Enhancing osteoblast differentiation through small molecule-incorporated engineered nanofibrous scaffold.

OBJECTIVE: This study aimed to investigate the effect of small molecules incorporated into the engineered nanofibrous scaffold to enhance the osteoblast differentiation MATERIALS AND METHODS: Poly-ε-caprolactone (PCL) nanofiber matrices with lithium chloride (LiCl) were fabricated using the electrospinning technique. Scaffolds were characterized using scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX). Scaffolds were seeded with MC3T3-E1 cells and assessed using Western blots (ß-catenin), alamarBlue assay (proliferation), qPCR (osteoblast differentiation), and mineralization (Alizarin Red staining). RESULTS: We observed LiCl nanofiber scaffolds induced concentration-dependent cell proliferation that correlated with an increased ß-catenin expression indicating sustained Wnt signaling. Next, we examined osteoblast differentiation markers such as osteocalcin (OCN) and Runt-related transcription factor 2 (Runx2) and noted increased expression in LiCl nanofiber scaffolds. We also noted increased bone morphogenetic protein (BMP-2, 4, and 7) expressions suggesting activated Wnt can promote cures to further osteogenic differentiation. Finally, Alizarin Red staining demonstrated increased mineral deposition in LiCl-incorporated nanofiber scaffolds. CONCLUSIONS: Together, these results indicated that LiCl-incorporated nanofiber scaffolds enhance osteoblast differentiation. CLINICAL RELEVANCE: Small molecule-incorporated nanofibrous scaffolds are an innovative clinical tool for bone tissue engineering.

Fibrin-Enhanced Canonical Wnt Signaling Directs Plasminogen Expression in Cementoblasts.

Cementum is a mineralized layer on the tooth's root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin) enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of ß-catenin, and T-cell factor (TCF) optimal motif (TOP) reporter activity than the cells on tissue culture dishes (OCCM30-TCD), indicating that the OCCM30-fibrin enhanced canonical Wnt/ß-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1)-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or ß-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/ß-catenin complex in the plasminogen proximal promoter regions (-900 to +54). Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation.

Advances in Regenerative Dentistry: A Systematic Review of Harnessing Wnt/ß-Catenin in Dentin-Pulp Regeneration.

Dentin pulp has a complex function as a major unit in maintaining the vitality of teeth. In this sense, the Wnt/ß-Catenin pathway has a vital part in tooth development, maintenance, repair, and regeneration by controlling physiological activities such as growth, differentiation, and migration. This pathway consists of a network of proteins, such as Wnt signaling molecules, which interact with receptors of targeted cells and play a role in development and adult tissue homeostasis. The Wnt signals are specific spatiotemporally, suggesting its intricate mechanism in development, regulation, repair, and regeneration by the formation of tertiary dentin. This review provides an overview of the recent advances in the Wnt/ß-Catenin signaling pathway in dentin and pulp regeneration, how different proteins, molecules, and ligands influence this pathway, either upregulating or silencing it, and how it may be used in the future for clinical dentistry, in vital pulp therapy as an effective treatment for dental caries, as an alternative approach for root canal therapy, and to provide a path for therapeutic and regenerative dentistry.

Tideglusib-incorporated nanofibrous scaffolds potently induce odontogenic differentiation.

Pulp-Dentin regeneration is a key aspect of maintain tooth vitality and enabling good oral-systemic health. This study aimed to investigate a nanofibrous scaffold loaded with a small molecule i.e. Tideglusib to promote odontogenic differentiation. Tideglusib (GSK-3ß inhibitor) interaction with GSK-3ß was determined using molecular docking and stabilization of ß-catenin was examined by confocal microscopy. 3D nanofibrous scaffolds were fabricated through electrospinning and their physicochemical characterizations were performed. Scaffolds were seeded with mesenchymal stem cells or pre-odontoblast cells to determine the cells proliferation and odontogenic differentiation. Our results showed that Tideglusib (TG) binds with GSK-3ß at Cys199 residue. Stabilization and nuclear translocation of ß-catenin was increased in the odontoblast cells treated with TG. SEM analysis revealed that nanofibers exhibited controlled architectural features that effectively mimicked the natural ECM. UV-Vis spectroscopy demonstrated that TG was incorporated successfully and released in a controlled manner. Both kinds of biomimetic nanofibrous matrices (PCLF-TG100, PCLF-TG1000) significantly stimulated cells proliferation. Furthermore, these scaffolds significantly induced dentinogenic markers (ALP, and DSPP) expression and biomineralization. In contrast to current pulp capping material driving dentin repair, the sophisticated, polymeric scaffold systems with soluble and insoluble spatiotemporal cues described here can direct stem cell differentiation and dentin regeneration. Hence, bioactive small molecule-incorporated nanofibrous scaffold suggests an innovative clinical tool for dentin tissue engineering.

Drug/bioactive eluting chitosan composite foams for osteochondral tissue engineering.

Joint defects associated with a variety of etiologies often extend deep into the subchondral bone leading to functional impairment and joint immobility, and it is a very challenging task to regenerate the bone-cartilage interface offering significant opportunities for biomaterial-based interventions to improve the quality of life of patients. Herein drug-/bioactive-loaded porous tissue scaffolds incorporating nano-hydroxyapatite (nHAp), chitosan (CS) and either hydroxypropyl methylcellulose (HPMC) or Bombyx mori silk fibroin (SF) are fabricated through freeze drying method as subchondral bone substitute. A combination of spectroscopy and microscopy (Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), energy dispersive X-ray (EDX), and X-ray fluorescence (XRF) were used to analyze the structure of the porous biomaterials. The compressive mechanical properties of these scaffolds are biomimetic of cancellous bone tissues and capable of releasing drugs/bioactives (exemplified with triamcinolone acetonide, TA, or transforming growth factor-ß1, TGF-ß1, respectively) over a period of days. Mouse preosteoblast MC3T3-E1 cells were observed to adhere and proliferate on the tissue scaffolds as confirmed by the cell attachment, live-dead assay and alamarBlue™ assay. Interestingly, RT-qPCR analysis showed that the TA downregulated inflammatory biomarkers and upregulated the bone-specific biomarkers, suggesting such tissue scaffolds have long-term potential for clinical application.

Precision-engineered niche for directed differentiation of MSCs to lineage-restricted mineralized tissues.

The major difference between tissue healing and regeneration is the extent of instructional cues available to precisely direct the biological response. A classic example is reparative or osteodentin that is seen in response to physicochemical injury to the pulp-dentin complex. Dentin regeneration can direct the differentiation of dental stem cells using concerted actions of both soluble (biomolecules, agonists, and antagonists) and insoluble (matrix topology) cues. The major purpose of this study was to examine the synergistic combination of two discrete biomaterial approaches by utilizing nanofiber scaffolds in discrete configurations (aligned or random) with incorporated polymeric microspheres capable of controlled release of growth factors. Further, to ensure appropriate disinfection for clinical use, Radio-Frequency Glow Discharge (RFGD) treatments were utilized, followed by seeding with a mesenchymal stem cell (MSC) line. SEM analysis revealed electrospinning generated controlled architectural features that significantly improved MSC adhesion and proliferation on the aligned nanofiber scaffolds compared to randomly oriented scaffolds. These responses were further enhanced by RFGD pre-treatments. These enhanced cell adhesion and proliferative responses could be attributed to matrix-induced Wnt signaling that was abrogated by pre-treatments with anti-Wnt3a neutralizing antibodies. Next, we incorporated controlled-release microspheres within these electrospun scaffolds with either TGF-ß1 or BMP4. We observed that these scaffolds could selectively induce dentinogenic or osteogenic markers (DSPP, Runx2, and BSP) and mineralization. This work demonstrates the utility of a novel, modular combinatorial scaffold system capable of lineage-restricted differentiation into bone or dentin. Future validation of this scaffold system in vivo as a pulp capping agent represents an innovative dentin regenerative approach capable of preserving tooth pulp vitality.

Nanofibrous topography-driven altered responsiveness to Wnt5a mediates the three-dimensional polarization of odontoblasts.

Cell differentiation with the proper three-dimensional (3-D) structure is critical for cells to carry out their cellular functions in tissues. Odontoblasts derived from neural crest cells are elongated and polarized with the cell process, which is decisive for one directional tubular dentin formation. Here, we report that the fibrous topography of scaffolds directs odontoblast-lineage cells to differentiate to have the 3-D structure of odontoblasts through an altered responsiveness to Wnt family member 5A (Wnt5a). In a pulp-exposure animal model, the scaffolds with the nanofibrous topography supported the regeneration of tubular dentin with odontoblast processes. In cultures of pre-odontoblast cells, the nanofibrous topography heightened the cells on the z-axis. The cells on nanofibrous substrate (FIBER) formed stress fiber cytoskeletons on a conventional tissue culture plate (TCP). Differential activation of Cell division control protein 42 (Cdc42) on FIBER and Ras homolog family member A (RhoA) on TCP led to these differences. The signal from Wnt5a-Cdc42 in the cells on FIBER mediated the phosphorylation of JNK and the polarity growth signaling. Taken together, the nanofibrous topography of the scaffolds led to the 3-D structural differentiation of odontoblasts in vitro and in vivo, implying its application for dentin regeneration. Furthermore, the results on the altered activation of Cdc42 by Wnt5a on FIBER provide evidence that the topography of the scaffolds can cause a distinctive cell responsiveness to their micro-environments.

Redox signaling induces laminin receptor ribosomal protein-SA expression to improve cell adhesion following radiofrequency glow discharge treatments.

Current biomaterials effectively replace biological structures but are limited by infections and long-term material failures. This study examined the molecular mechanisms of radio frequency glow discharge treatments (RFGDT) in mediating the disinfection of biomaterial surfaces and concurrently promoting cell attachment and proliferation. Dental biomaterials were subjected to RFGDT, and viability of oral microbial species, namely Streptococcus mutants (SM), Streptococcus gordonii (SG), Moraxella catarrhalis (MC), and Porphyromonas gingivalis (PG), were assessed. Cell attachment and survival of a pre-odontoblast cell line, MDPC-23, was examined. Finally, mechanistic investigations into redox generation and biological signaling were investigated. Based on their compositions, dental biomaterials induced reactive oxygen species (ROS) following dose-dependent RFGDT. Reduced microbial viability was evident following RFGDT in the catalase-negative (SM and SG) species more prominently than catalase-positive (MC and PG) species. Cell adhesion assays noted improved MDPC-23 attachment and survival. Pretreatments with N-acetylcysteine (NAC) and catalase abrogated these responses. Immunoassays noted redox-induced downstream expression of a laminin receptor, Ribosomal Protein SA, following RFGDT. Thus, RFGDT-induced redox mediates antimicrobial and improves cell responses such as adhesion and proliferation. These observations together provide a mechanistic rationale for the clinical utility of RFGDT with dental biomaterials for regenerative clinical applications.

Label-free quantitation of the changes in salivary proteome associated with the chronic consumption of the betel nut (Areca catechu).

Areca nut or betel nut chewing is most frequently used in Pakistan and is associated with a high risk for oral cancer. Until now, however, there has not been any research conducted on the long-term effect(s) of betel nut chewing on the saliva proteome. In the present study, initially, the changes in the saliva proteome associated with betel nut chewing were investigated. Secondly, the analysis was focused on the changes in salivary proteome with respect to prolonged usage of betel nuts. After extraction, the saliva proteins were digested into peptides and these were subsequently analyzed using mass spectrometry. Data are available via ProteomeXchange with identifier PXD029768. Label-free quantitation of saliva samples revealed a total of 12 proteins that were differentially expressed between betel nut addicts (BNAs), and the control group. The study groups were further divided into three subgroups, the BNA-1, BNA-2, and BNA-3 groups, with respect to the extent of consumption of betel nuts in terms of years. The data analysis revealed a more detailed profiling of proteins expressed after five, ten, and more than ten years of betel nut consumption. A total of 30, 17, and 22 proteins were found to be differentially expressed when divided into the BNA-1, BNA-2, and BNA-3 groups. The present study shows that the chronic usage of betel nuts leads to the expression of proteins, such as SPARC1, profilin, and SBSN, which are known to be involved in head and neck cancers.

Surface-modified polymeric nanoparticles for drug delivery to cancer cells.

INTRODUCTION: The utilization of polymeric nanoparticles, as drug payloads, has been extensively prevailed in cancer therapy. However, the precise distribution of these nanocarriers is restrained by various physiological and cellular obstacles. Nanoparticles must avoid nonspecific interactions with healthy cells and in vivo compartments to circumvent these barriers. Since in vivo interactions of nanoparticles are mainly dependent on surface properties of nanoparticles, efficient control on surface constituents is necessary for the determination of nanoparticles' fate in the body. AREAS COVERED: In this review, the surface-modified polymeric nanoparticles and their utilization in cancer treatment were elaborated. First, the interaction of nanoparticles with numerous in vivo barriers was highlighted. Second, different strategies to overcome these obstacles were described. Third, some inspiring examples of surface-modified nanoparticles were presented. Later, fabrication and characterization methods of surface-modified nanoparticles were discussed. Finally, the applications of these nanoparticles in different routes of treatments were explored. EXPERT OPINION: Surface modification of anticancer drug-loaded polymeric nanoparticles can enhance the efficacy, selective targeting, and biodistribution of the anticancer drug at the tumor site.

Nanoscale and Macroscale Scaffolds with Controlled-Release Polymeric Systems for Dental Craniomaxillofacial Tissue Engineering.

Tremendous progress in stem cell biology has resulted in a major current focus on effective modalities to promote directed cellular behavior for clinical therapy. The fundamental principles of tissue engineering are aimed at providing soluble and insoluble biological cues to promote these directed biological responses. Better understanding of extracellular matrix functions is ensuring optimal adhesive substrates to promote cell mobility and a suitable physical niche to direct stem cell responses. Further, appreciation of the roles of matrix constituents as morphogen cues, termed matrikines or matricryptins, are also now being directly exploited in biomaterial design. These insoluble topological cues can be presented at both micro- and nanoscales with specific fabrication techniques. Progress in development and molecular biology has described key roles for a range of biological molecules, such as proteins, lipids, and nucleic acids, to serve as morphogens promoting directed behavior in stem cells. Controlled-release systems involving encapsulation of bioactive agents within polymeric carriers are enabling utilization of soluble cues. Using our efforts at dental craniofacial tissue engineering, this narrative review focuses on outlining specific biomaterial fabrication techniques, such as electrospinning, gas foaming, and 3D printing used in combination with polymeric nano- or microspheres. These avenues are providing unprecedented therapeutic opportunities for precision bioengineering for regenerative applications.

Fibrous Topography-Potentiated Canonical Wnt Signaling Directs the Odontoblastic Differentiation of Dental Pulp-Derived Stem Cells.

Nanofibrous engineered matrices have significant potential in cellular differentiation and tissue regeneration. Stem cells require specific extracellular signals that lead to the induction of different lineages. However, the mechanisms by which the nanofibrous matrix promotes mesenchymal stem cell (MSC) differentiation are largely unknown. Here, we investigated the mechanisms that underlie nanofibrous matrix-induced odontoblastic differentiation of human dental pulp MSCs (DP-MSCs). An electrospun polystyrene nanofibrous (PSF) matrix was prepared, and the cell responses to the PSF matrix were assessed in comparison with those on conventional tissue culture dishes. The PSF matrix promoted the expression of Wnt3a, Wnt5a, Wnt10a, BMP2, BMP4, and BMP7 in the DP-MSCs, concomitant with the induction of odontoblast/osteoblast differentiation markers, dentin sialophosphoprotein (DSPP), osteocalcin, and bone sialoprotein, whose levels were further enhanced by treatment with recombinant Wnt3a. The DP-MSCs cultured on the PSF matrix also exhibited a high alkaline phosphatase activity and intense Alizarin Red staining, indicating that the PSF matrix promotes odontoblast differentiation. Besides inducing the expression of Wnt3a, the PSF matrix maintained high levels of ß-catenin protein and enhanced its translocation to the nucleus, leading to its transcriptional activity. Forced expression of LEF1 or treatments with LiCl further enhanced the DSPP expression. Blocking the Wnt3a-initiated signaling abrogated the PSF-induced DSPP expression. Furthermore, the cells on the PSF matrix increased the DSPP promoter activity. The ß-catenin complex was bound to the conserved motifs on the DSPP promoter dictating its transcription. Transplantations of the preodontoblast-seeded PSF matrix to the subcutaneous tissues of nude mice confirmed the association of the PSF matrix with the Wnt3a and DSPP expressions in vivo. Taken together, these results demonstrate the nanofibrous engineered matrix strongly supports odontoblastic differentiation of DP-MSCs by enhancing Wnt/ß-catenin signaling.

Effects of the incorporation of ε-aminocaproic acid/chitosan particles to fibrin on cementoblast differentiation and cementum regeneration.

Cementum formation on the exposed tooth-root surface is a critical process in periodontal regeneration. Although various therapeutic approaches have been developed, regeneration of integrated and functional periodontal complexes is still wanting. Here, we found that the OCCM30 cementoblasts cultured on fibrin matrix express substantial levels of matrix proteinases, leading to the degradation of fibrin and the apoptosis of OCCM30 cells, which was reversed upon treatment with a proteinase inhibitor, ε-aminocaproic acid (ACA). Based on these findings, ACA-releasing chitosan particles (ACP) were fabricated and ACP-incorporated fibrin (fibrin-ACP) promoted the differentiation of cementoblasts in vitro, as confirmed by bio-mineralization and expressions of molecules associated with mineralization. In a periodontal defect model of beagle dogs, fibrin-ACP resulted in substantial cementum formation on the exposed root dentin in vivo, compared to fibrin-only and enamel matrix derivative (EMD) which is used clinically for periodontal regeneration. Remarkably, the fibrin-ACP developed structural integrations of the cementum-periodontal ligament-bone complex by the Sharpey's fiber insertion. In addition, fibrin-ACP promoted alveolar bone regeneration through increased bone volume of tooth roof-of-furcation defects and root coverage. Therefore, fibrin-ACP can promote cementogenesis and osteogenesis by controlling biodegradability of fibrin, implicating the feasibility of its therapeutic use to improve periodontal regeneration. STATEMENT OF SIGNIFICANCE: Cementum, the mineralized layer on root dentin surfaces, functions to anchor fibrous connective tissues on tooth-root surfaces with the collagenous Sharpey's fibers integration, of which are essential for periodontal functioning restoration in the complex. Through the cementum-responsible fiber insertions on tooth-root surfaces, PDLs transmit various mechanical responses to periodontal complexes against masticatory/occlusal stimulations to support teeth. In this study, periodontal tissue regeneration was enhanced by use of modified fibrin biomaterial which significantly promoted cementogenesis within the periodontal complex with structural integration by collagenous Sharpey's fiber insertions in vivo by controlling fibrin degradation and consequent cementoblast apoptosis. Furthermore, the modified fibrin could improve repair and regeneration of tooth roof-of-furcation defects, which has spatial curvatures and geometrical difficulties and hardly regenerates periodontal tissues.

The prolyl hydroxylase inhibitor dimethyloxalylglycine enhances dentin sialophoshoprotein expression through VEGF-induced Runx2 stabilization.

Prolyl hydroxylase (PHD) inhibitors are suggested as therapeutic agents for tissue regeneration based on their ability to induce pro-angiogenic responses. In this study, we examined the effect of the PHD inhibitor dimethyloxalylglycine (DMOG) on odontoblast maturation and sought to determine the underlying mechanism using MDPC-23 odontoblast-like cells. DMOG significantly enhanced matrix mineralization, confirmed by alizarin red staining and by measurement of the calcium content. DMOG dose-dependently increased alkaline phosphatase activity and the expressions of dentin sialophosphoprotein (Dspp) and osteocalcin. To determine the underlying events leading to DMOG-induced Dspp expression, we analyzed the effect of DMOG on Runx2. Knockdown of Runx2 using siRNAs decreased Dspp expression and prevented DMOG-induced Dspp expression. DMOG enhanced the transcriptional activity and level of Runx2 protein but not Runx2 transcript, and this enhancement was linked to the inhibitory effects of DMOG on the degradation of Runx2 protein. The vascular endothelial growth factor (VEGF) siRNAs profoundly decreased the Runx2 protein levels and inhibited the DMOG-increased Runx2 protein. Recombinant VEGF protein treatment significantly and dose-dependently increased the transcriptional activity and level of the Runx2 protein but not Runx2 transcript. Dspp expression was also enhanced by VEGF. Last, we examined the involvement of the Erk mitogen-activated protein kinase and Pin1 pathway in VEGF-enhanced Runx2 because this pathway can regulate the stability and activity of the Runx2 protein. VEGF stimulated Erk activation, and the inhibitors of Erk and Pin1 hampered VEGF-enhanced Runx2 protein. Taken together, the results of this study provide evidence that DMOG can enhance Dspp expression through VEGF-induced stabilization of Runx2 protein, and thus, suggest that DMOG can be used as a therapeutic tool for enhancing odontoblast maturation in dental procedures.