Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in Vitro redifferentiation

Background: If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. ----- ----- Hypothesis: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture).----- ----- Study Design: Controlled laboratory study.----- ----- Methods: Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes).----- ----- Results: Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone.----- ----- Conclusion: Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion.

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.

Porous zirconia scaffold modified with mesoporous bioglass coating

Porous yttria-stabilized zirconia (YSZ) has been regarded as a potential candidate for bone substitute as its high mechanical strength. However, porous YSZ bodies are biologically inert to bone tissue. It is therefore necessary to introduce bioactive coatings onto the walls of the porous structures to enhance the bioactivity. In this study, the porous zirconia scaffolds were prepared by infiltration of Acrylonitrile Butadiene Styrene (ABS) scaffolds with 3 mol% yttria stabilized zirconia slurry. After sintering, a method of sol-gel dip coating was involved to make coating layer of mesoporous bioglass (MBGs). The porous zirconia without the coating had high porosities of 60.1% to 63.8%, and most macropores were interconnected with pore sizes of 0.5-0.8mm. The porous zirconia had compressive strengths of 9.07-9.90MPa. Moreover, the average coating thickness was about 7μm. There is no significant change of compressive strength for the porous zirconia with mesoporous biogalss coating. The bone marrow stromal cell (BMSC) proliferation test showed both uncoated and coated zirconia scaffolds have good biocompatibility. The scanning electron microscope (SEM) micrographs and the compositional analysis graphs demonstrated that after testing in the simulated body fluid (SBF) for 7 days, the apatite formation occurred on the coating surface. Thus, porous zirconia-based ceramics were modified with bioactive coating of mesoporous bioglass for potential biomedical applications.

Mesoporous bioactive glasses as drug delivery and bone tissue regeneration platforms

The use of mesoporous bioactive glasses (MBG) for drug delivery and bone tissue regeneration has grown significantly over the past 5 years. In this review, we highlight the recent advances made in the preparation of MBG particles, spheres, fibers and scaffolds. The advantages of MBG for drug delivery and bone scaffold applications are related to this material’s well-ordered mesopore channel structure, superior bioactivity, and the application for the delivery of both hydrophilic and hydrophobic drugs. A brief forward-looking perspective on the potential clinical applications of MBG in regenerative medicine is also discussed.

Multifunctional magnetic mesoporous bioactive glass scaffolds with a hierarchical pore structure

Hyperthermia and local drug delivery have been proposed the potential therapeutic approaches for bone defects resulting from malignant bone tumors. Development of bioactive materials with magnetic and drug-delivery properties may potentially meet this target. The aim of this study is to develop a multifunctional mesoporous bioactive glass (MBG) scaffold system for both hyperthermia and local-drug delivery application potentially. For this aim, Iron (Fe) containing MBG (Fe-MBG) scaffolds with hierarchically large pores (300-500 µm) and fingerprint-like mesopores (4.5 nm) have been successfully prepared. The effect of Fe on the mesopore structure, physiochemical, magnetism, drug delivery and biological properties of MBG scaffolds has been systematically investigated. The results showed that the morphology of the mesopore varied from straight channels to curved fingerprint-like channels after incorporated parts of Fe into MBG scaffolds. The magnetism magnitude of MBG scaffolds can be tailored by controlling Fe contents. Furthermore, the incorporating of Fe into mesoporous MBG glass scaffolds enhanced the mitochondrial activity and bone-relative gene (ALP and OCN) expression of human bone marrow mesenchymal stem cells (BMSCs) on the scaffolds. The obtained Fe-MBG scaffolds also possessed high specific surface areas and sustained drug delivery. Therefore, Fe-MBG scaffolds are magnetic, degradable and bioactive. The multifunction of Fe-MBG scaffolds indicates that there is a great potential for Fe-MBG scaffolds to be used for the therapy and regeneration of large-bone defects caused by malignant bone tumors through the combination of hyperthermia, local drug delivery and their osteoconductivity.

A cell migration device that maintains a defined surface with no cellular damage during wound edge generation

Studying the rate of cell migration provides insight into fundamental cell biology as well as a tool to assess the functionality of synthetic surfaces and soluble environments used in tissue engineering. The traditional tools used to study cell migration include the fence and wound healing assays. In this paper we describe the development of a microchannel based device for the study of cell migration on defined surfaces. We demonstrate that this device provides a superior tool, relative to the previously mentioned assays, for assessing the propagation rate of cell wave fronts. The significant advantage provided by this technology is the ability to maintain a virgin surface prior to the commencement of the cell migration assay. Here, the device is used to assess rates of mouse fibroblasts (NIH 3T3) and human osteosarcoma (SaOS2) cell migration on surfaces functionalized with various extracellular matrix proteins as a demonstration that confining cell migration within a microchannel produces consistent and robust data. The device design enables rapid and simplistic assessment of multiple repeats on a single chip, where surfaces have not been previously exposed to cells or cellular secretions.