Interleukin-1 receptor antagonist delivered directly and by gene therapy inhibits matrix degradation in the intact degenerate human intervertebral disc: an in situ zymographic and gene therapy study

Data implicate IL-1 in the altered matrix biology that characterizes human intervertebral disc (IVD) degeneration. In the current study we investigated the enzymic mechanism by which IL-1 induces matrix degradation in degeneration of the human IVD, and whether the IL-1 inhibitor IL-1 receptor antagonist (IL-1Ra) will inhibit degradation. A combination of in situ zymography (ISZ) and immunohistochemistry was used to examine the effects of IL-1 and IL-1Ra on matrix degradation and metal-dependent protease (MDP) expression in explants of non-degenerate and degenerate human IVDs. ISZ employed three substrates (gelatin, collagen, casein) and different challenges (IL-1β, IL-1Ra and enzyme inhibitors). Immunohistochemistry was undertaken for MDPs. In addition, IL-1Ra was introduced into degenerate IVD explants using genetically engineered constructs. The novel findings from this study are: IL-1Ra delivered directly onto explants of degenerate IVDs eliminates matrix degradation as assessed by multi-substrate ISZ; there is a direct relationship between matrix degradation assessed by ISZ and MDP expression defined by immunohistochemistry; single injections of IVD cells engineered to over-express IL-1Ra significantly inhibit MDP expression for two weeks. Our findings show that IL-1 is a key cytokine driving matrix degradation in the degenerate IVD. Furthermore, IL-1Ra delivered directly or by gene therapy inhibits IVD matrix degradation. IL-1Ra could be used therapeutically to inhibit degeneration of the IVD

Bi-Directional Exchange of Membrane Components Occurs during Co-Culture of Mesenchymal Stem Cells and Nucleus Pulposus Cells

Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells

Collagen-like Osteoclast-Associated Receptor (OSCAR)-Binding Motifs Show a Co-Stimulatory Effect on Osteoclastogenesis in a Peptide Hydrogel System

Osteoclastogenesis, one of the dynamic pathways underlying bone remodelling, is a complex process that includes many stages. This complexity, while offering a wealth of therapeutic opportunities, represents a substantial challenge in unravelling the underlying mechanisms. As such, there is a high demand for robust model systems to understand osteoclastogenesis. Hydrogels seeded with osteoclast precursors and decorated with peptides or proteins mimicking bone’s extracellular matrix could provide a useful synthetic tool to study pre-osteoclast-matrix interactions and their effect on osteoclastogenesis. For instance, fibrillar collagens have been shown to provide a co-stimulatory pathway for osteoclastogenesis through interaction with the osteoclast-associated receptor (OSCAR), a regulator of osteoclastogenesis expressed on the surface of pre-osteoclast cells. Based on this rationale, here we design two OSCAR-binding peptides and one recombinant OSCAR-binding protein, and we combine them with peptide-based hydrogels to study their effect on osteoclastogenesis. The OSCAR-binding peptides adopt the collagen triple-helical conformation and interact with OSCAR, as shown by circular dichroism spectropolarimetry and surface plasmon resonance. Furthermore, they have a positive effect on osteoclastogenesis, as demonstrated by appropriate gene expression and tartrate-resistant acid phosphatase staining typical of osteoclast formation. Combination of the OSCAR-binding peptides or the OSCAR-binding recombinant protein with peptide-based hydrogels enhances osteoclast differentiation when compared to the non-modified hydrogels, as demonstrated by multi-nucleation and by F-actin staining showing a characteristic osteoclast-like morphology. We envisage that these hydrogels could be used as a platform to study osteoclastogenesis and, in particular, to investigate the effect of costimulatory pathways involving OSCAR

Down-regulation of human osteoblast PTH/PTHrP receptor mRNA in end-stage renal failure

Down-regulation of human osteoblast PTH/PTHrP receptor mRNA in end-stage renal failure.BackgroundResistance to the action of parathyroid hormone (PTH) has been demonstrated in end-stage renal failure and is considered to be important in the pathogenesis of secondary hyperparathyroidism. The mechanism of resistance is unknown. However, altered regulation of cellular PTH/PTH-related protein (PTH/PTHrP) receptor (PTH1R) has been assumed to be important.MethodsWe have used in situ hybridization to examine PTH1R mRNA expression by osteoblasts in human bone and have compared the expression in high- and low-turnover renal bone disease, high-turnover nonrenal bone disease (healing fracture callus and Pagetic bone), and normal bone. Bone biopsies were formalin fixed, ethylenediaminetetraacetic acid decalcified, and paraffin wax embedded. A 1.8kb PTH1R cDNA probe, labeled with 35S, was used, and the hybridization signal was revealed by autoradiography. The density of signal over osteoblasts was quantitated using a semiautomated Leica™ image analysis software package.ResultsThe mean density of PTH1R mRNA signal over osteoblasts in renal high-turnover bone was only 36% of that found in nonrenal high-turnover bone (P < 0.05) and 51% of that found in normal bone (P < 0.05). Osteoblast PTH1R mRNA signal in adynamic bone from individuals with diabetes mellitus was 28% of normal bone (P < 0.05) and 54% of that found in renal high-turnover bone (P < 0.05).ConclusionsThese results demonstrate a down-regulation of osteoblast PTH1R mRNA in end-stage renal failure in comparison to normal and high-turnover bone from otherwise healthy individuals, and provide an insight into the mechanisms of “skeletal resistance” to the actions of PTH

Mesenchymal stem cells in regenerative medicine: focus on articular cartilage and intervertebral disc regeneration

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Designing Peptide/Graphene Hybrid Hydrogels through Fine-Tuning of Molecular Interactions

A recent strategy that has emerged for the design of increasingly functional hydrogels is the incorporation of nanofillers in order to exploit their specific properties to either modify the performance of the hydrogel or add functionality. The emergence of carbon nanomaterials in particular has provided great opportunity for the use of graphene derivatives (GDs) in biomedical applications. The key challenge when designing hybrid materials is the understanding of the molecular interactions between the matrix (peptide nanofibers) and the nanofiller (here GDs) and how these affect the final properties of the bulk material. For the purpose of this work, three gelling β-sheet-forming, self-assembling peptides with varying physiochemical properties and five GDs with varying surface chemistries were chosen to formulate novel hybrid hydrogels. First the peptide hydrogels and the GDs were characterized; subsequently, the molecular interaction between peptides nanofibers and GDs were probed before formulating and mechanically characterizing the hybrid hydrogels. We show how the interplay between electrostatic interactions, which can be attractive or repulsive, and hydrophobic (and π–π in the case of peptide containing phenylalanine) interactions, which are always attractive, play a key role on the final properties of the hybrid hydrogels. The shear modulus of the hydrid hydrogels is shown to be related to the strength of fiber adhesion to the flakes, the overall hydrophobicity of the peptides, as well as the type of fibrillar network formed. Finally, the cytotoxicity of the hybrid hydrogel formed at pH 6 was also investigated by encapsulating and culturing human mesemchymal stem cells (hMSC) over 14 days. This work clearly shows how interactions between peptides and GDs can be used to tailor the mechanical properties of the resulting hydrogels, allowing the incorporation of GD nanofillers in a controlled way and opening the possibility to exploit their intrinsic properties to design novel hybrid peptide hydrogels for biomedical applications