Surface functionalization of hydroxyapatite nanoparticles for biomedical applications.

This review completely covers the various aspects of hydroxyapatite (HAp) nanoparticles and their role in different biological situations, and provides the surface and interface contents on (i) hydroxyapatite nanoparticles and their hybridization with organic molecules, (ii) surface designing of hydroxyapatite nanoparticles to provide their biocompatibility and photofunction, and (iii) coating technology of hydroxyapatite nanoparticles. In particular, we summarized how the HAp nanoparticles interact with the different ions and molecules and highlighted the potential for hybridization between HAp nanoparticles and organic molecules, which is driven by the interactions of the HAp nanoparticle surface ions with several functional groups of biological molecules. In addition, we highlighted the studies focusing on the interfacial interactions between the HAp nanoparticles and proteins for exploring the enhanced biocompatibility. Such studies focus on how these interactions affect the hydration layers and protein adsorption. However, the hydration layer state involves diverse molecular interactions that can alter the shape of the adsorbed proteins, thereby affecting cell adhesion and spreading on the surfaces. We also summarized the relationship between the surface properties of the HAp nanoparticles and the hydration layer. Furthermore, we spotlighted the cytocompatible photoluminescent probes that can be developed by designing HAp/organic nanohybrid structures. We then emphasized the importance of photofunctionalization in theranostics, which involves the integration of diagnostics and therapy based on the surface design of the HAp nanoparticles. Furthermore, the coating techniques using HAp nanoparticles and HAp nanoparticle/polymer composites were outlined for fusing base biomaterials with biological tissues. The advantages of HAp/biocompatible polymer composite coatings include the ability to effectively cover porous or irregularly shaped surfaces while controlling the thickness of the coating layer, and the addition of HAp nanoparticles to the polymer matrix improves the mechanical properties, increases the roughness, and forms the morphologies that mimic bone nanostructures. Therefore, the fundamental design of hydroxyapatite nanoparticles and their surfaces was suggested from various aspects for biomedical applications.

Functional evaluation of mineral trioxide aggregate cement with choline dihydrogen phosphate.

To improve the cytocompatibility of mineral trioxide aggregate (MTA) cement and its ability for reparative dentin formation, the effect of adding choline dihydrogen phosphate (CDHP), which is reported to be biocompatible, to MTA cement was investigated. The L929 cell proliferation showed that the addition of CDHP improved cell viability. The addition of CDHP shortened the setting time of MTA cement, with a significant decrease in consistency above 0.4 g/mL. Diametral tensile strength of the set cement was improved by the addition of 0.4 g/mL CDHP. Solubility was judged to be within the range of clinical application. The spontaneous precipitation of low crystalline hydroxyapatite was examined by immersing the set cement in phosphate buffer saline, and it was found that the ability of the cement with 0.4 g/mL of CDHP was significantly improved compared with that of the cement without CDHP.

Investigation into the Photochemical Properties of Methylene Blue-Immobilized Hydroxyapatite Nanoparticles for Theranostic Application.

In the biomedical field, there has been a requirement for developing theranostic nanomaterials with higher biosafety, leading to both diagnosis and therapy. Methylene blue (MB+) is an organic dye with both photoluminescence (PL) and photosensitization abilities to generate singlet oxygen (1O2). However, MB+ easily loses its generation ability by hydrogen reduction in vivo or by forming aggregates. In this study, MB+ immobilized on biocompatible hydroxyapatite (HA) nanoparticles was applied for the bifunctions of efficient PL and photosensitization. The MB+-immobilized HA nanoparticles (MH) formed aggregates with sizes of 80-100 nm in phosphate buffer (PB). The generation amount and efficiency of 1O2 from the nanoparticles in PB seem to depend on the immobilized MB+ amount and the percentage of the monomer, respectively. Considering the larger immobilized amount and percentage of the MB+ monomer, it was found that there was MH with the lower generation amount and efficiency of 1O2 to exhibit the highest PL intensity. The photofunctional measurement of MB+ revealed the state of MB+ molecules on the HA surface, and it was suggested that the MB+ molecules immobilized on the MH surface would form more hydrogen bonds to change their excitation states. In the cellular experiments, the Hela cancer cells reacted with the nanoparticles and showed red-color PL, indicating cellular imaging. Furthermore, the adherent cell coverage decreased by 1O2 generation, indicating the importance of the immobilization amount of the MB+ monomer. Therefore, theranostic nanomaterials with biosafety were successfully synthesized to show two photofunctions, which provide both cellular imaging and photodynamic therapy by the nanohybrid system between HA and MB+.

Effective Immobilization of Monomeric Methylene Blue on Hydroxyapatite Nanoparticles by Controlling Inorganic-Organic Interfacial Interactions.

We successfully synthesized methylene blue (MB+)-immobilized hydroxyapatite (HM) nanoparticles by changing the initial P/Ca molar ratio. The immobilized amount of MB+ increased with increasing the initial P/Ca molar ratio from 0.6 to 4.0, and the HM had an elliptical shape (long length, 21-24 nm; short length, 11-13 nm) irrespective of the initial P/Ca molar ratio. Upon increasing the initial P/Ca molar ratio, the number of carbonate ions on the HM surface decreased, which would be owing to the electrostatic repulsion by the surface phosphate ions (i.e., P-O-), the surface P-OH mainly dissociated to form P-O-, and the electrostatic interaction of P-O- with MB+ enhanced. The bonding of MB+ with surface P-OH and Ca2+ sites of hydroxyapatite would be hydrogen-bonding and Lewis acid-base interactions, respectively. The optimum synthesis condition for MB+ immobilization at the monomer state was found to be the initial P/Ca molar ratio of 2.0. Here, the existence percentage of the MB+ monomer and the molecular occupancy of the surface carbonate ion at the initial P/Ca molar ratio of 2.0 were higher than those at 4.0 with no significant difference in the immobilized amount of MB+, indicating that MB+ at the initial P/Ca molar ratio of 4.0 is more aggregated than that at 2.0. These results suggested that a part of carbonate ions has a role as a spacer to suppress MB+ aggregation. Accordingly, the interfacial interactions between the MB+ monomer and the hydroxyapatite surface were clarified, which can effectively be controlled by the initial P/Ca molar ratio. These findings will provide fundamental and useful knowledge for the design of calcium phosphate-organic nanohybrids. We believe that these particles will be the base materials to realize diagnostic and/or therapeutic functions through the molecular state control by optimizing the synthesis conditions.

Nanostructural control of transparent hydroxyapatite nanoparticle films using a citric acid coordination technique.

Hydroxyapatite (HA), as the main mineral component in hard tissues, has good biocompatibility. In particular, HA films are widely used as bioactive coatings for artificial bones and dental implants in biomedical fields. However, it is currently difficult to prepare a nanostructure-controlled HA film by a wet process for further applications. Herein, we report the synthesis of HA nanoparticles coordinated by citric acid (Cit/HA) based on the interactions between carboxylate and calcium ions to control the sizes and shapes of the hybrid nanoparticles, to improve their dispersibility in water and to eventually form uniform transparent films with nanospaces, and investigated the film formation mechanism. As compared with the well-known rod-like HA nanoparticles (size: 48 × 15 nm2), we successfully synthesized spherical and negatively charged Cit/HA nanoparticles (size: 25 × 23 nm2) to achieve highly transparent Cit/HA films using the spin-coating technique. The Cit/HA films had uniform and crack-free appearance. About the nanostructures, we found that the Cit/HA film surfaces had meso-scaled nanospaces with a diameter of 4.2 nm based on the regular arrangement of spherical nanoparticles, instead of the HA film with a nanospace diameter of 24.5 nm formed by non-uniform accumulation. Therefore, we successfully achieved the control of the nanospace sizes of the films with the nanoparticle arrangement and realized transparent nanoparticle film formation in a very simple way, which will provide more convenient bioceramic films for biomedical applications.

Primary rhegmatogenous retinal detachment: evaluation of a minimally restricted face-down positioning after pars plana vitrectomy and gas tamponade.

AIM: To evaluate the safety and efficacy of a minimally restricted face-down postoperative positioning following pars plana vitrectomy (PPV) with gas tamponade for primary rhegmatogenous retinal detachment (RRD). METHODS: Patients with primary RRD treated with PPV and gas tamponade and followed up for at least 6mo were selected for the study. All phakic eyes underwent simultaneous cataract surgery. The patients were required to be in a postoperative position that prevented downward flow of retinal tears. Patients with macular detachment were positioned face-down for only a couple of hours. The patients were assessed for preoperative and postoperative best-corrected visual acuity (BCVA), anatomical retinal reattachment rate, and postoperative complications. RESULTS: In total, 40 eyes of 39 patients with primary RRD were included in the study. A single tear was present in 30 eyes (75.0%), multiple retinal tears were present in nine eyes (22.5%), and oral dialysis was present in one eye (2.5%). The anatomical success rate was 90.0% (36 cases) after the primary surgery, and the final anatomical success rate was 100%. The BCVA improved significantly (P<0.001) from 0.75 logarithm angle of resolution (logMAR) preoperatively to 0.12 logMAR at the final visit. Postoperative complications included intraocular pressure elevation (≥25 mm Hg) in 11 patients (27.5%), fibrin formation in two patients (5.0%), pupillary capture of the intraocular lens in two patients (5.0%), and posterior synechia in one patient (2.5%). CONCLUSION: A minimally restricted face-down and flexible postoperative positioning after PPV and gas tamponade for primary RRD is effective and safe.

Preparation of Monodispersed Nanoporous Eu(III)/Titania Loaded with Ibuprofen: Optimum Loading, Luminescence, and Sustained Release.

Functional nanomaterials are one of the potential carriers for drug delivery, whereas there are many prerequisites for this purpose. The carrier should be monodispersed, be fluorescent, and have a proper nanostructure to keep/release drug molecules to achieve controlled release, although preparing a nanomaterial which fulfills all the demands is still very challenging. In this paper, we show the preparation of monodispersed nanoporous amorphous titania submicron particles with fluorescent property. They adsorb a model drug molecule-ibuprofen-with their surface coverage up to 100%. Such a perfect loading does not decrease the fluorescent intensity because of any quenching effects but even maximize it. We also demonstrate the release behavior of IBU into simulated body fluid. Interestingly, the present carrier releases most of IBU in 6 h, whereas that modified with the polyethylene glycol moiety takes 48 h to finish releasing IBU, indicating its potential for controlled release applications.

Visual Acuity Recovery After Macular Hole Closure Associated With Foveal Avascular Zone Change.

Purpose: To evaluate changes in the foveal avascular zone (FAZ) area during the postoperative period of macular hole (MH) surgery using the optical coherence tomography angiography (OCTA) and to investigate its relationship to visual acuity (VA). Methods: Consecutive unilateral MH patients who underwent successful MH closure with at least a six-month observation period were studied retrospectively. To evaluate the FAZ area, OCTA images were obtained at the preoperative visit, the first postoperative visit, and the six-month visit. Main outcome measures were postoperative FAZ change and its relationship to VA change after MH closure. Results: Fifty-one cases were studied. The FAZ area was 0.42 ± 0.11 mm2 at the preoperative visit, 0.25 ± 0.091 mm2 at the first postoperative visit and 0.31 ± 0.11 mm2 at the six-month visit. FAZ area at the first postoperative visit was significantly smaller (P < 0.0001) than at the preoperative visit. FAZ area at the six-month visit was significantly greater (P < 0.0001) than at the first postoperative visit, but still significantly smaller (P = 0.0002) compared to the normal fellow eye. The postoperative FAZ area enlargement from the first postoperative visit to the six-month visit was significantly correlated with the postoperative VA recovery (P = 0.0322) and the postoperative photoreceptor reconstruction (P = 0.0213). Conclusions: The FAZ area once decreases along with MH closure; it thereafter increases toward the normal value over time. The postoperative FAZ change was correlated with the VA recovery. Translational Relevance: This study suggests that the postoperative FAZ area enlargement might be a potential biomarker indicating foveal reconstruction after MH closure.

Assessment of noninvasive positive pressure ventilation in healthy young volunteers using salivary stress biomarkers.

AIM: We performed a stress assessment of noninvasive positive pressure ventilation (NPPV) using the salivary biomarkers chromogranin A. MATERIALS & METHODS: Twenty healthy volunteers were subjected to NPPV for 30 min. We collected saliva samples before and after NPPV and evaluated chromogranin A. RESULTS: We collected saliva samples from 13 volunteers for enzyme measurement. Of the 13 volunteers, 11 showed elevated chromogranin A levels, which were significantly higher after NPPV than before NPPV (p < 0.01). The chromogranin A increase group displayed significantly increased leak flow and reduced respiratory rate and absolute humidity compared with the chromogranin A reduction group. CONCLUSION: The increase of leak volume might be a stress factor in patients receiving NPPV.

Surface-Engineered Design of Efficient Luminescent Europium(III) Complex-Based Hydroxyapatite Nanocrystals for Rapid HeLa Cancer Cell Imaging.

We synthesized hydroxyapatite nanocrystals under the existence of tris(2,2,6,6-tetramethyl-3,5-heptanedionato)europium(III) (EuTH) complex to form inorganic/organic hybrid nanocrystal (EHA). Then, the folic acid derivative (folate N-hydroxysuccinimidyl ester (FA-NHS)) as the targeting ligand for the HeLa cancer cells was immobilized on the EHA by the mediation of both 3-aminopropyltriethoxysilane and methyltriethoxysilane molecules. Here, we investigated the photofunctions based on the interfacial interactions between the FA-NHS and EHA nanohybrids for preparing the novel bioimaging nanomaterials. As a result, the photofunctions could be changed by the FA-NHS molecular occupancy on the EHA. When the molecular occupancy ratio to the EHA surfaces is at around 3-5%, the intense luminescence from the f-f transition of the Eu3+ ions as well as the charge transfer between the EuTH-FA-NHS was observed to exhibit higher quantum efficiency. Moreover, effective dispersibility in phosphate-buffered saline was confirmed with immobilizing the positively charged FA-NHS. The cytotoxicity against the HeLa cells was also evaluated to verify whether the nanohybrids can be the candidate for cell imaging. The affinity and noncytotoxicity between the FA-NHS-immobilized EHA nanohybrids and cells were monitored for 3 days. Red luminescence from the cells could be observed, and the labels with following the cellular shapes were achieved by an additional culture time of 1 h after injecting the FA-NHS-immobilized EHA nanohybrids to the spheres, indicating the rapid bioimaging process. Therefore, this is the first successful report to describe the synthesis of inorganic-organic nanohybrid systems for controlling the EuTH-FA-NHS interactions. The photofunction of the interfacial interactions was successfully designed to provide "efficient luminescent ability" as well as "rapid targeting to the cancer cells" in one particle.

SILICONE MICROTUBE-ASSISTED SCLERAL FIXATION OF A POSTERIOR CHAMBER INTRAOCULAR LENS.

PURPOSE: To report a novel technique for intrascleral fixation of a posterior chamber intraocular lens using a silicone microtube to manipulate the haptics into position. METHODS: Intrascleral fixation was performed in six eyes and the results evaluated in this retrospective case series. A silicone microtube with a 0.2-mm external diameter was passed from a sclerocorneal incision through the chamber and a sclerotomy made using a 30-gauge needle. The tips of the intraocular lens haptics were connected to the silicone microtube outside the eye. After the intraocular lens was injected into the posterior chamber, the haptics were drawn through the scleral incision through their attachment to the silicone microtube. RESULTS: The mean postoperative corrected visual acuity was 0.62 logarithm of the minimum angle of resolution (20/43) with a mean refraction error of -0.06 ± 0.4 diopter, which did not differ significantly (P = 0.53) from the expected value. The postoperative complications included transient ocular hypotension, vitreous hemorrhage, and choroidal detachment. CONCLUSION: Our technique using a silicone microtube reduces the number of intraocular procedures compared with previous methods using forceps or needles for moving the intraocular lens haptics from the posterior chamber to the outside through sclerotomies.

Preparation of Eu(iii) acetylacetonate-doped well-defined titania particles with efficient photoluminescence properties.

The aggregation/dispersion of luminescent species is a critical factor that determines their luminescence properties. In this study, europium(iii) acetylacetonate (Eu(acac)3) was doped into a titania matrix to form Eu(acac)3-doped titania particles with well-defined size and shape through a microreactor-based sol-gel approach. The morphology and structure of the as-synthesized products were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, scanning transmission electron microscopy and X-ray diffraction measurements. The Eu/Ti value of the products varied in the range from 0.125 to 5.0 and the resulting luminescence properties were examined. It should be noted that there was an optimum Eu/Ti value that exhibited the strongest luminescence. A possible reason for this phenomenon can be explained on the basis of a balance between the inter-molecular distance of Eu(acac)3 and its doped amount. The effects of the crystal phase of the titania matrix on luminescence behavior were also investigated. As a result, Eu(acac)3-doped amorphous titania demonstrated more efficient luminescence than that after calcined at 550 °C for 6 h to convert amorphous to anatase probably because of the aggregation of Eu species on the crystallite surface. The stability of the present Eu(acac)3-doped titania was confirmed by preparing thin films on a glass substrate and by applying UV/ozone treatment. As compared to bare Eu(acac)3, degradation in luminescence was suppressed in the case of Eu(acac)3-doped titania. Thus, the present titania-based hybrid with controlled Eu(acac)3 doping is useful as a stable, luminescent material for optical, biological and environmental applications.

The mobility of proton carriers in phosphate glasses depends on polymerization of the phosphate framework.

Proton conducting phosphate glasses were prepared by electrochemical substitution of sodium ions with protons applied to glasses with the compositions xNaO1/2-1WO3-8NbO5/2-5LaO3/2-(86 - x)PO5/2 (x = 28, 32, 35, 38, and 40). The mobilities of proton carriers in the glasses were studied in terms of the polymerization degree of the phosphate framework. The proton mobility at 200 °C increased as the depolymerization of the phosphate framework developed up to x = 38, and decreased at x = 40. On the basis of Raman and infrared spectra measurements of the O-H stretching vibration region, the decreasing mobility at x > 38 was attributed to the increasing concentration of protons trapped by non-bridging oxygen in P2O74- ions, owing to strong O-H bonding. We found that the highly polymerized phosphate framework decreased the mobility of proton carriers, not because of suppression of the proton dissociation from oxygen atoms but rather the suppression of the proton migration. The compositions at which the phosphate framework was sufficiently depolymerized and did not contain P2O74- ions as a main component, achieved high mobility of proton carriers in phosphate glasses.

Efficient penetration into aqueous humor by administration of oral and topical levofloxacin.

PURPOSE: We investigated whether an optimized combination of oral and topical levofloxacin would lead to higher levofloxacin concentrations in aqueous humor. METHODS: Fifteen patients with cataracts in both eyes began topical treatment at 1 week before the first surgery and oral treatment at 1 day before the first surgery. On the day of surgery, they received oral and topical levofloxacin at 4 h and 1 h before surgery, respectively. Two days after the first operation, we performed cataract surgery on the second eye with the same drug administration protocol. RESULTS: Postsurgery concentrations of levofloxacin in the aqueous humor of the first and second eyes were 2.87±0.89 µg/mL (mean±standard deviation, n=15) and 3.76±1.32 µg/mL, respectively; the levofloxacin level in the second eye was significantly higher than that in the first eye (P=0.0085). CONCLUSIONS: Our protocol to achieve high aqueous humor concentrations of levofloxacin may be favorable in preventing endophthalmitis after eye surgery.

Increase in matrix metalloproteinase-2 level in the chicken retina after laser photocoagulation.

BACKGROUND AND OBJECTIVE: We investigated the levels of matrix metalloproteinase-2 (MMP-2), which has been implicated in various vitreoretinal diseases, in the retina after laser photocoagulation (LPC). MATERIALS AND METHODS: The time course of MMP-2 expression in 2-day-old chicken retinas before and 6 hours, 12 hours, 1 day, 2 days, 4 days, 8 days, 16 days, and 32 days after LPC was determined by real-time PCR and gelatin zymography. The basal level of MMP-2 in the retina and vitreous was also measured by gelatin zymography. MMP-2 localization in the retina was examined by immunohistochemistry. The localization of MMP-2 mRNA was determined by fluorescent in situ hybridization. The internal limiting membrane (ILM) was observed by scanning electron microscopy. RESULTS: MMP-2 mRNA expression in the retina peaked at day 4, but gelatin zymography showed that MMP-2 peaked 6 hours after LPC and the significant increase in the level of active MMP-2 lasted for more than 4 days. The concentration of MMP-2 in the vitreous was significantly higher than that in the retina. A distinct MMP-2 signal around the ILM was identified 6 hours after LPC, but MMP-2 mRNA was not detected there. Electron microscopy showed a damaged retinal surface after LPC. CONCLUSION AND OUTLOOK: The significant increase in retinal MMP-2 which lasted for more than 4 days after LPC may be induced by influx from the vitreous into the retina. This MMP-2 dynamics may contribute to pathological processes in the retina after LPC.

Distinct response to heparin by two chicken brain type creatine kinase subunits.

In the chicken, two creatine kinase-type B (B-CK) isoproteins, Ba- and Bb-CK, both of which are derived from a single copy gene by alternative splicing, dimerize in neural tissues. The two isoproteins contain distinct N-terminal portions, but their functional difference remains unknown. We investigated the binding affinities of Ba- and Bb-CK to heparin, hyaluronan and chondroitin sulfates, and examined the influence of these glycosaminoglycans on enzyme activity. Chicken retinal samples analyzed by Western blotting and amino acid sequence study after two-dimensional gel electrophoresis showed that heparin binds Bb-CK, but not Ba-CK, while hyaluronan and chondroitin sulfates showed no interaction with either isoprotein. Using fusion proteins covering the distinct N-terminal portions, we also showed that heparin did not react with the N-terminus of Ba-CK, but did react with that of Bb-CK. Site-directed mutagenesis of basic amino acids found in the N-terminal portion of Bb-CK identified three basic amino acids critical for this binding. Furthermore, heparin dose-dependently inhibited the enzymatic activities of Ba-CK; Bb-CK activities were less intensely inhibited. Hyaluronan and chondroitin sulfates had no effects on the activities of these enzymes. Thus, the N-terminal portion of B-CK is critical to mediate its affinity to heparin and control enzyme activity, which may be important for regulating energy metabolism in neural tissues such as brain and retina, unique organs abundant in heparan sulfates.

Competitive binding of heparin with hyaluronan to a specific motif in SPACR.

The critical hyaluronan binding motif (HABM) in sialoprotein associated with cones and rods (SPACR) has already been determined. As sialoproteoglycan associated with cones and rods, another interphotoreceptor matrix molecule, binds to chondroitin sulfate and heparin with or without the employment of HABMs, respectively, we evaluated and compared the binding of these glycosaminoglycans to SPACR. A western blotting study in combination with inhibition assays showed that heparin bound specifically to SPACR. A series of GST fusion proteins covering the whole SPACR molecule narrowed down the region responsible for the binding. Finally, a site-directed mutagenesis assay demonstrated that the critical HABM also acts as a specific binding site for heparin. These results were supported with mutual inhibitions by hyaluronan and heparin in analyses using GST fusion proteins and native SPACR derived from retina. Thus, these glycosaminoglycans bind to SPACR in a different manner than to sialoproteoglycan associated with cones and rods. The competitive binding between hyaluronan and heparin to SPACR, mediated through the identical HABM, may dominate the functions of SPACR, in turn involving physiological and pathological processes involved in retinal development, aging and other related disorders.

Characterization of a motif for specific binding to hyaluronan in chicken SPACR.

The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA-binding motif found near the carboxyl-terminus of SPACR was mutated by site-directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA-affinity column under both reducing and non-reducing conditions. These results revealed that the specific putative HA-binding motif is located near the carboxyl-terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.

Versican and fibrillin-1 form a major hyaluronan-binding complex in the ciliary body.

PURPOSE: In this study, biochemistry, molecular biology, immunohistochemistry, and electron microscopy techniques were used to examine whether versican, which is known to bind fibrillin-1, interacts with fibrillin-1 in the ciliary body and vitreous, and whether the versican in this complex binds to hyaluronan. METHODS: The new polyclonal antibodies against the amino and carboxyl termini of versican were raised and characterized. The mRNA expression levels of versican and fibrillin-1 were analyzed by RT-PCR and real-time PCR, and their protein levels were evaluated by Western blot analysis and immunohistochemistry. Isolation of versican bound to fibrillin-1-containing microfibrils from ciliary bodies was performed by extraction studies. Slot-blot analyses and rotary shadowing electron microscopy were applied to identify versican associated with fibrillin-1-containing microfibrils after gel filtration chromatography and density gradient centrifugation. RESULTS: The newly prepared polyclonal antibodies recognized amino and carboxyl termini of chicken versican. Versican, principally V0 and V1, was found to be securely bound to fibrillin-1-containing microfibrils, forming a major hyaluronan-binding structure in the ciliary nonpigmented epithelium. In addition, Western blot analysis revealed two cleaved complexes, the carboxyl-terminal end of versican bound to fibrillin microfibrils and the amino terminal end of versican bound to hyaluronan in the vitreous body. CONCLUSIONS: Fibrillin-1, versican, and hyaluronan form a unique complex in the ciliary nonpigmented epithelium, and two cleavage products of this complex were shown to exist in the vitreous body. This newly clarified fibrillin-versican-hyaluronan (FiVerHy) complex and its cleavage products may be indispensable for the physiological properties important to the ciliary body and vitreous.