versatile elastomeric polymers. Their segmented block copolymeric nature, comprising of hard and soft segments allows for an almost limitless potential to control physical properties and mechanical behaviour. Here we show direct 3D printing of biocompatible thermoplastic polyurethanes with Fused Deposition Modelling, with a view to presenting cell independent in-situ tissue substitutes. This method can expeditiously and economically produce heterogenous, biomimetic elastic tissue substitutes with controlled porosity to potentially facilitate vascularisation. The flexibility of this application is shown here with tubular constructs as exemplars. We demonstrate how these 3D printed constructs can be post-processed to incorporate bioactive molecules. This efficacious strategy, when combined with the privileges of digital healthcare, can be used to produce bespoke elastic tissue substitutes in-situ, independent of extensive cell culture and may be developed as a point-of-care therapy approach.

Quantitative Parameters of Interdental Gingiva in Chronic Periodontitis Patients with IFN-γ Gene Polymorphism.

Chronic periodontitis (CP), an infectious disease resulting in inflammation within the periodontal tissue, is the main cause of adult tooth loss. CP is a multi-factorial disorder and the interaction between multiple genetic and environmental factors results in the manifestation of this disease. Recent researches in periodontitis has focused on cytokine gene polymorphisms that play important role in periodontal inflammation, but few studies investigated histological change that occur during CP in the supporting tissue of teeth. The aims of this study were to investigate the association of IFN-γ +874 A/T polymorphisms and quantitative parameters of interdental gingiva in CP patients. The study samples were interdental gingiva biopsies from 60 individuals including 38 patients and 22 healthy subjects. After determination of IFN-γ +874 A/T gene polymorphism by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), patients were divided in three subgroups: 10 AA, 18 AT and 10 TT. After slides preparation, quantitative parameters were estimated by Cavalieri's point-counting method. Statistical analyses were performed using Mann-Whitney and Kruskal-Wallis test to compare differences between groups. The volume density (Vv) of epithelium, connective tissue and its components were significantly different between the control and CP groups (P&lt;0.05). Statistically significant differences in the Vv of collagenous and non-collagenous matrix of interdental gingiva between AA, AT and TT groups were found (P&lt;0.05). Result of present study shows that IFN-γ +874 A/T is strongly associated with some quantitative parameters of connective tissue constituents of interdental papilla in CP patients.

Effect of hyaluronic acid on morphological changes to dentin surfaces and subsequent effect on periodontal ligament cell survival, attachment, and spreading.

Hyaluronic acid (HA) is a natural constituent of connective tissues and plays an important role in their development, maintenance, and regeneration. Recently, HA has been shown to improve wound healing. However, no basic in vitro study to date has investigated its mode of action. Therefore, the purpose of this study was to examine morphological changes of dentin surfaces following HA coating and thereafter investigate the influence of periodontal ligament (PDL) cell survival, attachment, and spreading to dentin discs. HA was coated onto dentin discs utilizing either non-cross-linked (HA) or cross-linked (HA cl) delivery systems. Morphological changes to dentin discs were then assessed using scanning electron microscopy (SEM). Thereafter, human PDL cells were seeded under three in vitro conditions including (1) dilution of HA (1:100), (2) dilution of HA (1:10), and (3) HA coated directly to dentin discs. Samples were then investigated for PDL cell survival, attachment, and spreading using a live/dead assay, cell adhesion assay, and SEM imaging, respectively. While control dentin discs demonstrated smooth surfaces both at low and high magnification, the coating of HA altered surface texture of dentin discs by increasing surface roughness. HA cl further revealed greater surface texture/roughness likely due to the cross-linking carrier system. Thereafter, PDL cells were seeded on control and HA coated dentin discs and demonstrated a near 100% survival rate for all samples demonstrating high biocompatibility of HA at dilutions of both 1:100 and 1:10. Interestingly, non-cross-linked HA significantly increased cell numbers at 8h, whereas cross-linked HA improved cell spreading as qualitatively assessed by SEM. The results from the present study demonstrate that both carrier systems for HA were extremely biocompatible and demonstrated either improved cell numbers or cell spreading onto dentin discs. Future in vitro and animal research is necessary to further characterize the optimal delivery system of HA for improved clinical use. HA is a highly biocompatible material that may improve PDL cell attachment or spreading on dentin.

Periodontal Disease and Potential Association with Systemic Diseases and Conditions (Mini-review)

Periodontitis, the most common chronic inflammatory disease known world-wide, is characterized by pathologically-excessive degradation of collagen and other connective tissue constituents and accelerated resorption of the alveolar bone in the periodontal supporting structures of the teeth including the gingiva, periodontal ligament and the alveolar bone. Nationwide, the prevalence of periodontal disease, in some form, is known to affect up to 50% of the adult population and is a substantial inflammatory burden which can be detrimental to over-all systemic health. In this regard, this common dental disease, chronic periodontitis, has, over the past few decades, been increasingly linked to a variety of medical diseases such as cardiovascular disease (CVD) and stroke, increased severity of diabetes, low birth weight babies (controversial), bacterial pneumonia, rheumatoid arthritis, osteoporosis, Crohn’s disease, HIV Diseases, and head and neck cancer. Decades ago it was thought that all adults were essentially equally susceptible to periodontal disease. More recently, various risk factors such as genetic factors have been identified which significantly impact the susceptibility to periodontitis. Therefore, identifying individuals at risk for development of severe periodontitis provides a way for early interventions. Keywords: Alveolar bone, lipopolysaccharide (LPS), periodontal disease, periodontitis, systemic diseases, tooth loss.

Association between TGF-Beta1 (-509) C/T Gene Polymorphism and Tissue Degradation Level In Chronic Periodontitis: A Stereological Study

Background: The recent years has seen enormous research attempts in the study for genetic factors that may predispose individuals to periodontitis, as one of the most common chronic infectious disease in humans. Objectives: The aim of the present study was to determine the breakdown of interdental gingiva tissues in chronic periodontitis patients with TGF-β1 (-509) C/T polymorphisms. Patients and Methods: A total of 60 gingival samples, including 45 patient and 15 healthy subjects, were selected for this study. Following determination of TGF-β1 (-509) C/T gene polymorphism by Tetra primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR), the patients were divided to three subgroups according to their genotype (6 TT, 21 TC and 18 CC). After tissue processing, sectioning and staining, quantitative parameters were estimated by Cavalieri’s point-counting estimation. Results: The Volume density (Vʋ) of epithelium, connective tissue, collagenous and non-collagenous matrix and blood vessels had statistically significant differences between the control and chronic periodontitis (CP) groups (P 0.05). Conclusions: The results of the present study showed that TGF-β1 (-509) C/T is strongly associated with quantitative parameters of connective tissue constituents of interdental papilla in CP patients.

콜라겐 코팅된 블록공중합(PCL/PCL-PEG) 나노섬유의 제조 및 특성분석

Electrospun polymeric nanofibers have been extensively studied for biomedical materials because of their unique structures and relatively easy fabrication with biocompatible polymers. The amount of surface exposed amine groups increases as the blend ratio of block copolymer increases. Cell attachments on the nanofibers change according to the ratio of the block copolymer ((Poly(e-caprolactone, PCL), Poly(e-caprolactone)-Poly (ethylen glycol-)) in the blend. We assume that the PEG and amine moiety plays a significant role in biocompatibility of nanofiber surfaces. Collagen was used as a grafting material on the composite nanofibers to enhance the cell adhesion because the collagen is a major constituent of connective tissue.

Elastin development-associated extracellular matrix constituents of subepithelial connective tissue in human pterygium.

We evaluated the expression of several extracellular matrix constituents implicated in the synthesis and reticulation of elastin in human pterygium, according to age and sex of the patients. Pterygia and normal conjunctiva samples were divided into groups according to age (<50/≥50 years) and sex. Tissue was subjected to immunohistochemical staining with anti-lysyl oxidase (LOX), lyxyl oxidase-like 1 (LOXL-1), fibulin (FBLN)-5 and FBLN4, and fibrillin-1 (FBN1) antibodies. Specific primers for the same constituents were used in a quantitative real-time PCR (qRT-PCR) analysis to determine gene expression. The LOXL-1 (P = 0.0002), FBLN5 (P = 0.0035), and FBN1 (P < 0.0001) mRNAs were significantly higher in pterygium than conjunctiva. No differences were found for LOX and FBLN4 mRNA. The expression of LOXL-1 was not affected by age or sex; however, pterygium from men and patients over 50 years old exhibited significantly higher FBLN5/FBN1 expression than did controls. The LOX gene expression was higher in the pathologic samples from the over 50-year-olds compared to the conjunctiva (P = 0.0396) and in men's versus women's pterygium (P = 0.0173). In general, the levels of LOX, LOXL-1, and FBLN5 decreased with age in healthy samples, while the pathology seemed to have increased expression of the three proteins, and even more so in older patients. The FBLN4 and FBN1 immunostaining was slight in all samples, displaying no differences between groups. Several extracellular matrix constituents, LOXs, FBN1, and FBLN5, implicated in the development of elastin, are overexpressed in the subepithelial connective tissue extracellular matrix of human pterygium, supporting our hypothesis that elastic synthesis and reticulation is dysregulated in this type of pathology.

Water ice is a soft matrix for the structural characterization of glycosaminoglycans by infrared matrix-assisted laser desorption/ionization.

Glycosaminoglycans (GAGs) are a class of heterogeneous, often highly sulfated glycans that form linear chains consisting of up to 100 monosaccharide building blocks and more. GAGs are ubiquitous constituents of connective tissue, cartilage, and the extracellular matrix, where they have key functions in many important biological processes. For their characterization by mass spectrometry (MS) and tandem MS, the high molecular weight polymers are usually enzymatically digested to oligomers with a low degree of polymerization (dp), typically disaccharides. However, owing to their lability elimination of sulfate groups upon desorption/ionization is often encountered leading to a loss of information on the analyte. Here, we demonstrate that, in particular, water ice constitutes an extremely mild matrix for the analysis of highly sulfated GAG disaccharides by infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry. Depending on the degree of sulfation, next to the singly charged ionic species doubly- and even triply charged ions are formed. An unambiguous assignment of the sulfation sites becomes possible by subjecting sodium adducts of the GAGs to low-energy collision-induced dissociation tandem MS. These ionic species exhibit a remarkable stability of the sulfate substituents, allowing the formation of fragment ions retaining their sulfation that arise from either cross-ring cleavages or rupture of the glycosidic bonds, thereby allowing an unambiguous assignment of the sulfation sites.

Soft Texture of Atlantic Salmon Fillets Is Associated with Glycogen Accumulation

Atlantic salmon (Salmo salar L.) with soft fillets are not suited for manufacturing high quality products. Therefore fillets with insufficient firmness are downgraded, leading to severe economic losses to the farming and processing industries. In the current study, morphological characteristics of salmon fillets ranging from soft to hard were analysed. Different microscopic techniques were applied, including novel methods in this field of research: morphometric image analysis, periodic acid Schiff staining, immunofluorescence microscopy, transmission electron microscopy and fourier transform infrared microscopy. The results showed that the myocytes of soft muscle had detached cells with mitochondrial dysfunctions, large glycogen aggregates and enlarged inter cellular areas, void of extracellular matrix proteins, including lower amounts of sulfated glycoproteins. Myofibre-myofibre detachment and disappearance of the endomysium in soft muscles coincided with deterioration of important connective tissue constituents such as Collagen type I (Col I), Perlecan and Aggrecan. In summary our investigations show for the first time an association between soft flesh of Atlantic salmon and massive intracellular glycogen accumulation coinciding with degenerated mitochondria, myocyte detachment and altered extracellular matrix protein distribution. The results are important for further understanding the etiology of soft salmon.

Connective tissue lung: different composition between male and female

Abnormal elasticity of lung tissue has a major impact on the clinical progression and outcome of many lung diseases including respiratory distress syndrome, asthma, emphysema and pneumothorax post thoracic injury. The major components of lung connective tissue are elastin, collagen and together provide the lung with its elasticity and tensile strength. Elastin is composed of flexible cross-linked polypeptides and has a linear stress-strain relation up to 200% strain; collagen has a more organized structure with both crystalline and amorphous phases and exhibits a highly unlinear stress-strain curve. The thoracic injury is responsible for about 25% of death for injury and in particular it represent 12% of all death in man in comparison to 7% of dealt in female. This different in death rate is due to the different hormone levels between man and female. On the other hand female sex hormones, estrogens (e.g. 17b-estradiol) exert important biological actions, both protective and undesirable. In particular in addition to their primary function as reproductive hormones, estrogens (e.g. 17b-estradiol) influence a wide range of other physiological processes in humans and other mammals, including cardiovascular, respiratory, and neuronal function, and bone density.. With special respect to the cardiorespiratory system, estrogens promote lung development and differentiation and exhibit pulmonary and cardiovascular protective properties. However, very little is known about the different composition of the two major constituents of the connective tissue of the female and men lung. We have studied by CLSM the elastin an collagen in man and female lung. All proteins tested are present in lung tissue of male and female and they are localized in the peribronchiolar and in the interalveolar septum. The reaction to elastin shows very differences staining in the basal laminae of small and medium bronchioles in two sexes, in particular these structures stained very strongly in female but not in male. Opposite behavior we have showed to collagen type IV reactions. In male the collagen type IV is localized as a continuous network throughout the lung parenchymal tissue and in the basal laminae it shows strongly staining.

PReS-FINAL-2031: Hyaluronic acid is a marker of active arthritis, but not systemic inflammation in systemic juvenile idiopathic arthritis

Systemic juvenile idiopathic arthritis (JIA) is a subtype of childhood arthritis associated with significant systemic inflammation as well as arthritis. Hyaluronic acid as a constituent of connective tissue has previously been described as a marker for arthritis in JIA in an Asian population. Mutations in hyaluronic acid synthase 2 leading to accumulation of hyaluronic acid have been found to be associated with a periodic fever syndrome in Shar-Pei dogs. Hyaluronic acid has therefore been suspected as a risk factor for systemic inflammation in humans.

Age-dependent alterations of decorin glycosaminoglycans in human skin

Proteoglycans, a family of glycosaminoglycan (GAG) conjugated proteins, are important constituents of human skin connective tissue (dermis) and are essential for maintaining mechanical strength of the skin. Age-related alterations of dermal proteoglycans have not been fully elucidated. We quantified transcripts of 20 known interstitial proteoglycans in human skin and found that decorin was the most highly expressed. Decorin was predominantly produced by dermal fibroblasts. Decorin was localized in dermal extracellular matrix with GAG bound to type I collagen fibrils. Analysis of decorin extracted from young (21–30 years) and aged (>80 years) sun-protected human buttock skin revealed that decorin molecular size in aged skin is significantly smaller than in young skin. The average size of decorin protein did not alter, indicating size of GAG chain is reduced in aged, compared to young skin. This age-dependent alteration of decorin GAG may contribute to skin fragility of elderly people.

SAT0433 Hyaluronic Acid is a Marker of Active Arthritis, but not Systemic Inflammation in Systemic Juvenile Idiopathic Arthritis

BackgroundSystemic juvenile idiopathic arthritis (JIA) is a subtype of childhood arthritis associated with significant systemic inflammation as well as arthritis. Hyaluronic acid as a constituent of connective tissue has previously...

Evaluation ofIn VivoWound Healing Activity ofBacopa monnieraon Different Wound Model in Rats

Wound healing effects of 50% ethanol extract of dried whole plant of Bacopa monniera (BME) was studied on wound models in rats. BME (25 mg/kg) was administered orally, once daily for 10 days (incision and dead space wound models) or for 21 days or more (excision wound model) in rats. BME was studied for its in vitro antimicrobial and in vivo wound breaking strength, WBS (incision model), rate of contraction, period of epithelization, histology of skin (excision model), granulation tissue free radicals (nitric oxide and lipid peroxidation), antioxidants (catalase, superoxide dismutase, and reduced glutathione), acute inflammatory marker (myeloperoxidase), connective tissue markers (hydroxyproline, hexosamine, and hexuronic acid), and deep connective tissue histology (dead space wound). BME showed antimicrobial activity against skin pathogens, enhanced WBS, rate of contraction, skin collagen tissue formation, and early epithelization period with low scar area indicating enhanced healing. Healing effect was further substantiated by decreased free radicals and myeloperoxidase and enhanced antioxidants and connective tissue markers with histological evidence of more collagen formation in skin and deeper connective tissues. BME decreased myeloperoxidase and free radical generated tissue damage, promoting antioxidant status, faster collagen deposition, other connective tissue constituent formation, and antibacterial activity.

447: Amniochorion apoptosis and autophagy: novel insights into the mechanisms of PPROM after fetoscopic laser surgery (FLS) for twin twin transfusion syndrome (TTTS)

into the mechanisms of PPROM after fetoscopic laser surgery (FLS) for twin twin transfusion syndrome (TTTS) Ramesha Papanna, Lovepreet Mann, Kenneth Moise, Themis Kyriakides, Guomao Zhao, Pedro Argoti, Catalin Buhimschi, Irina Buhimschi Yale University, Ob/Gyn & Reprod. Sci., New Haven, CT, University of Texas Medical School at Houston, Obstetrics, Gynecology and Reproductive Sciences, Houston, TX, Yale University, Biomedical Engineering & Pathology, New Haven, CT OBJECTIVE: Laser photocoagulation of chorionic plate vessels has emerged as an effective treatment of TTTS. This therapy poses major post-intervention challenges such as PPROM. Traditionally, this complication is interpreted as an iatrogenic mechanic event. We tested the hypothesis that following FLS, fetal membranes become susceptible to PPROM through activation of apoptosis and autophagy pathways in the amniochorion cells. STUDY DESIGN: Extra-placental membranes from 31 pregnancies that underwent FLS for TTTS (GA procedure: 20 3w, GA birth: 30 4w, PPROM n 9) were prospectively collected at birth. Trocar insertion site (recipient sac), fetal membranes of the recipient and donor sacs, and the inter-twin membrane were evaluated for tissue morphology and connective tissue constituents using Masson’s trichrome and Movat’s staining. Collagen histochemistry was assessed with Sirius red and quantified by image analysis. Amniochorion sections were stained using various markers of proliferation (Ki67), mesenchymal and epithelial features (vimentin, cytokeratin), autophagy (beclin-1, LC3) and apoptosis (DNA fragmentation). CRL tissues came from monochorionic/diamniotic twin (idiopathic PTB, n 4) and healthy term singleton (elective CS, n 8) gestations. RESULTS: Histological hallmarks of healing (angiogenesis, collagen deposition, granulation) or cellular proliferation (Ki67 staining) were absent at the trocar insertion site. Compared to CRLs, there was global loss of physiologic collagen organization in both amnion and chorion of the recipient and the inter-twin membrane, but not the donor sac (P .001). The recipient amniochorion showed features of autophagy (beclin-1 & LC3 in clusters) and higher apoptotic index (highest at the trocar insertion site) compared to the donor sac (P .03). CONCLUSION: FLS for TTTS is associated with generalized alteration of structural integrity and amniochorion cellular damage of the recipient’s sac that likely contribute to PPROM.