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.

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.

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.

Biomaterials in Gastroenterology: A Critical Overview.

In spite of the large diversity of diagnostic and interventional devices associated with gastrointestinal endoscopic procedures, there is little information on the impact of the biomaterials (metals, polymers) contained in these devices upon body tissues and, indirectly, upon the treatment outcomes. Other biomaterials for gastroenterology, such as adhesives and certain hemostatic agents, have been investigated to a greater extent, but the information is fragmentary. Much of this situation is due to the paucity of details disclosed by the manufacturers of the devices. Moreover, for most of the applications in the gastrointestinal (GI) tract, there are no studies available on the biocompatibility of the device materials when in intimate contact with mucosae and other components of the GI tract. We have summarized the current situation with a focus on aspects of biomaterials and biocompatibility related to the device materials and other agents, with an emphasis on the GI endoscopic procedures. Procedures and devices used for the control of bleeding, for polypectomy, in bariatrics, and for stenting are discussed, particularly dwelling upon the biomaterial-related features of each application. There are indications that research is progressing steadily in this field, and the establishment of the subdiscipline of "gastroenterologic biomaterials" is not merely a remote projection. Upon the completion of this article, the gastroenterologist should be able to understand the nature of biomaterials and to achieve a suitable and beneficial perception of their significance in gastroenterology. Likewise, the biomaterialist should become aware of the specific tasks that the biomaterials must fulfil when placed within the GI tract, and regard such applications as both a challenge and an incentive for progressing the research in this field.

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.

Hydrogels with Lotus Leaf Topography: Investigating Surface Properties and Cell Adhesion.

The interactions of cells with the surface of materials is known to be influenced by a range of factors that include chemistry and roughness; however, it is often difficult to probe these factors individually without also changing the others. Here we investigate the role of roughness on cell adhesion while maintaining the same underlying chemistry. This was achieved by using a polymerization in mold technique to prepare poly(hydroxymethyl methacrylate) hydrogels with either a flat topography or a topography that replicated the microscale features of lotus leaves. These materials were then assessed for cell adhesion, and atomic force microscopy and contact angle analysis were then used to probe the physical reasons for the differing behavior in relation to cell adhesion.

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.

Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.

The New Zealand White rabbit has been widely used as a model of limbal stem cell deficiency (LSCD). Current techniques for experimental induction of LSCD utilize caustic chemicals, or organic solvents applied in conjunction with a surgical limbectomy. While generally successful in depleting epithelial progenitors, the depth and severity of injury is difficult to control using chemical-based methods. Moreover, the anterior chamber can be easily perforated while surgically excising the corneal limbus. In the interest of creating a safer and more defined LSCD model, we have therefore evaluated a mechanical debridement technique based upon use of the AlgerBrush II rotating burr. An initial comparison of debridement techniques was conducted in situ using 24 eyes in freshly acquired New Zealand White rabbit cadavers. Techniques for comparison (4 eyes each) included: (1) non-wounded control, (2) surgical limbectomy followed by treatment with 100% (v/v) n-heptanol to remove the corneal epithelium (1-2 min), (3) treatment of both limbus and cornea with n-heptanol alone, (4) treatment of both limbus and cornea with 20% (v/v) ethanol (2-3 min), (5) a 2.5-mm rounded burr applied to both the limbus and cornea, and (6) a 1-mm pointed burr applied to the limbus, followed by the 2.5-mm rounded burr applied to the cornea. All corneas were excised and processed for histology immediately following debridement. A panel of four assessors subsequently scored the degree of epithelial debridement within the cornea and limbus using masked slides. The 2.5-mm burr most consistently removed the corneal and limbal epithelia. Islands of limbal epithelial cells were occasionally retained following surgical limbectomy/heptanol treatment, or use of the 1-mm burr. Limbal epithelial cells were consistently retained following treatment with either ethanol or n-heptanol alone, with ethanol being the least effective treatment overall. The 2.5-mm burr method was subsequently evaluated in the right eye of 3 live rabbits by weekly clinical assessments (photography and slit lamp examination) for up to 5 weeks, followed by histological analyses (hematoxylin & eosin stain, periodic acid-Schiff stain and immunohistochemistry for keratin 3 and 13). All 3 eyes that had been completely debrided using the 2.5-mm burr displayed symptoms of ocular surface failure as defined by retention of a prominent epithelial defect (∼40% of corneal surface at 5 weeks), corneal neovascularization (2-3 quadrants), reduced corneal transparency and conjunctivalization of the corneal surface (demonstrated by the presence of goblet cells and/or staining for keratin 13). In conclusion, our findings indicate that the AlgerBrush II rotating burr is an effective method for the establishment of ocular surface failure in New Zealand White rabbits. In particular, we recommend use of the 2.5-mm rotating burr for improved efficiency of epithelial debridement and safety compared to surgical limbectomy.

Isolation of microvascular endothelial cells from cadaveric corneal limbus.

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with foetal bovine serum (10% v/v), D-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 µg/mL), hydrocortisone (1 µg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF(+) cells formed aligned cell-to-cell 'trains' when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32-80 years) or duration in eye bank corneal preservation medium prior to use (3-10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p < 0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

Therapeutic strategies based on non-ionizing radiation to prevent venous neointimal hyperplasia: the relevance for stenosed arteriovenous fistula, and the role of vascular compliance.

We have reviewed the development and current status of therapies based on exposure to non-ionizing radiation (with a photon energy less than 10 eV) aimed at suppressing the venous neointimal hyperplasia, and consequentially at avoiding stenosis in arteriovenous grafts. Due to the drawbacks associated with the medical use of ionizing radiation, prominently the radiation-induced cardiovascular disease, the availability of procedures using non-ionizing radiation is becoming a noteworthy objective for the current research. Further, the focus of the review was the use of such procedures for improving the vascular access function and assuring the clinical success of arteriovenous fistulae in hemodialysis patients. Following a brief discussion of the physical principles underlying radiotherapy, the current methods based on non-ionizing radiation, either in use or under development, were described in detail. There are currently five such techniques, including photodynamic therapy (PDT), far-infrared therapy, photochemical tissue passivation (PTP), Alucent vascular scaffolding, and adventitial photocrosslinking. The last three are contingent on the mechanical stiffening achievable by the exogenous photochemical crosslinking of tissular collagen, a process that leads to the decrease of venous compliance. As there are conflicting opinions on the role of compliance mismatch between arterial and venous conduits in a graft, this aspect was also considered in our review.

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.

Exogenous Crosslinking of Tendons as a Strategy for Mechanical Augmentation and Repair: A Narrative Review.

Collagens constitute a family of triple-helical proteins with a high level of structural polymorphism and a broad diversity of structural and chemical characteristics. Collagens are designed to form supporting aggregates in the extracellular spaces of our body, but they can be isolated from animal sources and processed to become available as biomaterials with wide applications in biomedicine and bioengineering. Collagens can be conveniently modified chemically, and their propensity for participating in crosslinking reactions is an important feature. While the crosslinking promoted by a variety of agents provides a range of collagen-based products, there has been minor interest for therapies based on the crosslinking of collagen while located within living connective tissues, known as exogenous crosslinking. Currently, there is only one such treatment in ocular therapeutics (for keratoconus), and another two in development, all based on mechanical augmentation of tissues due to ultraviolet (UV)-induced crosslinking. As seen in this review, there was some interest to employ exogenous crosslinking in order to reinforce mechanically the lax tendons with an aim to arrest tear propagation, stabilize the tissue, and facilitate the healing. Here we reviewed in details both the early stages and the actual status of the experimental research dedicated to the topic. Many results have not been encouraging, however there is sufficient evidence that tendons can be mechanically reinforced by chemical or photochemical exogenous crosslinking. We also compare the exogenous crosslinking using chemical agents, which was predominant in the literature reviewed, to that promoted by UV radiation, which was rather neglected but might have some advantages.

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.

Oxygen Permeability of Silk Fibroin Hydrogels and Their Use as Materials for Contact Lenses: A Purposeful Analysis.

Fibroin is a fibrous protein that can be conveniently isolated from the silk cocoons produced by the larvae of Bombyx mori silk moth. In its form as a hydrogel, Bombyx mori silk fibroin (BMSF) has been employed in a variety of biomedical applications. When used as substrates for biomaterial-cells constructs in tissue engineering, the oxygen transport characteristics of the BMSF membranes have proved so far to be adequate. However, over the past three decades the BMSF hydrogels have been proposed episodically as materials for the manufacture of contact lenses, an application that depends on substantially elevated oxygen permeability. This review will show that the literature published on the oxygen permeability of BMSF is both limited and controversial. Additionally, there is no evidence that contact lenses made from BMSF have ever reached commercialization. The existing literature is discussed critically, leading to the conclusion that BMSF hydrogels are unsuitable as materials for contact lenses, while also attempting to explain the scarcity of data regarding the oxygen permeability of BMSF. To the author's knowledge, this review covers all publications related to the topic.

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.

Biodegradation of poly(2-hydroxyethyl methacrylate) (PHEMA) and poly{(2-hydroxyethyl methacrylate)-co-[poly(ethylene glycol) methyl ether methacrylate]} hydrogels containing peptide-based cross-linking agents.

PHEMA-peptide and P[HEMA-co-(MeO-PEGMA)]-peptide conjugate hydrogels [where PHEMA = poly(2-hydroxyethyl methacrylate; PEGMA = poly(ethylene glycol) methacrylate] were readily prepared via photoinitiated free-radical polymerization in water. The PHEMA-peptide hydrogels were opaque and had a heterogeneous morphology of interconnected polymer droplets, characteristic of polymers that separate from the aqueous phase during the polymerization experiment. The P[HEMA-co-(MeO-PEGMA)]-peptide conjugates were transparent gels with a homogeneous morphology when formed in water, but when formed in aqueous NaCl solutions the P[HEMA-co-(MeO-PEGMA)]-peptide conjugates were also opaque and exhibited the heterogeneous morphology of interconnected polymer droplets. When incubated in solutions containing activated papain, P[HEMA-co-(MeO-PEGMA)]-peptide conjugates underwent degradation that was characterized by macroscopic changes to sample shape and size, sample weight, and microscopic structure. PHEMA-peptide conjugates did not undergo any significant degradation when incubated with papain, although ninhydrin-staining experiments suggested that some peptide cross-linker groups were cleaved during the incubation. The difference in degradation behavior of PHEMA-peptide and P[HEMA-co-(MeO-PEGMA)]-peptide conjugates is attributed to differences in aqueous solubility of PHEMA and P[HEMA-co-(MeO-PEGMA)].

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.

The behavior of aged regenerated Bombyx mori silk fibroin solutions studied by (1)H NMR and rheology.

As part of a project to utilize the regenerated silk fibroin (RSF) membranes as a supporting matrix for the attachment and growth of corneal stem/progenitor cells in the development of tissue engineered constructs for the surgical restoration of the ocular surface, the behavior of the aged RSF solutions has been investigated. The solutions were produced from domesticated silkworm (Bombyx mori) cocoons according to a protocol involving successive dissolution steps, filtration and dialysis. The solutions were kept at 4 degrees C in a refrigerator for a certain period of time until near the gelation time. The changes in molecular conformation were studied by solution-state (1)H NMR, while the flow of the solutions was characterized by rheological method. Upon ageing turbidity developed in solutions and the viscosity continuously decreased prior to a drastic increased near the gelation time. The (1)H resonances of aged solutions showed a consistent downfield shift as compared to the (1)H resonances of the fresh solution. Shear thinning with anomalous short recovery within a certain range of low shear rates occurred in both fresh and aged solutions. While the solutions behave as pseudo-plastic materials, the chain conformation in aged solutions adopted all secondary configurations with beta-strand being predominant.