Revolutionizing Endodontics: Innovative Approaches for Treating Mature Teeth With Closed Apices and Apical Lesions: A Report of Two Cases.

INTRODUCTION: Modern tissue engineering strategies have elucidated the potential of regenerative endodontic treatment (RET) as an alternative for treating mature teeth. METHODS: Here, we report two cases in which cell-based RET (CB-RET) using encapsulated allogeneic umbilical cord mesenchymal stem cells (UC-MSCs) in a platelet-poor plasma (PPP)-based scaffold was used in two mature teeth with pulp necrosis and apical periodontitis. RESULTS: After 5 years of follow-up, the healing response was satisfactory in both cases, with evidence of pulp revitalization. CONCLUSIONS: This is the first study to report the success of an extended, 5-year follow-up for allogeneic CB-RET. This report presents an innovative and sustainable solution to challenging endodontic scenarios.

An advanced biphasic porous and injectable scaffold displays a fine balance between mechanical strength and remodeling capabilities essential for cartilage regeneration.

An important challenge in tissue engineering is the regeneration of functional articular cartilage (AC). In the field, biomimetic hydrogels are being extensively studied as scaffolds that recapitulate microenvironmental features or as mechanical supports for transplanted cells. New advanced hydrogel formulations based on salmon methacrylate gelatin (sGelMA), a cold-adapted biomaterial, are presented in this work. The psychrophilic nature of this biomaterial provides rheological advantages allowing the fabrication of scaffolds with high concentrations of the biopolymer and high mechanical strength, suitable for formulating injectable hydrogels with high mechanical strength for cartilage regeneration. However, highly intricate cell-laden scaffolds derived from highly concentrated sGelMA solutions could be deleterious for cells and scaffold remodeling. On this account, the current study proposes the use of sGelMA supplemented with a mesophilic sacrificial porogenic component. The cytocompatibility of different sGelMA-based formulations is tested through the encapsulation of osteoarthritic chondrocytes (OACs) and stimulated to synthesize extracellular matrix (ECM) components in vitro and in vivo. The sGelMA-derived scaffolds reach high levels of stiffness, and the inclusion of porogens impacts positively the scaffold degradability and molecular diffusion, improved fitness of OACs, increased the expression of cartilage-related genes, increased glycosaminoglycan (GAG) synthesis, and improved remodeling toward cartilage-like tissues. Altogether, these data support the use of sGelMA solutions in combination with mammalian solid gelatin beads for highly injectable formulations for cartilage regeneration, strengthening the importance of the balance between mechanical properties and remodeling capabilities.

A novel porous hydrogel based on hybrid gelation for injectable and tough scaffold implantation and tissue engineering applications.

Although there have been many advances in injectable hydrogels as scaffolds for tissue engineering or as payload-containing vehicles, the lack of adequate microporosity for the desired cell behavior, tissue integration, and successful tissue generation remains an important drawback. Herein, we describe an effective porous injectable system that allowsin vivoformation of pores through conventional syringe injection at room temperature. This system is based on the differential melting profiles of photocrosslinkable salmon gelatin and physically crosslinked porogens of porcine gelatin (PG), in which PG porogens are solid beads, while salmon methacrylamide gelatin remains liquid during the injection procedure. After injection and photocrosslinking, the porogens were degraded in response to the physiological temperature, enabling the generation of a homogeneous porous structure within the hydrogel. The resultant porogen-containing formulations exhibited controlled gelation kinetics within a broad temperature window (18.5 ± 0.5-28.8 ± 0.8 °C), low viscosity (133 ± 1.4-188 ± 16 cP), low force requirements for injectability (17 ± 0.3-39 ± 1 N), robust mechanical properties after photo-crosslinking (100.9 ± 3.4-332 ± 13.2 kPa), and favorable cytocompatibility (>70% cell viability). Remarkably,in vivosubcutaneous injection demonstrated the suitability of the system with appropriate viscosity and swift crosslinking to generate porous hydrogels. The resulting injected porous constructs showed favorable biocompatibility and facilitated cell infiltration for desirable potential tissue remodeling. Finally, the porogen-containing formulations exhibited favorable handling, easy deposition, and good shape fidelity when used as bioinks in 3D bioprinting technology. This injectable porous system serves as a platform for various biomedical applications, thereby inspiring future advances in cell therapy and tissue engineering.

Delivery of affordable and scalable encapsulated allogenic/autologous mesenchymal stem cells in coagulated platelet poor plasma for dental pulp regeneration.

The main goal of regenerative endodontics procedures (REPs) is to revitalize teeth by the regeneration of healthy dental pulp. In this study, we evaluated the potential of combining a natural and accessible biomaterial based on Platelet Poor Plasma (PPP) as a support for dental pulp stem cells (DPSC) and umbilical cord mesenchymal stem cells (UC-MSC). A comparison study between the two cell sources revealed compatibility with the PPP based scaffold with differences noted in the proliferation and angiogenic properties in vitro. Additionally, the release of growth factors including VEGF, HGF and DMP-1, was detected in the media of cultured PPP and was enhanced by the presence of the encapsulated MSCs. Dentin-Discs from human molars were filled with PPP alone or with MSCs and implanted subcutaneously for 4 weeks in mice. Histological analysis of the MSC-PPP implants revealed a newly formed dentin-like structure evidenced by the expression of Dentin sialophosphoprotein (DSPP). Finally, DPSC induced more vessel formation around the dental discs. This study provides evidence of a cost-effective, xenofree scaffold that is compatible with either autologous or allogenic strategy for dental pulp regeneration. This attempt if successfully implemented, could make REPs treatment widely accessible, contributing in improving global health conditions.

Allogeneic Cellular Therapy in a Mature Tooth with Apical Periodontitis and Accidental Root Perforation: A Case Report.

INTRODUCTION: Cell therapy in regenerative endodontics introduces an alternative option to classic treatment strategies for complex endodontic cases. The aim of this case report was to describe cell-based therapy using allogeneic umbilical cord mesenchymal stem cells (UC-MSCs) encapsulated in a bioscaffold for a complex case of a mature permanent tooth with apical periodontitis and accidental root perforation. METHODS: A healthy 19-year-old man undergoing orthodontic treatment was referred for endodontic treatment in tooth #7; he was diagnosed with apical periodontitis during a previously initiated treatment associated with accidental perforation of the radicular cervical third. The root perforation was sealed with glass ionomer and composite resin, and the root canal was instrumented, disinfected, and dressed with calcium hydroxide. After 3 weeks, allogeneic UC-MSCs were encapsulated in platelet-poor plasma and then implanted into the root canal, and Biodentine (Septodont, Saint-Maur-des-Fosses, France) was placed below the cementoenamel junction. Finally, the tooth was restored with composite resin. RESULTS: Follow-up examinations were performed 6 months and 1 year later. The examinations included periapical radiography, cone-beam computed tomographic imaging, and sensitivity and vitality tests. Radiographic and cone-beam computed tomographic images indicated remission of the apical lesion. Clinical evaluations revealed normal responses to percussion and palpation tests; the tooth was responsive to the electric pulp test, and the vitality test indicated low blood perfusion units. CONCLUSIONS: This case report reveals the potential use of allogeneic cellular therapy using encapsulated UC-MSCS in a platelet-poor plasma scaffold for a complex case of a permanent tooth with apical periodontitis and root perforation.

Personalized Cell Therapy for Pulpitis Using Autologous Dental Pulp Stem Cells and Leukocyte Platelet-rich Fibrin: A Case Report.

INTRODUCTION: Regenerative endodontic procedures have emerged as a new treatment. The aim of this case report was to describe a regenerative autologous cellular therapy using mesenchymal stem cells from inflamed dental pulp and leukocyte platelet-rich fibrin (L-PRF) in a mature tooth. METHODS: A healthy 50-year-old man consulting for spontaneous dental pain was referred for endodontic treatment in tooth #28, which was diagnosed with symptomatic irreversible pulpitis. Inflamed dental pulp was extracted and transported to a good manufacturing practice laboratory for the isolation and culture of dental pulp stem cells (DPSCs). L-PRF was obtained from the patient's blood and was introduced into the instrumented and disinfected root canal, and expanded DPSCs were inoculated into the clot. The cervical part of the root canal was sealed with Biodentine (Septodont, Saint-Maur-des-Fosses, France) and a composite resin. RESULTS: Follow-up examinations were performed 6 months and 3 years later. The examinations included periapical radiographs (to measure the periapical index [PAI]), cone-beam computed tomographic (CBCT) imaging, sensitivity, and vitality tests. Clinical evaluations revealed normal responses to percussion and palpation tests. The tooth had a delayed response to cold, and the electric pulp test was responsive. The PAI and CBCT imaging revealed that the periapical area remained normal with a PAI score of 1 and a CBCT PAI score of 0. The vitality test performed indicated low blood perfusion units. CONCLUSIONS: This case study reveals the potential use of a patient's own DPSCs and L-PRF as an alternative procedure for the treatment of pulpitis in mature permanent teeth. It also paves the way for the design of personalized cell-based clinical trials in regenerative endodontics.

Cold-adaptation of a methacrylamide gelatin towards the expansion of the biomaterial toolbox for specialized functionalities in tissue engineering.

Tissue regeneration is witnessing a significant surge in advanced medicine. It requires the interaction of scaffolds with different cell types for efficient tissue formation post-implantation. The presence of tissue subtypes in more complex organs demands the co-existence of different biomaterials showing different hydrolysis rate for specialized cell-dependent remodeling. To expand the available toolbox of biomaterials with sufficient mechanical strength and variable rate of enzymatic degradation, a cold-adapted methacrylamide gelatin was developed from salmon skin. Compared with mammalian methacrylamide gelatin (GelMA), hydrogels derived from salmon GelMA displayed similar mechanical properties than the former. Nevertheless, salmon gelatin and salmon GelMA-derived hydrogels presented characteristics common of cold-adaptation, such as reduced activation energy for collagenase, increased enzymatic hydrolysis turnover of hydrogels, increased interconnected polypeptides molecular mobility and lower physical gelation capability. These properties resulted in increased cell-remodeling rate in vitro and in vivo, proving the potential and biological tolerance of this mechanically adequate cold-adapted biomaterial as alternative scaffold subtypes with improved cell invasion and tissue fusion capacity.

The clinical spectrum associated with a chromosome 17 short arm proximal duplication (dup 17p11.2) in three patients.

The p11.2-p12 region of human chromosome 17 is gene rich and composed of at least two genomically unstable domains: the Smith-Magenis syndrome region (17p11.2) and the Charcot-Marie-Tooth region (17p12), both of which are flanked by several low-copy repeat sequences. Homologous recombination between these flanking repeats results in either deletion- or duplication-associated phenotypes caused by a gene dosage effect. We report on the clinical phenotype of three patients presenting with either a 17p11.2 or 17p11.2p12 duplication, revealed by chromosome analysis and confirmed by fluorescent in situ hybridization analysis, high resolution genomic analysis of the 17p region using oligonucleotide array comparative genomic hybridization, and molecular studies with microsatellite markers. Two patients carry the 17p11.2 duplication, while the third one shows a larger duplication including the 17p12 region. The facial features observed in our patients include triangular face, full cheeks, smooth philtrum, thin upper lip, dental malocclusion, irregular eyebrows, and sparse hair, all of which are consistent with the pure proximal dup 17p phenotype. The patients' other clinical features are compared with previously published cases.

Gingival Mesenchymal Stem Cells Outperform Haploidentical Dental Pulp-derived Mesenchymal Stem Cells in Proliferation Rate, Migration Ability, and Angiogenic Potential.

High donor variation makes comparison studies between different dental sources dubious. Dental tissues offer a rare opportunity for comparing the biological characteristics of haploidentical mesenchymal stem cells (MSCs) isolated from the same donor. The objective was to identify the optimal dental source of MSCs through a biological and functional comparison of haploidentical MSCs from gingival (GMSCs) and dental pulp stem cells (DPSCs) focusing mainly on their angiogenic potential. The comparison study included (1) surface markers expression, (2) mesodermal differentiation capacity (chondrogenic, adipogenic, and osteogenic), (3) proliferation, (4) migration potential, (5) ability to form colony units, and (6) angiogenic potential in vitro and in vivo. Comparative analysis showed no difference in the immunophenotypic profile nor for the trilineage differentiation potential. Proliferation of GMSCs was higher than DPSCs at day 6 (2.6-fold higher, P < 0.05). GMSCs showed superior migratory capacity compared to DPSCs at 4, 8, and 12 h (2.1-, 1.5-, and 1.2-fold higher, respectively, P < 0.05). Furthermore, GMSCs formed a higher number of colony units for both cell concentrations (1.7- and 1.4-fold higher for 150 and 250 starting cells, respectively, P < 0.05). GMSCs showed an improved angiogenic capacity compared to DPSCs (total tube lengths 1.17-fold higher and 1.5-fold total loops, P < 0.05). This was correlated with an enhanced release of vascular growth factor under hypoxic conditions. Finally, in the plug transplantation assay evaluating the angiogenesis in vivo, the DPSC and GMSC hemoglobin content was 3.9- and 4-fold higher, respectively, when compared to the control (Matrigel alone). GMSCs were superior to their haploidentical DPSCs in proliferation, migration, and angiogenic potentials. This study positions GMSCs in the forefront of dental cell sources for applications in regenerative medicine.

Layer-by-layer approach for a uniformed fabrication of a cell patterned vessel-like construct.

Successful tissue engineered small diameter blood vessels (SDBV) require manufacturing systems capable of precisely controlling different key elements, such as material composition, geometry and spatial location of specialized biomaterials and cells types. We report in this work an automated methodology that enables the manufacture of multilayer cylindrical constructs for SDBV fabrication that uses a layer-by-layer deposition approach while controlling variables such as dipping and spinning speed of a rod and biomaterial viscosity. Different biomaterials including methacrylated gelatin, alginate and chitosan were tested using this procedure to build different parts of the constructs. The system was capable of controlling dimensions of lumen from 0.5 mm up to 6 mm diameter and individual layers from 1 µm up to 400 µm thick. A cellular component was successfully added to the biomaterial in the absence of significant cytotoxic effect which was assessed by viability and proliferation assays. Additionally, cells showed a homogenous distribution with well-defined concentric patterns across the multilayer vessel grafts. The challenging generation of inner endothelial cells of approximately 20-30 µm of thickness was achieved. Preliminary experimental evidences of microstructural alignment of the biomaterial were obtained when the dipping approach was combined with the rod rotation. The study demonstrated the wide versatility and scalability of the automated system to easily and rapidly fabricate complex cellularized multilayer vascular grafts with structural configuration that resembles natural blood vessels.

Efficient suppression of murine arthritis by combined anticytokine small interfering RNA lipoplexes.

OBJECTIVE: Blocking tumor necrosis factor (TNF) effectively inhibits inflammation and joint damage in rheumatoid arthritis (RA), but 40% of RA patients respond only transiently or not at all to the current anti-TNF biotherapies. The purpose of this study was to develop an alternative targeted therapy for this subgroup of RA patients. As proof of concept, we tested the efficiency of an RNA interference (RNAi)-based intervention that targets proinflammatory cytokines in suppressing murine collagen-induced arthritis (CIA). METHODS: Two synthetic short interfering RNA (siRNA) sequences were designed for each of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and IL-18. Their silencing specificity was assessed according to lipopolysaccharide-induced messenger RNA expression in J774.1 mouse macrophages as compared with control siRNA. For in vivo administration, siRNA were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA and were injected intravenously (0.5 mg/kg) into DBA/1 mice with CIA. RESULTS: Weekly injections of anti-IL-1, anti-IL-6, or anti-IL-18 siRNA-based lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling and destruction of cartilage and bone, both in the preventative and the curative settings. The most striking therapeutic effect was observed when the 3 siRNA were delivered in combination. The siRNA lipoplex cocktail reduced all pathologic features of RA, including inflammation, joint destruction, and the Th1 response, and overall parameters of RA were improved as compared with anti-TNF siRNA lipoplex-based treatment. CONCLUSION: Our results present a novel option for in vivo RNAi-based antiinflammatory immunotherapy. Our findings indicate that intravenous administration of a lipoplex cocktail containing several anticytokine siRNA is a promising novel antiinflammatory therapy for RA, as well as a useful and simple tool for understanding the pathophysiology of RA and for evaluating new therapeutic candidates.

Efficient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor alpha in experimental arthritis.

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) is among the most prominent cytokines in rheumatoid arthritis (RA) and is secreted mainly by macrophages. A direct method for restoring the immunologic balance in RA is use of small interfering RNA (siRNA) for silencing the TNFalpha transcript. The aim of this study was to determine the therapeutic effect of systemic administration of TNFalpha siRNA in an experimental model of RA, optimizing its delivery using new liposome formulations. METHODS: Murine macrophages were transfected with siRNA targeting TNFalpha, and expression was measured. The therapeutic effect in collagen-induced arthritis (CIA) was assessed after intravenous delivery of TNFalpha siRNA. Delivery was optimized using a carrier DNA for complexation with the cationic liposome RPR209120/DOPE. Levels of TNFalpha and other cytokines were measured in sera and joint tissue-conditioned media. Biodistribution was determined using a fluorescent siRNA. RESULTS: In vitro, TNFalpha siRNA efficiently and specifically modulated the expression of TNFalpha at both the messenger RNA and protein levels. In vivo, complete cure of CIA was observed when TNFalpha siRNA was administered weekly, complexed with the liposome and combined with carrier DNA. Inhibition (50-70%) of articular and systemic TNFalpha secretion was detected in the siRNA-injected groups, which correlated with a decrease in the levels of interleukin-6 and monocyte chemotactic protein 1. The main organs targeted by siRNA were the liver and spleen; the addition of liposome RPR209120 and carrier DNA significantly increased organ uptake. CONCLUSION: We demonstrated the efficiency of systemic delivery of siRNA designed to silence TNFalpha in CIA, using a liposome carrier system as a way to address the methodologic limitations in vivo.