Laser-Induced Blood Coagulation for Surgical Application: A Scoping Review.

There is a lack of evidence-based reviews on the effects of laser irradiation on blood coagulation in the literature, despite a large number of clinical trials. We therefore evaluated the available evidence on laser irradiation parameters used in coagulation of blood to optimize physical parameters. We performed a literature search for recent scientific studies indexed between 2017 and 2023 using the databases of PubMed and ScienceDirect. Articles were selected based on defined inclusion and exclusion criteria, and 78 publications in total were eventually included in this scoping review. The following were found to produce significant benefits in blood coagulation for surgical application: (1) dentistry and oral surgeries: 980 nm, 27 s, 2 W, 1502.7 W/cm2, 26.5 J, 622 J/cm2, 400 µm; (2) urogenital disorders: 532 nm, 4 s, 40 W, 10600 W/cm2, 1.3 J, 424 J/cm2, 600 µm; (3) ophthalmic disorders: 810 nm, 1 s, 1 W, 3540 W/cm2, 0.75 J, 1326 J/cm2, 100 µm; (4) embryological surgeries: 1064 nm, 10 s, 25 W, 35400 W/cm2, 262.5 J, 371000 J/cm2, 332.5 µm; (5) dermatological disorders: 1064 nm, 20 W, 2440 W/cm2, 0.1 J, 24 J/cm2, 670 µm; (6) gastrointestinal disorders: 532 nm, 3 s, 20 W, 1051 W/cm2, 120 J, 26500 J/cm2, 760 µm; (7) neurological surgeries: 2.5 s, 1.5 W, 1035 W/cm2, 2 J, 1584 J/cm2, 385 µm; (8) pulmonary disorders: 1320 nm, 5s, 35 W, 12450 W/cm2, 250 J, 65000 J/cm2, 700 µm (9) cardiovascular disorders: 1064 nm, 16.5 s, 5 W, 1980.5 W/cm2, 900 J, 760 J/cm2, 400 µm. In conclusion, our scoping review identifies that combining data from all clinically heterogeneous studies suggests that laser irradiation reflects an effective method for inducing blood coagulation in several medical fields.

Nanostructured Porous Silicon for Bone Tissue Engineering: Kinetics of Particle Degradation and Si-Controlled Release.

Nanostructured porous silicon (pSi) is a synthetic silicon-based material. Its biocompatibility and bioresorbability in body fluids make pSi an appealing biomaterial for tissue engineering, with surfaces characteristics facilitating human cell adhesion and differentiation. The resorption kinetics of such porous biomaterials is crucial for in vivo bone regeneration, in order to adapt biomaterial resorption to tissue formation, and to control the release of loaded bioactive molecules. We investigated pSi as a bioactive scaffold for bone tissue engineering, with an emphasis on kinetics of pSi resorption and silicon release. PSi particles and chips were fabricated from crystalline silicon, and functionalized by oxidation and chemical grafting of amine groups to mimic biological structures. Materials resorption over time was investigated with Raman spectroscopy, infrared spectroscopy, and Scanning Electron Microscopy. Silicon release was followed by mass spectrometry. Particle degradation and inclusion in newly formed bone were studied in vivo. The in vitro experiments revealed that non-oxidized pSi had an accelerated initial dissolution in ddH2O and an inhibition of initial Si release in SBF. This high reactivity also led to transformation towards amorphous non-resorbable silica when incubated in SBF. PSi resorption started immediately with a maximal dissolution in the first 24 h. Later, the dissolution rate decreased over time. In comparison, the resorption process of oxidized pSi seemed delayed, but more continuous. This delayed dissolution increased the bioactivity and stability, leading to enhanced bone formation in vivo. Delayed pSi degradation provided a constant surge of silicic acid over time and promoted bone regeneration, demonstrating the high potential of pSi for bone tissue engineering: Oxidized pSi were almost completely resorbed after 2 months of healing, with remaining partially dissolved particles surrounded by newly formed bone. On the contrary, non-oxidized particles were still obviously present after 2 months with limited bone regeneration. This delayed resorption is consistent with the in vitro observations in SBF, and particles' transformation towards silica.

Biomechanical characterization of a fibrinogen-blood hydrogel for human dental pulp regeneration.

In dental practice, Regenerative Endodontic Treatment (RET) is applied as an alternative to classical endodontic treatments of immature necrotic teeth. This procedure, also known as dental pulp revitalization, relies on the formation of a blood clot inside the root canal leading to the formation of a reparative vascularized tissue similar to dental pulp, which would provide vitality to the affected tooth. Despite the benefit of this technique, it lacks reproducibility due to the fast degradation and poor mechanical properties of blood clots. This work presents a method for constructing a fibrinogen-blood hydrogel that mimics the viscoelastic properties of human dental pulp while preserving the biological properties of blood for application in RET. By varying the blood and fibrinogen concentrations, gels with different biomechanical and biological properties were obtained. Rheology and atomic force microscopy (AFM) were combined to study the viscoelastic properties. AFM was used to evaluate the elasticity of human dental pulp. The degradation and swelling rates were assessed by measuring weight changes. The biomimetic properties of the gels were demonstrated by studying the cell survival and proliferation of dental pulp cells (DPCs) for 14 days. The formation of an extracellular matrix (ECM) was assessed by multiphoton microscopy (MPM). The angiogenic potential was evaluated by an ex vivo aortic ring assay, in which the endothelial cells were observed by histological staining after migration. The results show that the Fbg-blood gel prepared with 9 mg ml-1 fibrinogen and 50% blood of the Fbg solution volume has similar elasticity to human dental pulp and adequate degradation and swelling rates. It also allows cell survival and ECM secretion and enhances endothelial cell migration and formation of neovessel-like structures.

The Secretome of Human Dental Pulp Stem Cells and Its Components GDF15 and HB-EGF Protect Amyotrophic Lateral Sclerosis Motoneurons against Death.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable paralytic disorder caused by the progressive death of upper and lower motoneurons. Although numerous strategies have been developed to slow disease progression and improve life quality, to date only a few therapeutic treatments are available with still unsatisfactory therapeutic benefits. The secretome of dental pulp stem cells (DPSCs) contains numerous neurotrophic factors that could promote motoneuron survival. Accordingly, DPSCs confer neuroprotective benefits to the SOD1G93A mouse model of ALS. However, the mode of action of DPSC secretome on motoneurons remains largely unknown. Here, we used conditioned medium of human DPSCs (DPSCs-CM) and assessed its effect on survival, axonal length, and electrical activity of cultured wildtype and SOD1G93A motoneurons. To further understand the role of individual factors secreted by DPSCs and to circumvent the secretome variability bias, we focused on GDF15 and HB-EGF whose neuroprotective properties remain elusive in the ALS pathogenic context. DPSCs-CM rescues motoneurons from trophic factor deprivation-induced death, promotes axon outgrowth of wildtype but not SOD1G93A mutant motoneurons, and has no impact on the spontaneous electrical activity of wildtype or mutant motoneurons. Both GDF15 and HB-EGF protect SOD1G93A motoneurons against nitric oxide-induced death, but not against death induced by trophic factor deprivation. GDF15 and HB-EGF receptors were found to be expressed in the spinal cord, with a two-fold increase in expression for the GDF15 low-affinity receptor in SOD1G93A mice. Therefore, the secretome of DPSCs appears as a new potential therapeutic candidate for ALS.

Towards the Standardization of Mesenchymal Stem Cell Secretome-Derived Product Manufacturing for Tissue Regeneration.

Mesenchymal stem cell secretome or conditioned medium (MSC-CM) is a combination of biomolecules and growth factors in cell culture growth medium, secreted by mesenchymal stem cells (MSCs), and the starting point of several derived products. MSC-CM and its derivatives could be applied after injuries and could mediate most of the beneficial regenerative effects of MSCs without the possible side effects of using MSCs themselves. However, before the clinical application of these promising biopharmaceuticals, several issues such as manufacturing protocols and quality control must be addressed. This review aims to underline the influence of the procedure for conditioned medium production on the quality of the secretome and its derivatives and highlights the questions considering cell sources and donors, cell expansion, cell passage number and confluency, conditioning period, cell culture medium, microenvironment cues, and secretome-derived product purification. A high degree of variability in MSC secretomes is revealed based on these parameters, confirming the need to standardize and optimize protocols. Understanding how bioprocessing and manufacturing conditions interact to determine the quantity, quality, and profile of MSC-CM is essential to the development of good manufacturing practice (GMP)-compliant procedures suitable for replacing mesenchymal stem cells in regenerative medicine.

Improving dental epithelial junction on dental implants with bioengineered peptides.

Introduction: The functionalization of titanium (Ti) and titanium alloys (Ti6Al4V) implant surfaces via material-specific peptides influence host/biomaterial interaction. The impact of using peptides as molecular linkers between cells and implant material to improve keratinocyte adhesion is reported. Results: The metal binding peptides (MBP-1, MBP-2) SVSVGMKPSPRP and WDPPTLKRPVSP were selected via phage display and combined with laminin-5 or E-cadherin epithelial cell specific peptides (CSP-1, CSP-2) to engineer four metal-cell specific peptides (MCSPs). Single-cell force spectroscopy and cell adhesion experiments were performed to select the most promising candidate. In vivo tests using the dental implant for rats showed that the selected bi functional peptide not only enabled stable cell adhesion on the trans-gingival part of the dental implant but also arrested the unwanted apical migration of epithelial cells. Conclusion: The results demonstrated the outstanding performance of the bioengineered peptide in improving epithelial adhesion to Ti based implants and pointed towards promising new opportunities for applications in clinical practice.

Allogenic Stem Cells Carried by Porous Silicon Scaffolds for Active Bone Regeneration In Vivo.

To date, bone regeneration techniques use many biomaterials for bone grafting with limited efficiencies. For this purpose, tissue engineering combining biomaterials and stem cells is an important avenue of development to improve bone regeneration. Among potentially usable non-toxic and bioresorbable scaffolds, porous silicon (pSi) is an interesting biomaterial for bone engineering. The possibility of modifying its surface can allow a better cellular adhesion as well as a control of its rate of resorption. Moreover, release of silicic acid upon resorption of its nanostructure has been previously proved to enhance stem cell osteodifferentiation by inducing calcium phosphate formation. In the present study, we used a rat tail model to experiment bone tissue engineering with a critical size defect. Two groups with five rats per group of male Wistar rats were used. In each rat, four vertebrae were used for biomaterial implantation. Randomized bone defects were filled with pSi particles alone or pSi particles carrying dental pulp stem cells (DPSC). Regeneration was evaluated in comparison to empty defect and defects filled with xenogenic bone substitute (Bio-Oss®). Fluorescence microscopy and SEM evaluations showed adhesion of DPSCs on pSi particles with cells exhibiting distribution throughout the biomaterial. Histological analyzes revealed the formation of a collagen network when the defects were filled with pSi, unlike the positive control using Bio-Oss®. Overall bone formation was objectivated with µCT analysis and showed a higher bone mineral density with pSi particles combining DPSC. Immunohistochemical assays confirmed the increased expression of bone markers (osteocalcin) when pSi particles carried DPSC. Surprisingly, no grafted cells remained in the regenerated area after one month of healing, even though the grafting of DPSC clearly increased bone regeneration for both bone marker expression and overall bone formation objectivated with µCT. In conclusion, our results show that the association of pSi with DPSCs in vivo leads to greater bone formation, compared to a pSi graft without DPSCs. Our results highlight the paracrine role of grafted stem cells by recruitment and stimulation of endogenous cells.

Prevention and repair of orthodontically induced root resorption using ultrasound: a scoping review.

INTRODUCTION: This review summarizes the available recent literature on different mechanisms and parameters of pulsed ultrasound (US) that have been used during orthodontic treatments to prevent and repair root resorption. AREAS COVERED: A literature search was conducted between January (2002) and September (2022) in the following databases: PubMed, Google-Scholar, Embase, and The-Cochrane-Library. After exclusions, a total of 19 papers were included in the present review. The most used US parameters with positive outcomes were frequency of 1.5 MHz, pulse repetition frequency of 1000 Hz, output intensity of 30 mW/cm2, duration of application of 20 min and total number sessions were 14 with a repetition interval of 1 day. The suggested mechanisms induced by US were alteration of cementoblasts, osteoblasts, osteoclasts, alkaline-phosphatase (ALP), runt-related-gene-2 (Runx2), osteoprotegerin (OPG), type-I-collagen (Col-I), C-telopeptide-type-I-collagen (CTX-I), hepatocyte-growth-factor (HGF), bone morphogenetic protein-2 (BMP-2), cyclooxygenase-2 (Cox-2), calcium (Ca2+), receptor activator of nuclear factor-kappa-B ligand (RANKL), and receptor activator of nuclear factor-kappa-B (RANK). EXPERT OPINION: Understanding mechanisms and deciding which parameters of US that can be used during orthodontic treatment to prevent and repair root resorption is a great challenge. This work summarizes all the available data that can aid this process and suggest that US is an effective noninvasive method not only in prevention and repairing of orthodontic induced root resorption but also in accelerating teeth movement.

Properties of dentin, enamel and their junction, studied with Brillouin scattering and compared to Raman microscopy.

OBJECTIVE: Dentin, enamel and the transition zone, called the dentin-enamel junction (DEJ), have an organization and properties that play a critical role in tooth resilience and in stopping the propagation of cracks. Understanding their chemical and micro-biomechanical properties is then of foremost importance. The aim of this study is to apply Brillouin microscopy on a complex biological structure, that is, the DEJ, and to compare these results with those obtained with Raman microscopy. DESIGN: Both techniques allow noncontact measurements at the microscopic scale. Brillouin microscopy is based on the interaction between acoustic phonons and laser photons and gives a relation between the frequency shift of the scattered light and the stiffness of the sample. Raman spectra contain peaks related to specific chemical bonds. RESULTS: Comparison of the Brillouin and Raman cartographies reveals correlations between mechanical and chemical properties. Indeed, the shapes of the phosphate content and stiffness curves are similar. The two spectroscopies give compatible values for the mean distance between two tubules, i.e., 4-6 µm. Moreover, for the first time, the daily cross striations of enamel could be studied, indicating a relationship between the variation in the phosphate concentration and the variation in the rigidity within the enamel prisms. CONCLUSIONS: We demonstrate here the possibility of using Brillouin scattering microscopy to both study complex biological materials such as the enamel-dentin junction and visualize secondary structures. Correlations between the chemical composition and mechanical properties could help in better understanding the tissue histology.

In vitro, ex vivo, and in vivo models for dental pulp regeneration.

Based on the concept of tissue engineering (Cells-Scaffold-Bioactive molecules), regenerative endodontics appeared as a new notion for dental endodontic treatment. Its approaches aim to preserve dental pulp vitality (pulp capping) or to regenerate a vascularized pulp-like tissue inside necrotic root canals by cell homing. To improve the methods of tissue engineering for pulp regeneration, numerous studies using in vitro, ex vivo, and in vivo models have been performed. This review explores the evolution of laboratory models used in such studies and classifies them according to different criteria. It starts from the initial two-dimensional in vitro models that allowed characterization of stem cell behavior, through 3D culture matrices combined with dental tissue and finally arrives at the more challenging ex vivo and in vivo models. The travel which follows the elaboration of such models reveals the difficulty in establishing reproducible laboratory models for dental pulp regeneration. The development of well-established protocols and new laboratory ex vivo and in vivo models in the field of pulp regeneration would lead to consistent results, reduction of animal experimentation, and facilitation of the translation to clinical practice.

Enamel and dentin in Enamel renal syndrome: A confocal Raman microscopy view.

Enamel Renal Syndrome (ERS) is a rare genetic disorder caused by biallelic mutations in Family with sequence similarity 20A (FAM20A) gene encoding the secretory pathway pseudokinase FAM20A. ERS is characterized by hypoplastic amelogenesis imperfecta (AI), impaired tooth eruption, intra-pulpal calcifications, gingival fibromatosis and nephrocalcinosis of various severity. Previous studies showed that the hypoplastic enamel was also hypomineralized but its chemical composition has not been extensively studied. Furthermore it is currently unclear whether dentinal defects are associated with AI in ERS patients. The objective of the study was to provide a structural and chemical analysis of enamel, dentin and dentin enamel junction (DEJ) in ERS patients carrying four, previously reported, distinct mutations in FAM20A. Chemical cartography obtained with Raman microscopy showed that compared to control samples, ERS enamel composition was severely altered and a cementum-like structure was observed in some cases. Chemical composition of peripulpal dentin was also affected and usual gradient of phosphate intensity, shown in DEJ profile, was absent in ERS samples. DEJ and dentinal anomalies were further confirmed by scanning electron microscopy analysis. In conclusion, our study shows that enamel formation is severely compromised in ERS patients and provides evidence that dentinal defects are an additional feature of the ERS dental phenotype.

Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy.

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

Trans-Cinnamaldehyde Eluting Porous Silicon Microparticles Mitigate Cariogenic Biofilms.

Dental caries, a preventable disease, is caused by highly-adherent, acid-producing biofilms composed of bacteria and yeasts. Current caries-preventive approaches are ineffective in controlling biofilm development. Recent studies demonstrate definite advantages in using natural compounds such as trans-cinnamaldehyde in thwarting biofilm assembly, and yet, the remarkable difficulty in delivering such hydrophobic bioactive molecules prevents further development. To address this critical challenge, we have developed an innovative platform composed of components with a proven track record of safety. We fabricated and thoroughly characterised porous silicon (pSi) microparticles to carry and deliver the natural phenyl propanoid trans-cinnamaldehyde (TC). We investigated its effects on preventing the development of cross-kingdom biofilms (Streptococcus mutans and Candida albicans), typical of dental caries found in children. The prepared pSi microparticles were roughly cubic in structure with 70-75% porosity, to which the TC (pSi-TC) was loaded with about 45% efficiency. The pSi-TC particles exhibited a controlled release of the cargo over a 14-day period. Notably, pSi-TC significantly inhibited biofilms, specifically downregulating the glucan synthesis pathways, leading to reduced adhesion to the substrate. Acid production, a vital virulent trait for caries development, was also hindered by pSi-TC. This pioneering study highlights the potential to develop the novel pSi-TC as a dental caries-preventive material.

Dental stem cell-conditioned medium for tissue regeneration: Optimization of production and storage.

BACKGROUND: Mesenchymal stem cells (MSC) effects on tissue regeneration are mainly mediated by their secreted substances (secretome), inducing their paracrine activity. This Conditioned medium (CM), including soluble factors (proteins, nucleic acids, lipids) and extracellular vesicles is emerging as a potential alternative to cell therapy. However, the manufacturing of CM suffers from variable procedures and protocols leading to varying results between studies. Besides, there is no well-defined optimized procedure targeting specific applications in regenerative medicine. AIM: To focus on conditioned medium produced from dental MSC (DMSC-CM), we reviewed the current parameters and manufacturing protocols, in order to propose a standardization and optimization of these manufacturing procedures. METHODS: We have selected all publications investigating the effects of dental MSC secretome in in vitro and in vivo models of tissue regeneration, in accordance with the PRISMA guidelines. RESULTS: A total of 351 results were identified. And based on the inclusion criteria described above, 118 unique articles were included in the systematic review. DMSC-CM production was considered at three stages: before CM recovery (cell sources for CM), during CM production (culture conditions) and after production (CM treatment). CONCLUSION: No clear consensus could be recovered as evidence-based methods, but we were able to describe the most commonly used protocols: donors under 30 years of age, dental pulp stem cells and exfoliated deciduous tooth stem cells with cell passage between 1 and 5, at a confluence of 70% to 80%. CM were often collected during 48 h, and stored at -80 °C. It is important to point out that the preconditioning environment had a significant impact on DMSC-CM content and efficiency.

Fabrication of Radio-Opaque and Macroporous Injectable Calcium Phosphate Cement.

The aim of this work was the development of injectable radio-opaque and macroporous calcium phosphate cement (CPC) to be used as a bone substitute for the treatment of pathologic vertebral fractures. A CPC was first rendered radio-opaque by the incorporation of zirconium dioxide (ZrO2). In order to create macroporosity, poly lactic-co-glycolic acid (PLGA) microspheres around 100 µm were homogeneously incorporated into the CPC as observed by scanning electron microscopy. Physicochemical analyses by X-ray diffraction and Fourier transform infrared spectroscopy confirmed the brushite phase of the cement. The mechanical properties of the CPC/PLGA cement containing 30% PLGA (wt/wt) were characterized by a compressive strength of 2 MPa and a Young's modulus of 1 GPa. The CPC/PLGA exhibited initial and final setting times of 7 and 12 min, respectively. Although the incorporation of PLGA microspheres increased the force necessary to inject the cement and decreased the percentage of injected mass as a function of time, the CPC/PLGA appeared fully injectable at 4 min. Moreover, in comparison with CPC, CPC/PLGA showed a full degradation in 6 weeks (with 100% mass loss), and this was associated with an acidification of the medium containing the CPC/PLGA sample (pH of 3.5 after 6 weeks). A cell viability test validated CPC/PLGA biocompatibility, and in vivo analyses using a bone defect assay in the caudal vertebrae of Wistar rats showed the good opacity of the CPC through the tail and a significant increased degradation of the CPC/PLGA cement a month after implantation. In conclusion, this injectable CPC scaffold appears to be an interesting material for bone substitution.

Confocal Raman data analysis of tufts and spindles at the human dentin-enamel junction.

OBJECTIVE: The aim of this article is to analyze the chemical mapping of tufts and spindles of the human dental enamel using confocal Raman microscopy measuring length, structuration and composition of spindles and tufts. DESIGN: we used Raman diffusion, based on the interaction between photons and optic phonons, to reveal chemical bound. Adult molars were selected and longitudinally sectioned. Areas of 120 * 120 µm were scanned near the dentin-enamel junction and grooves. Spectra were collected and phosphate and proteins peak intensities images were reconstructed, related to HPA concentration. Images of Phosphate (PO43-, 960 cm-1) and protein (CH, 2800/3000 cm-1) intensities have been reconstructed. K-mean cluster has been calculated to compare centroid spectra from enamel, dentin and tuft or spindle. RESULTS: intensity profile revealed spindles as less mineralized areas than enamel, from 5 to 10 µm large. In the groove of molar, long tufts were found, more than 150 µm. CONCLUSIONS: Confocal Raman microscopy is a very interesting tool to characterize chemically secondary structure of enamel. The size of a tuft in the groove allows us make the hypothesis that they could play a role in long term resilience of mechanical stress.

Adsorption of proteins on TiO2 particles influences their aggregation and cell penetration.

TiO2 nanoparticles known as E171 are one controversial food additive due to its potential toxicity. In this work, the main hypothesis is that the proteins adsorbed on the TiO2 nanoparticles prevent their aggregation and favor the cell penetration. To do so, the TiO2 nanoparticles were coated with gelatin and ß-lactoglobulin to reach interfacial concentrations about 0.25 mg/mg and 0.32 mg/mg, respectively. The measurement of NP size showed that the protein coating improve the colloidal stability of TiO2 nanoparticles. The FTIR analysis suggests that the ß-lactoglobulin structure is modified after adsorption. The penetration of TiO2 penetration inside human intestinal epithelial cells was shown and quantify by using confocal Raman microscopy. The promoting role of the protein coating on the cell penetration was demonstrated for both the gelatin and ß-lactoglobulin. Finally, the results allow establishing a correlation between the ability of proteins to prevent NP aggregation and the cell penetration.

Identification of secreted factors in dental pulp cell-conditioned medium optimized for neuronal growth.

With their potent regenerative and protective capacities, stem cell-derived conditioned media emerged as an effective alternative to cell therapy, and have a prospect to be manufactured as pharmaceutical products for tissue regeneration applications. Our study investigates the neuroregenerative potential of human dental pulp cells (DPCs) conditioned medium (CM) and defines an optimization strategy of DPC-CM for enhanced neuronal outgrowth. Primary sensory neurons from mouse dorsal root ganglia were cultured with or without DPC-CM, and the lengths of ßIII-tubulin positive neurites were measured. The impacts of several manufacturing features as the duration of cell conditioning, CM storage, and preconditioning of DPCs with some factors on CM functional activity were assessed on neurite length. We observed that DPC-CM significantly enhanced neurites outgrowth of sensory neurons in a concentration-dependent manner. The frozen storage of DPC-CM had no impact on experimental outcomes and 48 h of DPC conditioning is optimal for an effective activity of CM. To further understand the regenerative feature of DPC-CM, we studied DPC secretome by human growth factor antibody array analysis and revealed the presence of several factors involved in either neurogenesis, neuroprotection, angiogenesis, and osteogenesis. The conditioning of DPCs with the B-27 supplement enhanced significantly the neuroregenerative effect of their secretome by changing its composition in growth factors. Here, we show that DPC-CM significantly stimulate neurite outgrowth in primary sensory neurons. Moreover, we identified secreted protein candidates that can potentially promote this promising regenerative feature of DPC-CM.

Effects of green light photobiomodulation on Dental Pulp Stem Cells: enhanced proliferation and improved wound healing by cytoskeleton reorganization and cell softening.

Photobiomodulation (PBM) has been shown to improve cell proliferation and cell migration. Many cell types have been investigated, with most studies using deep penetrating red light irradiation. Considering the interest of surface biostimulation of oral mesenchymal cells after surgical wound, the present study aimed to assess green light irradiation effects on Dental Pulp Stem Cells' (DPSC) proliferation and migration. To understand the mechanisms underlying these effects, we investigated cytoskeleton organization and subsequent cell shape and stiffness. A 532-nm wavelength Nd:YAG laser (30 mW) was applied between 30 and 600 s on DPSC in vitro. Cell proliferation was analyzed at 24, 48, and 72 h after irradiation, by cell counting and enzymatic activity quantification (paranitrophenylphosphate phosphatase (pNPP) test). A wound healing assay was used to study cell migration after irradiation. Effects of PBM on cytoskeleton organization and cell shape were assessed by actin filaments staining. Elasticity changes after irradiation were quantified in terms of Young's modulus measured using Atomic Force Microscopy (AFM) force spectroscopy. Green light significantly improved DPSC proliferation with a maximal effect obtained after 300-s irradiation (energy fluence 5 J/cm2). This irradiation had a significant impact on cell migration, improving wound healing after 24 h. These results were concomitant with a decrease of cells' Young's modulus after irradiation. This cell softening was explained by actin cytoskeleton reorganization, with diminution of cell circularity and more abundant pseudopodia. This study highlights the interest of green laser PMB for the proliferation and migration of mesenchymal stem cells, with encouraging results for clinical application, especially for surgical wound healing procedures.