RMeS - Regenerative Medicine and Skeleton

Methylation of cytosine residues within the CpG dinucleotide in mammalian cells is an important mediator of gene expression, genome stability, X-chromosome inactivation, genomic imprinting, chromatin structure, and embryonic development. The majority of CpG sites in mammalian cells is methylated in a nonrandom fashion, raising the question of how DNA methylation is distributed along the genome. Here, we focused on the functions of DNA methyltransferase-3b (Dnmt3b), of which deregulated activity is linked to several human pathologies. We generated Dnmt3b hypomorphic mutant mice with reduced catalytic activity, which first revealed a deregulation of Hox genes expression, consistent with the observed homeotic transformations of the posterior axis. In addition, analysis of deregulated expression programs in Dnmt3b mutant embryos, using DNA microarrays, highlighted illegitimate activation of several germ-line genes in somatic tissues that appeared to be linked directly to their hypomethylation in mutant embryos. We provide evidence that these genes are direct targets of Dnmt3b. Moreover, the recruitment of Dnmt3b to their proximal promoter is dependant on the binding of the E2F6 transcriptional repressor, which emerges as a common hallmark in the promoters of genes found to be up-regulated as a consequence of impaired Dnmt3b activity. Therefore, our results unraveled a coordinated regulation of genes involved in meiosis, through E2F6-dependant methylation and transcriptional silencing in somatic tissues.

A wide variety of biomaterials have been developed as both stabilizing structures for the injured bone and inducers of bone neoformation. They differ in chemical composition, shape, porosity, and mechanical properties. The most extensively employed and studied subset of bioceramics are calcium phosphate materials (CaPs). These materials, when transplanted alongside mesenchymal stem cells (MSCs), lead to ectopic (intramuscular and subcutaneous) and orthotopic bone formation in preclinical studies, and effective fracture healing in clinical trials. Human MSC transplantation in pre-clinical and clinical trials reveals very low engraftment in spite of successful clinical outcomes and their therapeutic actions are thought to be primarily through paracrine mechanisms. The beneficial role of transplanted MSC could rely on their strong immunomodulatory effect since, even without long-term engraftment, they have the ability to alter both the innate and adaptive immune response which is critical to facilitate new bone formation. This study presents the current knowledge of the immune response to the implantation of CaP biomaterials alone or in combination with MSC. In particular the central role of monocyte-derived cells, both macrophages and osteoclasts, in MSC-CaP mediated bone formation is emphasized. Biomaterial properties, such as macroporosity and surface microstructure, dictate the host response, and the ultimate bone healing cascade. Understanding intercellular communications throughout the inflammation, its resolution and the bone regeneration phase, is crucial to improve the current therapeutic strategies or develop new approaches.

Modern drug discovery methods led to evolving new agents with significant therapeutic potential. However, their properties, such as solubility and administration-related challenges, may hinder their benefits. Moreover, advances in biotechnology resulted in the development of a new generation of molecules with a short half-life that necessitates frequent administration. In this context, controlled release systems are required to enhance treatment efficacy and improve patient compliance. Innovative drug delivery systems are promising tools that protect therapeutic proteins and peptides against proteolytic degradation where controlled delivery is achievable. The present review provides an overview of different approaches used for drug delivery.

Abstract Objectives The purpose of this systematic review and meta‐analysis was to compare implant‐supported removable partial dentures (ISRPDs) with distal extension removable partial dentures (DERPDs) in terms of patient‐reported outcome measures (PROMs: patients' quality of life and satisfaction) and to determine mechanical and biological complications associated with ISRPDs. Material and Methods An electronic search was performed on four databases to identify studies treating Kennedy class I or II edentulous patients and which compared ISRPDs with DERPDs in terms of PROMS and studies, which evaluated mechanical and biological complications associated ISRPDs. Two authors independently extracted data on quality of life, patient satisfaction, and biomechanical complications from these studies. The risk of bias was assessed for each study, and for PROMs, the authors performed a meta‐analysis by using a random‐effects model. Results Thirteen articles were included based on the selection criteria. The difference in mean scores for quality of life (30.5 ± 1.8; 95% confidence interval [CI], 24.9–36.1) and patient satisfaction (−20.8 ± 0.2; 95% CI, −23.7 to −17.8) between treatments with conventional and implant‐supported removable dentures was statistically significant ( p < .05). Implant‐supported removable dentures improved patients' overall quality of life and satisfaction. Some mechanical and biological complications, such as clasp adjustment, abutment or implant loosening, marginal bone resorption, and peri‐implant mucositis, were noted in ISRPDs during patient follow‐up. Studies assessing PROMs were very heterogeneous ( I 2 = 65%, p = .85; I 2 = 75%, p = .88). Conclusions ISRPDs significantly improved quality of life and patient satisfaction. Some mechanical and biological complications have been associated with ISRPDs treatment, requiring regular monitoring of patients to avoid the occurrence of these complications.

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic rare diseases disrupting enamel development (Smith et al., Front Physiol, 2017a, 8, 333). The clinical enamel phenotypes can be described as hypoplastic, hypomineralized or hypomature and serve as a basis, together with the mode of inheritance, to Witkop’s classification (Witkop, J Oral Pathol, 1988, 17, 547–553). AI can be described in isolation or associated with others symptoms in syndromes. Its occurrence was estimated to range from 1/700 to 1/14,000. More than 70 genes have currently been identified as causative. Objectives: We analyzed using next-generation sequencing (NGS) a heterogeneous cohort of AI patients in order to determine the molecular etiology of AI and to improve diagnosis and disease management. Methods: Individuals presenting with so called “isolated” or syndromic AI were enrolled and examined at the Reference Centre for Rare Oral and Dental Diseases (O-Rares) using D4/phenodent protocol ( www.phenodent.org ). Families gave written informed consents for both phenotyping and molecular analysis and diagnosis using a dedicated NGS panel named GenoDENT. This panel explores currently simultaneously 567 genes. The study is registered under NCT01746121 and NCT02397824 ( https://clinicaltrials.gov/ ). Results: GenoDENT obtained a 60% diagnostic rate. We reported genetics results for 221 persons divided between 115 AI index cases and their 106 associated relatives from a total of 111 families. From this index cohort, 73% were diagnosed with non-syndromic amelogenesis imperfecta and 27% with syndromic amelogenesis imperfecta. Each individual was classified according to the AI phenotype. Type I hypoplastic AI represented 61 individuals (53%), Type II hypomature AI affected 31 individuals (27%), Type III hypomineralized AI was diagnosed in 18 individuals (16%) and Type IV hypoplastic-hypomature AI with taurodontism concerned 5 individuals (4%). We validated the genetic diagnosis, with class 4 (likely pathogenic) or class 5 (pathogenic) variants, for 81% of the cohort, and identified candidate variants (variant of uncertain significance or VUS) for 19% of index cases. Among the 151 sequenced variants, 47 are newly reported and classified as class 4 or 5. The most frequently discovered genotypes were associated with MMP20 and FAM83H for isolated AI. FAM20A and LTBP3 genes were the most frequent genes identified for syndromic AI. Patients negative to the panel were resolved with exome sequencing elucidating for example the gene involved ie ACP4 or digenic inheritance. Conclusion: NGS GenoDENT panel is a validated and cost-efficient technique offering new perspectives to understand underlying molecular mechanisms of AI. Discovering variants in genes involved in syndromic AI ( CNNM4, WDR72, FAM20A … ) transformed patient overall care. Unravelling the genetic basis of AI sheds light on Witkop’s AI classification.

Tendon disorders affect people of all ages, from elite and recreational athletes and workers to elderly patients. After an acute injury, 3 successive phases are described to achieve healing: an inflammatory phase followed by a proliferative phase, and finally by a remodeling phase. Despite this process, healed tendon fails to recover its original mechanical properties. In this review, we proposed to describe the key factors involved in the process such as cells, transcription factors, extracellular matrix components, cytokines and growth factors and vascularization among others. A better understanding of this healing process could help provide new therapeutic approaches to improve patients' recovery while tendon disorders management remains a medical challenge.

Several studies have recently highlighted the implication of B cells in physiopathogenesis of periodontal disease by showing that a B cell deficiency leads to improved periodontal parameters. However, the detailed profiles of circulating B cell subsets have not yet been investigated in patients with severe periodontitis (SP). We hypothesised that an abnormal distribution of B cell subsets could be detected in the blood of patients with severe periodontal lesions, as already reported for patients with chronic inflammatory diseases as systemic autoimmune diseases. Fifteen subjects with SP and 13 subjects without periodontitis, according to the definition proposed by the CDC periodontal disease surveillance work group, were enrolled in this pilot observational study. Two flow cytometry panels were designed to analyse the circulating B and B1 cell subset distribution in association with the RANKL expression. A significantly higher percentage of CD27+ memory B cells was observed in patients with SP. Among these CD27+ B cells, the proportion of the switched memory subset was significantly higher. At the same time, human B1 cells, which were previously associated with a regulatory function (CD20+CD69-CD43+CD27+CD11b+), decreased in SP patients. The RANKL expression increased in every B cell subset from the SP patients and was significantly greater in activated B cells than in the subjects without periodontitis. These preliminary results demonstrate the altered distribution of B cells in the context of severe periodontitis. Further investigations with a larger cohort of patients can elucidate if the analysis of the B cell compartment distribution can reflect the periodontal disease activity and be a reliable marker for its prognosis (clinical trial registration number: NCT02833285, B cell functions in periodontitis).

Drug delivery systems are proposed for the in situ controlled delivery of therapeutic molecules in the scope of tissue engineering. We propose herein silica nanofibers as carriers for the loading and release of bioactive proteins. The influence of pH, time and concentration on the amount of adsorbed proteins was studied. The interactions allowing loading were then studied by means of electron microscopy, zeta potential measurements, electron energy loss spectroscopy and attenuated total reflectance Fourier transform infrared analysis. Release profiles were determined and biological activities were enzymatically assessed. The first part of the work was carried out with lysozyme as a model protein, and then bioactive growth factors TGF-β1 and GDF-5 were used because their significance in human adipose stromal cell differentiation towards intervertebral disc nucleopulpocytes was previously assessed. It is demonstrated that protein-silica nanofiber interactions are mainly driven by hydrogen bonds and local electrostatic interactions. The present data thus provide a better understanding of the adsorption phenomenon involved, as well as a method to control protein adsorption and release. It is worth pointing out that the kinetic release of growth factors, up to 28 days, and their biological activity maintenance seem to be compatible with intervertebral disc regenerative medicine.

Since low-back pain is increasing in ageing populations, current research efforts are focused on obtaining a better understanding of the pathophysiology of intervertebral disc degeneration and on developing new therapeutic strategies. This requires adequate and clinically relevant models of the disease process. Ex vivo models can provide insights into isolated aspects of the degenerative/regenerative processes involved; although, ultimately, in vivo models are needed for preclinical translational studies. Such models have been developed in numerous animal species with significant variations in size and disc physiology and their number is considerable. Importantly, the choice of the model has to be tailored to the aim of the study. Given the number of available options, it is important to have a good understanding of the various models of disc degeneration and to be fully aware of their advantages and limitations. After comparing the anatomy and histology of intervertebral discs in animals and humans, the present study provides an overview of the different models of in vivo disc degeneration. It also provides a comprehensive guide with suggested criteria to select the most appropriate animal model in a question-driven manner.

The cellular microenvironment plays a major role in the biological functions of cells. Thus, biomaterials, especially hydrogels, which can be design to mimic the cellular microenvironment, are being increasingly used for cell encapsulation, delivery, and 3D culture, with the hope of controlling cell functions. Yet, much remains to be understood about the effects of cell-material interactions, and advanced synthetic strategies need to be developed to independently control the mechanical and biochemical properties of hydrogels. To address this challenge, we designed a new hyaluronic acid (HA)-based hydrogel platform using a click and bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. This approach facilitates the synthesis of hydrogels that are easy to synthesize and sterilize, have minimal swelling, are stable long term, and are cytocompatible. It provides bioorthogonal HA gels over an uncommonly large range of stiffness (0.5-45 kPa), all forming within 1-15 min. More importantly, our approach offers a versatile one-pot procedure to independently tune the hydrogel composition (e.g., polymer and adhesive peptides). Using this platform, we investigate the independent effects of polymer type, stiffness, and adhesion on the secretory properties of human adipose-derived stromal cells (hASCs) and demonstrate that HA can enhance the secretion of immunomodulatory factors by hASCs.

The coronavirus disease 2019 (COVID-19) is an acute infectious disease leading to a global pandemic. Dental professionals (dental staff, academics) and students are one part of the healthcare workers that face the greatest severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) risk. Consequently, several measures were imposed to limit the spread of the virus, the overload of the health system, and to protect the dental professionals and students. This brief communication focuses on chronology of COVID-19 related events, the temporary modifications, and the potential impacts on dental education in France.

The 5- and 10-year implant success rates in dentistry are nearly 90%. Prevalence of peri-implant diseases is 10% for peri-implantitis and 50% for peri-implant mucositis. To better understand these inflammatory pathologies of infectious origin, it is important to know if the composition of the peri-implant microbiota is comparable with the periodontal microbiota in healthy and pathological conditions. New generation sequencing (NGS) is a recent metagenomic method that analyzes the overall microorganisms present in an ecological niche by exploiting their genome. These methods are of two types: 16S rRNA sequencing and the shotgun technique. For several years, they have been used to explore the oral, periodontal, and, more specifically, peri-implant microbiota. The aim of this systematic review is to analyze the recent results of these new explorations by comparing the periodontal and peri-implant microbiota in patients with healthy and diseased sites and to explore the microbiological characteristics of peri-implantitis. A better knowledge of the composition of the peri-implant microbiota would enable us to optimize our therapeutic strategies. An electronic systematic search was performed using the medical databases PubMed/Medline, Cochrane Library, and ScienceDirect, and Periodontology 2000. The selected articles were published between January 2015 and March 2021. Inclusion criteria included clinical studies comparing healthy and pathological periodontal and peri-implant microbiota exclusively using 16S rRNA sequencing or shotgun sequencing, with enrolled populations free of systemic pathology, and studies without substantial bias. Eight articles were selected and reviewed. All of them used 16S rRNA sequencing exclusively. The assessment of these articles demonstrates the specific character of the peri-implant microbiota in comparison with the periodontal microbiota in healthy and pathological conditions. Indeed, peri-implant diseases are defined by dysbiotic bacterial communities that vary from one individual to another, including known periodontopathogens such as Porphyromonas gingivalis (P.g.) and genera less mentioned in the periodontal disease pattern such as Filifactor alocis. Examination of peri-implant microbiota with 16S rRNA sequencing reveals differences between the periodontal and peri-implant microbiota under healthy and pathological conditions in terms of diversity and composition. The pattern of dysbiotic drift is preserved in periodontal and peri-implant diseases, but when comparing the different types of pathological sites, the peri-implant microbiota has a specificity in the presence of bacteria proper to peri-implantitis and different relative proportions of the microorganisms present.

Abstract. This study focuses on daytime low-level clouds (LLCs) that occur within the first 2 km of the atmosphere over West Africa (WA). These daytime LLCs play a major role in the earth's radiative balance, yet their understanding is still relatively low in WA. We use the state-of-the-art ERA5 dataset to understand their occurrence and associated drivers as well as their impact on the incoming surface solar radiation in the two contrasting Guinean and Sahelian regions of WA. The diurnal cycle of the daytime occurrence of three LLC classes namely No LCC, LLC Class-1 (LLCs with lower fraction), and LLC Class-2 (LLCs with higher fraction) is first studied. The monthly evolutions of hourly and long-lasting LLC (for at least 6 consecutive hours) events are then analyzed as well as the synoptic-scale moisture flux associated with the long-lasting LLC events. Finally, the impact of LLC on the surface heat fluxes and the incoming solar irradiance is investigated. During the summer months in the Guinean region, LLC Class-1 occurrence is low, while LLC Class-2 is frequent (occurrence frequency around 75 % in August). In the Sahel, LLC Class-1 is dominant in the summer months (occurrence frequency more than 80 % from June to October); however the peak occurrence frequency of Class-2 is also in the summer. In both regions, events with No LLC do not present any specific correlation with the time of the day. However, a diurnal evolution that appears to be strongly different from one region to the other is noted for the occurrence of LLC Class-2. LLC occurrence in both regions is associated with high moisture flux driven by strong southwesterly winds from the Gulf of Guinea and significant background moisture levels. LLC Class-2 in particular leads to a significant reduction in the upward transfer of energy and a net downward energy transfer caused by the release of large amounts of energy in the atmosphere during the cloud formation. In July, August, and September (JAS), most of the LLC Class-2 events may likely be the low-level stratiform clouds that occur frequently over the Guinean region, while they may be deep convective clouds in the Sahel. Additionally, LLC Class-2 causes high attenuation of the incoming solar radiation, especially during JAS, where about 49 % and 44 % of the downwelling surface shortwave radiation is lost on average in Guinea and the Sahel, respectively.

Objective: To describe the feasibility and dye diffusion of selective perineural injection of the 7th and 8th cervical nerve (C7 and C8) ramus ventralis under ultrasonographic guidance in horses. Study design: Prospective experimental pilot cadaver study. Animals: Four equine cadavers of similar body weight (425-510 kg) and neck conformation. Methods: Five C7 and five C8 rami were perineurally injected with a dye solution. Anatomic dissections including vertebral canal opening were conducted to confirm nerve dye staining and describe the extent of color diffusion. Results: The ramus ventralis of the spinal cervical nerves was visualized in all cadavers. All the injections were successful in staining a portion of the nerve trunk. Eight rami had a uniform transversal staining of the nerve trunk that longitudinally covered a distance greater than 2 cm. One C7 and one C8 nerve trunk showed incomplete transversal staining with a more concentrated color on its half cranial aspect and a longitudinal coverage of less than 2 cm. Five injections resulted in dye extending proximally and medially into the epidural space. Volume had no appreciable effect on the extent of nerve staining. A greater proportion of epidural diffusion was found with injections done within less than one cm distally to the articular processes. All injections were considered to be selective for the targeted nerve. Conclusion and clinical relevance: Ultrasonography-guided perineural injection of C7 and C8 ramus ventralis is a feasible technique that may have multiple applications in multimodal analgesia in horses. Further clinical study will be necessary to determine the appropriate drug, dosage and volume to inject and to confirm its usefulness.

Osteoarthritis (OA) is an incurable, painful, and debilitating joint disease affecting over 500 million people worldwide. The OA joint tissues are infiltrated by various immune cells, particularly macrophages, which are able to induce or perpetuate inflammation. Notably, synovitis and its macrophage component represent a target of interest for developing treatments. In this review, we describe the latest advances in understanding the heterogeneity of macrophage origins, phenotypes, and functions in the OA joint and the effect of current symptomatic therapies on these cells. We then highlight the therapeutic potential of anticytokines/chemokines, nano- and microdrug delivery, and future strategies to modulate macrophage functions in OA.

This prospective randomized clinical trial aimed to evaluate the effect of low-level laser therapy on tooth movement during Class II intermaxillary elastics treatment. Forty-two patients with Class II malocclusion were included, and their maxillary quadrants were allocated into two groups: treatment with an active diode laser and a placebo group. In each group, the time taken to obtain Class I occlusion after 6 months, rate of movement, total displacement of the maxillary canine to Class I occlusion and pain were recorded. The time to reach Class I occlusion in the active laser group (2.46 ± 2.1 months) was not significantly different from that in the placebo group (2.48 ± 2.0 months) (p = 0.938). Interestingly, the total distance of movement on the active laser side (2.27 ± 1.5 mm) was significantly greater than that on the placebo side (1.64 ± 1.3 mm) (p = 0.009). The pain levels on days 1, 2 and 3 were not significantly different between the laser and placebo sections. The rate of distance change toward Class I occlusion in the laser group (1.1 ± 0.7 mm/month) was significantly higher than that in the placebo group (0.74 ± 0.6 mm/month) (p = 0.037). Low-level laser therapy (970 nm) did not reduce the time needed to obtain Class I occlusion, but a significant acceleration in tooth movement was observed in the irradiated group.Trial registration: NCT02181439. Registered 04 July 2014- https://www.clinicaltrials.gov/ct2/results?term=cinelaser .

Modelling rare neurogenetic diseases to develop new therapeutic strategies is highly challenging. The use of human-induced pluripotent stem cells (hiPSCs) is a powerful approach to obtain specialized cells from patients. For hereditary peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) Type II, spinal motor neurons (MNs) are impaired but are very difficult to study. Although several protocols are available to differentiate hiPSCs into neurons, their efficiency is still poor for CMT patients. Thus, our goal was to develop a robust, easy, and reproducible protocol to obtain MNs from CMT patient hiPSCs. The presented protocol generates MNs within 20 days, with a success rate of 80%, using specifically chosen molecules, such as Sonic Hedgehog or retinoic acid. The timing and concentrations of the factors used to induce differentiation are crucial and are given hereby. We then assessed the MNs by optic microscopy, immunocytochemistry (Islet1/2, HB9, Tuj1, and PGP9.5), and electrophysiological recordings. This method of generating MNs from CMT patients in vitro shows promise for the further development of assays to understand the pathological mechanisms of CMT and for drug screening.

BACKGROUND: One of the major reasons to stop antibiotic prophylaxis (AP) to prevent infective endocarditis (IE) in the United Kingdom but not in the rest of the world was that it would result in more deaths from fatal adverse drug reactions (ADRs) than the number of IE deaths. The main aim of this study was to quantify and describe the ADRs with amoxicillin or clindamycin for IE AP. The second aim was to infer a crude incidence of anaphylaxis associated with amoxicillin for IE AP. STUDY DESIGN: The Medical Dictionary for Regulatory Activities (MedDRA) was used to group ADRs for IE AP using the broad Standardized MedDRA Queries "Anaphylactic reaction, Amoxicillin, Clindamycin, Clostridium Difficile infection" to the French Pharmacovigilance Database System. From this first-line collection, we selected all cases occurring for IE AP and ultimately, the cases for IE AP for a dental procedure. Then, each case was analyzed. RESULTS: Of 11639 first-line recorded ADRs, 100 were for IE AP but no fatal anaphylaxis to amoxicillin or clindamycin and no C. difficile infection associated with clindamycin were identified. Only 17 cases of anaphylaxis to amoxicillin related to dental procedures were highlighted. The estimation of the crude incidence rate of anaphylaxis associated with amoxicillin for IE AP for invasive dental procedure was 1/57 000 (95% CI 0.2-0.6). CONCLUSIONS: Fatal or severe ADRs with amoxicillin or clindamycin is not a rational argument to stop IE AP before invasive dental procedures.

The in vivo resorption rate of two injectable apatitic calcium phosphate cements used in clinics (Graftys® HBS and NORIAN®) was compared, using a good laboratory practice (GLP) study based on an animal model of critical-sized bone defect. To rationalize the markedly different biological properties observed for both cements, key physical features were investigated, including permeability and water-accessible porosity, total porosity measured by mercury intrusion and gravimetry, and microstructure. Due to a different concept for creating porosity between the two cements investigated in this study, a markedly different microstructural arrangement of apatite crystals was observed in the intergranular space, which was found to significantly influence both the mechanical strength and in vivo degradation of the two calcium phosphate cements.

Over the last two decades, click chemistry and bioorthogonal chemistry have revolutionized the field of biomedical engineering. Extensive investigations have been conducted and allowed for unforeseeable developments in many different fields, including imaging, drug delivery, 3D cell culture, and bioprinting. The properties of advanced click and bioorthogonal chemical tools have now exceeded the expectations set by the researchers who first introduced the two concepts, calling for reevaluation. More importantly, their developments in distinct research contexts have led to conceptual divergences, which can now be a source of misunderstandings and disagreements. This perspective article aims to take a fresh look at the two concepts, with particular attention to their combination for applications in biological environments. After a short historical introduction, the definitions of click chemistry and bioorthogonal chemistry are revised, and the concept of ideal click chemistry is introduced. Chemical reactions that are commonly considered as click and bioorthogonal reactions are discussed in light of this new perspective. Finally, current challenges and new chemical concepts are presented, offering a new framework for future research.