Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

Lithiated porous silicon nanowires stimulate periodontal regeneration.

Periodontal disease is a significant burden for oral health, causing progressive and irreversible damage to the support structure of the tooth. This complex structure, the periodontium, is composed of interconnected soft and mineralised tissues, posing a challenge for regenerative approaches. Materials combining silicon and lithium are widely studied in periodontal regeneration, as they stimulate bone repair via silicic acid release while providing regenerative stimuli through lithium activation of the Wnt/ß-catenin pathway. Yet, existing materials for combined lithium and silicon release have limited control over ion release amounts and kinetics. Porous silicon can provide controlled silicic acid release, inducing osteogenesis to support bone regeneration. Prelithiation, a strategy developed for battery technology, can introduce large, controllable amounts of lithium within porous silicon, but yields a highly reactive material, unsuitable for biomedicine. This work debuts a strategy to lithiate porous silicon nanowires (LipSiNs) which generates a biocompatible and bioresorbable material. LipSiNs incorporate lithium to between 1% and 40% of silicon content, releasing lithium and silicic acid in a tailorable fashion from days to weeks. LipSiNs combine osteogenic, cementogenic and Wnt/ß-catenin stimuli to regenerate bone, cementum and periodontal ligament fibres in a murine periodontal defect.

Investigating trial design variability in trials of disease-modifying therapies in Parkinson's disease: a scoping review protocol.

INTRODUCTION: Parkinson's disease (PD) is a debilitating neurological disorder for which the identification of disease-modifying interventions represents a major unmet need. Diverse trial designs have attempted to mitigate challenges of population heterogeneity, efficacious symptomatic therapy and lack of outcome measures that are objective and sensitive to change in a disease modification setting. It is not clear whether consensus is emerging regarding trial design choices. Here, we report the protocol of a scoping review that will provide a contemporary update on trial design variability for disease-modifying interventions in PD. METHODS AND ANALYSIS: The Population, Intervention, Comparator, Outcome and Study design (PICOS) framework will be used to structure the review, inform study selection and analysis. The databases MEDLINE, Web of Science, Cochrane and the trial registry ClinicalTrials.gov will be systematically searched to identify published studies and registry entries in English. Two independent reviewers will screen study titles, abstracts and full text for eligibility, with disagreements being resolved through discussion or by a third reviewer where necessary. Data on general study information, eligibility criteria, outcome measures, trial design, retention and statistically significant findings will be extracted into a standardised form. Extracted data will be presented in a descriptive analysis. We will report our findings using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Scoping Review extension. ETHICS AND DISSEMINATION: This work will provide an overview of variation and emerging trends in trial design choices for disease-modifying trials of PD. Due to the nature of this study, there are no ethical or safety considerations. We plan to publish our findings in a peer-reviewed journal.

Repurposing Metformin for periodontal disease management as a form of oral-systemic preventive medicine.

BACKGROUND: Despite the improvements in treatment over the last decades, periodontal disease (PD) affects millions of people around the world and the only treatment available is based on controlling microbial load. Diabetes is known to increase the risk of PD establishment and progression, and recently, glucose metabolism modulation by pharmaceutical or dietarian means has been emphasised as a significant modulator of non-communicable disease development. METHODS: The impact of pharmaceutically controlling glucose metabolism in non-diabetic animals and humans (REBEC, UTN code: U1111-1276-1942) was investigated by repurposing Metformin, as a mean to manage periodontal disease and its associated systemic risk factors. RESULTS: We found that glucose metabolism control via use of Metformin aimed at PD management resulted in significant prevention of bone loss during induced periodontal disease and age-related bone loss in vivo. Metformin also influenced the bacterial species present in the oral environment and impacted the metabolic epithelial and stromal responses to bacterial dysbiosis at a single cell level. Systemically, Metformin controlled blood glucose levels and age-related weight gain when used long-term. Translationally, our pilot randomized control trial indicated that systemic Metformin was safe to use in non-diabetic patients and affected the periodontal tissues. During the medication window, patients showed stable levels of systemic blood glucose, lower circulating hsCRP and lower insulin levels after periodontal treatment when compared to placebo. Finally, patients treated with Metformin had improved periodontal parameters when compared to placebo treated patients. CONCLUSION: This is the first study to demonstrate that systemic interventions using Metformin in non-diabetic individuals aimed at PD prevention have oral-systemic effects constituting a possible novel form of preventive medicine for oral-systemic disease management.

Markers of dental pulp stem cells in in vivo developmental context.

Teeth and their associated tissues contain several populations of mesenchymal stem cells, one of which is represented by dental pulp stem cells (DPSCs). These cells have mainly been characterised in vitro and numerous positive and negati ve markers for these cells have been suggested. To investigate the presence and localization of these molecules during development, forming dental pulp was examined using the mouse first mandibular molar as a model. The stages corresponding to postnatal (P) days 0, 7, 14, and 21 were investigated. The expression was monitored using customised PCR Arrays. Additionally, in situ localization of the key trio of markers (Cd73, Cd90, Cd105 coded by genes Nt5e, Thy1, Eng) was performed at prenatal and postnatal stages using immunohistochemistry. The expression panel of 24 genes assigned as in vitro markers of DPSCs or mesenchymal stem cells (MSCs) revealed their developmental dynamics during formation of dental pulp mesenchyme. Among the positive markers, Vcam1, Fgf2, Nes were identified as increasing and Cd44, Cd59b, Mcam, Alcam as decreasing between perinatal vs. postnatal stages towards adulthood. Within the panel of negative DPSC markers, Cd14, Itgb2, Ptprc displayed increased and Cd24a decreased levels at later stages of pulp formation. Within the key trio of markers, Nt5e did not show any significant expression difference within the investigated period. Thy1 displayed a strong decrease between P0 and P7 while Eng increased between these stages. In situ localization of Cd73, Cd90 and Cd105 showed them overlap in differentiated odontoblasts and in the sub-odontoblastic layer that is speculated to host odontoblast progenitors. The highly prevalent expression of particularly Cd73 and Cd90 opens the question of potential multiple functions of these molecules. The results from this study add to the in vitro based knowledge by showing dynamics in the expression of DPSC/MSC markers during dental pulp formation in an in vivo context and thus with respect to the natural environment important for commitment of stem cells.

Spatially resolved transcriptomics reveals pro-inflammatory fibroblast involved in lymphocyte recruitment through CXCL8 and CXCL10.

The interplay among different cells in a tissue is essential for maintaining homeostasis. Although disease states have been traditionally attributed to individual cell types, increasing evidence and new therapeutic options have demonstrated the primary role of multicellular functions to understand health and disease, opening new avenues to understand pathogenesis and develop new treatment strategies. We recently described the cellular composition and dynamics of the human oral mucosa; however, the spatial arrangement of cells is needed to better understand a morphologically complex tissue. Here, we link single-cell RNA sequencing, spatial transcriptomics, and high-resolution multiplex fluorescence in situ hybridisation to characterise human oral mucosa in health and oral chronic inflammatory disease. We deconvolved expression for resolution enhancement of spatial transcriptomic data and defined highly specialised epithelial and stromal compartments describing location-specific immune programs. Furthermore, we spatially mapped a rare pathogenic fibroblast population localised in a highly immunogenic region, responsible for lymphocyte recruitment through CXCL8 and CXCL10 and with a possible role in pathological angiogenesis through ALOX5AP. Collectively, our study provides a comprehensive reference for the study of oral chronic disease pathogenesis.

Ift88 regulates enamel formation via involving Shh signaling.

OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

Enhanced bone tissue regeneration with hydrogel-based scaffolds by embedding parathyroid hormone in mesoporous bioactive glass.

OBJECTIVES: To evaluate hydrogel-based scaffolds embedded with parathyroid hormone (PTH)-loaded mesoporous bioactive glass (MBG) on the enhancement of bone tissue regeneration in vitro. MATERIALS AND METHODS: MBG was produced via sol-gel technique followed by PTH solution imbibition. PTH-loaded MBG was blended into the hydrogels and submitted to a lyophilisation process associated with a chemical crosslinking reaction to the production of the scaffolds. Characterisation of the MBG and PTH-loaded MBG scaffolds, including the scanning electron microscope (SEM) connected with an X-ray detector (EDX), Fourier transform infrared (FTIR), compression strength, rheological measurements, swelling and degradation rates, and PTH release analysis, were performed. Also, bioactivity using simulated-body fluid (SBF), biocompatibility (MTT), and osteogenic differentiation analyses (von Kossa and Alizarin Red stainings, and µ-computed tomography, µCT) of the scaffolds were carried out. RESULTS: SEM images demonstrated MBG particles dispersed into the hydrogel-based scaffold structure, which was homogeneously porous and well interconnected. EDX and FTIR revealed large amounts of carbon, oxygen, sodium, and silica in the scaffold composition. Bioactivity experiments revealed changes on sample surfaces over the analysed period, indicating the formation of carbonated hydroxyapatite; however, the chemical composition remained stable. PTH-loaded hydrogel-based scaffolds were biocompatible for stem cells from human-exfoliated deciduous teeth (SHED). A high quantity of calcium deposits on the extracellular matrix of SHED was found for PTH-loaded hydrogel-based scaffolds. µCT images showed MBG particles dispersed into the scaffolds' structure, and a porous, lamellar, and interconnected hydrogel architecture. CONCLUSIONS: PTH-loaded hydrogel-based scaffolds demonstrated consistent morphology and physicochemical properties for bone tissue regeneration, as well as bioactivity, biocompatibility, and osteoinductivity in vitro. Thus, the scaffolds presented here are recommended for future studies on 3D printing. CLINICAL RELEVANCE: Bone tissue regeneration is still a challenge for several approaches to oral and maxillofacial surgeries, though tissue engineering applying SHED, scaffolds, and osteoinductive mediators might help to overcome this clinical issue.

Potential of extracellular space for tissue regeneration in dentistry.

With the proven relationship between oral and general health and the growing aging population, it is pivotal to provide accessible therapeutic approaches to regenerate oral tissues and restore clinical function. However, despite sharing many core concepts with medicine, dentistry has fallen behind the progress in precision medicine and regenerative treatments. Stem cell therapies are a promising avenue for tissue regeneration, however, ethical, safety and cost issues may limit their clinical use. With the significance of paracrine signalling in stem cell and tissue regeneration, extracellular space comprising of the cell secretome, and the extracellular matrix can serve as a potent source for tissue regeneration. Extravesicles are secreted and naturally occurring vesicles with biologically active cargo that can be harvested from the extracellular space. These vesicles have shown great potential as disease biomarkers and can be used in regenerative medicine. As a cell free therapy, secretome and extracellular vesicles can be stored and transferred easily and pose less ethical and safety risks in clinical application. Since there are currently many reviews on the secretome and the biogenesis, characterization and function of extracellular vesicles, here we look at the therapeutic potential of extracellular space to drive oral tissue regeneration and the current state of the field in comparison to regenerative medicine.

Telocytes regulate macrophages in periodontal disease.

Telocytes (TCs) or interstitial cells are characterised in vivo by their long projections that contact other cell types. Although telocytes can be found in many different tissues including the heart, lung, and intestine, their tissue-specific roles are poorly understood. Here we identify a specific cell signalling role for telocytes in the periodontium whereby telocytes regulate macrophage activity. We performed scRNA-seq and lineage tracing to identify telocytes and macrophages in mouse periodontium in homeostasis and periodontitis and carried out hepatocyte growth factor (HGF) signalling inhibition experiments using tivantinib. We show that telocytes are quiescent in homeostasis; however, they proliferate and serve as a major source of HGF in periodontitis. Macrophages receive telocyte-derived HGF signals and shift from an M1 to an M1/M2 state. Our results reveal the source of HGF signals in periodontal tissue and provide new insights into the function of telocytes in regulating macrophage behaviour in periodontitis through HGF/Met cell signalling, which may provide a novel approach in periodontitis treatment.

Expression of periodontitis susceptibility genes in human gingiva using single-cell RNA sequencing.

OBJECTIVE: Single-cell transcriptomics was used to determine the possible cell-type specificity of periodontitis susceptibility genes. BACKGROUND: The last decade has witnessed remarkable advances in the field of human genomics. Despite many advances, the genetic factors associated with or contributing to the periodontitis pathogenesis have only been identified to a limited extent and are often poorly validated. Confirming whether a given single nucleotide polymorphism has an association with periodontitis requires a robust mechanistic explanation on the functional consequences of a given genetic variant. METHODS: We globally assessed the expression of 26 disease-associated genes identified by GWAS within the gingival mucosa. A total of 12 411 cells from 4 different donors were analysed. Differentially expressed genes were analysed using Seurat, a non-parametric Wilcoxon rank sum test. The minimum threshold for significance was defined as p < .05. RESULTS: This exploration at a cellular-level suggests diverse populations contributing to disease pathogenesis, with macrophages expressing a higher number of the analysed disease-associated genes. IL1B, PTGS2, FCGR2A, IL10 and IL1A specifically showed a more restricted expression in the myeloid lineages. CONCLUSION: This short report combines human genetics and single-cell genomics to better understand periodontitis by mapping variants to predict their cells of action and putative functions. These findings seem to suggest that innate cell dysfunction is linked to disease susceptibility.

Modulators of Wnt Signaling Pathway Implied in Dentin Pulp Complex Engineering: A Literature Review.

The main goal of vital pulp therapy (VPT) is to preserve the vitality of the pulp tissue, even when it is exposed due to bacterial invasion, iatrogenic mechanical preparation, or trauma. The type of new dentin formed as a result of VPT can differ in its cellular origin, its microstructure, and its barrier function. It is generally agreed that the new dentin produced by odontoblasts (reactionary dentin) has a tubular structure, while the dentin produced by pulp cells (reparative dentin) does not or has less. Thus, even VPT aims to maintain the vitality of the pulp. It does not regenerate the dentin pulp complex integrity. Therefore, many studies have sought to identify new therapeutic strategies to successfully regenerate the dentin pulp complex. Among them is a Wnt protein-based strategy based on the fact that Wnt proteins seem to be powerful stem cell factors that allow control of the self-renewal and proliferation of multiple adult stem cell populations, suitable for homeostasis maintenance, tissue healing, and regeneration promotion. Thus, this review outlines the different agents targeting the Wnt signaling that could be applied in a tooth environment, and could be a potential therapy for dentin pulp complex and bone regeneration.

Oral stem cells, decoding and mapping the resident cells populations.

The teeth and their supporting tissues provide an easily accessible source of oral stem cells. These different stem cell populations have been extensively studied for their properties, such as high plasticity and clonogenicity, expressing stem cell markers and potency for multilineage differentiation in vitro. Such cells with stem cell properties have been derived and characterised from the dental pulp tissue, the apical papilla region of roots in development, as well as the supporting tissue of periodontal ligament that anchors the tooth within the alveolar socket and the soft gingival tissue. Studying the dental pulp stem cell populations in a continuously growing mouse incisor model, as a traceable in vivo model, enables the researchers to study the properties, origin and behaviour of mesenchymal stem cells. On the other side, the oral mucosa with its remarkable scarless wound healing phenotype, offers a model to study a well-coordinated system of healing because of coordinated actions between epithelial, mesenchymal and immune cells populations. Although described as homogeneous cell populations following their in vitro expansion, the increasing application of approaches that allow tracing of individual cells over time, along with single-cell RNA-sequencing, reveal that different oral stem cells are indeed diverse populations and there is a highly organised map of cell populations according to their location in resident tissues, elucidating diverse stem cell niches within the oral tissues. This review covers the current knowledge of diverse oral stem cells, focusing on the new approaches in studying these cells. These approaches "decode" and "map" the resident cells populations of diverse oral tissues and contribute to a better understanding of the "stem cells niche architecture and interactions. Considering the high accessibility and simplicity in obtaining these diverse stem cells, the new findings offer potential in development of translational tissue engineering approaches and innovative therapeutic solutions.

Weight loss outcomes achieved by adults accessing an online programme offered as part of Public Health England's 'Better Health' campaign.

Effective use of health technology may offer a scalable solution to the obesity pandemic. Online digital programmes provide a convenient and flexible way for more people to access regular support. This service evaluation aims to determine whether adults accessing an online weight management programme via a national campaign are successful in losing weight.Data was analysed for adults registering with Slimming World's online programme using a discounted membership offered as part of PHE's 'Better Health' campaign between July and December 2020. Last-weight carried forward was used to calculate weight outcomes for participants who had the opportunity to complete 12-weeks and recorded ≥ one weight besides baseline. Engagement was determined using number of online weekly weights recorded with high engagers having weight data for ≥ 9 occasions. Socioeconomic status was assessed using postcode data. Resubscription and uploaded weight data were used to determine numbers who continued beyond the offer period.Twenty-seven thousand two hundred forty-eight adults (5.3% males) with mean age 41.0 ± 11.4 years met inclusion criteria. Mean baseline BMI was 33.4 ± 6.8 kg/m2 (29.2% 30-34.9, 18.3% 35-39.9 and 15.1% > 40 kg/m2). Mean weight loss at 12 weeks was 2.7 (± 3) kg representing a mean loss of 3% (± 3.1) body weight with 42.3% achieving ≥ 3% and 22.1% weight loss ≥ 5%. Median number of weigh-ins was six. Men had greater weight losses compared to women (p < 0.001). High engagers, both men and women, achieved greater weight losses (p < 0.001). Absolute weight loss was associated with joining BMI (rs = -0.15, p < 0.001) but for % weight change only small differences were seen (max effect size = 0.03) with no differences in weight change for high engagers between different baseline BMI categories (p > 0.05). 30.9% were in the lowest two IMD quintiles and absolute and percentage weight change did not differ across deprivation quintiles (p > 0.05). 34.9% continued to access the online support after the offer period.This service evaluation shows that an online programme, offered as part of a national campaign, can offer effective support to a large number of people with different starting BMIs and from different socioeconomic backgrounds. An increased level of engagement leads to better weight losses.

Single Cell RNA-Seq: Cell Isolation and Data Analysis.

Single-cell RNA-sequencing technologies have revolutionized the way that researchers can interrogate cellular relationships and the level of detail by which tissue architecture can be characterized. Multiple cell capturing methods have been developed that, when coupled to next-generation sequencing, can yield cell-to-cell specific information regarding gene expression profiles. One of the commonalities between all of the cell capturing techniques to succeed is the necessity to submit samples with a high cell viability. In addition, these cells should have undergone minimal processing to limit induced stress responses so that their transcriptomes, when sequenced, closely reflect their transcriptomes in vivo at the time of isolation. Below we present a streamlined protocol to isolate fresh cells from tissues in vivo. We also share extensive notes to highlight considerations researchers should take into account before beginning their cell isolation protocol.

Making the head: Caspases in life and death.

The term apoptosis, as a way of programmed cell death, was coined a half century ago and since its discovery the process has been extensively investigated. The anatomy and physiology of the head are complex and thus apoptosis has mostly been followed in separate structures, tissues or cell types. This review aims to provide a comprehensive overview of recent knowledge concerning apoptosis-related molecules involved in the development of structures of head with a particular focus on caspases, cysteine proteases having a key position in apoptotic pathways. Since many classical apoptosis-related molecules, including caspases, are emerging in several non-apoptotic processes, these were also considered. The largest organ of the head region is the brain and its development has been extensively investigated, including the roles of apoptosis and related molecules. Neurogenesis research also includes sensory organs such as the eye and ear, efferent nervous system and associated muscles and glands. Caspases have been also associated with normal function of the skin and hair follicles. Regarding mineralised tissues within craniofacial morphogenesis, apoptosis in bones has been of interest along with palate fusion and tooth development. Finally, the role of apoptosis and caspases in angiogenesis, necessary for any tissue/organ development and maintenance/homeostasis, are discussed. Additionally, this review points to abnormalities of development resulting from improper expression/activation of apoptosis-related molecules.

Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance.

Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

Isolation of Single Cells from Mouse Periodontal Ligament.

The periodontal ligament (PDL) is an essential tissue connecting teeth and bone. It is a complex tissue specifically designed to absorb the forces of mastication; analysis of its multiple cell populations is important to understand its function and the cell changes associated with periodontal disease. Cells in the periodontal ligament are not fully understood due to their physical location and small tissue size. It is challenging to isolate thin layers of cells compared with many other more substantial tissues. Here, we provide a straightforward protocol for the isolation of periodontal ligament cells from mice.

Tunable Cross-Linking and Adhesion of Gelatin Hydrogels via Bioorthogonal Click Chemistry.

Engineering cytocompatible hydrogels with tunable physico-mechanical properties as a biomimetic three-dimensional extracellular matrix (ECM) is fundamental to guide cell response and target tissue regeneration or development of in vitro models. Gelatin represents an optimal choice given its ECM biomimetic properties; however, gelatin cross-linking is required to ensure structural stability at physiological temperature (i.e., T > Tsol-gel gelatin). Here, we use a previously developed cross-linking reaction between tetrazine (Tz)- and norbornene (Nb) modified gelatin derivatives to prepare gelatin hydrogels and we demonstrate the possible tuning of their properties by varying their degree of modification (DOM) and the Tz/Nb ratio (R). The percentage DOM of the gelatin derivatives was tuned between 5 and 15%. Hydrogels prepared with higher DOM cross-linked faster (i.e., 10-20 min) compared to hydrogels prepared with lower DOM (i.e., 60-70 min). A higher DOM and equimolar Tz/Nb ratio R resulted in hydrogels with lower weight variation after immersion in PBS at 37 °C. The mechanical properties of the hydrogels were tuned by varying DOM and R by 1 order of magnitude, achieving elastic modulus E values ranging from 0.5 (low DOM and nonequimolar Tz/Nb ratio) to 5 kPa (high DOM and equimolar Tz/Nb ratio). Human dental pulp stem cells were embedded in the hydrogels and successfully 3D cultured in the hydrogels (percentage viable cells >85%). An increase in metabolic activity and a more elongated cell morphology was detected for cells cultured in hydrogels with lower mechanical properties (E < 1 kPa). Hydrogels prepared with an excess of Tz or Nb were successfully adhered and remained in contact during in vitro cultures, highlighting the potential use of these hydrogels as compartmentalized coculture systems. The successful tuning of the gelatin hydrogel properties here developed by controlling their bioorthogonal cross-linking is promising for tissue engineering and in vitro modeling applications.

A modified glass ionomer cement to mediate dentine repair.

OBJECTIVES: Glass ionomer cements (GIC) can be used to protect dentine following caries removal. However, GIC have little biological activity on biological repair processes, which means that neo-dentine formation remains reliant on limited endogenous regenerative processes. Wnt/ß-catenin signalling is known to play a central role in stimulating tertiary dentine formation following tooth damage and can be stimulated by a range of glycogen synthase kinase (GSK3) antagonists, including lithium ions. METHODS: Here, we created lithium-containing bioactive glass (BG) by substituting lithium for sodium ions in 45S5 BG. We then replaced between 10 and 40% of the powder phase of a commercial GIC with the lithium-substituted BG to create a range of formulations of 'LithGlassGIC'. In vitro physical properties of the resulting glasses were characterised and their ability to stimulate reactionary dentine formation in mouse molars in vivo was tested. RESULTS: Lithium release from LithGlassGIC increased with increasing lithium content in the cement. In common with unmodified commercial GIC, all formations of LithGlassGIC showed in vitro toxicity when measured using an indirect cell culture assay based on ISO10993:5, precluding direct pulp contact. However, in a murine non-exposed pulp model of tooth damage, LithGlassGIC quickly released lithium ions, which could be transiently detected in the saliva and blood. LithGlassGIC also enhanced the formation of tertiary dentine, resulting in a thickening of the dentine at the damage site that restored lost dentine volume. Dentine regeneration was likely mediated by upregulation of Wnt/ß-catenin activity, as LithGlassGIC placed in TCF/Lef:H2B-GFP reporter mice showed enhanced GFP activity. SIGNIFICANCE: We conclude that LithGlassGIC acts as a biological restorative material that promotes tertiary dentine formation and restores tooth structure.