Matthew W. Grol

Expression, signaling, and function of P2X7 receptors in bone

Nucleotides released from cells in response to mechanical stimulation or injury may serve as paracrine regulators of bone cell function. Extracellular nucleotides bind to multiple subtypes of P2 receptors on osteoblasts (the cells responsible for bone formation) and osteoclasts (cells with the unique ability to resorb mineralized tissues). Both cell lineages express the P2X7 receptor subtype. The skeletal phenotype of mice with targeted disruption of P2rx7 points to interesting roles for this receptor in the regulation of bone formation and resorption, as well as the response of the skeleton to mechanical stimulation. This paper reviews recent work on the expression of P2X7 receptors in bone, their associated signal transduction mechanisms and roles in regulating bone formation and resorption. Areas for future research in this field are also discussed.

Finite-element modeling of viscoelastic cells during high-frequency cyclic strain.

Mechanotransduction refers to the mechanisms by which cells sense and respond to local loads and forces. The process of mechanotransduction plays an important role both in maintaining tissue viability and in remodeling to repair damage; moreover, it may be involved in the initiation and progression of diseases such as osteoarthritis and osteoporosis. An understanding of the mechanisms by which cells respond to surrounding tissue matrices or artificial biomaterials is crucial in regenerative medicine and in influencing cellular differentiation. Recent studies have shown that some cells may be most sensitive to low-amplitude, high-frequency (i.e., 1–100 Hz) mechanical stimulation. Advances in finite-element modeling have made it possible to simulate high-frequency mechanical loading of cells. We have developed a viscoelastic finite-element model of an osteoblastic cell (including cytoskeletal actin stress fibers), attached to an elastomeric membrane undergoing cyclic isotropic radial strain with a peak value of 1,000 µstrain. The results indicate that cells experience significant stress and strain amplification when undergoing high-frequency strain, with peak values of cytoplasmic strain five times higher at 45 Hz than at 1 Hz, and peak Von Mises stress in the nucleus increased by a factor of two. Focal stress and strain amplification in cells undergoing high-frequency mechanical stimulation may play an important role in mechanotransduction.

P2 receptor networks regulate signaling duration over a wide dynamic range of ATP concentrations

Summary The primordial intercellular signaling molecule ATP acts through two families of cell-surface P2 receptors – the P2Y family of G-protein-coupled receptors and the P2X family of ligand-gated cation channels. Multiple P2 receptors are expressed in a variety of cell types. However, the significance of these networks of receptors in any biological system remains unknown. Using osteoblasts as a model system, we found that a low concentration of ATP (10 µM, ATPlow) induced transient elevation of cytosolic Ca2+, whereas a high concentration of ATP (1 mM, ATPhigh) elicited more sustained elevation. Moreover, graded increases in the Ca2+ signal were achieved over a remarkable million-fold range of ATP concentrations (1 nM to 1 mM). Next, we demonstrated that ATPlow caused transient nuclear localization of the Ca2+-regulated transcription factor NFATc1; whereas, ATPhigh elicited more sustained localization. When stimulated with ATPhigh, osteoblasts from P2X7 loss-of-function mice showed only transient Ca2+-NFATc1 signaling; in contrast, sustained signaling was observed in wild-type cells. Additional experiments revealed a role for P2Y receptors in mediating transient signaling induced by low ATP concentrations. Thus, distinct P2 receptors with varying affinities for ATP account for this wide range of sensitivity to extracellular nucleotides. Finally, ATPhigh, but not ATPlow, was shown to elicit robust expression of the NFAT target gene Ptgs2 (encoding COX-2), consistent with a crucial role for the duration of Ca2+-NFAT signaling in regulating target gene expression. Taken together, ensembles of P2 receptors provide a mechanism by which cells sense ATP over a wide concentration range and transduce this input into distinct cellular signals.

Reinforcement of flowable dental composites with titanium dioxide nanotubes

OBJECTIVES Flowable dental composites are used as restorative materials due to their excellent esthetics and rheology. However, they suffer from inferior mechanical properties compared to conventional composites. The aim of this study was to reinforce a flowable dental composite with TiO2 nanotubes (n-TiO2) and to assess the effect of n-TiO2 surface modifications on the mechanical properties of the reinforced composite. METHODS n-TiO2 were synthesized using an alkaline hydrothermal process and then functionalized with silane or methacrylic acid (MA). Nanotubes were characterized by scanning and transmission electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy. Commercially available flowable composite (Filtek™ Supreme Ultra Flowable Restorative, 3M ESPE) was reinforced with varying amounts of nanotubes (0-5wt%). Flowability of the resulting composites was evaluated using a Gillmore needle method. Dynamic Youngs modulus (E) was measured using an ultrasonic technique. Fracture toughness (KIc) was assessed using a notchless triangular prism and radiopacity was quantified. Viability of NIH/3T3 fibroblasts was evaluated following incubation on composite specimens for 24h. RESULTS Electron microscopy revealed a tubular morphology of n-TiO2. All reinforced composites exhibited significantly greater values of E than unreinforced composite. Composites reinforced with 3wt% n-TiO2 functionalized with MA exhibited the greatest values of E and KIc. Cytotoxicity assays revealed that reinforced composites were biocompatible. Taken together, flowable composites reinforced with n-TiO2 exhibited mechanical properties superior to those of unreinforced composite, with minimal effects on flowability and radiopacity. SIGNIFICANCE n-TiO2-reinforced flowable composites are promising materials for use in dental restorations.

Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations

Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

Gene therapy for repair and regeneration of bone and cartilage

HighlightsGene therapy uses vectors to deliver therapeutic nucleic acids to diseased tissues.Classic approaches target inflammation and tissue remodeling to promote repair.Monotherapies targeting IL‐1Ra, TGF‐&bgr; and others are being evaluated for OA and RA.Combinatorial and inducible strategies are emerging to address diseases complexity.miRNAs may offer a means to modulate multiple targets with a single therapeutic. &NA; Gene therapy refers to the use of viral and non‐viral vectors to deliver nucleic acids to tissues of interest using direct (in vivo) or transduced cell‐mediated (ex vivo) approaches. Over the past few decades, strategies have been adopted to express therapeutic transgenes at sites of injury to promote or facilitate repair of bone and cartilage. Targets of interest have typically included secreted proteins such as growth factors and anti‐inflammatory mediators; however, work has also begun to focus intracellularly on signaling components, transcription factors and small, regulatory nucleic acids such as microRNAs (miRNAs). In recent years, a number of single therapeutic gene approaches (termed ‘monotherapies’) have proven effective in preclinical models of disease, and several are being evaluated in clinical trials. In particular, an ex vivo TGF‐&bgr;1 gene therapy was approved in Korea in 2017 for treatment of moderate‐to‐severe osteoarthritis (OA). The ability to utilize viral vectors for context‐specific and combinatorial gene therapy is also being investigated, and these strategies are likely to be important in more robustly addressing the complexities of tissue repair and regeneration in skeletal disease. In this review, we provide an overview of viral gene therapies being developed for treatment of bone and cartilage pathologies, with an emphasis on emerging combinatorial strategies as well as those targeting intracellular mediators such as miRNAs.

Disease-Modifying Osteoarthritis Treatment With Interleukin-1 Receptor Antagonist Gene Therapy in Small and Large Animal Models

Gene therapy holds great promise for the treatment of osteoarthritis (OA) because a single intraarticular injection can lead to long‐term expression of therapeutic proteins within the joint. This study was undertaken to investigate the use of a helper‐dependent adenovirus (HDAd)–mediated intraarticular gene therapy approach for long‐term expression of interleukin‐1 receptor antagonist (IL‐1Ra) as sustained symptomatic and disease‐modifying therapy for OA.

Chapter 8 – Prospects of Gene Therapy for Skeletal Diseases

Gene therapy refers to the use of viral and nonviral vectors to deliver nucleic acids to target tissues. First employed to replace defective genes in monogenetic diseases, such as inborn errors of metabolism and blood disorders, gene therapy has more recently been adopted to express transgenes at sites of injury to promote or facilitate repair. In this chapter, we focus on recent achievements in gene therapy for inherited and acquired disorders of and injuries to connective tissues, such as bone, cartilage, and tendon. The basic principles of in vivo and ex vivo gene therapy will be described. Furthermore, an overview will be given of the viral and nonviral vectors used in the field and their associated immunological concerns. A number of skeletal disorders and injury types that have been treated using gene therapy will be discussed, including candidate genes and approaches. Finally, an outlook for the future of gene therapy for skeletal pathologies will be given.

Combinatorial Prg4 and Il-1ra gene therapy protects against hyperalgesia and cartilage degeneration in post-traumatic osteoarthritis

Osteoarthritis (OA) is a degenerative disease of synovial joints characterized by progressive loss of articular cartilage, subchondral bone remodeling, and intra-articular inflammation with synovitis that results in chronic pain and motor impairment. Despite the economic and health impacts, current medical therapies are targeted at symptomatic relief of OA and fail to alter its progression. Given the complexity of OA pathogenesis, we hypothesized that a combinatorial gene therapy approach, designed to inhibit inflammation with interleukin-1 receptor antagonist (IL-1Ra) while promoting chondroprotection using lubricin (PRG4), would improve preservation of the joint compared to monotherapy alone. Employing two surgical techniques to model mild, moderate and severe posttraumatic OA, we found that combined delivery of helper-dependent adenoviruses (HDVs), expressing IL-1Ra and PRG4, preserved articular cartilage better than either monotherapy in both models as demonstrated by preservation of articular cartilage volume and surface area. This improved protection was associated with increased expression of proanabolic and cartilage matrix genes together with decreased expression of catabolic genes and inflammatory mediators. In addition to improvements in joint tissues, this combinatorial gene therapy prolonged protection against thermal hyperalgesia compared to either monotherapy. Taken together, our results show that a combinatorial strategy is superior to monotherapeutic approaches for treatment of posttraumatic OA.