Strategies for zonal cartilage repair using hydrogels

Articular cartilage is a highly hydrated tissue with depth-dependent cellular and matrix properties that provide low-friction load bearing in joints. However, the structure and function are frequently lost and there is insufficient repair response to regenerate high-quality cartilage. Several hydrogel-based tissue-engineering strategies have recently been developed to form constructs with biomimetic zonal variations to improve cartilage repair. Modular hydrogel systems allow for systematic control over hydrogel properties, and advanced fabrication techniques allow for control over construct organization. These technologies have great potential to address many unanswered questions involved in prescribing zonal properties to tissue-engineered constructs for cartilage repair.

Combining electrospun scaffolds with electrosprayed hydrogels leads to three-dimensional cellularization of hybrid constructs

A common problem in the design of tissue engineered scaffolds using electrospun scaffolds is the poor cellular infiltration into the structure. To tackle this issue, three approaches to scaffold design using electrospinning were investigated: selective leaching of a water-soluble fiber phase (poly ethylene oxide (PEO) or gelatin), the use of micron-sized fibers as the scaffold, and a combination of micron-sized fibers with codeposition of a hyaluronic acid-derivative hydrogel, Heprasil. These designs were achieved by modifying a conventional electrospinning system with two charged capillaries and a rotating mandrel collector. Three types of scaffolds were fabricated: medical grade poly(epsilon-caprolactone)/collagen (mPCL/Col) cospun with PEO or gelatin, mPCL/Col meshes with micron-sized fibers, and mPCL/Col microfibers cosprayed with Heprasil. All three scaffold types supported attachment and proliferation of human fetal osteoblasts. However, selective leaching only marginally improved cellular infiltration when compared to meshes obtained by conventional electrospinning. Better cell penetration was seen in mPCL/Col microfibers, and this effect was more pronounced when Heprasil regions were present in the structure. Thus, such techniques could be further exploited for the design of cell permeable fibrous meshes for tissue engineering applications.

A study of water binding, mobility and dehydration in contact lens hydrogels using NMR

Hydrogel polymers are used for the manufacture of soft (or disposable) contact lenses worldwide today, but have a tendency to dehydrate on the eye. In vitro methods that can probe the potential for a given hydrogel polymer to dehydrate in vivo are much sought after. Nuclear magnetic resonance (NMR) has been shown to be effective in characterising water mobility and binding in similar systems (Barbieri, Quaglia et al., 1998, Larsen, Huff et al., 1990, Peschier, Bouwstra et al., 1993), predominantly through measurement of the spin-lattice relaxation time (T1), the spinspin relaxation time (T2) and the water diffusion coefficient (D). The aim of this work was to use NMR to quantify the molecular behaviour of water in a series of commercially available contact lens hydrogels, and relate these measurements to the binding and mobility of the water, and ultimately the potential for the hydrogel to dehydrate. As a preliminary study, in vitro evaporation rates were measured for a set of commercial contact lens hydrogels. Following this, comprehensive measurement of the temperature and water content dependencies of T1, T2 and D was performed for a series of commercial hydrogels that spanned the spectrum of equilibrium water content (EWC) and common compositions of contact lenses that are manufactured today. To quantify material differences, the data were then modelled based on theory that had been used for similar systems in the literature (Walker, Balmer et al., 1989, Hills, Takacs et al., 1989). The differences were related to differences in water binding and mobility. The evaporative results suggested that the EWC of the material was important in determining a material's potential to dehydrate in this way. Similarly, the NMR water self-diffusion coefficient was also found to be largely (if not wholly) determined by the WC. A specific binding model confirmed that the we was the dominant factor in determining the diffusive behaviour, but also suggested that subtle differences existed between the materials used, based on their equilibrium we (EWC). However, an alternative modified free volume model suggested that only the current water content of the material was important in determining the diffusive behaviour, and not the equilibrium water content. It was shown that T2 relaxation was dominated by chemical exchange between water and exchangeable polymer protons for materials that contained exchangeable polymer protons. The data was analysed using a proton exchange model, and the results were again reasonably correlated with EWC. Specifically, it was found that the average water mobility increased with increasing EWe approaching that of free water. The T1 relaxation was also shown to be reasonably well described by the same model. The main conclusion that can be drawn from this work is that the hydrogel EWe is an important parameter, which largely determines the behaviour of water in the gel. Higher EWe results in a hydrogel with water that behaves more like bulk water on average, or is less strongly 'bound' on average, compared with a lower EWe material. Based on the set of materials used, significant differences due to composition (for materials of the same or similar water content) could not be found. Similar studies could be used in the future to highlight hydrogels that deviate significantly from this 'average' behaviour, and may therefore have the least/greatest potential to dehydrate on the eye.

The influence of fibrin based hydrogels on the chondrogenic differentiation of human bone marrow stromal cells

Mesenchymal Stem Cells (MSC) are frequently incorporated into osteochondral implants and cell seeding is often facilitated with hydrogels which exert a profound influence on the chondrogenic differentiation of MSC. An attempt was made to elucidate this effect by comparing the chondrogenic differentiation of Bone Marrow Stromal Cells (BMSC) in fibrin and fibrin alginate composites. A biphasic osteochondral model which simulated the native in vivo environment was employed in the study. In the first stage of the experiment, BMSC was encapsulated in fibrin, Fibrin Alginate 0.3% (FA0.3) and 0.6% (FA0.6). Chondrogenic differentiation within these cell-hydrogel pellets was compared against that of standard cell pellets under inductive conditions and the matrices which supported chondrogenesis were used in the cartilage phase of biphasic constructs. Neo-cartilage growth was monitored in these cocultures. It was observed that hydrogel encapsulation influenced mesenchymal condensation which preceded chondrogenic differentiation. Early cell agglomeration was observed in fibrin as compared to fibrin alginate composites. These fibrin encapsulated cells differentiated into chondrocytes which secreted aggrecan and collagen II. When the alginate content rose from 0.3 to 0.6%, chondrogenic differentiation declined with a reduction in the expression of collagen II and aggrecan. Fibrin and FA0.3 were tested in the cartilage phase of the biphasic osteochondral constructs and the former supported superior cartilage growth with higher cellularity, total Glycosaminoglycan (GAG) and collagen II levels. The FA0.3 cartilage phase was found to be fragmented and partially calcified. The use of fibrin for cartilage repair was advocated as it facilitated BMSC chondrogenesis and cartilaginous growth in an osteochondral environment.

Reinforced hydrogels for silicone copolymer delivery for scar remediation

Hypertrophic scars are formed by collagen overproduction in wounded areas and often occur in victims of severe burns. There are several methods for hypertrophic scar remediation and silicone gel therapy is one of the more successful methods. Research by others has shown that the activity of these gels may be due to migration of amphiphilic silicone oligomers from the gel and into the dermis, down-regulating production of collagen by fibroblasts. Normal silicone oil (PDMS) does not produce the same effect on fibroblasts. The main purpose of this project is the introduction of a particular amphiphilic silicone rake copolymer into an appropriate network which can absorb and release the silicone copolymer on the scarred area. Hydrogels are polymeric crosslinked networks which can swell in water or a drug solution, and gradually release the drug when applied to the skin. The application of gel enhances the effectiveness of the therapy, reduces the period of treatment and can be comfortable for patients to use. Polyethylene glycol (PEG) based networks have been applied in this research, because the amphiphilic silicone rake copolymer to be used as a therapy has polyethylene oxide (PEO) as a side chain. These PEO side chains have very similar chemical structure to a PEG gel chain so enhancing both the compatibility and the diffusion of the amphiphilic silicone rake copolymer into and out of the gel. Synthesis of PEG-based networks has been performed by two methods: in situ silsesquioxane formation as crosslink with a sol-gel reaction under different conditions and UV curing. PEG networks have low mechanical properties which is a fundamental limitation of the polymer backbone. For mechanical properties enhancement, composite networks were synthesized using nano-silica with different surface modification. The chemical structure of in situ silsesquioxane in the dry network has been examined by Solid State NMR, Differential Scanning Calorimetry (DSC) and swelling measurements in water. Mechanical properties of dry networks were tested by Dynamic Mechanical Thermal Analysis (DMTA) to determine modulus and interfacial interaction between silica and the network. In this way a family of self-reinforced networks has been produced that have been shown to absorb and deliver the active amphiphilic silicone- PEO rake copolymer.

Hylaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues : in vitro and in vivo evaluation in a PLDLLA-TCP-PCL-composite system

Smart matrices are required in bone tissueengineered grafts that provide an optimal environment for cells and retain osteo-inductive factors for sustained biological activity. We hypothesized that a slow-degrading heparin-incorporated hyaluronan (HA) hydrogel can preserve BMP-2; while an arterio–venous (A–V) loop can support axial vascularization to provide nutrition for a bioartificial bone graft. HA was evaluated for osteoblast growth and BMP-2 release. Porous PLDLLA–TCP–PCL scaffolds were produced by rapid prototyping technology and applied in vivo along with HA-hydrogel, loaded with either primary osteoblasts or BMP-2. A microsurgically created A–V loop was placed around the scaffold, encased in an isolation chamber in Lewis rats. HA-hydrogel supported growth of osteoblasts over 8 weeks and allowed sustained release of BMP-2 over 35 days. The A–V loop provided an angiogenic stimulus with the formation of vascularized tissue in the scaffolds. Bone-specific genes were detected by real time RT-PCR after 8 weeks. However, no significant amount of bone was observed histologically. The heterotopic isolation chamber in combination with absent biomechanical stimulation might explain the insufficient bone formation despite adequate expression of bone-related genes. Optimization of the interplay of osteogenic cells and osteo-inductive factors might eventually generate sufficient amounts of axially vascularized bone grafts for reconstructive surgery.

Investigation of electrophoretic loading and enhanced mechanical properties of hydrogels for delivery of therapeutic proteins

Hydrogels, which are three-dimensional crosslinked hydrophilic polymers, have been used and studied widely as vehicles for drug delivery due to their good biocompatibility. Traditional methods to load therapeutic proteins into hydrogels have some disadvantages. Biological activity of drugs or proteins can be compromised during polymerization process or the process of loading protein can be really timeconsuming. Therefore, different loading methods have been investigated. Based on the theory of electrophoresis, an electrochemical gradient can be used to transport proteins into hydrogels. Therefore, an electrophoretic method was used to load protein in this study. Chemically and radiation crosslinked polyacrylamide was used to set up the model to load protein electrophoretically into hydrogels. Different methods to prepare the polymers have been studied and have shown the effect of the crosslinker (bisacrylamide) concentration on the protein loading and release behaviour. The mechanism of protein release from the hydrogels was anomalous diffusion (i.e. the process was non-Fickian). The UV-Vis spectra of proteins before and after reduction show that the bioactivities of proteins after release from hydrogel were maintained. Due to the concern of cytotoxicity of residual monomer in polyacrylamide, poly(2-hydroxyethyl- methacrylate) (pHEMA) was used as the second tested material. In order to control the pore size, a polyethylene glycol (PEG) porogen was introduced to the pHEMA. The hydrogel disintegrated after immersion in water indicating that the swelling forces exceeded the strength of the material. In order to understand the cause of the disintegration, several different conditions of crosslinker concentration and preparation method were studied. However, the disintegration of the hydrogel still occurred after immersion in water principally due to osmotic forces. A hydrogel suitable for drug delivery needs to be biocompatible and also robust. Therefore, an approach to improving the mechanical properties of the porogen-containing pHEMA hydrogel by introduction of an inter-penetrating network (IPN) into the hydrogel system has been researched. A double network was formed by the introduction of further HEMA solution into the system by both electrophoresis and slow diffusion. Raman spectroscopy was used to observe the diffusion of HEMA into the hydrogel prior to further crosslinking by ã-irradiation. The protein loading and release behaviour from the hydrogel showing enhanced mechanical property was also studied. Biocompatibility is a very important factor for the biomedical application of hydrogels. Different hydrogels have been studied on both a three-dimensional HSE model and a HSE wound model for their biocompatibilities. They did not show any detrimental effect to the keratinocyte cells. From the results reported above, these hydrogels show good biocompatibility in both models. Due to the advantage of the hydrogels such as the ability to absorb and deliver protein or drugs, they have potential to be used as topical materials for wound healing or other biomedical applications.