Multifunctional biomimetic materials for corneal regeneration

The cornea is the outermost layer of the eye, which is responsible for transmitting 95% of the incident light to the retina for vision and provides 70% of the focusing power of the eye. Corneal disease is a primary cause of blindness worldwide. Replacing the pathologic cornea with a donor cornea is the most accepted treatment, but there is a severe shortage of donor tissue, resulting in an extensive waiting list for transplantation of over 10 million people. In this thesis, we worked on the development of artificial corneas to solve the donor shortage issue. Although an artificial cornea made from carbodiimide crosslinked recombinant human collagen developed within our lab was successfully transplanted into 10 patients in a clinical trial, this material was not tough enough to withstand severe disease conditions where inflammation is present, and where enzymes secreted can cause premature implant degradation. To improve mechanical strength and material stability, a secondary network of 2-methacryloyloxyethyl phosphorylcholine (MPC) biopolymer was incorporated within the collagen hydrogel, forming an interpenetrating network (IPN). High resolution transmission electron microscopy showed that the implants comprised loosely bundled collagen filaments. X-ray scattering further revealed that the collagen fibrils within the implants were uniaxially oriented, whereas a biaxial alignment is present within the human cornea. This fibril arrangement resulted in highly transparent implants that transmitted virtually all incoming light of visible spectra together with a large proportion of UV light. This study is critical in a sense that it strongly suggests that all patients transplanted with this artificial cornea should take the precaution to use UV protection prior to re-growth of the epithelium, which is known to absorb harmful UV rays. To determine the utility of the implants for clinical use, we showed that they could be cut with a femtosecond laser. Laser excision of diseased patient tissue avoids damage to the surrounding healthy tissue, thereby circumventing excessive, undesirable inflammatory responses associated with the manual surgical technique while the cutting of a matched implant allows for precise host-graft apposition and seamless regeneration. We also showed that the surface of the implants could be modified to enhance rapid and stable epithelial growth. We demonstrated that we could pattern the implants surfaces using microcontact printing with fibronectin as “ink”. The dimensions of the patterned stripes were important in controlling corneal epithelial cell behavior including proliferation. This is important to ensure rapid wound healing and hence, an overall superior clinical outcome. In all of the above materials, the collagen was crosslinked with N-(3-dimethylaminopropyl)- N'-ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS). EDC is a zero-length crosslinker and while it produces a sufficiently robust hydrogel for clinical implantation, suturability was still an issue. To enhance suturability, we evaluated the effects of an epoxy-based crosslinker, 1,4-Butanediol diglycidyl ether (BDDGE), which has been shown to result in collagen hydrogels with enhanced elasticity. As neuronal ingrowth into the hydrogels and epithelial cell coverage are important considerations in achieving regeneration, we examined the effects of incorporation of short cell adhesive laminin peptides within the BDDGE-crosslinked hydrogels. We showed that incorporation of YIGSR and IKVAV peptides enhanced the proliferation of corneal epithelial cells and neuronal progenitor cells, respectively. Although artificial corneas made from collagen have been successfully tested in the clinic, animal-derived collagens, in general, come from very heterogeneous sources and carry a risk of pathogen transmission. Use of recombinant human collagens mitigates those issues but just like native collagens; they are large macromolecules, relatively inert and therefore difficult to chemically alter to design in new functionalities. They are difficult and hence expensive to produce. Collagen-like peptides (CLP), also known as collagen mimetic peptides, are relatively short sequences that have been designed to replicate and reproduce the function of full-length collagen. We examined the safety and efficacy of one such CLP that we had conjugated to polyethylene glycol-maleimide (PEG) as implants for promoting corneal regeneration in mini-pig models. This CLP-PEG implants promoted the regeneration of corneal epithelial and stromal cells from endogenous progenitors, as well as cornea nerves to form a stable neo-cornea. The use of fully synthetic materials that can be produced under a tightly controlled environment such as CLP-PEG mitigates safety issues associated with native collagen from animal or human sources, as well as makes production sufficiently costeffective to allow for future scale-up

Phytochemical investigations of Campsis radicans L.

Petroleum ether, dichloromethane and ethyl acetate soluble fractions were obtained through partitioning the crude methanolic extract of the leaves of Campsis radicans L. (Family. Bignoniaceae) followed by the chromatographic separation of secondary metabolites from them. A total of five triterpene compounds i.e., corosolic acid methyl ester (1), β-amyrin (2), arjunolic acid (3), maslinic acid (4) and 28-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-2α,3α,19α-trihydroxy-12-en-28-ursolic acid (5) were isolated from the dichloromethane fractions and their structures were characterized by 1H NMR spectroscopy and compared the NMR data with published values

Tendencies of globalization of development of international tourism

Проаналізовано сучасні тенденції розвитку міжнародного туризму як провідного сегмента світового ринку, однієї із найважливіших складових процесу глобалізації. Визначено передумови глобалізації світового туристичного ринку, окреслено перспективи розвитку системи міжнародного туризму в сучасних умовах. Зміст міжнародного туризму як економічної категорії представлено як функціональну спрямованість його впливу на формування сучасної глобальної економічної системи, що проявляється в єдності його функцій.In the article modern progress of international tourism trends are analysed as a leading world market segment, one of major constituents of process of globalization. Pre-conditions of globalization of world tourist market are certain, the prospects of development of the system of international tourism are outlined in modern terms. The table of contents of international tourism as an economic category is presented as a functional orientation of his influence on forming of the modern global economic system that shows up in unity of his functions. Passing of international tourism to the global stage of the development was caused by the specific of the market state of affairs of the concrete historical stage, aspiring of large tourist companies to minimization of prime price of tourist package and increase of guarantees of grant of the announced tourist services of the proper quality; and also by the height of investment attractiveness of tourist business as a result of mass character of international tourism and increase of effect of scale in him. Global international tourism certainly as the socio-economic phenomenon that depends already not on domestic, but from external factors, maintenance and quantity of that determined by a global world concord the members of that are related to each other in all spheres of public life, including an economy, policy, ideology, culture, social sphere, ecology, safety. By the basic components of globalization of international tourism following: expansion of international economic connections in a tourist production, increase of internationalization of factors of tourist production, distribution of multinationals corporations in tourist business

Collagen-Based Bioengineered Substitutes of Donor Corneal Allograft Implantation: Assessment and Hypotheses

To fabricate donor corneal substitutes based on carbodiimide cross-linked porcine collagen, to study their in vitro and in vivo properties, and to elaborate new implantation techniques for the donor corneal collagen-based substitutes, this study had been performed. Bioengineered substitutes of corneal stroma (BSCS) were fabricated by cross-linking porcine type I collagen with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide, as previously described. Their refractive indices were measured using an Abbe refractometer. The mechanical properties were evaluated by their ability to tolerate interrupted stitches placed during deep lamellar keratoplasty performed on isolated rabbit eyes. BSCS were then implanted into one cornea of 8 rabbits and were followed-up for 12 months. Our BSCS had refractive indices of 1.24-1.3 (human cornea 1.37-1.38), and tolerated the placement of 12 interrupted stitches well. A new technique, the BSCS “stitchless†implantation, was developed. When implanted into rabbit corneas, BSCS remained stably integrated and clear during the 12 month follow-up. Non-intensive opacities within corneal layers (grade 1.5 on a scale of 0 to 4) were observed in 2/8 eyes during the 1st postoperative week, and in one eye the opacity resolved. In the 2nd eye a fine opacity (grade 1) remained. Light microscopy confirmed the integrity of the implants and the absence of inflammation in corneal stroma. The current data suggest that the BSCS fabricated in the Ukraine by cross-linking collagen is a good alternative to human donor corneas if medical grade porcine collagen is used. In addition, the new “stitchless†technique of BSCS implantation may decrease corneal substitute damage and accelerate its epithelialisation

Crosslinker-free collagen gelation for corneal regeneration

Development of an artificial cornea can potentially fulfil the demand of donor corneas for transplantation as the number of donors is far less than needed to treat corneal blindness. Collagen-based artificial corneas stand out as a regenerative option, having promising clinical outcomes. Collagen crosslinked with chemical crosslinkers which modify the parent functional groups of collagen. However, crosslinkers are usually cytotoxic, so crosslinkers need to be removed from implants completely before application in humans. In addition, crosslinked products are mechanically weak and susceptible to enzymatic degradation. We developed a crosslinker free supramolecular gelation strategy using pyrene conjugated dipeptide amphiphile (PyKC) consisting of lysine and cysteine; in which collagen molecules are intertwined inside the PyKC network without any functional group modification of the collagen. The newly developed collagen implants (Coll-PyKC) are optically transparent and can effectively block UV light, are mechanically and enzymatically stable, and can be sutured. The Coll-PyKC implants support the growth and function of all corneal cells, trigger anti-inflammatory differentiation while suppressing the pro-inflammatory differentiation of human monocytes. Coll-PyKC implants can restrict human adenovirus propagation. Therefore, this crosslinker-free strategy can be used for the repair, healing, and regeneration of the cornea, and potentially other damaged organs of the body

Phosphorylcholine and KR12-Containing Corneal Implants in HSV-1-Infected Rabbit Corneas

Severe HSV-1 infection can cause blindness due to tissue damage from severe inflammation. Due to the high risk of graft failure in HSV-1-infected individuals, cornea transplantation to restore vision is often contraindicated. We tested the capacity for cell-free biosynthetic implants made from recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) to suppress inflammation and promote tissue regeneration in the damaged corneas. To block viral reactivation, we incorporated silica dioxide nanoparticles releasing KR12, the small bioactive core fragment of LL37, an innate cationic host defense peptide produced by corneal cells. KR12 is more reactive and smaller than LL37, so more KR12 molecules can be incorporated into nanoparticles for delivery. Unlike LL37, which was cytotoxic, KR12 was cell-friendly and showed little cytotoxicity at doses that blocked HSV-1 activity in vitro, instead enabling rapid wound closure in cultures of human epithelial cells. Composite implants released KR12 for up to 3 weeks in vitro. The implant was also tested in vivo on HSV-1-infected rabbit corneas where it was grafted by anterior lamellar keratoplasty. Adding KR12 to RHCIII-MPC did not reduce HSV-1 viral loads or the inflammation resulting in neovascularization. Nevertheless, the composite implants reduced viral spread sufficiently to allow stable corneal epithelium, stroma, and nerve regeneration over a 6-month observation period

Collagen analogs with phosphorylcholine are inflammation-suppressing scaffolds for corneal regeneration from alkali burns in mini-pigs

The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas

The quest for anti-inflammatory and anti-infective biomaterials in clinical translation

Biomaterials are now being used or evaluated clinically as implants to supplement the severe shortage of available human donor organs. To date however, such implants have mainly been developed as scaffolds to promote the regeneration of failing organs due to old age or congenital malformations. In the real world, however, infection or immunological issues often compromise patients. For example, bacterial and viral infections can result in uncontrolled immunopathological damage and lead to organ failure. Hence, there is a need for biomaterials and implants that not only promote regeneration but also address issues that are specific to compromised patients such as infection and inflammation. Different strategies are needed to address the regeneration of organs that have been damaged by infection or inflammation for successful clinical translation. Therefore, the real quest is for multi-functional biomaterials with combined properties that can combat infections, modulate inflammation and promote regeneration at the same time. These strategies will necessitate the inclusion of methodologies for management of the cellular and signaling components elicited within the local microenvironment. In the development of such biomaterials, strategies range from the inclusion of materials that have intrinsic anti-inflammatory properties, such as the synthetic lipid polymer, 2-methacryloyloxyethyl phosphorylcholine (MPC), to silver nanoparticles that have anti-bacterial properties, to inclusion of nano- and micro-particles in biomaterials composites that deliver active drugs. In this present review, we present examples of both kinds of materials in each group along with their pros and cons. Thus, as a promising next generation strategy to aid or replace tissue/organ transplantation, an integrated smart programmable platform is needed for regenerative medicine applications to create and/or restore normal function at the cell and tissue levels. Therefore, now it is an utmost important to develop integrative biomaterials based on multi-functional biopolymers and nanosystem for their practical and successful clinical translation

Bioengineered Corneas Grafted as Alternatives to Human Donor Corneas in Three High-Risk Patients

Corneas with severe pathologies have a high risk of rejection when conventionally grafted with human donor tissues. In this early observational study, we grafted bioengineered corneal implants made from recombinant human collagen and synthetic phosphorylcholine polymer into three patients for whom donor cornea transplantation carried a high risk of transplant failure. These patients suffered from corneal ulcers and recurrent erosions preoperatively. The implants provided relief from pain and discomfort, restored corneal integrity by promoting endogenous regeneration of corneal tissues, and improved vision in two of three patients. Such implants could in the future be alternatives to donor corneas for high-risk patients, and therefore, merits further testing in a clinical trial.Funding Agencies|Swedish Research Council (EU Nanomedicine project "I-CARE") [521-2012-5706]; Integrative Regenerative Medicine Centre, Linkoping, Sweden; Swedish Institute fellowship</p

Phytochemical Investigations of Campsis Radicans L.

Petroleum ether, dichloromethane and ethyl acetate soluble fractions were obtained through partitioning the crude methanolic extract of the leaves of Campsis radicans L. (Family. Bignoniaceae) followed by the chromatographic separation of secondary metabolites from them. A total of five triterpene compounds i.e., corosolic acid methyl ester (1), β-amyrin (2), arjunolic acid (3), maslinic acid (4) and 28-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-2α,3α,19α-trihydroxy-12-en-28-ursolic acid (5) were isolated from the dichloromethane fractions and their structures were characterized by 1H NMR spectroscopy and compared the NMR data with published values