GSK3 Inhibitor-Induced Dentinogenesis Using a Hydrogel.

Small-molecule drugs targeting glycogen synthase kinase 3 (GSK3) as inhibitors of the protein kinase activity are able to stimulate reparative dentine formation. To develop this approach into a viable clinical treatment for exposed pulp lesions, we synthesized a novel, small-molecule noncompetitive adenosine triphosphate (ATP) drug that can be incorporated into a biodegradable hydrogel for placement by syringe into the tooth. This new drug, named NP928, belongs to the thiadiazolidinone (TDZD) family and has equivalent activity to similar drugs of this family such as tideglusib. However, NP928 is more water soluble than other TDZD drugs, making it more suitable for direct delivery into pulp lesions. We have previously reported that biodegradable marine collagen sponges can successfully deliver TDZD drugs to pulp lesions, but this involves in-theater preparation of the material, which is not ideal in a clinical context. To improve surgical handling and delivery, here we incorporated NP928 into a specifically tailored hydrogel that can be placed by syringe into a damaged tooth. This hydrogel is based on biodegradable hyaluronic acid and can be gelled in situ upon dental blue light exposure, similarly to other common dental materials. NP928 released from hyaluronic acid-based hydrogels upregulated Wnt/ß-catenin activity in pulp stem cells and fostered reparative dentine formation compared to marine collagen sponges delivering equivalent concentrations of NP928. This drug-hydrogel combination has the potential to be rapidly developed into a therapeutic procedure that is amenable to general dental practice.

Translation Approach for Dentine Regeneration Using GSK-3 Antagonists.

The canonical Wnt/ß-catenin signaling pathway is crucial for reparative dentinogenesis following tooth damage, and the modulation of this pathway affects the rate and extent of reparative dentine formation in damaged mice molars by triggering the natural process of dentinogenesis. Pharmacological stimulation of Wnt/ß-catenin signaling activity by small-molecule GSK-3 inhibitor drugs following pulp exposure in mouse molars results in reparative dentinogenesis. The creation of similar but larger lesions in rat molars shows that the adenosine triphosphate (ATP)-competitive GSK-3 inhibitor, CHIR99021 (CHIR), and the ATP noncompetitive inhibitor, Tideglusib (TG), can equally enhance reparative dentine formation to fully repair an area of dentine damage up to 10 times larger, mimicking the size of small lesions in humans. To assess the chemical composition of this newly formed dentine and to compare its structure with surrounding native dentine and alveolar bone, Raman microspectroscopy analysis is used. We show that the newly formed dentine comprises equal carbonate to phosphate ratios and mineral to matrix ratios to that of native dentine, both being significantly different from bone. For an effective dentine repair, the activity of the drugs needs to be restricted to the region of damage. To investigate the range of drug-induced Wnt-activity within the dental pulp, RNA of short-term induced (24-h) molars is extracted from separated roots and crowns, and quantitative Axin2 expression is assayed. We show that the activation of Wnt/ß-catenin signaling is highly restricted to pulp cells in the immediate location of the damage in the coronal pulp tissue with no drug action detected in the root pulp. These results provide further evidence that this simple method of enhancement of natural reparative dentinogenesis has the potential to be translated into a clinical direct capping approach.

Multiscale analyses reveal native-like lamellar bone repair and near perfect bone-contact with porous strontium-loaded bioactive glass.

The efficient healing of critical-sized bone defects using synthetic biomaterial-based strategies is promising but remains challenging as it requires the development of biomaterials that combine a 3D porous architecture and a robust biological activity. Bioactive glasses (BGs) are attractive candidates as they stimulate a biological response that favors osteogenesis and vascularization, but amorphous 3D porous BGs are difficult to produce because conventional compositions crystallize during processing. Here, we rationally designed a porous, strontium-releasing, bioactive glass-based scaffold (pSrBG) whose composition was tailored to deliver strontium and whose properties were optimized to retain an amorphous phase, induce tissue infiltration and encourage bone formation. The hypothesis was that it would allow the repair of a critical-sized defect in an ovine model with newly-formed bone exhibiting physiological matrix composition and structural architecture. Histological and histomorphometric analyses combined with indentation testing showed pSrBG encouraged near perfect bone-to-material contact and the formation of well-organized lamellar bone. Analysis of bone quality by a combination of Raman spectral imaging, small-angle X-ray scattering, X-ray fluorescence and focused ion beam-scanning electron microscopy demonstrated that the repaired tissue was akin to that of normal, healthy bone, and incorporated small amounts of strontium in the newly formed bone mineral. These data show the potential of pSrBG to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in large animal models.

Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro.

Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

Composition of Mineral Produced by Dental Mesenchymal Stem Cells.

Mesenchymal stem cells isolated from different dental tissues have been described to have osteogenic/odontogenic-like differentiation capacity, but little attention has been paid to the biochemical composition of the material that each produces. Here, we used Raman spectroscopy to analyze the mineralized materials produced in vitro by different dental cell populations, and we compared them with the biochemical composition of native dental tissues. We show that different dental stem cell populations produce materials that differ in their mineral and matrix composition and that these differ from those of native dental tissues. In vitro, BCMP (bone chip mass population), SCAP (stem cells from apical papilla), and SHED (stem cells from human-exfoliated deciduous teeth) cells produce a more highly mineralized matrix when compared with that produced by PDL (periodontal ligament), DPA (dental pulp adult), and GF (gingival fibroblast) cells. Principal component analyses of Raman spectra further demonstrated that the crystallinity and carbonate substitution environments in the material produced by each cell type varied, with DPA cells, for example, producing a more carbonate-substituted mineral and with SCAP, SHED, and GF cells creating a less crystalline material when compared with other dental stem cells and native tissues. These variations in mineral composition reveal intrinsic differences in the various cell populations, which may in turn affect their specific clinical applications.

Inadequate fine-tuning of protein synthesis and failure of amino acid homeostasis following inhibition of the ATPase VCP/p97.

The cellular mechanisms that control protein degradation may constitute a non-oncogenic cancer cell vulnerability and, therefore, a therapeutic target. Although this proposition is supported by the clinical success of proteasome inhibitors in some malignancies, most cancers are resistant to proteasome inhibition. The ATPase valosin-containing protein (VCP; p97) is an essential regulator of protein degradation in multiple pathways and has emerged as a target for cancer therapy. We found that pharmacological depletion of VCP enzymatic activity with mechanistically different inhibitors robustly induced proteotoxic stress in solid cancer and multiple myeloma cells, including cells that were insensitive, adapted, or clinically resistant to proteasome inhibition. VCP inhibition had an impact on two key regulators of protein synthesis, eukaryotic initiation factor 2α (eIF2α) and mechanistic target of rapamycin complex 1 (mTORC1), and attenuated global protein synthesis. However, a block on protein translation that was itself cytotoxic alleviated stress signaling and reduced cell death triggered by VCP inhibition. Some of the proteotoxic effects of VCP depletion depended on the eIF2α phosphatase, protein phosphatase 1 regulatory subunit 15A (PPP1R15A)/PP1c, but not on mTORC1, although there appeared to be cross-talk between them. Thus, cancer cell death following VCP inhibition was linked to inadequate fine-tuning of protein synthesis and activity of PPP1R15A/PP1c. VCP inhibitors also perturbed intracellular amino acid levels, activated eukaryotic translation initiation factor 2α kinase 4 (EIF2AK4), and enhanced cellular dependence on amino acid supplies, consistent with a failure of amino acid homeostasis. Many of the observed effects of VCP inhibition differed from the effects triggered by proteasome inhibition or by protein misfolding. Thus, depletion of VCP enzymatic activity triggers cancer cell death in part through inadequate regulation of protein synthesis and amino acid metabolism. The data provide novel insights into the maintenance of intracellular proteostasis by VCP and may have implications for the development of anti-cancer therapies.

Surface properties and ion release from fluoride-containing bioactive glasses promote osteoblast differentiation and mineralization in vitro.

Bioactive glasses (BG) are suitable for bone regeneration applications as they bond with bone and can be tailored to release therapeutic ions. Fluoride, which is widely recognized to prevent dental caries, is efficacious in promoting bone formation and preventing osteoporosis-related fractures when administered at appropriate doses. To take advantage of these properties, we created BG incorporating increasing levels of fluoride whilst holding their silicate structure constant, and tested their effects on human osteoblasts in vitro. Our results demonstrate that, whilst cell proliferation was highest on low-fluoride-containing BG, markers for differentiation and mineralization were highest on BG with the highest fluoride contents, a likely effect of a combination of surface effects and ion release. Furthermore, osteoblasts exposed to the dissolution products of fluoride-containing BG or early doses of sodium fluoride showed increased alkaline phosphatase activity, a marker for bone mineralization, suggesting that fluoride can direct osteoblast differentiation. Taken together, these results suggest that BG that can release therapeutic levels of fluoride may find use in a range of bone regeneration applications.

Short collagen fibers provide control of contraction and permeability in fibroblast-seeded collagen gels.

Tissue engineering may allow for the reconstruction of breast, facial, skin, and other soft tissue defects in the human body. Cell-seeded collagen gels are a logical choice for creating soft tissues because they are biodegradable, mimic the natural tissue, and provide a three-dimensional environment for the cells. The main drawback associated with this approach, however, is the subsequent contraction of the gel by the constituent cells, which severely reduces permeability, initiates apoptosis, and precludes control of the resulting shape and size of the construct. In this study, type I collagen gels were seeded with fibroblasts and cast either with or without the addition of short collagen fibers. Gel contraction was monitored and permeability was assessed after 7 and 14 days in culture. The addition of short collagen fibers both significantly limited contraction and increased permeability of fibroblast-seeded collagen gels. The addition of short collagen fibers had no detrimental effect on cell proliferation, and there were a high number of viable fibroblasts in gels with fibers and gels without fibers. Gels containing short collagen fibers demonstrated permeabilities that were 100 to 1000 times greater than controls and also closely maintained their casting dimensions (never less than 96% of original). By limiting contraction and maintaining permeability, the incorporation of short collagen fibers should enable the creation of larger constructs by allowing for greater nutrient diffusion, and permit the creation of more complicated shapes during gel casting.