In vitro biocompatibility of new PVA-based hydrogels as vitreous body substitutes

In order to synthesize injectable hydrogels suitable as vitreous body substitutes, a new method based on the use of trisodium trimetaphosphate (STMP) to cross-link PVA was recently proposed. Hydrogels with different molar ratios between STMP and PVA were realised. The aim of the present study was the evaluation of the biocompatibility of the different STMP/PVA hydrogels synthesised by analysing the effects of their in vitro interaction with cultures of mouse fibroblasts NIH3T3, primary human microvascular endothelial cells adult (HMVECad) and human lens cells. Cytotoxicity of hydrogels was first evaluated by analysing cell density and proliferation. Morphological and morphometric analysis of cell in contact with hydrogels was then performed using light microscopy and scanning electron microscopy, respectively. Moreover, cell adhesion and growth onto the hydrogels surface was evaluated and correlated to the amount of adsorbed proteins. At last, the biocompatibility of the sheared STMP/PVA 1:8 hydrogel was tested. The in vitro data of all the STMP/PVA hydrogels demonstrated their good biocompatibility, and indicated that the 1:8 sample was the most promising as vitreous body substitute.     

In vitro evaluation of in situ gels as short term vitreous substitutes

Dysfunction of the vitreous humor, present in the posterior cavity of the eye, leads to its detachment from the retina and vision loss. In this study, biopolymers were evaluated as in situ gels for short term vitreous substitution. Biophysical characterization revealed that the viscosity of the vitreous was >4000 cP at a shear rate of 0.15/s and it formed a gel with elastic modulus G' greater than the viscous modulus G'. Biopolymers of gellan and hyaluronic acid (8:2 w/w, 1% concentration) were low viscosity liquids at 37 degrees C and gelation was triggered both by the addition of 0.18 mM CaCl(2) as well as ocular temperature, thus making them feasible as in situ gels. Gelation was confirmed by viscoelastic moduli where G' was greater than G', similar to the vitreous and unlike that of silicone oil, a common vitreous substitute. The gels had a viscosity >5000 cP at a shear rate of 0.512/s, excellent light transmittance and absence of syneresis. Contact angle studies with water and simulated ocular fluids showed that gellan hyaluronic acid gels had similar wetting properties to that of vitreous with contact angles of 27 degrees +/- 1 degrees , 36.7 degrees +/- 1.6 degrees , and 33.7 degrees +/- 0.5 degrees for water, simulated tear fluid, and simulated aqueous humor, respectively. The results of this study suggest that biopolymers of gellan and hylauronic acid are suitable as in situ gels, have biophysical properties similar to that of the vitreous, and may be promising as alternatives to silicone oil as short-term vitreous substitutes.     

Poly(I-vinyl-2-pyrrolidinone) hydrogels as vitreous substitutes: Histopathological evaluation in the animal eye

A homopolymer of 1-vinyl-2 pyrrolidinone and its copolymer with 2-hydroxyethyl methacrylate, both cross-linked with divinyl glycol, were produced as possible substitutes for the vitreous body of the eye. The hydrated polymers behaved like viscoelastic gels, displaying excellent physical and optical properties. The sterile gels (0.7-1.5 ml) were injected into the vitreous cavity of rabbits, which previously underwent gas-mediated vitrectomy. Clinically, the eyes were quiet, with the exception of transient opacities in the vitreous. After 4 weeks, the operated eyes were enucleated and subjected to histopathological analysis using light and transmission electron microscopy. The common feature in all sections was the invasion of inflammatory cells. Vacuoles containing granular material, assumed to be polymer, were seen in the intercellular spaces of the neural retina, in the retinal pigment epithelium cells, and in macrophages. These findings indicated the fragmentation and phagocytosis of synthetic gels. It appeared that the biodegradation of the internalized polymers did not proceed further, however, the fate of polymers and their usefulness as vitreous substitutes should be investigated through long-term experiments.     

Methacrylated gelatin hydrogels as corneal stroma substitutes: in vivo study

Abstract

Methacrylated gelatin (GelMA) hydrogels were prepared to serve as corneal stroma equivalents. They were highly transparent (ca. 95% at 700?nm), mechanically strong and withstood handling and had high human corneal keratocyte viability (98%) after 21?days of culture period. In order to test the in vivo performance of the cell free GelMA hydrogels a pilot in vivo study was carried out using eyes of two white New Zealand rabbits. Hydrogel was implanted in a mid-stromal pocket created and without suture fixation, and observed for 8?weeks under a slit lamp. No edema, ulcer formation, inflammation or infection was observed in both the control (sham) and hydrogel implanted corneas. Corneal vascularization on week 3 was treated with one dose of anti-VEGF application. Hematoxylin and Eosin staining showed that the hydrogel was integrated with the host tissue with only a minimal foreign body reaction. Results demonstrated some degradation in the construct within 8?weeks as evidenced by the decrease of the diameter of the hydrogel from 4?mm to 2.6?mm. High transparency, adequate mechanical strength, biocompatibility and well integration with the host tissue, indicates that this hydrogel is a viable alternative to the current methods for the treatment of corneal blindness and deserves testing on larger number of rabbits and more extensively using microscopy, histology and immune histochemistry.     

In situ crosslinkable hyaluronan hydrogels for tissue engineering

We describe the development of an injectable, cell-containing hydrogel that supports cell proliferation and growth to permit in vivo engineering of new tissues. Two thiolated hyaluronan (HA) derivatives were coupled to four alpha,beta-unsaturated ester and amide derivatives of poly(ethylene glycol) (PEG) 3400. The relative chemical reactivity with cysteine decreased in the order PEG-diacrylate (PEGDA)>PEG-dimethacrylate>PEG-diacrylamide>PEG-dimethacrylamide. The 3-thiopropanoyl hydrazide derivative (HA-DTPH) was more reactive than the 4-thiobutanoyl hydrazide, HA-DTBH. The crosslinking of HA-DTPH with PEGDA in a molar ratio of 2:1 occurred in approximately 9 min, suitable for an in situ crosslinking applications. The in vitro cytocompatibility and in vivo biocompatibility were evaluated using T31 human tracheal scar fibroblasts, which were suspended in medium in HA-DTPH prior to addition of the PEGDA solution. The majority of cells survived crosslinking and the cell density increased tenfold during the 4-week culture period in vitro. Cell-loaded hydrogels were also implanted subcutaneously in the flanks of nude mice, and after immunohistochemistry showed that the encapsulated cells retained the fibroblast phenotype and secreted extracellular matrix in vivo. These results confirm the potential utility of the HA-DTPH-PEGDA hydrogel as an in situ crosslinkable, injectable material for tissue engineering.     

Enzyme-mediated fast in situ formation of hydrogels from dextran-tyramine conjugates

Dextran hydrogels were formed in situ by enzymatic crosslinking of dextran-tyramine conjugates and their mechanical, swelling and degradation properties were evaluated. Two types of dextran-tyramine conjugates (M(n,dextran)=14k, M(w)/M(n)=1.45), i.e. dextran-tyramine linked by a urethane bond (denoted as Dex-TA) or by an ester-containing diglycolic group (denoted as Dex-DG-TA), with different degrees of substitution (DS) were prepared. Hydrogels were rapidly formed under physiological conditions from Dex-TA DS 10 or 15 and Dex-DG-TA DS 10 at or above a concentration of 2.5 wt% in the presence of H(2)O(2) and horseradish peroxidase (HRP). The gelation time ranged from 5s to 9 min depending on the polymer concentration and HRP/TA and H(2)O(2)/TA ratios. Rheological analysis showed that these hydrogels are highly elastic. The storage modulus (G'), which varied from 3 to 41 kPa, increased with increasing polymer concentration, increasing HRP/TA ratio and decreasing H(2)O(2)/TA ratio. The swelling/degradation studies showed that under physiological conditions, Dex-TA hydrogels are rather stable with less than 25% loss of gel weight in 5 months, whereas Dex-DG-TA hydrogels are completely degraded within 4-10d. These results demonstrate that enzymatic crosslinking is an efficient way to obtain fast in situ formation of hydrogels. These dextran-based hydrogels are promising for use as injectable systems for biomedical applications including tissue engineering and protein delivery.     

In situ forming IPN hydrogels of calcium alginate and dextran-HEMA for biomedical applications

In situ forming hydrogels, which allow for the modulation of physico-chemical properties, and in which cell response can be tailored, are providing new opportunities for biomedical applications. Here, we describe interpenetrating polymer networks (IPNs) based on a physical network of calcium alginate (Alg-Ca), interpenetrated with a chemical one based on hydroxyethyl-methacrylate-derivatized dextran (dex-HEMA). IPNs with different concentration and degree of substitution of dex-HEMA were characterized and evaluated for protein release as well as for the behavior of embedded cells. The results demonstrated that the properties of the semi-IPNs, which are obtained by dissolution of dex-HEMA chains into the Alg-Ca hydrogels, would allow for injection of these hydrogels. Degradation times of the IPNs after photocross-linking could be tailored from 15 to 180 days by the concentration and the degree of substitution of dex-HEMA. Further, after an initial burst release, bovine serum albumin was gradually released from the IPNs over approximately 15 days. Encapsulation of expanded chondrocytes in the IPNs revealed that cells remained viable and, depending on the composition, were able to redifferentiate, as was demonstrated by the deposition of collagen type II. These results demonstrate that these IPNs are attractive materials for pharmaceutical and biomedical applications due to their tailorable mechanical and degradation characteristics, their release kinetics and biocompatibility.     

In situ detection of superoxide anions within porcine articular cartilage.

Cartilage was isolated from pig articular joints, and the production of reactive oxygen species (ROS) by chondrocytes embedded within the cartilage was assessed by two methods: the reduction of nitro blue tetrazolium and by the use of diaminobenzidine in the presence of manganese ions. Little constitutive generation of ROS was seen, but it could be detected after the addition of the calcium ionophore ionomycin. Further, the response seen was extremely heterogeneous; some cells showed a far greater release of ROS than others. Cells arranged in the columnar arrays of the deep zone were the most active, while those furthest from the cartilage/bone interface (i.e. nearer to the outer face of the cartilage) were unresponsive. Chondrocytes cultured in alginate beads also showed a similar heterogeneity in their response, suggesting that the isolation of these cells and the measurement of ROS production in a population is not representative of the true situation.     

In situ hybridization and immunohistochemistry for the detection of porcine cytomegalovirus

To establish in situ hybridization and immunohistochemistry based-assays for the detection of porcine cytomegalovirus, routinely processed renal tissue sections from 34 diseased piglets suspected of having the infection were obtained and examined. Using hematoxylin and eosin, porcine cytomegalovirus inclusion bodies were found in the nucleus of renal epithelial cells and capillary endothelial cells in the renal medulla in 30 cases. Inclusion bodies corresponding to porcine cytomegalovirus mRNA after in situ hybridization or porcine cytomegalovirus antigens after immunohistochemistry were easily determined. The cells were characterized by cytomegaly and basophilic intranuclear inclusion bodies. Using in situ hybridization, porcine cytomegalovirus mRNA were clearly detected in the nucleus and cytoplasm of the cells in 28 of the 30 (93.3%) cases. Using immunohistochemistry, porcine cytomegalovirus antigens were clearly detected in the cytoplasm of the cells in 21 of the 30 (70.0%) cases. Higher specificities and increased intensity of staining was observed with minimal background using in situ hybridization and immunohistochemistry compared with hematoxylin and eosin. Thus, the two established methods are useful and helpful tools for detecting the presence of a porcine cytomegalovirus infection.     

In situ formation of silver nanoparticles in photocrosslinking polymers

Nanocomposites of cross-linked methacrylate polymers with silver nanoparticles have been synthesized by coupling photoinitiated free radical polymerization of dimethacrylates with in situ silver ion reduction. A polymerizable methacrylate bearing a secondary amino functional group was used to increase the solubility of the silver salt in the hydrophobic resin system. Fourier transform infrared spectroscopy (FTIR) revealed that the silver ion reduction had no significant effect on the degree of vinyl conversion of the methacrylate. X-ray photoelectron spectroscopy (XPS) measurements showed an increased silver concentration at the composite surface compared to the expected concentration based on the total amount of silver salt added. Furthermore, the surface silver concentration leveled off when the silver salt mass fractions were 0.08% or greater. Composites with low concentrations of silver salt (< 0.08% by mass) exhibited comparable mechanical properties to those containing no silver. Transmission electron microscopy (TEM) confirmed that the silver nanoparticles formed within the polymer matrix were nanocrystalline in nature and primarily ≈ 3 nm in diameter, with some large particle aggregates. Composites containing silver nanoparticles were shown to reduce bacterial colonization with as little as 0.03% (by mass) silver salt, while additional amounts of silver salt did not further decrease their surface colonization. With a substantial effect on bacterial growth and minimal effects on mechanical properties, the in situ formation of silver nanoparticles within methacrylate materials is a promising technique for synthesizing antibacterial nanocomposites for biomedical applications.     

Granuloma formation and parasite disintegration in porcine cysticercosis: comparison with human neurocysticercosis

Taenia solium cysticerci infect human beings and pigs, causing cysticercosis. In this study the pig was used as a model to characterize the immune response against cysticerci, given the difficulties in analysing the developing immune response in infected human brains. Metacestodes in different stages of viability or degeneration were isolated from the brain, heart and skeletal muscle of naturally infected swine, and the adjacent tissue was examined histologically. The immune response elicited by the cysticerci was classified into four separate stages. In stage I the parasites were surrounded by a thin layer of collagen type I, and by stage II there was a sparse inflammatory infiltrate. In stage III, granuloma formation was evident, and by stage IV the parasite was surrounded by an eosinophil-rich infiltrate and its vesicular membrane had begun to degenerate. The final stage, IV, was detected mainly in the heart but not in the brain. The granulomatous reaction in swine resembled that described previously in human patients, but differed in the abundance of eosinophils, the relative paucity of plasma cells, and the discrete deposition of collagen. These differences were probably due to the fact that in pigs the immune response can be examined earlier than in human patients, in whom sampling is inevitably made at a more chronic stage.     

In situ cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model

We studied the efficacy of an in situ cross-linked hyaluronic acid hydrogel (HAX) in preventing post-surgical peritoneal adhesions, using a rabbit sidewall defect-cecum abrasion model. Two cross-linkable precursors were prepared by modifying hyaluronic acid with adipic dihydrazide and aldehyde, respectively. The hydrogel precursors cross-linked to form a flexible hydrogel upon mixing. The hydrogel was biodegradable and provided a durable physical barrier, which was highly effective in reducing the formation of post-operative adhesions. Ten out of 12 animals in the untreated control group developed fibrous adhesions requiring sharp dissection, while only 2 out of 8 animals treated with HAX gels showed such adhesions, and those occurred in locations that were not covered by the hydrogel. We also studied means by which gel degradation time can be modulated by varying the precursor concentration and molecular weight.     

In situ crosslinkable heparin-containing poly(ethylene glycol) hydrogels for sustained anticoagulant release

Low-molecular weight heparin (LMWH) is widely used in anticoagulation therapies and for the prevention of thrombosis. LMWH is administered by subcutaneous injection usually once or twice per day. This frequent and invasive delivery modality leads to compliance issues for individuals on prolonged therapeutic courses, particularly pediatric patients. Here, we report a long-term delivery method for LMWH via subcutaneous injection of long-lasting hydrogels. LMWH is modified with reactive maleimide groups so that it can be crosslinked into continuous networks with four-arm thiolated poly(ethylene glycol) (PEG-SH). Maleimide-modified LMWH (Mal-LMWH) retains bioactivity as indicated by prolonged coagulation time. Hydrogels comprising PEG-SH and Mal-LMWH degrade via hydrolysis, releasing bioactive LMWH by first-order kinetics with little initial burst release. Separately dissolved Mal-LMWH and PEG-SH solutions were co-injected subcutaneously in New Zealand White rabbits. The injected solutions successfully formed hydrogels in situ and released LMWH as measured via chromogenic assays on plasma samples, with accumulation of LMWH occurring at day 2 and rising to near-therapeutic dose equivalency by day 5. These results demonstrate the feasibility of using LMWH-containing, crosslinked hydrogels for sustained and controlled release of anticoagulants.     

Dextran-based in situ cross-linked injectable hydrogels to prevent peritoneal adhesions

Peritoneal adhesions are serious sequelae of surgery, and can cause significant morbidity and/or mortality due to pain, infertility, and bowel obstruction. We have designed and synthesized novel dextran (DX)-based injectable hydrogels for adhesion prevention, which are formed by mixing hydrazide-modified carboxymethyldextran (CMDX-ADH) with aldehyde-modified DX (DX-CHO) or carboxymethylcellulose (CMC-CHO). At high polymer concentrations, hydrogels formed very quickly upon mixing, e.g. 5% CMDX-ADH with 6% DX-CHO (=CMDX-DX; 1.8 s) and 5% CMDX-ADH with 6% CMC-CHO (=CMDX-CMC; 5.8 s). CMDX-DX shrank after gelling, while CMDX-CMC swelled. CMDX-ADH and CMC-CHO showed minimal to mild cytotoxicity to mesothelial cells and macrophages in vitro, while DX-CHO was very cytotoxic. However, all cross-linked gels had very mild cytotoxicity. When applied in a rabbit sidewall defect-bowel abrasion model of adhesion formation, CMDX-CMC greatly reduced the formation of adhesions while CMDX-DX worsened them.     

In situ photocrosslinked hyaluronic acid and poly (γ-glutamic acid) hydrogels as injectable drug carriers for load-bearing tissue application

Abstract

Due to the syringeability of precursor solution and convenience of open surgical treatment, injectable hydrogels have gained growing attention in drug delivery application. For load-bearing tissue, the excellent mechanical property is an important requirement for delivery vehicles to resist external stress and loads. Herein, we prepared mechanically robust injectable hydrogels (HA/γ-PGA hydrogels for short) using methacrylate-functionalized hyaluronic acid and poly (γ-glutamic acid) via photopolymerization. The HA/γ-PGA hydrogels showed outstanding anti-compression ability and could suffer a more than 80% strain. Meanwhile, after 5 cycles of compression, HA/γ-PGA hydrogels could still recover quickly against external stress, showing excellent shape recovery capability. Moreover, the mechanical properties could be modulated easily by changing the molar ratio of HA to γ-PGA. The drug release behavior was also evaluated and the drug-loaded HA/γ-PGA hydrogels showed a weak burst release and sustained release behavior. Additionally, HA/γ-PGA hydrogels also exhibited superior biocompatibility. Therefore, HA/γ-PGA hydrogels have great potential as injectable drug carriers for load-bearing tissue application.     

In vivo response to dynamic hyaluronic acid hydrogels

Tissue-specific elasticity arises in part from developmental changes in extracellular matrix over time, e.g. ～10-fold myocardial stiffening in the chicken embryo. When this time-dependent stiffening has been mimicked in vitro with thiolated hyaluronic acid (HA-SH) hydrogels, improved cardiomyocyte maturation has been observed. However, host interactions, matrix polymerization, and the stiffening kinetics remain uncertain in vivo, and each plays a critical role in therapeutic applications using HA-SH. Hematological and histological analysis of subcutaneously injected HA-SH hydrogels showed minimal systemic immune response and host cell infiltration. Most importantly, subcutaneously injected HA-SH hydrogels exhibited time-dependent porosity and stiffness changes at a rate similar to hydrogels polymerized in vitro. When injected intramyocardially host cells begin to actively degrade HA-SH hydrogels within 1 week post-injection, continuing this process while producing matrix to nearly replace the hydrogel within 1 month post-injection. While non-thiolated HA did not degrade after injection into the myocardium, it also did not elicit an immune response, unlike HA-SH, where visible granulomas and macrophage infiltration were present 1 month post-injection, likely due to reactive thiol groups. Altogether these data suggest that the HA-SH hydrogel responds appropriately in a less vascularized niche and stiffens as had been demonstrated in vitro, but in more vascularized tissues, in vivo applicability appears limited.     

In situ gold nanoparticles formation: contrast agent for dental optical coherence tomography

In this work we demonstrate the potential use of gold nanoparticles as contrast agents for the optical coherence tomography (OCT) imaging technique in dentistry. Here, a new in situ photothermal reduction procedure was developed, producing spherical gold nanoparticles inside dentinal layers and tubules. Gold ions were dispersed in the primer of commercially available dental bonding systems. After the application and permeation in dentin by the modified adhesive systems, the dental bonding materials were photopolymerized concurrently with the formation of gold nanoparticles. The gold nanoparticles were visualized by scanning electron microscopy (SEM). The SEM images show the presence of gold nanospheres in the hybrid layer and dentinal tubules. The diameter of the gold nanoparticles was determined to be in the range of 40 to 120 nm. Optical coherence tomography images were obtained in two- and three-dimensions. The distribution of nanoparticles was analyzed and the extended depth of nanosphere production was determined. The results show that the OCT technique, using in situ formed gold nanoparticles as contrast enhancers, can be used to visualize dentin structures in a non-invasive and non-destructive way.     

Evaluation of an in situ chemically crosslinked hydrogel as a long-term vitreous substitute material

Currently there is no material that can be used as a long-term vitreous substitute, and this remains an unmet clinical need in ophthalmology. In this study, we developed an injectable, in situ chemically crosslinked hydrogel system and evaluated it in a rabbit model. The system consisted of two components, both based on multi-functional poly(ethylene glycol) (PEG) but with complementarily reactive end groups of thiol and active vinyl groups, respectively. The two components are mixed and injected as a solution mixture, react in vivo via the Michael addition route and form a chemically crosslinked hydrogel in situ. The linkages between the end groups and the backbone PEG chains are specially designed to ensure that the final network structure is hydrolysis-resistant. In the rabbit study and with an optimized operation protocol, we demonstrated that the hydrogel indeed formed in situ after injection, and remained transparent and stable during the study period of 9 months without significant adverse reactions. In addition, the hydrogel formed in situ showed rheological properties very similar to the natural vitreous. Therefore, our study demonstrated that this in situ chemically crosslinked PEG gel system is suitable as a potential long-term vitreous substitute.     

In situ formation of antigen-antibody complexes in the mouse glomerulus.

Intravenous administration of heat-aggregated bovine serum albumin (BSA) to mice resulted in localization of micro-particulate BSA in the mesangial region of the glomerulus. Subsequent intravenous administration of rabbit anti-BSA antibody at a time when no free antigen remained in the circulation produced localization of antibody and fixation of mouse complement within the glomerulus in a pattern corresponding to that of the heat-aggregated BSA. This apparent in situ formation of immune complexes in the glomerulus is similar to that of the classical Arthus phenomenon in the skin.     

In situ formation and collagen-alginate composite encapsulation of pancreatic islet spheroids

In this study, we suggest in situ islet spheroid formation and encapsulation on a single platform without replating as a method for producing mono-disperse spheroids and minimizing damage to spheroids during encapsulation. Using this approach, the size of spheroid can be controlled by modulating the size of the concave well. Here, we used 300 μm concave wells to reduce spheroid size and thereby eliminating the central necrosis caused by large volume. As the encapsulation material, we used alginate and collagen-alginate composite (CAC), and evaluated their suitability through diverse in vitro tests, including measurements of viability, oxygen consumption rate (OCR), hypoxic damage to encapsulated spheroids, and insulin secretion. For in situ encapsulation, alginate or CAC was spread over a concave microwell array containing spheroids, and CaCl₂ solution was diffused through a nano-porous dialysis membrane to achieve uniform polymerization, forming convex structures. By this process, the formation of uniform-size islet spheroids and their encapsulation without an intervening replating step was successfully performed. As a control, intact islets were evaluated concurrently. The in vitro test demonstrated excellent performance of CAC-encapsulated spheroids, and on the basis of these results, we transplanted the islet spheroids-encapsulated with CAC into the intraperitoneal cavity of mice with induced diabetes for 4 weeks, and evaluated subsequent glucose control. Intact islets were also transplanted as control to investigate the effect of encapsulation. Transplanted CAC-encapsulated islet spheroids maintained glucose levels below 200 mg/dL for 4 weeks, at which they were still active. At the end of the implantation experiment, we carried out intraperitoneal glucose tolerance test (IPGTT) in mice to investigate whether the implanted islets remained responsive to glucose. The glucose level in mice with CAC-encapsulated islet spheroids dropped below 200 mg/dL 60 min after glucose injection and was stably maintained. In conclusion, the proposed encapsulation method enhances the viability and function of islet spheroids, and protects these spheroids from immune attack.