Photochemical Crosslinking of Tarsal Collagen as a Treatment for Eyelid Laxity: Evaluation in Ex Vivo Human Tissue.

PURPOSE: Experimental investigation in human eyelids to confirm that exposing excised tarsal plates to ultraviolet-A radiation can induce a stiffening effect through the riboflavin-photosensitized crosslinking of tarsal collagen. METHODS: Thirteen tarsal plates excised from nonfrozen human cadavers were irradiated with ultraviolet-A rays (365 nm wavelength) at an irradiance of 75 mW/cm2 for 3 minutes, equivalent to a radiation fluence of 13.5 J/cm2, in the presence of a riboflavin derivative as a photosensitizer. The tensile stress (strength) and Young's modulus (stiffness) of both nonirradiated and irradiated specimens were measured with the BioTester 5000 in the uniaxial mode at a strain of 10% and analyzed statistically. Individual specimens excised from 2 cadavers were also examined by routine histopathologic protocols to assess the effect of radiation on the Meibomian glands and collagen organization. RESULTS: The irradiation enhanced both stiffness and strength of the human tarsal specimens, the difference between the test samples and controls being statistically significant (p < 0.0002 for n = 13). Histology indicated no damage to tarsal connective tissue or to Meibomian glands, and revealed a more compact packing of the collagen network located around the glands, which may be beneficial. The existence of collagen compaction was also supported by the reduction of samples' thickness after irradiation (p = 0.0645). CONCLUSIONS: The irradiation of tarsal tissue with ultraviolet-A light of tarsus appears to be a safe and effective method for reducing eyelid laxity in human patients.

Effect of Ultraviolet Radiation on the Enzymolytic and Biomechanical Profiles of Abdominal Aortic Adventitia Tissue.

BACKGROUND: The abdominal aortic aneurysm (AAA) is defined as an increase in aortic diameter by more than 50% and is associated with a high risk of rupture and mortality without treatment. The aim of this study is to analyze the role of aortic adventitial collagen photocrosslinking by UV-A irradiation on the biomechanical profile of the aortic wall. METHODS: This experimental study is structured in two parts: the first part includes in vitro uniaxial biomechanical evaluation of porcine adventitial tissue subjected to either short-term elastolysis or long-term collagenolysis in an attempt to duplicate two extreme situations as putative stages of aneurysmal degeneration. In the second part, we included biaxial biomechanical evaluation of in vitro human abdominal aortic adventitia and human AAA adventitia specimens. Biomechanical profiles were examined for porcine and human aortic tissue before and after irradiation with UV-A light (365 nm wavelength). RESULTS: On the porcine aortic sample, the enhancing effect of irradiation was evident both on the tissue subjected to elastolysis, which had a high collagen-to-elastin ratio, and on the tissue subjected to prolonged collagenolysis despite being considerably depleted in collagen. Further, the effect of irradiation was conclusively demonstrated in the human adventitia samples, where significant post-irradiation increases in Cauchy stress (longitudinal axis: p = 0.001, circumferential axis: p = 0.004) and Young's modulus (longitudinal axis: p = 0.03, circumferential axis: p = 0.004) were recorded. Moreover, we have a stronger increase in the strengthening of the AAA adventitia samples following the exposure to UV-A irradiation (p = 0.007) and a statistically significant but not very important increase (p = 0.021) regarding the stiffness in the circumferential axis. CONCLUSIONS: The favorable effect of UV irradiation on the strength and stiffness of degraded aortic adventitia in experimental situations mimicking early and later stages of aneurysmal degeneration is essential for the development and potential success of procedures to prevent aneurysmal ruptures. The experiments on human normal and aneurysmal adventitial tissue confirmed the validity and potential success of a procedure based on exposure to UV-A radiation.

Computed Tomography Angiography Markers and Intraluminal Thrombus Morphology as Predictors of Abdominal Aortic Aneurysm Rupture.

Background: Abdominal aortic aneurysm (AAA) is a complex vascular disease characterized by progressive and irreversible local dilatation of the aortic wall. Currently, the indication for repair is linked to the transverse diameter of the abdominal aorta, using computed tomography angiography imagery, which is one of the most used markers for aneurysmal growth. This study aims to verify the predictive role of imaging markers and underlying risk factors in AAA rupture. Methods: The present study was designed as an observational, analytical, retrospective cohort study and included 220 patients over 18 years of age with a diagnosis of AAA, confirmed by computed tomography angiography (CTA), admitted to Vascular Surgery Clinic of Mures County Emergency Hospital in Targu Mures, Romania, between January 2018 and September 2022. Results: Patients with a ruptured AAA had higher incidences of AH (p = 0.006), IHD (p = 0.001), AF (p < 0.0001), and MI (p < 0.0001), and higher incidences of all risk factors (tobacco (p = 0.001), obesity (p = 0.02), and dyslipidemia (p < 0.0001)). Multivariate analysis showed that a high baseline value of all imaging ratios markers was a strong independent predictor of AAA rupture (for all p < 0.0001). Moreover, a higher baseline value of DAmax (OR:3.91; p = 0.001), SAmax (OR:7.21; p < 0.001), and SLumenmax (OR:34.61; p < 0.001), as well as lower baseline values of DArenal (OR:7.09; p < 0.001), DACT (OR:12.71; p < 0.001), DAfemoral (OR:2.56; p = 0.005), SArenal (OR:4.56; p < 0.001), SACT (OR:3.81; p < 0.001), and SThrombusmax (OR:5.27; p < 0.001) were independent predictors of AAA rupture. In addition, AH (OR:3.33; p = 0.02), MI (OR:3.06; p = 0.002), and PAD (OR:2.71; p = 0.004) were all independent predictors of AAA rupture. In contrast, higher baseline values of SAmax/Lumenmax (OR:0.13; p < 0.001) and ezetimibe (OR:0.45; p = 0.03) were protective factors against AAA rupture. Conclusions: According to our findings, a higher baseline value of all imaging markers ratios at CTA strongly predicts AAA rupture and AH, MI, and PAD highly predicted the risk of rupture in AAA patients. Furthermore, the diameter of the abdominal aorta at different levels has better accuracy and a higher predictive role of rupture than the maximal diameter of AAA.

Study of the Antioxidative Effects of Bombyx mori Silk Sericin in Cultures of Murine Retinal Photoreceptor Cells.

The availability of natural substances able to fulfill the role of antioxidants in a physiologic environment is important for the development of therapies against diseases associated with excessive production of reactive oxygen species and ensuing oxidative stress. Antioxidant properties have been reported episodically for sericin, a proteinaceous constituent of the silk thread in the cocoons generated by the larvae of the Lepidoptera order. We investigated the sericin fractions isolated from the cocoons spun by the domesticated (Bombyx mori) silkworm. Three fractions were isolated and evaluated, including two peptidoid fractions, the crude sericin and the purified (dialyzed) sericin, and the non-peptidoid methanolic extract of the crude fraction. When subjected to Trolox equivalent antioxidant capacity (TEAC) assay, the extract showed much higher antioxidant capacity as compared to the crude or purified sericin fractions. The three fractions were also evaluated in cultures of murine retinal photoreceptor cells (661 W), a cell line that is highly susceptible to oxidants and is crucially involved in the retinopathies primarily caused by oxidative stress. The extract displayed a significant dose-dependent protective effect on the cultured cells exposed to hydrogen peroxide. In identical conditions, the crude sericin showed a certain level of antioxidative activity at a higher concentration, while the purified sericin did not show any activity. We concluded that the non-peptidoid components accompanying sericin were chiefly responsible for the previously reported antioxidant capacity associated with sericin fractions, a conclusion supported by the qualitative detection of flavonoids in the extract but not in the purified sericin fraction.

Membranes Prepared from Recombinant RGD-Silk Fibroin as Substrates for Human Corneal Cells.

A recombinant formulation of silk fibroin containing the arginine-glycine-aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring Bombyx mori silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal-epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.

A study of the permeation and water-structuring behavioural properties of PEG modified hydrated silk fibroin membranes.

The potential of naturally occurring substances as a source of biomedical materials is well-recognised and is being increasingly exploited. Silk fibroin membranes derived fromBombyx morisilk cocoons exemplify this, for example as substrata for the growth of ocular cells with the aim of generating biomaterial-cell constructs for tissue engineering. This study investigated the transport properties of selected silk fibroin membranes under conditions that allowed equilibrium hydration of the membranes to be maintained. The behaviour of natural fibroin membranes was compared with fibroin membranes that have been chemically modified with poly(ethylene glycol). The permeation of the smaller hydrated sodium ion was higher than that of the hydrated calcium ion for all three ethanol treated membranes investigated. The PEG and HRP-modified C membrane, which had the highest water content at 59.6 ± 1.5% exhibited the highest permeation of the three membranes at 95.7 ± 2.8 × 10-8cm2s-1compared with 17.9 ± 0.9 × 10-8cm2s-1and 8.7 ± 1.7 × 10-8cm2s-1for membranes A and B respectively for the NaCl permeant. Poly(ethylene glycol) was used to increase permeability while exploiting the crosslinking capabilities of horseradish peroxidase to increase the compressive strength of the membrane. Importantly, we have established that the permeation behaviour of water-soluble permeants with hydrated radii in the sub-nanometer range is analogous to that of conventional hydrogel polymers.

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes.

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet's membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

Optimization of silk fibroin membranes for retinal implantation.

Silk fibroin membrane displays potential for ocular tissue reconstruction as demonstrated by its ability to support a functioning retinal pigment epithelium (RPE) in vitro. Nevertheless, translation of these findings to the clinic will require the use of membranes that can be readily handled and implanted into diseased retinas, with minimal impact on the surrounding healthy tissue. To this end, we optimized the physical properties of fibroin membranes to enable surgical handling during implantation into the retina, without compromising biocompatibility or permeability. Our central hypothesis is that optimal strength and permeability can be achieved by combining the porogenic properties of poly(ethylene glycol) (PEG) with the crosslinking properties of horseradish peroxidase (HRP). Our study reveals that PEG used in conjunction with HRP enables the production of fibroin membranes with superior handling properties to conventional fibroin membranes. More specifically, the modified membranes could be more easily implanted into the retinas of rats and displayed good evidence of biocompatibility. Moreover, the modified membranes retained the ability to support construction of functional RPE derived from pluripotent stem cells. These findings pave the way for preclinical studies of RPE-implantation using the optimized fibroin membranes.

Further Investigations on the Crosslinking of Tarsal Collagen as a Treatment for Eyelid Laxity: Optimizing the Procedure in Animal Tissue.

PURPOSE: A follow-up experimental study on the exposure of animal tarsal plate to ultraviolet-A radiation aimed at establishing an optimum range for safe irradiation conditions. METHODS: Sheep tarsus specimens were excised postmortem and then subjected to irradiation with ultraviolet-A rays (wavelength 365 nm) at higher irradiances than those reported in an initial study, using a laboratory radiation source. The mechanical properties (tensile strength and Young's modulus) of irradiated and nonirradiated samples were evaluated in a mechanical tester. The test and control specimens were examined histologically with an aim to assess the effects of radiation upon the meibomian glands and tarsal collagen networks, and to establish a safe range for the exposure irradiance level. RESULTS: As expected, irradiation induced both stiffening and strengthening of the tarsal plate specimens. At an irradiance of 50 mW/cm for 3-minute exposure, these effects were at their maximum level, after which a decline in mechanical characteristics were observed. No destruction of the tarsal connective tissue or the meibomian glands were noticed up to an irradiance of 125 mW/cm for 3-minute exposure, corresponding to a fluence of 22.5 J/cm. Histology revealed that the collagen network surrounding the glands were packed more compactly following irradiation. At a fluence of 45 J/cm, massive destruction of periglandular collagen-rich network and meibocytes were demonstrated histologically. CONCLUSIONS: The study indicates that irradiation of tarsal collagen leading to tissue stiffening shall be carried out at levels of fluence between 10 and 15 J/cm, a region that is deemed safe. The exposure time can be adjusted according to the surgeon's decision.Safe irradiation conditions are established for the exposure of ex vivo ovine tarsus to ultraviolet-A radiation as a potentially effective treatment for eyelid laxity in human patients.

Effect of covering with cross-linked gelatin glue on tissue regeneration in a rat lung injury model.

OBJECTIVES: Thoracic reintervention is a common treatment; however, preventing adhesion of the lung to the thoracic cavity wall remains a problem. This study aimed to investigate the effect on pleural adhesion of covering the postoperative pleural injury site with cross-linked gelatin glue (gelatin plus glutaraldehyde, hereafter 'gelatin glue') and to evaluate the proliferation of healing cells on gelatin glue. METHODS: We created a rat incisional lung-wound model and compared the effects of sealing the wound with gelatin glue (group A, n = 5), fibrin glue (group B, n = 5) or fibrin glue with a polyglycolic acid sheet (group C, n = 5). Adhesions were assessed 28 days postoperatively and compared among the groups using the Karacam's scoring method. Lung-wound healing was studied histologically at day 7 postoperatively. Mesothelial cell proliferation was investigated on gelatin and fibrin glues in vitro. RESULTS: There were no or few adhesions of the chest wall in group A. The adhesion scores (mean ± standard deviation) were 1.2 ± 0.4, 2.6 ± 1.4 and 3.2 ± 1.2 in groups A, B and C, respectively (A vs C, P = 0.0496). During the healing process, the gelatin glue surface was covered by mesothelial-like cells. Proliferation of cultured mesothelial cells was promoted on the gelatin glue compared with the fibrin glue. CONCLUSIONS: Covering lung wounds with the gelatin glue reduced adhesions and promoted the growth of healing cells compared with the fibrin glue. These findings suggest that the gelatin glue may help prevent adhesions and thus be a therapeutically effective biomaterial in lung surgery.

Characterization of Human iPSC-RPE on a Prosthetic Bruch's Membrane Manufactured From Silk Fibroin.

Purpose: RPE cell transplantation as a potential treatment for AMD has been extensively investigated; however, in AMD, ultrastructural damage affects both the RPE and its underlying matrix support, the Bruch's membrane (BrM). An RPE monolayer supported by a surrogate scaffold could thus provide a more effective approach to cell-based therapy for AMD. Toward this goal, we aimed to establish a functional human induced pluripotent stem cell-derived (hiPSC)-RPE monolayer on a Bombyx mori silk fibroin (BMSF) scaffold. Methods: RPE differentiated from five distinct hiPSC lines were cultured on BMSF membrane coated with extracellular matrix (ECM, COL1), and either regular tissue culture plastic or Transwell coated with ECM (LAM-TCP). Morphologic, gene and protein expression, and functional characteristics of the hiPSC-RPE cultured on different membranes were compared in longitudinal experiments spanning 1 day to ≥3 months. Results: The hiPSC-RPE monolayers on ECM-coated BMSF and TCP could be maintained in culture for ≥3 months and displayed RPE-characteristic morphology, pigmentation, polarity, and expression of RPE signature genes and proteins. Furthermore, hiPSC-RPE on both ECM-coated BMSF and TCP displayed robust expression and secretion of several basement membrane proteins. Importantly, hiPSC-RPE cells on COL1-BMSF and LAM-TCP showed similar efficacy in the phagocytosis and degradation of photoreceptor outer segments. Conclusions: A biomaterial scaffold manufactured from silk fibroin supports the maturation and long-term survival of a functional hiPSC-RPE monolayer. This has significant implications for both in vitro disease modeling and in vivo cell replacement therapy.

Photochemically Induced Crosslinking of Tarsal Collagen as a Treatment for Eyelid Laxity: Assessing Potentiality in Animal Tissue.

PURPOSE: An experimental study to demonstrate in animal eyelids that the controlled exposure of excised tarsal plate to ultraviolet-A radiation can induce a rigidification effect due to photochemical crosslinking of the constitutive collagen. METHODS: Excised strips of sheep tarsus were irradiated with ultraviolet-A rays (wavelength 365 nm) at low and high irradiances, in the presence of riboflavin as a photosensitizer, using radiation sources available for corneal collagen crosslinking procedure. The tensile strength and Young's modulus (stiffness) of irradiated and control samples were measured in a mechanical tester and analyzed statistically. Histologic examination of the specimens was carried out to evaluate the effect of radiation on the meibomian glands and collagen organization. RESULTS: Mechanical evaluation showed that irradiation induced both stiffening and strengthening of the tarsal plate specimens, and this effect was enhanced at the higher levels of irradiance. The changes in mechanical properties can be attributed to a process of photochemically induced crosslinking of tarsal collagen. Histology revealed no changes in the meibomian glands or in the fibrous collagen system of the tarsus. CONCLUSIONS: These findings indicate that irradiation of tarsal collagen leading to tissue stiffening could be a safe procedure for treating lax eyelid conditions in human patients.

Low-voltage coagulation, polyglycolic acid sheets, and fibrin glue to control air leaks in lung surgery.

OBJECTIVE: We evaluated the efficacy of low-voltage coagulation (LVC) with polyglycolic acid (PGA) sheets (Neoveil, GUNZE Ltd., Japan) and fibrin glue to control intraoperative alveolar air leaks after lung surgery. METHODS: We included 176 patients with non-small cell lung cancer who underwent thoracoscopic lobectomies. When alveolar air leak was confirmed after lung resection, we applied LVC system to the pleural defect followed by layers of PGA sheets and fibrin glue (n = 40). We then analyzed postoperative air leaks (rate of occurrence and duration time). RESULTS: 73% of patients (29/40 cases) experienced no postoperative air leaks. Although 11 patients experienced air leaks after surgery, there were no prolonged air leaks (>7 days) (resolution time, 3.5 ± 1.4 days; range, 2-6 days). Two patients required drainage for late-onset air leaks, but their conditions improved without further treatment. There were no further adverse events. CONCLUSIONS: The use of LVC with PGA sheets and fibrin glue following pulmonary resection efficiently prevented both intraoperative air leaks and prolonged air leaks after lung surgery.

A comparative investigation of Bombyx mori silk fibroin hydrogels generated by chemical and enzymatic cross-linking.

Fibroin, the major proteinaceous component of the silk fiber produced by larvae of the domesticated silk moth (Bombyx mori), has been widely investigated as a biomaterial for potential applications in tissue engineering and regenerative medicine. Following sol-gel transition, silk fibroin solutions can generate hydrogels that present certain advantages when employed as biomaterials, especially if they are cross-linked. The subject of this study was the self-cross-linking of silk fibroin through a process induced by the enzyme horseradish peroxidase (HRP) in the presence of hydrogen peroxide, a method only recently proposed and scarcely reported. The hydrogels were prepared either by physical cross-linking, by cross-linking with a natural compound (genipin), or by enzymatic cross-linking. The products were comparatively characterized in regard to their synthesis and background chemical aspects, physical and optical properties, mechanical properties, secondary structure, swelling/deswelling behavior, enzymatic degradation, and compatibility as substrates for cell adhesion and proliferation. The study confirmed the advantages of the HRP-induced cross-linking, which included considerably shorter gelation times, enhanced elasticity of the resulting hydrogels, and improved cytocompatibility. Discrepancies between certain results of this investigation and those reported previously were discussed in detail.

A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell® ). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+ /K+ -ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd.

Characterization of Bombyx mori and Antheraea pernyi silk fibroins and their blends as potential biomaterials.

Fibroin proteins isolated from the cocoons of certain silk-producing insects have been widely investigated as biomaterials for tissue engineering applications. In this study, fibroins were isolated from cocoons of domesticated Bombyx mori (BM) and wild Antheraea pernyi (AP) silkworms following a degumming process. The object of this study was to obtain an assessment on certain properties of these fibroins in order that a concept might be had regarding the feasibility of using their blends as biomaterials. Membranes, 10-20 µm thick, which are water-insoluble, flexible and transparent, were prepared from pure fibroins and from their blends, and subjected to water vapor annealing in vacuum, with the aim of providing materials sufficiently strong for manipulation. The resulting materials were characterized by electrophoretic analysis and infrared spectrometry. The tensile properties of the membranes were measured and correlated with the results of infrared analysis. At low concentrations of any of the two fibroins, the mechanical characteristics of the membranes appeared to be adequate for surgical manipulation, as the modulus and strength surpassed those of BM silk fibroin alone. It was noticed that high concentrations of AP silk fibroin led to a significant reduction in the elasticity of membranes.

Optimization of Corneal Epithelial Progenitor Cell Growth on Bombyx mori Silk Fibroin Membranes.

Scaffolds prepared from silk fibroin derived from cocoons of the domesticated silkworm moth Bombyx mori have demonstrated potential to support the attachment and growth of human limbal epithelial (HLE) cells in vitro. In this study, we attempted to further optimize protocols to promote the expansion of HLE cells on B. mori silk fibroin- (BMSF-) based scaffolds. BMSF films were initially coated with different extracellular matrix proteins and then analysed for their impact on corneal epithelial cell adhesion, cell morphology, and culture confluency. Results showed that collagen I, collagen III, and collagen IV consistently improved HCE-T cell adherence, promoted an elongated cell morphology, and increased culture confluency. By contrast, ECM coating had no significant effect on the performance of primary HLE cells cultured on BMSF films. In the second part of this study, primary HLE cells were grown on BMSF films in the presence of medium (SHEM) supplemented with keratinocyte growth factor (KGF) and the Rho kinase inhibitor, Y-27632. The results demonstrated that SHEM medium supplemented with KGF and Y-27632 dramatically increased expression of corneal differentiation markers, keratin 3 and keratin 12, whereas expression of the progenitor marker, p63, did not appear to be significantly influenced by the choice of culture medium.

Further development of silk sericin as a biomaterial: comparative investigation of the procedures for its isolation from Bombyx mori silk cocoons.

There is significant research dedicated to fibroin and sericin, the two major proteinaceous components of the silk threads produced by the domesticated silkworm, Bombyx mori. While fibroin is accepted as an established biomaterial, sericin (BMSS) has been largely neglected in this respect on the account of a hypothetical allergenic activity. Research over the past decade, including our previous study (Prog Biomater 2:14, 2013), demonstrated the biocompatibility of sericin and feasibility of its use as a biomaterial. However, the current procedures for isolating BMSS from the raw silk cocoons can only provide degraded proteins, where the size and distribution of their molecular masses are significantly altered. Based on the plausible assumption that such effects can have a negative impact on the properties of sericin as a biomaterial, in this study we investigated comparatively four different extraction procedures in order to find the method that would cause the least hydrothermal degradation of BMSS. The products resulting from commonly used procedures (extraction in boiling water, alkaline extraction, and extraction in autoclave) were compared to those resulting from aqueous extraction in mild conditions as described a long time ago by Anderlini. The molecular mass distribution in BMSS resulting from each procedure was examined by electrophoretic analysis performed on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE), while the conformational changes pertaining to secondary structure of BMSS were evaluated by Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectrometry. The electrophoretograms indicated that the aqueous extraction in mild conditions conducted at 50 °C for durations up to 4 weeks, with/without stirring, afforded the least degraded BMSS. The infrared spectrometric analysis showed that BMSS resulting from the mild extraction method contained predominantly ß-sheet conformations, while the more degradative methods (alkaline, autoclave) led to BMSS where the random-coil conformations were preferential. The long-duration aqueous extraction at 50 °C (but not at 60 °C) appeared as a valid option for obtaining BMSS products where the hydrothermally induced fragmentation of the polypeptidic components is minimized.

Sealing Effect of Cross-Linked Gelatin Glue in the Rat Lung Air Leak Model.

BACKGROUND: Air leak is a common problem in pulmonary surgical procedures. In this study, we evaluated the efficacy and safety of gelatin glue (cross-linked with glutaraldehyde) in a rat model of lung air leak. METHODS: A model of pulmonary fistula was created in the rat lung with the use of a needle. The fistula was then sealed with either gelatin glue (group A), fibrin glue (group B), or fibrin glue with a polyglycolic acid sheet (group C). The seal breaking pressures were measured for each group, and the results were compared. To assess the biocompatibility of the gelatin glue, a model of lung damage was created with incision, and the gelatin glue was applied to seal the wound. Histologic analysis was then performed on the lung tissue. RESULTS: The seal breaking pressure in group A (47.88 ± 6.69 mm Hg) was significantly higher than that in group B (24.67 ± 3.24 mm Hg, p = 0.0302) or group C (28.67 ± 3.55 mm Hg, p = 0.0406). Histologically, the gelatin glue adhered firmly to the lung surface, and only localized tissue inflammation was observed. CONCLUSIONS: The sealing effect of gelatin glue was superior to that of fibrin glue, with or without a polyglycolic acid sheet. In addition, the gelatin glue only caused mild inflammation of the lung and was absorbed without any adverse foreign body response. These findings suggest that gelatin glue may be a therapeutically effective biomaterial for sealing lung wounds and restoring respiratory function.

Experimental use of crosslinked gelatin glue for arterial hemostasis in cardiovascular surgery.

BACKGROUND: Anastomotic needle hole bleeding is a frequently encountered problem in cardiovascular surgeries. OBJECTIVE: To examine the feasibility of crosslinked gelatin glue as an anastomotic needle hole sealant in comparison with fibrin glue. METHODS: The in vitro burst water pressures were measured for gelatin and fibrin glue sealed needle holes of expanded polytetrafluoroethylene (ePTFE) or collagen coated woven polyester grafts. For in vivo investigations, abdominal aorta-ePTFE graft anastomoses of heparinized beagle dogs were sealed by gelatin or fibrin glue and hemostatic efficacy was judged. The implanted sites were re-examined 4 weeks postoperatively. RESULTS: The in vitro burst water pressures of gelatin glue sealed needle holes of both grafts were higher than those sealed by fibrin glue. For in vivo canine studies, hemostasis was successful for all gelatin glue applied suture lines, but not two out of three fibrin glue treated sites when 3-0 polypropylene suture was employed. Although adhesions of surrounding tissues were intense for all sites 4 weeks postoperatively, inflammation was more severe for the fibrin glue group compared to those of gelatin glue. CONCLUSIONS: Gelatin glue was found to be an effective and safe sealant for accomplishing hemostasis of anastomotic needle holes of vascular grafts.