Effect of sustained insulin-releasing device made of poly(ethylene glycol) dimethacrylates on retinal function in streptozotocin-induced diabetic rats.

In this study, we developed a subcutaneous insulin-releasing device consisting of a disk-shaped capsule and drug formulation comprised of poly(ethylene glycol) dimethacrylates, then evaluated its efficacy on retinal function in streptozotocin (STZ)-induced diabetic rats. In vitro release studies showed that recombinant human insulin was released with a constant rate for more than 30 days. The device was able to maintain a basal level of blood glucose in diabetic rats for a prolonged period of more than 30 days, simultaneously preventing a decrease in body weight. For assessing the pharmacological effect of the device on retinal function in diabetic rats, electroretinograms were conducted for 12 weeks. The reduction in amplitude and delay in implicit time were attenuated by the device during the initial 4 weeks of application. The increase in gene expression of protein kinase C (PKC)-γ and caspase-3 in the diabetic retina was also attenuated by the device. Immunohistochemistry showed that the increase in glial fibrillary acidic protein expression in the diabetic retina was attenuated by the device. Histological evaluation of subcutaneous tissue around the device showed the biocompatibility of the device. In conclusion, the insulin-releasing device attenuated the reduction of retinal function in STZ-induced diabetic conditions for 4 weeks and the efficacy of the device might be partially related to PKC signaling in the retina. The long-term ability to control the blood glucose level might help to reduce the daily frequency of insulin injections.

Physicochemical and biological characterization of sustained isopropyl unoprostone-release device made of poly(ethyleneglycol) dimethacrylates.

Transscleral drug delivery is becoming increasingly popular to manage posterior eye diseases. To evaluate the clinical application of a transscleral, sustained, unoprostone (UNO)-release device (URD) constructed of photopolymerized tri(ethyleneglycol) dimethacrylate and poly(ethyleneglycol) dimethacrylate, we evaluated physicochemical and biological properties of this device. The URD consists of a drug-impermeable reservoir and a semi-permeable cover. The in vitro release rate of UNO from the URD increased with increasing temperatures from 20 to 45 °C. Scanning electron microscopy and atomic-force microscopy showed that the border between the reservoir and drug formulation was sharply defined but that between the cover and drug was poorly determined, indicating that UNO could permeate only through the cover. For stability tests, the URDs were sterilized with ethylene oxide gas and stored at 40 °C/75% for 3 and 6 months and at 25 °C/60% for 3, 6, 9, 12, 18, and 24 months; UNO content and release rate at 37 °C were then evaluated. There was no significant decrease in either UNO content or release rate after the storage conditions. Cytotoxicity was evaluated by examining the colony formation of Chinese hamster fibroblast V79 cells in a media extract of the URD without UNO. This extract did not affect colony formation of V79 cells, indicating the cytocompatibility of the URD. In conclusion, the URD was physically stable for 24 months and is potentially useful for clinical application.

Transscleral Controlled Delivery of Geranylgeranylaceton Using a Polymeric Device Protects Rat Retina Against Light Injury.

We evaluated the effects of a transscleral drug delivery device, consisting of a reservoir and controlled-release cover, which were made of photopolymerized polyethylene glycol dimethacrylate and triethylene glycol dimethacrylate, combined at different ratios. Geranylgeranylacetone (GGA), a heat-shock protein (HSP) inducer, was loaded into the device. The GGA was released from the device under zero-order kinetics. At both 1 week and 4 weeks after device implantation on rat sclera, HSP70 gene and protein expression were up-regulated in the sclera-choroid-retinal pigment epithelium fraction of rat eyes treated with the GGA-loaded device compared with rat eyes treated with saline-loaded devices or eyes of non-treated rats. Flash electroretinograms were recorded 4 days after white light exposure (8000 lx for 18 h). Electroretinographic amplitudes of the a- and b-waves were preserved significantly in rats treated with GGA-loaded devices compared with rats treated with saline-loaded devices. Histological examination showed that the outer nuclear layer thickness was preserved in rats that had the GGA-loaded device. These results may show that transscleral GGA delivery using our device may offer an alternative method to treat retinal diseases.

In vivo effects of isolated implantation of salmon-derived crosslinked atelocollagen sponge into an osteochondral defect.

We have developed crosslinked salmon-derived atelocollagen (SC) sponge, which has a denaturation temperature of 47°C. Sixty-four knees of 32 mature rabbits were randomly divided into 4 groups after creating an osteochondral defect in the femoral trochlea. Defects in Groups I, II, and III were filled with the crosslinked SC sponge, the crosslinked porcine collagen (PC) sponge, and the non-crosslinked PC sponge, respectively. In Group IV, defects were left untreated as the control. At 12 weeks after implantation, the histological score showed that Group I was significantly greater than Groups III (P = 0.0196) and IV (P = 0.0021). In addition, gene expression of type-2 collagen, aggrecan, and SOX9 was the greatest in Group I at 12 weeks. The fundamental in vivo properties of the crosslinked SC sponge showed that this is a promising biomaterial, specifically as a scaffold for cartilage tissue engineering.

Preparation and characterization of collagen microspheres for sustained release of VEGF.

In this study, we prepared injectable collagen microspheres for the sustained delivery of recombinant human vascular endothelial growth factor (rhVEGF) for tissue engineering. Collagen solution was formed into microspheres under a water-in-oil emulsion condition, followed by crosslinking with water-soluble carbodiimide. Various sizes of collagen microspheres in the range of 1-30 mum diameters could be obtained by controlling the surfactant concentration and rotating speed of the emulsified mixture. Particle size proportionally decreased with increasing the rotating speed (1.8 mum per 100 rpm increase in the range of 300-1,200 rpm) and surfactant concentration (3.1 mum per 0.1% increase in the range of 0.1-0.5%). The collagen microspheres showed a slight positive charge of 8.86 and 3.15 mV in phosphate-buffered saline and culture medium, respectively. Release study showed the sustained release of rhVEGF for 4 weeks. Released rhVEGF was able to induce capillary formation of human umbilical vein endothelial cells, indicating the maintenance of rhVEGF bioactivity after release. In conclusion, the results suggest that the collagen microspheres have potential for sustained release of rhVEGF.

A drug refillable device for transscleral sustained drug delivery to the retina.

Continuous drug administration with better adherence to treatment and less invasive procedures is important in treating retinal diseases such as age-related macular disease. In this study, we report a drug-refillable device consisting of a silicone reservoir and an injectable gelatin/chitosan gel (iGel). The silicone reservoir was fabricated with polydimethylsiloxane (PDMS) using a computer-aided design and manufacturing to have micropores at a releasing side for uniaxial release to the sclera. A stainless steel wire and sheet were combined in the side and bottom of the reservoir to ensure flexibility and to fit on the curvature of the eyeball and prevent irritation to the sclera through the bottom of the reservoir. The drug was injected and formulated in the reservoir by in situ crosslinking of gelatin/chitosan gel with the crosslinker; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. The in vitro release study using fluorescein molecules showed that the release rate from encapsulated iGel in the reservoir was slower than that from the original iGel. After reinjecting the iGel into the reservoir, the same release profile as the first injection was observed. The reservoir containing iGel was placed on the sclera of a rabbit and the distribution of 150 kDa fluorescein isothiocyanate-dextran (FD150) in the retina and choroid/retinal pigment epithelium (choroid/RPE) was studied. The cryosections showed that FD150 was observed in the choroid/RPE. Homogenates of the retina and choroid/RPE showed fluorescence during 12 weeks implantation, indicating the drug could be delivered to the retina by using the device. The drug filling was successful into the reservoir implanted on the sclera through the conjunctiva by using a needle. In conclusion, the refillable drug delivery device is a promising tool to administer drugs long-term by reinjection with less invasiveness to intraocular tissues.

Transscleral sustained ranibizumab delivery using an episcleral implantable device: Suppression of laser-induced choroidal neovascularization in rats.

Successful treatment of age-related macular diseases requires an effective controlled drug release system with less invasive route of administration in the eye to reduce the burden of frequent intravitreal injections for patients. In this study, we developed an episcleral implantable device for sustained release of ranibizumab, and evaluated its efficacy on suppression of laser-induced choroidal neovascularization (CNV) in rats. We tested both biodegradable and non-biodegradable sheet-type devices consisting of crosslinked gelatin/chitosan (Gel/CS) and photopolymerized poly(ethyleneglycol) dimethacrylate that incorporated collagen microparticles (PEGDM/COL). In vitro release studies of FITC-labeled albumin showed a constant release from PEGDM/COL sheets compared to Gel/CS sheets. The Gel/CS sheets gradually biodegraded in the sclera during the 24-week implantation; however, the PEGDM/COL sheets did not degrade. FITC-albumin was detected in the retina during 18 weeks implantation in the PEGDM/COL sheet-treated group, and was detected in the Gel/CS sheet-treated group during 6 weeks implantation. CNV was suppressed 18 weeks after application of ranibizumab-loaded PEGDM/COL sheets compared to a placebo PEGDM/COL sheet-treated group, and to the intravitreal ranibizumab-injected group. In conclusion, the PEGDM/COL sheet device suppressed CNV via a transscleral administration route for 18 weeks, indicating that prolonged sustained ranibizumab release could reduce the burden of repeated intravitreal injections.

Biological properties of crosslinked salmon collagen fibrillar gel as a scaffold for human umbilical vein endothelial cells.

Collagen derived from chum salmon (Oncorhynchus keta) was crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) during collagen fibrillogenesis and applied to an in vitro cell culture to evaluate its potential use as a scaffold for vascular tissue engineering. Human umbilical vein endothelial cells (HUVEC) were cultured on the crosslinked salmon collagen fibrillar gel (EDC-SC gel), and their growth rates and production levels of cytokines, including platelet-derived growth factor-BB and von Willebrand factor, were measured. Comparison was also made with bovine collagen gel crosslinked with EDC (EDC-BC gel). The growth and cytokine production of the HUVEC cultured on the EDC-SC gel were higher than those on the EDC-BC gel. In addition, HUVEC were found to attach to the EDC-BC gel through alpha2beta1 integrin for native collagen, whereas they attached to the EDC-SC gel through alphavbeta3 integrin for denatured collagen as well as the alpha2beta1 integrin, indicating that HUVEC recognized denatured domains in the EDC-SC gel. In conclusion, the EDC-SC gel can be used as a scaffold to support HUVEC growth, although the integrin-mediated attachment manner differs between the two gels.

A self-deploying drug release device using polymeric films.

Herein, we report a sheet-type device capable of self-deployment and sustained release of protein type drugs. The device consisted of a thin photopolymerized polyethylene glycol dimethacrylate (PEGDM) sheet and collagen microparticles (COLs), which were embedded in the sheet as drug carriers and for increased drug permeation. When the density of the COLs in the sheet was increased to be sufficiently interconnected, the drug permeability was increased. In addition, since protein type drugs electrostatically interacted with the COLs, a prolonged sustained release was possible. The PEGDM/COLs device was flexible enough to be rolled up, and the device maintained its structure due to van der Waals attractive forces between the sheet surfaces. When the device was immersed in water, the attractive forces acting between the sheet surfaces were relieved by water. Subsequently, the device unfolded by bending-stress relaxation. Moreover, the rolled-up device could be injected through a conventional syringe needle into water to recover its original shape. The developed sheet-type device provides the possibility of minimally invasive transplantation into diseased tissues and organs, and could provide better therapeutic outcomes and reduce possible side effects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 780-786, 2018.

Pharmacokinetic and Safety Evaluation of a Transscleral Sustained Unoprostone Release Device in Monkey Eyes.

Purpose: We evaluate the ocular tissue distribution and retinal toxicity of unoprostone (UNO) during 12 months, after transscleral sustained-UNO administration using a drug delivery device in monkey eyes. Methods: The device consisted of a reservoir, controlled-release cover, and a drug formulation of photopolymerized polyethylene glycol dimethacrylate. Six mg UNO was loaded into the device (length, 17 mm; width, 4.4 mm; height, 1 mm). The concentrations of M1, a primary metabolite of UNO, in the retina, choroid, vitreous, lens, aqueous humor, iris, ciliary body, and plasma were determined by liquid chromatography-tandem mass spectrometry at 3, 6, and 12 months after implantation. Retinal toxicity was evaluated by electroretinography (ERG), optical coherence tomography (OCT), and IOP at preimplantation, and at 6, 9, and 12 months after implantation. Focal ERGs were performed at 9 and 12 months after implantation. Results: M1 was detected in the choroid and retina with maximum peaks of 243.2 and 8.41 ng/g at 6 months, respectively. M1 in the ciliary body and iris was detected with maximum peaks of 7.66 and 10.4 ng/g at 6 and 12 months, respectively. Less than 1 ng/mL or ng/g of M1 was detected in the aqueous humor, vitreous, and lens. No changes were observed in retinal function as assessed by ERG, IOP, or macula thickness and retinal histology by OCT examinations during the 12-month period. No differences in focal ERG amplitudes, especially in the macula, were observed. Conclusions: The device provided intraocular sustained delivery of UNO for 12 months without producing severe retinal toxicity.

In vitro growth and differentiated activities of human periodontal ligament fibroblasts cultured on salmon collagen gel.

Marine-derived collagen is expected to be a much safer alternative to calf collagen, which in medical applications carries the risk of bovine spongiform encephalopathy. In this study, acid-soluble collagen was extracted from salmon skin and crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide during fibril formation to produce a crosslinked salmon collagen (SC) gel. The growth rates and the differentiated functions of human periodontal ligament fibroblasts (HPdLFs) cultured on the SC gel were investigated. Growth was faster on the SC gel than on porcine collagen (PC) gel. In addition, the HPdLFs cultured on the SC gel exhibited higher alkaline phosphatase (ALP) activity than those cultured on the PC gel. Quantitative RT-PCR revealed higher mRNA expression of type I collagen, ALP, and osteocalcin in the HPdLFs cultured on the SC gel. HPdLFs had a flat shape on the SC gel and a spindle shape on the PC gel, as revealed by observation with scanning electron microscopy and immunostaining with cytoskeletal protein and vinculin. The results showed that HPdLFs could grow and show highly differentiated activity on the SC gel as well as on the PC gel.

Preparation and characterization of multilayered hydroxyapatite/silk fibroin film.

We prepared multilayered films consisting of silk fibroin (SF) and hydroxyapatite (HAp) by alternating lamination using untreated SF and HAp-deposited SF films. Untreated SF films were prepared from a regenerated SF solution by air drying. HAp-deposited SF films were prepared by soaking methanol-treated SF films containing >5 wt% CaCl2 in a simulated body fluid with the ion concentration 1.5-fold higher than that of the standard one. The multilayered HAp/SF films had HAp layers with approximate thicknesses of 3-5 microm and SF layers with thicknesses of 40-70 microm. The bonding strength between the SF and HAp layers was significantly affected by temperature and compression time under the lamination method. The optimal conditions for achieving the maximum T-peel strength and beta-sheet contents were determined to be 130 degrees C for 4 min. The Young's modulus of the multilayered films (133.4 MPa) was higher than that of the films consisting of SF alone (92.5 MPa) under swollen conditions. The biocompatibility of the HAp-deposited SF films was analyzed by culturing of osteoblasts (MC3T3-E1) on a film. The results indicate that HAp-deposited SF films and SF films show similar degrees of cell adhesion and alkaline phosphatase activities.

Electrochemical manipulation of cell populations supported by biodegradable polymeric nanosheets for cell transplantation therapy.

We describe an electrochemical method of harvesting cells cultured on a biodegradable polymeric nanosheet (cell/nanosheet construct), which is stabilized on a self-assembled monolayer (SAM) of thiol molecules. A poly(lactic-co-glycolic acid) (PLGA) nanosheet was attached by hydrophobic interactions onto the surface of a SAM of l-cysteine coated onto a gold electrode. Retinal pigment epithelial cell lines (RPE-J cells) were cultured on the nanosheet to form a monolayer. An AA-size dry battery was used to apply a negative electrical potential, causing reductive desorption of the SAM from the gold surface. Within one minute of application of the voltage, the cell/nanosheet of several mm in diameter was successfully detached without the loss of cell viability in a gentle stream of the electrolyte solution. The use of a porous electrode shortened the detachment time due to the more efficient permeation of the electrolyte solution to the electrode surface. Cell transplantation following the harvesting process was demonstrated by the local delivery of RPE-J cell/nanosheet constructs into the subretinal space of rat eyes through a capillary needle. This nanosheet-based approach that allows the on-demand harvesting of cell/nanosheet constructs and their subsequent transplantation in a minimally-invasive manner could play an important role in cell transplantation therapy.

Controlled basic fibroblast growth factor release device made of poly(ethyleneglycol) dimethacrylates for creating a subcutaneous neovascular bed for cell transplantation.

Subcutaneous space is a potential site for the transplantation of cells such as islets for treatment of type 1 diabetes. To enhance engraftment, an optimal space for the growth of the transplanted cells is needed along with neovascularization. In this study, we developed a device using a photocurable resin, poly(ethyleneglycol) dimethacrylates (PEGDM), for controlled release of basic fibroblast growth factor (bFGF) to create a subcutaneous neovascular bed in rats. The device consists of a disk-shaped capsule with micropores and is composed of tri(ethyleneglycol) dimethacrylate (TEGDM) and a drug formulation of PEGDM. The release rate was tuned by changing the number of pores and the composition of water and PEGDM in the drug formulation. bFGF released from devices incubated in phosphate-buffered saline (PBS) enhanced the growth of fibroblasts, indicating bioactivity of bFGF after release. Histological evaluation showed a significant increase in the extent of vasculature that was dependent on the amount of bFGF loaded into the device. A perfusion study using fluorescein isothiocyanate dextran 2000 kDa showed linear and capillary staining patterns, indicating potent functional vasculature. In conclusion, the controlled bFGF releasing device could provide a neovascular bed with the required vascularization in the subcutaneous space. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3017-3024, 2017.

A polymeric device for controlled transscleral multi-drug delivery to the posterior segment of the eye.

The design of drug delivery systems that can deliver multiple drugs to the posterior segment of the eye is a challenging task in retinal disease treatments. We report a polymeric device for multi-drug transscleral delivery at independently controlled release rates. The device comprises a microfabricated reservoir, controlled-release cover and three different fluorescent formulations, which were made of photopolymeized tri(ethyleneglycol)dimethacrylate (TEGDM) and poly(ethyleneglycol)dimethacrylate (PEGDM). The release rate of each fluorescent is controlled by varying the PEGDM/TEGDM ratio in its formulation and the cover. The release kinetics appeared to be related to the swelling ratio of the PEGDM/TEGDM polymers. When the devices were implanted onto rat sclerae, fluorescence was observable in the ocular tissues during 4 weeks' implantation and distributed locally around the implantation site. Our polymeric system, which can administer multiple compounds with distinct kinetics, provides prolonged action and less invasive transscleral administration, and is expected to provide new tools for the treatment of posterior eye diseases with new therapeutic modalities.

Micropatterned polymeric nanosheets for local delivery of an engineered epithelial monolayer.

Like a carpet for cells, micropatterned polymeric nanosheets are developed toward local cell delivery. The nanosheets direct morphogenesis of retinal pigment epithelial (RPE) cells and allow for the injection of an engineered RPE monolayer through syringe needles without the loss of cell viability. Such an ultrathin carrier has the promise of a minimally invasive delivery of cells into narrow tissue spaces.

The morphology and functions of articular chondrocytes on a honeycomb-patterned surface.

The present study investigated the potential of a novel micropatterned substrate for neocartilage formation. Articular chondrocytes were cultured on poly( ɛ-caprolactone) materials whose surfaces were either flat or honeycomb-patterned. The latter was prepared using a novel self-organization technique, while the former, was prepared by spin-coating. The chondrocytes attached and proliferated on both surfaces. On the honeycomb films, chondrocytes were found at the top surface and encased within the 10 µm pores. Meanwhile, chondrocytes on the spin-coated surface flattened out. Accumulation of DNA and keratin sulphate was comparatively higher on the honeycomb films within the first 7 days. At their respective peaks, DNA concentration increased on the honeycomb and flat surfaces by approximately 210% and 400% of their day 1 values, respectively. However, cultures on the flat surface took longer to peak. Extracellular Matrix (ECM) concentrations peaked at 900% and 320% increases for the honeycomb and flat cultures. Type II collagen was upregulated on the honeycomb and flat surfaces by as much as 28% and 25% of their day 1 values, while aggrecan was downregulated with time, by 3.4% and 7.4%. These initial results demonstrate the potential usefulness of honeycomb-based scaffolds during early cultures neocartilage and soft tissue engineering.

A platform for controlled dual-drug delivery to the retina: protective effects against light-induced retinal damage in rats.

Controlled transscleral co-delivery of two drugs, edaravone (EDV) and unoprostone (UNO), using a platform that comprises a microfabricated reservoir, controlled-release cover, and drug formulations, which are made of photopolymerized poly(ethyleneglycol) dimethacrylates, shows synergistic retinal neuroprotection against light injury in rats when compared with single-drug-loaded devices. The device would offer a safer therapeutic method than intravitreal injections for retinal disease treatments.

Transscleral sustained vasohibin-1 delivery by a novel device suppressed experimentally-induced choroidal neovascularization.

We established a sustained vasohibin-1 (a 42-kDa protein), delivery device by a novel method using photopolymerization of a mixture of polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and collagen microparticles. We evaluated its effects in a model of rat laser-induced choroidal neovascularization (CNV) using a transscleral approach. We used variable concentrations of vasohibin-1 in the devices, and used an enzyme-linked immunosorbent assay and Western blotting to measure the released vasohibin-1 (0.31 nM/day when using the 10 µM vasohibin-1 delivery device [10VDD]). The released vasohibin-1 showed suppression activity comparable to native effects when evaluated using endothelial tube formation. We also used pelletized vasohibin-1 and fluorescein isothiocyanate-labeled 40 kDa dextran as controls. Strong fluorescein staining was observed on the sclera when the device was used for drug delivery, whereas pellet use produced strong staining in the conjunctiva and surrounding tissue, but not on the sclera. Vasohibin-1 was found in the sclera, choroid, retinal pigment epithelium (RPE), and neural retina after device implantation. Stronger immunoreactivity at the RPE and ganglion cell layers was observed than in other retinal regions. Significantly lower fluorescein angiography (FA) scores and smaller CNV areas in the flat mounts of RPE-choroid-sclera were observed for the 10VDD, VDD (1 µM vasohibin-1 delivery device), and vasohibin-1 intravitreal direct injection (0.24 µM) groups when compared to the pellet, non-vasohibin-1 delivery device, and intravitreal vehicle injection groups. Choroidal neovascularization can be treated with transscleral sustained protein delivery using our novel device. We offer a safer sustained protein release for treatment of retinal disease using the transscleral approach.

Preparation and characterization of collagen from soft-shelled turtle (Pelodiscus sinensis) skin for biomaterial applications.

Collagen was isolated from the skin of soft-shelled turtle (Pelodiscus sinensis) by acid solubilization with pepsin. The yield of soft-shelled turtle collagen (STC) was 12.1% on a dry weight basis. The electrophoresis assay showed that STC consisted of a alpha(1)alpha(2) heterodimer similar to porcine collagen (PC). Amino-acid composition analysis showed that the hydroxyproline content of STC was 7.8%, which was lower than that of PC (9.5%). The denaturation temperature of STC was 36 degrees C from optical rotation analysis. An accelerated fibrillogenesis of STC was observed in phosphate-buffered saline at 25 degrees C. The resulting STC fibrillar gel had microfibrillar network with fibril diameter of ca. 124 nm, as revealed by observation with scanning electron microscopy. The compressive moduli of the STC gel and the PC gel were 3.2 +/- 0.8 kPa and 3.6 +/- 0.3 kPa, respectively. The potential of the STC gel for biomaterial applications was investigated by in vitro cell culture. Human dermal fibroblasts were three-dimensionally cultured in the STC gel and their growth was evaluated by DNA content measurement. Steady growth was observed in the STC gel for a 6-day culture period, although the growth rate was slower than in the PC gel. In conclusion, STC could be used as a novel collagen source for biomaterial applications.