| BackgroundThe vitreous body is a clear, non-renewable gel body, which is mainly constituted by the99%water and1%inorganic salts, collagen and hyaluronic acid. Collagen fibers are three-dimensional mesh structure, attached by hyaluronic mucopolysaccharide, which can be combined with water molecules, so that the vitreous is substantially gelled. The physiological functions of vitreous body are supporting the retina, refraction, cell barriers and nutrition in eyes. Due to the non-renewable properties of natural vitreous body, when vitreoretinal diseases occur, such as:the trauma caused retinal detachment, traumatic proliferative vitreoretinopathy (T-PVR), proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and so on, vitrectomy surgery is needed. After intraoperative resection of natural vitreous body, suitable artificial vitreous substitutes must be filled into vitreous cavities to repair the damage, supporting retina, reconstructing visual function to prevent atrophy of eyeball. Currently, the vitreous substitutes commonly used in clinical include air, inert gas, heavy water, silicone oil, heavy silicone oil and so on. All of them are directly injected into the vitreous cavity, directly contacting with the retina, to support retina by the surface tension. The others are short-term tamponade in vivo in addition to silicone oil. However, after a long-term tamponade in vitreous cavity, a series of post-operative complications are likely to occur, such as cataract, secondary glaucoma, corneal degeneration, silicone oil emulsification and even migrate to subretina or optic nerve, causing demyelination of the optic nerve fibers, resulting in permanent loss of vision. Therefore, it is quite urgent to find an ideal vitreous substitute with good biocompatibility without serious complications for long-term tamponade.Hydrogel is known as one of the best artificial vitreous candidates because of its good biocompatibility, optical property and shock absorption performances. It can mimic the characteristics of natural vitreous, and has been a hot research focus at home and abroad since the1990s. Hydrogel mainly includes PVA [poly (vinyl alcohol)] hydrogel, PEG [poly (ethylene glycol)] hydrogel, PAA [poly (acrylic acid)] hydrogel, PAM [poly acrylamide] hydrogel, and so on. To meet the physical properties of natural vitreous, researchers have been trying to change different synthesis process of the hydrogel in vitro, and then injected into the vitreous cavity. Liquid polymeric substance may also be injected into the vitreous cavity which is crosslinked in situ to form hydrogel in vitreous cavity. In this way, it can avoid the structure destruction during injection of the hydrogel. Additionally, the hydrogel can load drugs, acting as a release vector of drug within the eye. There were no significant postoperative complications after a short-term tamponade in animal experiments. Because all the substitutes are directly injected into the vitreous cavity, directly contacting with the retina, the hydrogels are also easily involved in the metabolism and circulation within the eye, resulting in rapidly degradation and absorbtion in the eye. Therefore, the hydrogels can not be used as a long-term vitreous substitute and can not meet the needs of long-term retina supporting for the treatment of serious diseases such as severe retinal detachment. Hence, how to reduce the degradation of the hydrogel and extend the residence time in vitreous cavity is a very important scientific issue for researchers.Natural vitreous is surrounded by a "thin film", which is called vitreous cortex. The vitreous cortex structure can be destroyed due to age, trauma and retinopathy. As all the artificial vitreous are directly injected into vitreous cavity, they are easily involved in metabolism and circulation in the eyes. Therefore, it is necessary to find a natural vitreous capsule membrane surrounding the artificial vitreous to restrict the flow in the eye, to avoid or reduce the absorption and deterioration, thereby extending the residence time in vivo.Therefore, we first propose a new strategy for an alternative natural vitreous, which was the foldable capsular vitreous body (FCVB), mainly constituting ofa balloon, drainage pipes and drainage valve. The balloon is produced by the fine computer simulation vitreous. It connects with the drainage tube, drainage valve, and is made of silicone rubber membrane.The balloon is implanted into the vitreous cavity through a minimally invasive incision, and then the flowing medium, such as saline, silicone oil, hydrogel can be injected into balloon to support the retina.The foldable capsular vitreous body (FCVB) can avoid the serious defects of the current artificial vitreous. It can well simulate the natural vitreous structure, restore the structural support of retina, as well as the physiological functions of refraction and cell barrier of natural vitreous. And from animal experiments to clinical trials, a series of studies have confirmed that the FCVB tamponade with silicone oil shows excellent biocompatibility, which can effectively360degree support and promote reattachment of the retina, significantly reduce the silicone oil-induced complications such as silicone oil emulsion, secondary glaucoma, corneal degeneration, and so on. However, the FCVB tamponade with silicone oil still can not restore a natureal refractive properties and viscoelastic properties of natural vitreous.PurposeThis aim of the project was to evaluate a foldable capsular vitreous body (FCVB) injected with polyvinylalcohol (PVA) hydrogel as an artificial vitreous substitute. Different concentrations of PVA hydrogel were crosslinked by irradiation in vitro, then optical, physical, mechanical properties and cytotoxicity were tested to screen a best PVA hydrogel which may be the most proximateto natural vitreous performance. Comprehensive assessment of FCVB injected with PVA hydrogel as a vitreous substitute was received in the rabbit eye model, including optical properties, retinal supporting function and biocompatibility. The novel vitreous substitute of FCVB tamponade with PVA hydrogel can effectively prevent the PVA hydrogel from directly contacting with the retina, and can avoid the PVA hydrogel participating in the intraocular metabolism and flowing into the anterior chamber, as well as reducing the degradation and absorption, extending the residence time of the hydrogel in vitreous cavity, restoring the refractive performance and viscoelastic properties of the natural vitreous.Materials and MethodsPart one:The preparation of Polyvinyl alcohol (PVA) hydrogel1. The crosslinking of PVA hydrogel:PVA was purchased from Sigma Aldrich. Different concentrations (1%,3%,7%,w/v) PVA solution were crosslinked by y-irradiation (7kGy, Co60), and1%,3%,7%PVA hydrogels were harvested. Hydrogel was immersed in the double distilled water soaking48h, in order to remove the unreacted crosslinked PVA monomers.2. Parameters of physical and optical properties:Measuring water content, light transmittance, refractive rate, pH value, and swelling properties of PVA hydrogel.3. Rheological properties:The rheological analyses were carried out at37±0.1℃on a strain-controlled ARES-RFS rheometer (TA Instruments Inc., New Castle, DE) using a cone and plate geometry of50mm diameter and cone angle of0.04rad. The cross-linked hydrogels were injected onto the plate through a19-gauge needle.Dynamic strain sweep tests, at a1.0Hz oscillation frequency were performed on the hydrogels to ensure which strain amplitude was in the linear viscoelastic region. The mechanical properties of PVA hydrogels were studied by analyzing the storage modulus (G’) and the loss modulus (G") according to frequency of oscillatory shear stress. Dynamic frequency sweep test was conducted at a strain amplitude γ0=1.0%under oscillating frequency ranging from10to0.01Hz.The relative contribution of the elastic and viscous properties can be quantified by the loss tangent (tan8) which is the ratio of the loss to the storage modulus (tan8=G"/G’). The higher the tan δ, the more liquid-like the sample, with a value of1considered to be a threshold between liquid and gel behavior. Because of the sufficient resilience of the material, when subjected to shear stress, it is also desirable for vitreous substitute. This parameter was estimated by recording the loss tangent. The resilience (R) is an inverse measure of the damping property and usually estimated as R=1-2π tan8.In a creep experiment, a constant shear stress (σ0) of0.1Pa was imposed to the hydrogel samples for a nominal creep time of500s. In viscoelastic materials the strain (γ) response is linear. The ratio γ/σ0is called creep compliance J(t).The same analyses were also performed on a usual commercial silicone oil (Oxane5700; Bauch&Lomb, Rochester, NY) and a hyaluronic acid (HA)(Medical Hyaluronan Gel; Iviz, Shangdong, China)4. Cytotoxicity:The experiment groups included1%,3%, and7%(w/v) PVA hydrogels and a combination of FCVBs that were injected with1%,3%, and7%(w/v) PVA hydrogels. These groups were defined as1%PVA,3%PVA,7%PVA, FCVB+1%PVA, FCVB+3%PVA, and FCVB+7%PVA. The extraction medium was prepared by incubating the varying samples (1%PVA,3%PVA,7%PVA, FCVB+1%PVA, FCVB+3%PVA, and FCVB+7%PVA) with standard culture medium (Dulbecco’s modified Eagle’s medium with10%fetal calf serum) for72hours at37℃. The extraction medium (200μl) was tested on a monolayer of murine fibroblast L929cells. The murine fibroblast L929cells were seeded in96-well culture plates at a cell density of1.0×103cells/well and fed with standard culture medium for24hours at37℃in a5%CO2atmosphere. Then, cell cultures were incubated with the extraction medium and a negative control (standard culture medium) at the same conditions. After24,48, and72hours of incubation, the cytotoxicity was assessed via a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-mide [MTT, Sigma Aldrich, St. Louis, MO] assay. The absorbance was measured at570nm, and the cytotoxicity was calculated via the following formulae:cytotoxicity (%)=Optical Density (OD) of sample/Optical Density (OD) of control×100%.Part two:The research of FCVB injected with PVA hydrogel in vivo1. Animal Preparation:The rabbit FCVB was made of tailor-made modified liquid silicone rubber. The basic material was Dow Corning Class VI elastomers and the shape was manipulated according to the vitreous parameters of the rabbit by using a computer. The FCVB consists of a vitreous-shaped capsule, tube, and valve, as described in a previous study. The FCVBs were sterilized by heating in double-distilled water at100℃for30min prior to surgery.All procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Vision and Ophthalmic Research. Eighteen New Zealand albino rabbits weighing2.5to3.5kg were divided into three groups:the FCVBs were implanted into the vitreous cavity and then injected with PVA hydrogel and an FCVB (FCVB+PVA group, n=6), PVA hydrogel only (PVA group, n=6), or balanced salt solution (BSS)(BSS group, n=6).All surgeries were performed with sterile technique in the right eyes of the rabbits. After pupillary dilatation, the rabbits were anesthetized via an intramuscular injection of a mixture of ketamine hydrochloride (30mg/kg) and chlorpromazine hydrochloride (15mg/kg). Under the standard ophthalmic operating microscope, a standard three-port PPV,20-gauge Alcon Accurus vitrectomy system was used. The sclerotomy was created approximately3.5mm posterior to the limbs. A core vitrectomy was performed and followed by a gentle vitrectomy of the peripheral vitreous base. After removing the vitreous as much as possible, PVA hydrogel (1.1ml) or BSS (1.1ml) was directly injected into the vitreous cavity through a19-gauge needle. In the FCVB+PVA group, the lens was cut during vitrectomy for a better long-term observation of the fundus. At the end of the procedure, the FCVB was folded into three petals and implanted into the vitreous cavity through a3mm incision at12o’clock without fluid-air exchange. Then,1.4ml of PVA hydrogel was injected into the capsule through the tube-valve device fixed under the conjunctiva. After surgery, the eyes were treated with tobramycin as antibiotic eyedrops and dexamethasone sodium phosphate as steroid eyedrops. This was done three times a day for2weeks.2. Postoperative Examinations:A slit lamp and a fundus camera were used to examine and record the anterior segment, ocular media, and fundus at day3,7,14,30,60,90, and180postoperatively. The measurement of intraocular pressure (IOP) with a Tono-Pen was performed at day3,7,14,30,60,90, and180postoperatively. Also, to evaluate the anatomical position of the retina, a B-scan ultrasound was performed at day14,30,60,90, and180postoperatively.3. Electroretinogram (ERG) tests:A standardized full-field ERG was recorded preoperatively and90days and180days postoperatively on the right eye by using the Roland Ganzfeld system and PC-based signal acquisition and analysis software. The eyes were dilated with0.5%tropicamide and dark-adapted for at least30min. Rabbits were anesthetized as described for surgery. The positive unipolar contact lens was placed on the cornea with methylcellulose (2%). The negative needle electrode was inserted into the subcutaneous in the forehead, and the ground electrode was clipped to the earlobe with some electric gel. The bright flash response was elicited by using the ISCEV standard flash of2.4cds/m2. The photopic ERG was recorded after10min of continuous light adaptation with a background illumination of10cds/m2. The a-wave and b-wave amplitudes were evaluated at different time points.4. Pathologic Examination:The rabbits were euthanatized via an intramuscular injection of an overdose of ketamine and chlorpromazine (1:1) at90days postoperative (n=3, BSS group; n=3, PVA group; n=3, FCVB+PVA group) and180days postoperative (n=3, BSS group; n=3, PVA group; n=3, FCVB+PVA group). Then, the experimental (right) eyes were harvested from these animals. After enucleation, the PVA hydrogels were removed from the vitreous cavities in order to analyze their elastic and viscous properties. Then, the eyeballs were immediately fixed with10%paraformaldehyde, and they were embedded in paraffin and stained with hematoxylin and eosin (HE) for a light microscope study. 5. Statistical Analysis:All data were analyzed by the SPSS statistical software version13.0(SPSS, Cary, NC, USA). Data were expressed as means±SD. Statistical differences between groups were tested using a one-way analysis of variance (ANOVA). If significance was identified, post hoc analysis with Dunnett was used to confirm the significant changes. A P value of less than0.05was considered statistically significant.ResultsPart one:The preparation of Polyvinyl alcohol (PVA) hydrogel1. Physical Properties of the Hydrogels:The physical properties of the PVA hydrogels are as follow:the1%PVA hydrogel, with98.9%water content, pH value of7.22, density1.0039, refractive index1.3355, and light transmittance94.8%. The3%PVA hydrogel, with98.1%of water content, pH value7.25, density1.0144, refractive index1.3361, and light transmittance93.2%. The7%PVA hydrogel, with water content93.5%, pH value of7.41, density1.1147, refractive index1.3425, and light transmittance88.2%. After injection, minor fragmentation occurred in only the7%PVA hydrogel, not in the1%and3%PVA hydrogels. All the hydrogels have high water content and are transparent. The1%and3%PVA hydrogels have similar parameters for these physical features and were close to those of the natural human vitreous.2. Rheological measurements:The oscillatory shear studies were performed on the PVA hydrogels, HA, and silicone oil. The storage modulus (G’) represents the elastic or solid-like component, while the loss modulus (G") represents the viscous or liquid-like component. For all the hydrogels, the storage modulus (G’) was greater than the loss modulus (G") at all frequencies, which showed "gel-like" behavior. Also, the plots of G’ and G" were almost parallel, and there was no crossover in the range of frequencies used. The behaviour is like that of type IV gel. This class of gels would be most suitable for vitreous substitution.However, for silicon oil, the G" is greater than the G’ for all the frequencies analysed, which means that the oil has the appearance of a viscous solution. Meanwhile, the HA behaviour is like type III gel. The G’ is generally higher than G", but a G’-G" crossover occurs at low frequencies, which suggest that the HA behaves as an entanglement network and that the sample is mainly viscous rather than elastic.The values of the human vitreous are similar to those of the porcine vitreous. The average steady state moduli for the porcine vitreous are G’=2.8±0.9Pa and G"=0.7±0.4Pa. Therefore, the7%PVA hydrogel is not suitable as a substitute, due to the fact that its rheological parameters are much higher than porcine rheological parameters.The loss tangent plots of all samples are recorded as a function of frequency. The lower the loss tangent, the higher the resilience is. Therefore, according to resilience, the hydrogels can be ranked as follows:7%PVA>3%PVA>1%PVA. A material with a high resilience may perform better mechanically in a vitreous cavity.All the hydrogels were subjected to creep analysis. The compliance of the three analysed hydrogels increases initially and tends to level off and approach a compliance plateau after intermediate periods of time. Therefore, the creep behaviours of these hydrogels are similar to those of very lightly cross-linked amorphous polymers. This behaviour would indicate that there are prominent instantaneous elastic responses followed by substantial retarded elastic responses over time under constant shear stress. The values of the instantaneous elastic deformation of3%PVA is higher than those of1%PVA and7%PVA, indicating that the3%PVA is more elastic.Therefore, among the three hydrogels, the3%PVA hydrogel would be the best candidate as a vitreous substitute according to the rheological analysis of G’, G", loss tangent, and creep behaviour.Additionally, stable mechanical properties are essential for the hydrogel after injection. The storage modulus G’ and the loss modulus G" of the3%PVA hydrogel before and after injection through a-19gauge needle are compared at37℃. The G’ value decreased from6.3±0.9to6.1±1.3Pa, and the G" value increased from1.2±0.8to1.3±0.9Pa. The3%PVA hydrogel shows approximately the same rheological behavior before and after the injection, indicating that the injection does not affect the cross-linked structure of the hydrogel.3. Cytotoxicity tests:The in vitro cytotoxicity of the experimental groups (1%PVA,3%PVA,7%PVA, and FCVB+1%PVA, FCVB+3%PVA, FCVB+7%PVA) was tested on the L929mouse fibroblast cell. No changes in cell morphology, detachment, and membrane lysis were observed in the culture with the tested materials and the negative control. Cell cytotoxicity was evaluated via MTT assay. After24,48, and72hours of incubation, the OD values did not show a significant difference among the experimental groups (P>0.05). Also, there was no significant difference between the experimental groups and the control group (P>0.05). The extractions of the experimental groups induced neither cell viability reduction nor the inhibition of cell growth, resulting in no cytotoxic effects.To sum up, the3%PVA hydrogel had good rheological properties and good biocompatibility in vitro, so it was selected as a vitreous substitute for further analysis of the long-term biocompatibility and residence time in vivo.Part two:The research of FCVB injected with PVA hydrogel in vivo1. Slit lamp:Anterior chamber inflammation was visible in all groups postoperatively. However, the inflammation subsided on the third day after the operation in the BSS group and in the PVA group. Although eyes of FCVB+PVA group had relatively severe inflammation (fibrinous exudation) in the anterior chamber, they recovered within seven days with intensive anti-inflammatory treatments. With the exception of cataracts, no serious complications, such as corneal opacity, keratopathy, or posterior synechia, were observed over180days.The groups showed varying degrees of lens opacity. In the BSS group, one of six eyes developed cataracts during the180days of observation. In the PVA group, one eye and two eyes developed cataracts in90and180days, respectively. In the FCVB+PVA group, lensectomy was performed during PPV surgery. Our prior study revealed that severe cataracts occurred most frequently in FCVB implantated eyes and that this led to a failure to observe the fundus. In order to avoid lesions from a second surgery, the lens and vitreous were removed at the same time during vitrectomy.2. Funduscopic examination:Funduscopic examination revealed that there was no evidence of vitritis, uveitis, retinitis, endophthalmitis, vitreous hemorrhage, or retina detachment in the rabbit eyes of all groups. The retina and the optic nerve appeared to be normal. In the BSS group and the FCVB+PVA group, the vitreous cavity remained optically clear on postoperative days90and180. However, in the PVA group, though the eyes showed clarity in their vitreous cavities on postoperative day90, the vitreous cavities appeared to be relatively blurry on postoperative day180due to the complication of cataracts.3. B-scan ultrasonography:The B-scan ultrasonography showed no retinal detachment in any groups during follow-up. In the FCVB+PVA group, slightly reflective signals of a capsule-like membrane were observed in the vitreous cavity. The FCVB injected with PVA hydrogel was apparently in good contact with the inner retina and can support it well.4. IOP:A downward trend was observed at postoperative day3in the PVA+FCVB group, which may be due to a leakage of the aqueous humor at the incision for the FCVB implantation before the incision healed. There were upward trends in the IOP after3days in the other two groups, but the IOP reached a plateau level at day7,14,30,60, and90. No statistically significant differences were found in the IOP among the three groups preoperatively or at day7,14,30,60, and90postoperatively, but a significant difference was found in180days. The IOP of the PVA group significantly decreased in180days (P<0.05). This may be due to the fact that some of the PVA hydrogel was dissolved in the vitreous cavities.5. Electroretinography:Full-field ERG measurements were obtained pre-and post-operatively in all eyes. In the BSS group and PVA groups, the ERG recordings appeared to be similar in terms of the a-wave and b-wave amplitudes before and after operation. There were no significant differences between the eyes of the BSS group and the PVA group in terms of a-or b-wave amplitudes at postoperative day90(P>0.05) and day180(P>0.05). By contrast, in the FCVB+PVA group, a decrease in a-wave and b-wave amplitudes was evident postoperatively, and the differences were significant when compared to the BSS group or PVA group at postoperative day90(P<0.05) and day180(P<0.05).6. Histological findings:The eyes were enucleated at postoperative day90and day180. In the FCVB+PVA group, a gross examination of eye specimens showed that the capsular wall of the FCVB could fit perfectly with the retina in the vitreous cavity and that the FCVBs injected with3%PVA hydrogel remained transparent and well-rounded. In the PVA group, the vitreous cavities were still filled with3%PVA hydrogel at postoperative day90. Also, the transparency of the3%PVA hydrogel was still very clear, and the viscoelasticity did not seem to have obviously changed. However, the vitreous cavities were filled with3%PVA hydrogel and water-like solution at postoperative day180, and some of the3%PVA hydrogel was dissolved and degenerated. Although the3%PVA hydrogel remained transparent and showed no apparent fragmentation, the viscoelasticity appeared to be poorer than it was before implantation.The histological examination of H&E-stained retinal sections from the BSS group and PVA group showed that the integrity of the retinal layers was good and that no loss of tissue was evident. There was no evidence of pathological changes, such as deformations, degeneration, or inflammation. In the FCVB+PVA group eyes terminated on postoperative day90, the H&E-stained sections displayed a normal retinal morphology. In eyes terminated at day180, retinal disorder was seen. The retinas displayed an aggregation of the inner nuclear layer and the outer nuclear layer and a thinning of the ganglion cells layer. No signs of inflammation were seen. Additionally, in all three groups, there were no pathological changes in other parts of the eye, including the cornea and ciliary body.7. Oscillatory shear measurements after in vivo experiments:After sacrifice and enucleation, the3%PVA hydrogel of the vitreous cavity in the PVA group and the FCVB+PVA group was collected and subjected to oscillatory shear measurements, as described before. The mechanical spectrum of the hydrogels. In the FCVB+PVA group, on postoperative day90and day180, the G’ and G" values remained similar to those before surgery. In the PVA group, though a mild decrease in the G’ and G" values was observed at postoperative day90, a conspicuous decrease was observed at postoperative day180as compared to preoperative values. The G’ decreased from6.1±1.3to3.9±0.8Pa and G" decreased from1.3±0.9to0.8±0.5Pa. This indicated that the3%PVA hydrogel had undergone obvious biodegradation in the vitreous cavity after a180-day retention.Conclusions3%PVA hydrogel has similar optical, physical and rheological properties to natural vitreous, and shows good biocompatibility. After a long-term (180days) tamponade in the vitreous cavity, the FCVB injected with3%PVA hydrogel as a vitreous substitute reveals good optical, mechanical properties and biocompatibility, which can effectively support the retina and maintain the anatomical structure of the retina as well as effectively avoiding degradation of the PVA hydrogeland elongating the residence time. This new approach may develop into a valuable tool to improve the stability performance of PVA hydrogel as a vitreous substitute and to extend the application function of FCVB for long-term implantation in vitreous cavity. |
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