Investigation of the early apical release from endodontic hydrogels: A 3D printed model.

AIM: Regenerative Endodontic Procedures (REPs) using new materials such as hydrogels aim to replace current endodontic treatments, but numerous limitations are to overcome. Apical release was little explored in previous studies, especially regarding hydrogels that incorporate molecules, such as growth factors and antibiotics. Apical release is a key mechanism in achieving regeneration, as it could regulate disinfection or cell colonization. Few models exist for apical release, limiting the transfer of these devices from bench to bedside. This study aims to design a simple and standardized model to identify parameters that influence the early apical release kinetic of molecules from endodontic hydrogels. METHODOLOGY: Endodontic Release Inserts (ERI) were designed to mimic the situation of an immature incisor using three different diameters (Ø 0.5 to 2 mm) and to allow the study of the early release from a hydrogel in a 96-well plate. ERI was produced with a 3D printing machine. The kinetic release was investigated using 2 fluorescent, hydrophobic (BDP-500) and hydrophilic (Fluorescein) molecules, in different hydrogels (fibrin and agarose) and in various media (PBS or serum). The release kinetics were estimated by measuring the fluorescence at different time points (1 to 24 h). RESULTS: ERI use made it possible to report that apical diameters increase from 500 to 1000 µm was associated with an increase in release from 4.02 ± 1.63% to 11.53 ± 2.38% over 24 h. It also allowed us to report that bottom solution composition change from PBS to human serum was associated with an increase in the release of fatty acid molecules, whilst a decrease in the hydrogel concentration was associated with a variation in release kinetics. Moreover, nano-encapsulation of a molecule was associated with a decreased release over the first 24 h from 5.25 to 0%. CONCLUSION: ERI use enables investigation of the parameters influencing release kinetics from endodontic hydrogels. Further investigations are necessary to evaluate the interaction of these parameters with each other, in animal models and clinic.

Bioactive Endodontic Hydrogels: From Parameters to Personalized Medicine.

Regenerative endodontic procedures (REPs) aim at recreating dental pulp tissue using biomaterials such as hydrogels. Their bioactivity is mostly related to the nature of biomolecules or chemical compounds that compose the endodontic hydrogel. However, many other parameters, such as hydrogel concentration, bioactive molecules solubility, and apex size, were reported to influence the reciprocal host-biomaterial relationship and hydrogel behavior. The lack of knowledge regarding these various parameters, which should be considered, leads to the inability to predict the clinical outcome and suggests that the biological activity of endodontic hydrogel is impossible to anticipate and could hinder the bench-to-bedside transition. We describe, in this review, that most of these parameters could be identified, described, and studied. A second part of the review lists some challenges and perspectives, including development of future mathematical models that are able to explain, and eventually predict, the bioactivity of endodontic hydrogel used in a clinical setting.

Next generation antibacterial strategies for regenerative endodontic procedures: A scoping review.

BACKGROUND: The clinical results following regenerative endodontic procedures (REPs) vary according to numerous parameters, including the presence of bacteria. This limitation reduces the indications for REPs and calls for the development of next generation antibacterial strategies (NGAS) providing alternatives to current antibacterial strategies (CAS) such as double or triple antibiotic paste (DAP/TAP) and (Ca(OH)2). OBJECTIVES: The present scoping review aims to describe the current trends regarding the use of such strategies and highlight future perspectives. METHODS: Four databases (PUBMed, Cochrane, ClinicalTrials and Science Direct) were searched until 1st May 2023. RESULTS: A total of 918 records were identified, 133 were screened and assessed for eligibility, and 87 articles were included. The findings show that (1) clinical studies are only available for CAS, (2) although next generation strategies are the most studied approach since 2017, they are all at the pre-clinical stage, (3) most of the next generation strategies use galenic forms which offer cell support and colonization and which simultaneously contain antibacterial molecules as alternatives to CAS and to antibiotics in general, (4) standardization is required for future research, specifically regarding the bacterial strains studied, the use of biofilm studies and the cellular behaviour assessments. CONCLUSION: Although NGAS are promising strategies to improve REPs in the context of infection, the current evidence is mostly limited to pre-clinical studies. Further methodological improvement is required to allow relevant comparisons between studies and to reduce the time from bench to bedside.

A High-Throughput Search for SFXN1 Physical Partners Led to the Identification of ATAD3, HSD10 and TIM50.

Sideroflexins (SFXN, SLC56) are a family of evolutionarily conserved mitochondrial carriers potentially involved in iron homeostasis. One member of the SFXN family is SFXN1, recently identified as a human mitochondrial serine transporter. However, little is known about the SFXN1 interactome, necessitating a high-throughput search to better characterize SFXN1 mitochondrial functions. Via co-immunoprecipitation followed by shotgun mass spectrometry (coIP-MS), we identified 96 putative SFXN1 interactors in the MCF7 human cell line. Our in silico analysis of the SFXN1 interactome highlights biological processes linked to mitochondrial organization, electron transport chains and transmembrane transport. Among the potential physical partners, ATAD3A and 17ß-HSD10, two proteins associated with neurological disorders, were confirmed using different human cell lines. Nevertheless, further work will be needed to investigate the significance of these interactions.

A critical analysis of research methods and experimental models to study pulpitis.

Pulpitis is the inflammatory response of the dental pulp to a tooth insult, whether it is microbial, chemical, or physical in origin. It is traditionally referred to as reversible or irreversible, a classification for therapeutic purposes that determines the capability of the pulp to heal. Recently, new knowledge about dental pulp physiopathology led to orientate therapeutics towards more frequent preservation of pulp vitality. However, full adoption of these vital pulp therapies by dental practitioners will be achieved only following better understanding of cell and tissue mechanisms involved in pulpitis. The current narrative review aimed to discuss the contribution of the most significant experimental models developed to study pulpitis. Traditionally, in vitro two (2D)- or three (3D)-dimensional cell cultures or in vivo animal models were used to analyse the pulp response to pulpitis inducers at cell, tissue or organ level. In vitro, 2D cell cultures were mainly used to decipher the specific roles of key actors of pulp inflammation such as bacterial by-products, pro-inflammatory cytokines, odontoblasts or pulp stem cells. However, these simple models did not reproduce the 3D organisation of the pulp tissue and, with rare exceptions, did not consider interactions between resident cell types. In vitro, tissue/organ-based models were developed to better reflect the complexity of the pulp structure. Their major disadvantage is that they did not allow the analysis of blood supply and innervation participation. On the contrary, in vivo models have allowed researchers to identify key immune, vascular and nervous actors of pulpitis and to understand their function and interplay in the inflamed pulp. However, inflammation was mainly induced by iatrogenic dentine drilling associated with simple pulp exposure to the oral environment or stimulation by individual bacterial by-products for short periods. Clearly, these models did not reflect the long and progressive development of dental caries. Lastly, the substantial diversity of the existing models makes experimental data extrapolation to the clinical situation complicated. Therefore, improvement in the design and standardisation of future models, for example by using novel molecular biomarkers, databased models and artificial intelligence, will be an essential step in building an incremental knowledge of pulpitis in the future.

In vivo N-Terminomics Highlights Novel Functions of ADAMTS2 and ADAMTS14 in Skin Collagen Matrix Building.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2 and ADAMTS14 were originally known for their ability to cleave the aminopropeptides of fibrillar collagens. Previous work using N-terminomic approach (N-TAILS) in vitro led to the identification of new substrates, including some molecules involved in TGF-ß signaling. Here, N-TAILS was used to investigate the substrates of these two enzymes in vivo, by comparing the N-terminomes of the skin of wild type mice, mice deficient in ADAMTS2, in ADAMTS14 and in both ADAMTS2 and ADAMTS14. This study identified 68 potential extracellular and cell surface proteins, with the majority of them being cleaved by both enzymes. These analyses comfort their role in collagen matrix organization and suggest their implication in inflammatory processes. Regarding fibrillar collagen, this study demonstrates that both ADAMTS2 and ADAMTS14 are involved in the processing of the aminopropeptide of alpha1 and alpha2 type V collagen. It also revealed the existence of several cleavage sites in the Col1 domain and in the C-propeptide of type I collagens. In addition to collagens and other extracellular proteins, two major components of the cell cytoskeleton, actin and vimentin, were also identified as potential substrates. The latter data were confirmed in vitro using purified enzymes and could potentially indicate other functions for ADAMTS2 and 14. This original investigation of mouse skin degradomes by N-terminomic highlights the essential role of ADAMTS2 and ADAMTS14 in collagen matrix synthesis and turnover, and gives clues to better understand their functions in skin pathophysiology. Data are available via ProteomeXchange with identifier PXD022179.

Differential cleavage of lysyl oxidase by the metalloproteinases BMP1 and ADAMTS2/14 regulates collagen binding through a tyrosine sulfate domain.

Collagens are the main structural component of the extracellular matrix and provide biomechanical properties to connective tissues. A critical step in collagen fibril formation is the proteolytic removal of N- and C-terminal propeptides from procollagens by metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and BMP1 (bone morphogenetic protein 1)/Tolloid-like families, respectively. BMP1 also cleaves and activates the lysyl oxidase (LOX) precursor, the enzyme catalyzing the initial step in the formation of covalent collagen cross-links, an essential process for fibril stabilization. In this study, using murine skin fibroblasts and HEK293 cells, along with immunoprecipitation, LOX enzymatic activity, solid-phase binding assays, and proteomics analyses, we report that the LOX precursor is proteolytically processed by the procollagen N-proteinases ADAMTS2 and ADAMTS14 between Asp-218 and Tyr-219, 50 amino acids downstream of the BMP1 cleavage site. We noted that the LOX sequence between the BMP1- and ADAMTS-processing sites contains several conserved tyrosine residues, of which some are post-translationally modified by tyrosine O-sulfation and contribute to binding to collagen. Taken together, these findings unravel an additional level of regulation in the formation of collagen fibrils. They point to a mechanism that controls the binding of LOX to collagen and is based on differential BMP1- and ADAMTS2/14-mediated cleavage of a tyrosine-sulfated domain.

Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-ß binding protein 1, TGF-ß RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-ß activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

ADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis.

The only documented activity of a subclass of ADAMTS proteases comprising ADAMTS2, 3 and 14 is the cleavage of the aminopropeptide of fibrillar procollagens. A limited number of in vitro studies suggested that ADAMTS3 is mainly responsible for procollagen II processing in cartilage. Here, we created an ADAMTS3 knockout mouse (Adamts3(-/-)) model to determine in vivo the actual functions of ADAMTS3. Heterozygous Adamts3(+/-) mice were viable and fertile, but their intercrosses demonstrated lethality of Adamts3(-/-) embryos after 15 days of gestation. Procollagens I, II and III processing was unaffected in these embryos. However, a massive lymphedema caused by the lack of lymphatics development, an abnormal blood vessel structure in the placenta and a progressive liver destruction were observed. These phenotypes are most probably linked to dysregulation of the VEGF-C pathways. This study is the first demonstration that an aminoprocollagen peptidase is crucial for developmental processes independently of its primary role in collagen biology and has physiological functions potentially involved in several human diseases related to angiogenesis and lymphangiogenesis.

The procollagen N-proteinases ADAMTS2, 3 and 14 in pathophysiology.

Collagen fibers are the main components of most of the extracellular matrices where they provide a structural support to cells, tissues and organs. Fibril-forming procollagens are synthetized as individual chains that associate to form homo- or hetero-trimers. They are characterized by the presence of a central triple helical domain flanked by amino and carboxy propeptides. Although there are some exceptions, these two propeptides have to be proteolytically removed to allow the almost spontaneous assembly of the trimers into collagen fibrils and fibers. While the carboxy-propeptide is mainly cleaved by proteinases from the tolloid family, the amino-propeptide is usually processed by procollagen N-proteinases: ADAMTS2, 3 and 14. This review summarizes the current knowledge concerning this subfamily of ADAMTS enzymes and discusses their potential involvement in physiopathological processes that are not directly linked to fibrillar procollagen processing.

Directed evolution of a formate dehydrogenase for increased tolerance to ionic liquids reveals a new site for increasing the stability.

The formate dehydrogenase (FDH) from Candida boidinii is a well-known enzyme in biocatalysis for NADH regeneration. Nevertheless, it has low activity in a water-miscible ionic liquid (1,3-dimethylimidazolium dimethyl phosphate, [MMIm][Me2 PO4 ]). In this work, this enzyme was subjected to directed evolution by using error-prone PCR, and a mutant (N187S/T321S) displaying higher activity was obtained following selection based on the formazan-based colorimetric assay. The mutation N187S is responsible for improved activity both in aqueous solution and in [MMIm][Me2 PO4 ], through an enhancement of the kcat value by a factor of 5.8. Fluorescence experiments performed in the presence of a quenching agent revealed that the mutant does not unfold in the presence of 50 % (v/v) [MMIm][Me2 PO4 ] whereas the wild-type enzyme does. Molecular modelling revealed that the mutation is located at the monomer-monomer interface and causes an increase in the pKa of residue E163 from 4.8 to 5.5. Calculation of the pKa of this residue in other microbial FDHs showed that thermostable FDHs have a highly basic glutamate at this position (pKa up to 6.2). We have identified a new site for improving FDH thermostability and tolerance to ionic liquids, and it is linked to the local charge of the enzymes in this class.

Rapid electrochemical screening of NAD-dependent dehydrogenases in a 96-well format.

The electrochemical detection of dehydrogenase activity in crude cell lysates is performed simultaneously using 96 carbon electrodes modified with electrografted phenazines. The method is applied to the screening of a library of formate dehydrogenase mutants obtained by directed evolution.

Contribution of dynamic and static quenchers for the study of protein conformation in ionic liquids by steady-state fluorescence spectroscopy.

The study of protein conformation in ionic liquids (ILs) is crucial to understand enzymatic activity. Steady-state fluorescence is a proven, rapid and easy method to evaluate the protein structure in aqueous solutions, but it is discussed when used in ILs. In this work, the structure of the formate dehydrogenase from Candida boidinii (FDH, EC: 1.2.1.2) in three imidazolium-based ILs (dimethylimidazolium dimethylphosphate [MMIm][Me(2)PO(4)], 1-butyl-3-methylimidazolium acetate [BMIm][CH(3)COO], and dimethylimidazolium methylphosphonate [MMIm][CH(3)HPO(2)(OCH(3))]) is studied by fluorescence spectroscopy. The UV-vis spectroscopic analysis shows that the decrease of the FDH fluorescence is not only due to the high light absorption of these ILs. The Stern-Volmer analysis clearly shows that these ILs are quenchers of the indole fluorescence, while this quenching property is not found when imidazole is used. Fluorescence spectra of the FDH in the presence of the ILs show that a maximal ionic liquid concentration (MILc), which could be used for steady-state fluorescence study, should be defined. Therefore, FDH conformation could not be directly related to the decrease of its fluorescence in ILs. Nevertheless, the structure of the FDH could be evaluated with dynamic and static quenchers like iodide or acrylamide, used below the MILc, demonstrating the relevance of this parameter. The Stern-Volmer constants (K(SV)(Q)), calculated in the presence of the different ILs, demonstrate that these ILs are strong denaturing agents, each one acting with a different mechanism. This report provides a suitable and easy-to-apply method to study any enzyme structures in ILs by steady-state fluorescence.

Role of the netrin-like domain of procollagen C-proteinase enhancer-1 in the control of metalloproteinase activity.

The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.