Osteoarthritis (OA) is a leading cause of joint pain, deformity and functional limitation. An imbalance of anabolic and catabolic activity results in destruction of the extracellular matrix of articular cartilage. While there is evidence to support the role of DNA methylation in the pathogenesis of OA, the effect of other epigenetic modifications is yet to be described. This study looks at the effect of two novel epigenetic modifiers, PFI-1, a bromodomain inhibitor, and SGC707, a histone methytransferase inhibitor, on gene expression in the pathogenesis of OA. Chondrocytes were extracted from OA femoral heads (n=6), cultured and incubated with increasing concentrations of the compounds. Cells were treated with media alone (control), interleukin 1-beta (IL-1β) plus oncostatin M (OSM) alone, or in combination with PFI-1 or SGC707. Levels of expression of iNOS, COX2, IL8, IL1B, matrix metalloproteinase-13 (MMP13), RUNX2 and COL9A1 were measured using qRT-PCR. PFI-1 (0.5 and 5µM) suppressed expression of catabolic genes in OA chondrocytes, at basal levels and when co-stimulated with IL-1β+OSM. While there was a decrease in catabolic gene expression (iNOS, COX2, IL8, IL1B and MMP13), RUNX2 expression was also supressed. There was no effect on expression of COL9A1, an anabolic chondrocytic gene. SGC707 (0.1 and 1µM) did not induce a reduction in expression of all the catabolic genes, with a less predictable effect on gene expression than PFI-1. This study has demonstrated that the BET inhibitor PFI-1 has a potent protective effect against cartilage degradation, through its action as an epigenetic modifier in modulating the expression of catabolic genes in OA chondrocytes. This further validates the role of epigenetics in OA, with potential implications for therapeutic interventions.
Tissue loss, as a result of injury or disease, provides reduced quality of life for many and with an increasingly ageing population there is a greater requirement for skeletal repair strategies. An emerging attractive approach, tissue engineering, is based on the use of an appropriate source of progenitor cells, a scaffold conducive to cell attachment and maintenance of cell function and the delivery of appropriate growth factors. As a cell source, mesenchymal stem cells (MSCs) or marrow stromal cells derived from adult human tissues offer tremendous potential for tissue regeneration. However, to date, the plasticity, multipotentiality and characteristics of potential stem cells from fetal skeletal tissue remain poorly defined. We have examined, in preliminary studies, the multipotentiality and phenotypic properties of cell populations derived from human fetal femurs collected at 8–12 weeks post-conception in comparison to adult-derived mesenchymal stem cell populations including those isolated using STRO-1 immunoselection. Fetal cells were culture expanded from explants in basal media then maintained for periods of up to 28 days in monolayer cultures in adipogenic and osteogenic conditions. Cells were also maintained in chondrogenic conditions via the pellet culture method, maintained in established media conditions including TGF-â3, with cultures taken to 7, 14, 21 and 28 days. Adipocyte formation was confirmed by morphology: large amounts of lipid accumulation were observed by Oil Red O staining and aP2 (FABP-3) immunocytochemistry. Osteogenic differentiation was also confirmed by Type I Collagen immunocytochemistry. The growth of fetal cells on biomimetic scaffolds and their osteogenic activity was confirmed by confocal microscopy and Alkaline Phosphatase staining respectively. In chondrogenic conditions, chondrocytes were embedded within lacunae and extensive matrix deposition was observed using Alcian blue/Sirius red staining. The chondrogenic phenotype was confirmed by positive staining via SOX9 immunocytochemistry. Differentiation and proliferation were accelerated in fetal populations compared to adult-derived immunoselected MSCs. Plasticity of fetal cells has been demonstrated by the formation of large numbers of adipocytes within osteogenic populations. In summary we demonstrate the proliferative and multi-potential properties of fetal-derived chondrocytic cells in direct comparison to adult-derived MSCs including STRO-1 immunoselected populations. Given the demographic challenges and ethical issues surrounding current embryonic cell research, fetal cell populations may also provide a unique half-way model to address stem cell differentiation in comparison to adult cells. Elucidation of immunogenecity and selective differentiation will confirm the potential of these fetal cells as a unique alternate cell source for therapeutic approaches in the restoration of damaged or diseased tissue.
Polysaccharide (alginate and chitosan) capsules coated with a unique self-assembled semi-crystalline shell of calcium phosphate provide an enclosed biological system for the spatial and temporal delivery of human cells and bioactive factors. The aim of this study was to demonstrate plasmid DNA entrapment, delivery and transfection of adjacent cells inside capsules, embedded capsules and plated. Bacterial plasmid DNA and/or bone cells (SaOS) was added to solution of sodium alginate solution supplemented with phosphate ions and mixed thoroughly. Alginate droplets were fed through a syringe into a solution of chitosan supplemented with calcium ions. Guest capsules were inserted into soft, pliable host capsules soon after immersion in chitosan solution. Capsules were then immersed in 2mL DMEM 10% FCS in 6-well plastic plates for up to 7 days to enable transfection to occur. Encapsulated bone cells were stained with standard X-Gal to show transfected cells expressing beta-galactosidase. DNA delivery and transfection was demonstrated within capsules containing SaOS cells and plasmid, an admixture of SaOS bone cells and plasmid (51%) and from capsules containing DNA alone suspended in media over plated SaOS one cells. We also demonstrate capsule transfection of encapsulated cells in vivo. Transfection efficiency is highest when plasmid is entrapped and released from embedded capsules followed by plasmid/ SaOS admixture within capsules and lowest efficiency was observed with plated SaOS cells (with a transfection efficiency of 5%). The ability to regulate shell decomposition by manipulating the degree of mineralization and the strength of gelling, and release of capsule contents provides a mechanism for programmed release of gene modulated cells into the biological environment. The beta-galactosidase plasmid was found to be strongly associated with the chitosan/ calcium phosphate shell as shown by ethidium-homodimer-1 staining of encapsulated DNA and this may assist the transfer from gel to cell. Programmed non-viral delivery of genes using biomaterial constructs is an important approach to gene therapy and orchestrated tissue regeneration. These unique biomineralised polysaccharide capsules provide a facile technique, and an enclosed biomimetic micro-environments with specifiable degradation characteristics, for the safe encapsulation and delivery of functional quantities of plasmid DNA with the implicit therapeutic implications therein.
Joint pain, as a consequence of cartilage degeneration or trauma results in severe pain or disability for millions of individuals worldwide. However, the potential for cartilage to regenerate is limited and there is an absence of clinically viable cartilage formation regimes. Cartilage is composed of only one cell type, is avascular and has a relatively simple composition and structure, thus cartilage tissue engineering has tremendous potential. Therefore, to address this clinical need, we have adopted a tissue engineering approach to the generation of cartilage ex vivo from mesenchymal cell populations encapsulated in polysaccharide templates form alginate and chitosan that favours chondrogenesis, and cultured within perfused or rotating bioreactor systems. To drive the chondrogenic phenotype, alginate beads were encapsulated with isolated human bone marrow cells, human articular chondrocytes or a combination of both in a 2:1 ratio, with the addition of TGF-â3, and placed in either a Synthecon rotating-wall bioreactor, perfused at a flow rate of 1ml/hour, or held in static conditions for 28 days. Alcian Blue and Sirius Red staining indicated ordered, structured and even cell distribution within capsules from the rotating bioreactor system in comparison with perfused and static conditions. Furthermore, alginate beads encapsulated with mixed cell populations that were cultured under static and rotating-wall conditions revealed positive staining for both collagen and proteoglycan, and with areas that closely resembled the formation of osteoid. Cell viability, assessed using the fluorescent dye Cell Tracker Green, indicated a higher proportion of metabolically active cells in capsules from the rotating-wall bioreactor than perfused or static under the conditions examined. Immunohistochemistry indicated the expression of type II collagen, SOX9 and C-MYC in samples from all conditions after 28 days. C-MYC is implicated in cell proliferation and differentiation and type II collagen and SOX9 are cartilage-specific markers. Biochemical analysis revealed significantly increased (p <
0.05) protein in samples encapsulated with mixed cell populations compared with alginate samples that were encapsulated with either bone marrow or chondrocytes. There was also a significant increase in protein in all samples that were cultured in the rotating-wall bioreactor in comparison with perfused or static conditions after 28 days. A significant increase in DNA was observed in the rotating-wall than perfused or static for the bone marrow cultures. Interestingly in chondrocyte cultures perfused conditions were found to result in significantly higher DNA than rotating-wall and static, and static conditions resulted in significantly higher DNA for alginate encapsulated with mixed cell populations. The current studies outline a tissue engineering approach utilising progenitor populations, bioreactors and appropriate stimuli to promote the formation of cartilage within a unique innovative polysaccharide capsule structure, and indicate the potential of rotating-wall systems to promote cartilage formation. Understanding the conditions required for the generation of functional cartilage constructs using such bioreactor systems carries significant clinical potential.
The demographic challenges of an advancing aged population emphasise the need for innovative approaches to tissue reconstruction to augment and repair tissue lost as a consequence of trauma or degeneration. Currently, the demand for bone graft outstrips supply, a key issue in the field of revision hip surgery where impaction bone grafting of the femur and acetabulum has impressive results in the short and medium term but often requires up to 6 donated femoral heads. Spine and selected tumour and trauma cases are also eminently suitable for this mode of bone stock replacement. In the current study, we examined the histological and biochemical findings of two parallel in-vitro and in-vivo studies using human mesenchymal stem cells on synthetic scaffolds for possible bone augmentation. The first study confirmed that culture expanded bone marrow cells from 3 patients (mean age 76 +/−4) could be successfully seeded onto washed morsellised allograft. The seeded graft was then exposed to a force equivalent to a standard femoral impaction (impulse=474 J/m2) and cultured for 4 weeks in osteogenic media. Examination of cell viability using cell tracker green and ethidium homodimer-1 and confocal microscopy confirmed extensive cell proliferation and viability following impaction and culture. Alcian blue/ Sirius red confirmed matrix production, alkaline phosphatase immunocytochemistry production of enzyme activity and Goldners trichrome enhanced osteoid formation. The second study compared 3 scaffolds; bone allograft, a ß – Tricalcium Phosphate (ß-TCP) graft substitute and a 50:50 mixture of allograft and ß-TCP. The scaffolds were seeded with either immunoselected STRO-1+ human mesenchymal stem cells or unselected marrow cells. The scaffolds were similarly exposed to impaction forces and cultured for 4 weeks in vitro or in vivo, implanted subcutaneously in MF1nu/nu mice. Both studies demonstrated cellular viability, activity and osteogenesis as assessed using confocal microscopy, Goldners trichrome and alcian blue/Sirius cytochemistry. The demonstration of enhanced osteoid formation as a consequence of stem cell proliferation after impaction grafting augers well for the success of autologous stem cell implantation on impacted graft substitute with or without the addition of morsellised allograft. The implications therein for clinical use in the future await clinical trials.
