Effects of nerve growth factor from genipin-crosslinked gelatin in polycaprolactone conduit on peripheral nerve regeneration--in vitro and in vivo

The gelatin solution crosslinked by genipin (0, 0.1, 0.5, 1.0, and 1.5% w/w) was studied as a nerve growth factor (NGF) carrier (GGp0, GGp0.1, GGp0.5, GGp1.0, and GGp1.5) in a polycaprolactone conduit in large-gap nerve regeneration. The GGp0 and GGp0.1 displayed the highest activity of PC12 cells and inhibited the reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). No cytotoxicity was found in all groups by lactate dehydrogenase (LDH) release. The NGF-releasing characters were obtained by ELISA tests. A relatively fast release rate appeared during the first 10 days and then a subsequent slower release profile followed. NGF was higher in GGp0.1 than in GGp0 and GGp0.1 after 10 days. The bioactivity of the released NGF remains the same when measuring the neurite outgrowth of PC 12 cells. Finally, the controlled-release conduits were implanted into 12-mm long sciatic nerve gaps of rats. In addition, the best site of NGF carrier was determined either by filling carrier into the conduit lumen or by sucking carrier to the conduit wall. Four and 8 weeks after implantation, morphological analysis revealed that GGp0.1 conduits had markedly larger and more number of myelin axons in the midconduit and distal nerve. Further, sucking the carrier into the conduit wall was an efficient and convenient way to prevent the regeneration of axons and vessels from being impaired by the lumen's carrier. The genipin-crosslinked gelatin is a promising carrier in producing a high release concentration and a long release period of NGF to promote the regeneration over a large-gap nerve injury.     

Genipin-crosslinked gelatin microspheres as a drug carrier for intramuscular administration: in vitro and in vivo studies

Gelatin microspheres have been widely evaluated as a drug carrier. Nevertheless, gelatin dissolves rather rapidly in aqueous environments, making the use of the polymer difficult for the production of long-term delivery systems. This adverse aspect requires the use of a crosslinking agent in forming nonsoluble networks in microspheres. However, the use of crosslinking agents such as formaldehyde and glutaraldehyde can lead to toxic side effects owing to residual crosslinkers. In an attempt to overcome this problem, a naturally occurring crosslinking agent (genipin) was used to crosslink gelatin microspheres as a biodegradable drug-delivery system for intramuscular administration. Glutaraldehyde was used as a control. In the in vitro study, the morphology, dynamic swelling, and antienzymatic degradation of test microspheres were evaluated. In the in vivo study, the biocompatibility and degradability of test microspheres were implanted in the skeletal muscle of a rat model via intramuscular injection. The results obtained in the study suggested that crosslinking of gelatin microspheres with glutaraldehyde or genipin may produce distinct crosslinking structures. The water transport mechanism in both the glutaraldehyde- and genipin-crosslinked gelatin microspheres exhibit anomalous behavior ranging from Fickian to Case-II extremes. The increase of the swelling diameter for the genipin-crosslinked microspheres was significantly less than that observed for the glutaraldehyde-crosslinked microspheres. In the animal study, it was found that the degree in inflammatory reaction for tissues implanted with the genipin-crosslinked microspheres was significantly less than that implanted with the glutaraldehyde-crosslinked microspheres. Additionally, the degradation rate of the genipin-crosslinked microspheres was significantly slower than their glutaraldehyde-crosslinked counterparts. These results indicated that the genipin-crosslinked gelatin microspheres may be used as a long-acting drug carrier for intramuscular administration.     

Flexible chitin films as potential wound-dressing materials: wound model studies

Chitin films possessing increased flexibility, softness, transparency, and conformability have been prepared. These attributes enable the potential application of chitin films as occlusive, semipermeable film wound dressings similar to commercial products such as Opsite trade mark. The chitin films are generally nonabsorbent, exhibiting a total weight gain of only up to 120-160% in physiological fluid. Dry chitin films transpire water vapor at a rate of about 600 g/m(2)/24 h, similar to commercial polyurethane-based film dressings, but rises to 2400 g/m(2)/24 h, when wet, which is higher than the water vapor transmission rate of intact skin. The chitin films are nontoxic to human skin fibroblasts, maintaining 70-80% cell viability. Wound studies using a rat model showed no signs of allergenicity or the high inflammatory response associated with biodegradable biomaterials. The chitin films displayed accelerated wound-healing properties. Based on histological examination, wound sites dressed with the chitin films stabilized and healed faster, and appeared stronger than those dressed with Opsite trade mark and gauze dressings after 7 days of healing.     

Growth factor sequestration and enzyme-mediated release from genipin-crosslinked gelatin microspheres

Controlled release of growth factors allows the efficient, localized, and temporally-optimized delivery of bioactive molecules to potentiate natural physiological processes. This concept has been applied to treatments for pathological states, including chronic degeneration, wound healing, and tissue regeneration. Peptide microspheres are particularly suited for this application because of their low cost, ease of manufacture, and interaction with natural remodeling processes active during healing. The present study characterizes gelatin microspheres for the entrapment and delivery of growth factors, with a focus on tailored protein affinity, loading capacity, and degradation-mediated release. Genipin crosslinking in PBS and CHES buffers produced average microsphere sizes ranging from 15 to 30 microns with population distributions ranging from about 15 to 60 microns. Microsphere formulations were chosen based on properties important for controlled transient and spatial delivery, including size, consistency, and stability. The microsphere charge affinity was found to be dependent on gelatin type, with type A (GelA) carriers consistently having a lower negative charge than equivalent type B (GelB) carriers. A higher degree of crosslinking, representative of primary amine consumption, resulted in a greater negative net charge. Gelatin type was found to be the strongest determinant of degradation, with GelA carriers degrading at higher rates versus similarly crosslinked GelB carriers. Growth factor release was shown to depend upon microsphere degradation by proteolytic enzymes, while microspheres in inert buffers showed long-term retention of growth factors. These studies illuminate fabrication and processing parameters that can be used to control spatial and temporal release of growth factors from gelatin-based microspheres.     

An in vivo study of hydrogels based on physiologically clotted fibrin-gelatin composites as wound-dressing materials

In this study we have reported the efficacy of three biomaterials: (a) physiologically clotted fibrin-gelatin composite (PFG), (b) PFG graft copolymerized with 2-hydroxyethyl methacrylate (PFG-HEMA), and (c) PFG graft copolymerized with 2-hydroxypropyl methacrylate (PFG-HPMA) as temporary wound-dressing materials using the rat as an animal model. Full-thickness excision wounds were made on the back of female rats weighing about 150 +/- 10 g. The dressings were applied on the wounds and changed periodically at an interval of 4 days with the respective materials. The wounds treated with PFG-HEMA healed completely on 15th day after wound creation, whereas those treated with PFG and PFG-HPMA resulted in complete healing on the 17th day. The concentrations of collagen, hexosamine, and uronic acid in the granulation tissue were determined. The PFG and its graft copolymers acted as hydrogels, thereby absorbing excess exudates, while still maintaining a moist environment at the wound site. The enhanced wound healing in the experimental animals was reflected in the increased rate of wound contraction. The results of the histological and mechanical studies of the experimental groups revealed that reepithelialization and remodeling of the skin have been achieved by providing a moist environment at the wound site by the biomaterials and thereby hastening the migration of keratinocytes.     

Preparation and characterization of gelatin/hyaluronic acid cryogels for adipose tissue engineering: In vitro and in vivo studies

Macroporous elastic scaffolds containing gelatin (4% or 10%) and 0.25% hyaluronic acid (HA) were fabricated by cryogelation for application in adipose tissue engineering. These cryogels have interconnected pores (～200 μm), high porosity (>90%) and a high degree of cross-linking (>99%). The higher gelatin concentration reduced the pore size, porosity and swelling ratio of the cryogel but improved its swelling kinetics. Compressive mechanical testing of cryogel samples demonstrated non-linear stress–strain behavior and hysteresis loops during loading–unloading cycles, but total recovery from large strains. The presence of more gelatin increased the elastic modulus, toughness and storage modulus and yielded a cryogel that was highly elastic, with a loss tangent equal to 0.03. Porcine adipose-derived stem cells (ADSCs) were seeded in the cryogel scaffolds to assess their proliferation and differentiation. In vitro studies demonstrated a good proliferation rate and the adipogenic differentiation of the ADSCs in the cryogel scaffolds, as shown by their morphological change from a fibroblast-like shape to a spherical shape, decreased actin cytoskeleton content, growth arrest, secretion of the adipogenesis marker protein leptin, Oil Red O staining for triglycerides and expression of early (LPL and PPARγ) and late (aP2 and leptin) adipogenic marker genes. In vivo studies of ADSCs/cryogel constructs implanted in nude mice and pigs demonstrated adipose tissue and new capillary formation, the expression of PPARγ, leptin and CD31 in immunostained explants, and the continued expression of adipocyte-specific genes. Both the in vitro and in vivo studies indicated that the gelatin/HA cryogel provided a structural and chemical environment that enabled cell attachment and proliferation and supported the biological functions and adipogenesis of the ADSCs.     

A molecular dynamic analysis of gelatin as an amorphous material: prediction of mechanical properties of gelatin systems

Biomaterials are used in regenerative medicine for induced autoregeneration and tissue engineering. This is often challenging, however, due to difficulties in tailoring and controlling the respective material properties. Since functionalization is expected to offer better control, in this study gelatin chains were modified with physically interacting groups based on tyrosine with the aim of causing the formation of physical crosslinks. This method permits application-specific properties like swelling and better tailoring of mechanical properties. The design of the crosslink strategy was supported by molecular dynamic (MD) simulations of amorphous bulk models for gelatin and functionalized gelatins at different water contents (0.8 and 25 wt.-%). The results permitted predictions to be formulated about the expected crosslink density and its influence on equilibrium swelling behavior and on elastic material properties. The models of pure gelatin were used to validate the strategy by comparison between simulated and experimental data such as density, backbone conformation angle distribution, and X-ray scattering spectra. A key result of the simulations was the prediction that increasing the number of aromatic functions attached to the gelatin chain leads to an increase in the number of physical netpoints observed in the simulated bulk packing models. By comparison with the Flory-Rehner model, this suggested reduced equilibrium swelling of the functionalized materials in water, a prediction that was subsequently confirmed by our experimental work. The reduction and control of the equilibrium degree of swelling in water is a key criterion for the applicability of functionalized gelatins when used, for example, as matrices for induced autoregeneration of tissues.     

Heparinized polyurethanes: in vitro and in vivo studies

Heparin immobilization chemistry using alkyl spacer arms was adapted to optimize yield on polyurethane (PU) surfaces. The resultant biological activity of immobilized heparin (HI) was examined in vitro and in vivo, and compared with a heparin releasing (HR) system. Immobilized heparin retained its ability to bind and inactivate thrombin and Factor Xa; nonspecific coagulation factor binding was insignificant. Such activity cannot be attributed to the leakage of improperly bound heparin. Immobilized heparin-polyurethane catheters implanted in canine femoral and jugular veins for 1 h periods exhibited significant reduction in thrombus formation compared with untreated PU contralateral controls. Polyurethane catheters coated with a 9% heparin dispersion in PU (HR) system provided even greater improvement in antithrombogenicity.     

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.     

A fully degradable tracheal stent: in vitro and in vivo characterization of material degradation

We report on the testing of materials for a fully degradable tracheal stent. Such a stent has several advantages over currently used permanent stents made of metal or silicone polymers. However, the mode of degradation in the trachea is expected to be different from a fully submerged device, because of the uniqueness of the tracheal environment. A physical model was developed to allow an in-depth study of degradation of bioabsorbable polymers exposed to two differing media; namely 70 wt % water (gel) on one side and humidified air on the other, simulating conditions in a tracheal passage. Longitudinal microtome slices were obtained from both polymer surfaces and degradation kinetics data were derived from size exclusion chromatography. On the basis of the data obtained, it is observed that well-studied bulk-degrading polymers might show surface-eroding properties in such an environment. Generally, hydrophobic polymers retard the formation of a water concentration gradient and exhibit bulk-degradation kinetics. However, addition of specific plasticizers can influence the water uptake gradient, and force the polymer towards a pseudo "surface-eroding" behavior. In vivo studies in a rabbit model of degradable stents made from a selected polymer, demonstrate the feasibility of a fully bioabsorbable tracheal stent. This study aims to improve understanding of degradation of polymers under heterogeneous environments.     

Self-crosslinkable hydrogels composed of partially oxidized hyaluronan and gelatin: in vitro and in vivo responses

Self-crosslinkable hydrogels had been formulated from two precursors, partially oxidized hyaluronan (oHA) and gelatin. The physicochemical properties of the resulting hydrogels have been elucidated by instrumental analyses (FTIR, SEM, and rheometry). These hydrogels were highly porous with an average pore size of 60 microm, and evidently, accommodative to cell infiltration. Increasing the oxidation degree of oHA resulted in corresponding increases in hydrogels' storage moduli and decreases in water uptake. Dermal fibroblasts were used to study the cell-hydrogel interactions in vitro. Both the hydrogels and their degradation byproducts are biocompatible as indicated by long-term cell viability assay. In addition, significant amount of cells migrated into the hydrogels and they aligned into highly organized arrays. When cultured with cells, the hydrogels underwent degradation within 4 weeks depending on composition with obvious loss of cohesiveness over time. The good biocompatibility and biodegradability of oHA/gelatin hydrogel were further demonstrated in mice subdermal implantations. Lastly, in vitro and in vivo depositions of extracellular matrix in hydrogels by cells were demonstrated by SEM analyses.     

Isocyanate-terminated urethane prepolymer as bioadhesive material: evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays

Attempts have been made to evaluate the degree of bioadhesion and biocompatibility of a synthesized urethane prepolymer with specially tailored microstructure. Wetting behaviour and extent of interfacial adhesion of the prepared prepolymer towards biological substrates were examined by in vitro methods. The former was carried out by measuring the contact angle between drops of the prepolymer liquid and a biological surface, while the latter was determined from the force between the prepolymer and tissue model or mucus. The obtained results exhibited good tissue wettability and bioadhesion by the prepolymer. Preliminary evaluation of biocompatibility for the uncatalytically cured prepolymer films was performed by cytotoxicity and histotoxicity experiments. Results showed a significant growth for the adhered L929 fibroblast cells within a period of 5 days incubation. Also, no severe inflammatory tissue response towards the samples implanted in rabbit for 16 weeks was seen. These observations can support the potentiality of the designed urethane prepolymer to be applied as hemostatic agent.     

Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility,in vitro and in vivo assays

Attempts have been made to evaluate the degree of bioadhesion and biocompatibility of a synthesized urethane prepolymer with specially tailored microstructure. Wetting behaviour and extent of interfacial adhesion of the prepared prepolymer towards biological substrates were examined by in vitro methods. The former was carried out by measuring the contact angle between drops of the prepolymer liquid and a biological surface, while the latter was determined from the force between the prepolymer and tissue model or mucus. The obtained results exhibited good tissue wettability and bioadhesion by the prepolymer. Preliminary evaluation of biocompatibility for the uncatalytically cured prepolymer films was performed by cytotoxicity and histotoxicity experiments. Results showed a significant growth for the adhered L929 fibroblast cells within a period of 5 days incubation. Also, no severe inflammatory tissue response towards the samples implanted in rabbit for 16 weeks was seen. These observations can support the potentiality of the designed urethane prepolymer to be applied as hemostatic agent.     

Biodegradation of chitosan-tripolyphosphate beads: in vitro and in vivo studies

In this study, chitosan [(1 --> 4) linked 2-amino-2-deoxy-beta-D-glucopyranose] beads were prepared by interacting this polycation (> 90% deacetylated) with the tripolyphosphate (TPP) polyanion. The resulting chitosan-TPP beads (C) were modified either by coating with sodium alginate (CA) or by cross-linking with glutaraldehyde (CGA). The in vitro degradation of C beads was found to be faster than its CA and CGA counterparts. C beads degraded faster at pH 6.5, compared to pH 7.4 conditions. At pH 7.4, about 41%, 37% and 10% of dry mass loss after 12 months was determined for C, CA and CGA, respectively. At pH 6.5, the dry mass loss of CA and CGA after the same period of time was found to be 73% and 37%, respectively. However, C beads completely degraded at pH 6.5 after 8 months of in vitro incubation. The in vivo biodegradation experiments were performed on Wistar rats (n = 24) for a duration of 6 months. No sign of fibrotic capsule formation was observed around any of the implanted beads at 2 and 6 months post-transplantation. At 2 months, the in vivo-degradation was slow-going and the beads in all groups were intact; CGA beads had more tissue reaction than C and CA beads at this time point. While the C beads had almost completely degraded after 6 months, the biodegradation process in CA and CGA beads was progressing. Histomorphometric analysis revealed that the in vivo biodegradation was in the order of C (approximately 85%) > CA (approximately 50%) > CGA (approximately 25%) after 6 months. Neovascularization was observed at the vicinity of the bead implants close to major blood vessels, both at 2 and 6 months time-points.     

Tissue engineering auricular reconstruction: in vitro and in vivo studies

Although investigators have demonstrated that neocartilage can be constituted in a predetermined shape and in complex three-dimensional structures, such as a human ear, by using cell transplantation on polymer constructs, many unsolved problems still remain. The crucial issues for auricular tissue engineering consisted of optimal cell culture environment, choice of polymers, behavior of chondrocytes, study of cell-polymer constructs in an acceptable animal model, and long-term structural integrity. Here we describe our tissue engineering approaches for auricular reconstruction including auricular scaffold fabrication, in vitro chondrogenesis, in vivo immunocompromized xenograft and immunocompetent autologous animal models, and long-term follow-up. Though many current obstacles regarding auricular tissue engineering still exist, we demonstrate techniques of auricular scaffold fabrication with promising in vitro and in vivo neocartilage formation, optimal selection and application of animal models, and, to the best of our knowledge, the first report of different biodegradable biomaterial trials and the longest in vivo results (10 months) for auricular tissue engineering.     

Extracellular matrix-enriched polymeric scaffolds as a substrate for hepatocyte cultures: in vitro and in vivo studies

Tissue engineering is a promising approach to developing hepatic tissue suitable for the functional replacement of a failing liver. The aim of the present study was to investigate whether an extracellular cell matrix obtained from fibroblasts-cultured within scaffolds of hyaluronic acid (HYAFF) could influence the proliferation rate and survival of rat hepatocytes both during long-term culture and after in vivo transplantation. Cultures were evaluated by histological and morphological analysis, a proliferation assay and metabolic activity (albumin secretion). Hepatocytes cultured in extracellular matrix-enriched scaffolds exhibited a round cellular morphology and re-established cell-cell contacts, growing into aggregates of several cells along and/or among fibers in the fabric. Hepatocytes were able to secrete albumin up to 14 days in culture. In vivo results demonstrated the biocompatibility of HYAFF-11 implanted in nude mice, in which hepatocytes maintained small well-organised aggregates until the 35th day. In conclusion, the presence of a fibroblast-secreted extracellular matrix improved the biological properties of the hyaluronan scaffold, favoring the survival and morphological integrity of hepatocytes in vitro and in vivo.     

An in vivo evaluation of a biodegradable genipin-cross-linked gelatin peripheral nerve guide conduit material

We evaluated peripheral nerve regeneration using a biodegradable nerve conduit, which was made of genipin-cross-linked gelatin. The genipin-cross-linked gelatin conduit (GGC) was dark blue in appearance, which was concentric and round with a rough outer surface whereas its inner lumen was smooth. After subcutaneous implantation on the dorsal side of the rat, the GGC only evoked a mild tissue response, forming a thin tissue capsule surrounding the conduit. Biodegradability of the GGC and its effectiveness as a guidance channel were examined as it was used to repair a 10 mm gap in the rat sciatic nerve. As a result, tube fragmentation was not obvious until 6 weeks post-implantation and successful regeneration through the gap occurred in all the conduits at the three experimental periods of 4, 6, and 8 weeks. Histological observation showed that numerous regenerated nerve fibers, mostly unmyelinated and surrounded by Schwann cells, crossed through and beyond the gap region 6 weeks after operation. Peak amplitude and area under the muscle action potential curve both showed an increase as a function of the recovery period, indicating that the nerve had undergone adequate regeneration. Thus, the GGC can not only be an effective aids for regenerating nerves but can also lead to favorable nerve functional recovery.     

Porous collagen sponge wound dressings: in vivo and in vitro studies

Collagen-based materials can be formed into a three-dimensional sponge for use as a wound dressing and as a support for cell cultured skin components. Factors such as biocompatibility, morphological structure and addition of non-collagenous molecules to collagen are analyzed and discussed. Large pores or channels, interchannel communications and combinations of macromolecules of the connective tissue enhance wound tissue infiltration in vivo as well as cell growth in vitro into collagen sponges. The presence of such factors can be useful in patients with excised burn wounds and pressure skin ulcers.     

Anti-inflammatory effect of lemon mucilage: in vivo and in vitro studies

The mucilage extracted from a lemon juice centrifugation pulp was studied for its anti-inflammatory effect in rat. In vivo the lemon mucilage significantly inhibited carrageenan-induced edema in rat paw from 59% to 73.5% showing the highest effect at the third hour. In vitro, at the doses of 10^-8, 10^-6, 10^-4 or 10^-2 mg/mL the lemon mucilage stimulated the superoxide anion production in rat testing neutrophils in whole blood but inhibited it in FMLP stimulated cells at the dose of 10^-2 mg/mL. The neutrophils of rats receiving p.o. the lemon mucilage for 21 days showed a significant decrease of 45.5% in O₂^- generation after FMLP stimulation, and a not-significant increase after phorbol-12-myristate-13-acetate (PMA) or zymosan stimulation. Since the activity on zymosan- and PMA-induced O₂^- production was not significant, the inhibition exerted by FMLP in rat neutrophils occurred mainly through the blockade of phospholipase D.     

Mesenchymal progenitor cell-mediated inhibition of tumor growth in vivo and in vitro in gelatin matrix

A preformed gelatin matrix containing adherent rat colon carcinoma cells was transplanted subcutaneously into rats to analyze the outgrowth of the tumor and the inflammatory response. The gelatin matrix simplifies the precise localization of the tumor cells early after implantation and allows the gelatin piece with a growing tumor to be dissected for analysis in vitro, after various times in vivo. The immortalized mesenchymal progenitor cell line MPC1cE was cocultured with rat colon carcinoma cells in vivo in gelatin matrix. The mesenchymal progenitor cells inhibited the outgrowth of the rat colon carcinoma and a complete inhibition was seen if the number of mesenchymal progenitor cells were at least equal to the number of tumor cells. The mixture of tumor cells and mesenchymal progenitor cells induced more infiltration of monocytes and granulocytes than tumor cells or mesenchymal progenitor cells alone. Infiltration of T cells and CD31+ endothelial cells correlated to the presence of tumor cells and not to mesenchymal progenitor cells. These findings suggest that tumor cell culture in vivo in a gelatin matrix is effective for early localization of tumor cells in vivo and that mesenchymal progenitor cells effectively inhibit the growth of the tumor cells in vivo.