Circ_0003764 Regulates the Osteogenic Differentiation of Periodontal Ligament Stem Cells.

INTRODUCTION AND AIMS: Specific circular RNAs (circRNAs) have been proven to play crucial roles in osteogenesis in vitro and in vivo. This study aims to identify a certain circRNA involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and explore its regulatory role. METHODS: The expression of 5 candidate circRNAs (circ_0026344, circ_ACAP2, circ_0003764, circ_0008259, and circ_0060731) was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) after PDLSCs were cultured in the osteogenic induction medium or medium supplemented with tumour necrosis factor-α (TNF-α, 10 ng/mL) for 3 and 7 days. The circRNA significantly decreased in both 3 and 7 days of osteogenic induction in PDLSCs and markedly increased in TNF-α-induced PDLSCs for 3 and 7 days screened. Identified circRNA was knocked down or overexpressed, and the effect on the osteogenic differentiation of PDLSCs was investigated by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining. Cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were applied to detect the effect of the circRNA on the proliferation of PDLSCs. RESULTS: qRT-PCR results showed that the expression of circ_0003764 was significantly decreased when PDLSCs were cultured in the osteogenic induction medium for 3 or 7 days, whereas it was dramatically increased in TNF-α-induced PDLSCs. Knockdown of circ_0003764 promoted the expression of the osteogenesis-related genes (RUNX2, ALP, OCN) and proteins (RUNX2, OCN), enhanced the ALP activity, and elevated the mineralization by PDLSCs, as shown by ARS staining. However, with the overexpression of circ_0003764, the osteogenic differentiation capacity of PDLSCs was significantly reduced. The CCK-8 and EdU results indicated that circ_0003764 could inhibit the proliferation of PDLSCs. CONCLUSION: Circ_0003764 is involved in the osteogenesis process and inhibits the osteogenic differentiation and proliferation of PDLSCs. CLINICAL RELEVANCE: This study indicates that circ_0003764 can serve as a diagnostic and therapeutic target in bone regeneration-related diseases treated by PDLSCs-based tissue engineering.

Does crocin create new hope for the treatment of oral problems? A focus on periodontitis.

According to the World Health Organization (WHO) reports, oral health has an indispensable role in the maintenance of human public health. However, oral problems, especially periodontitis, are known as bad players in this issue. Periodontitis, as the most prevalent oral disease, is a type of chronic illness mediated by bacterial pathogens and immune system reactions, which is linked with the destruction of tooth-protecting tissues, such as alveolar bone and periodontal ligament. Periodontitis has a high prevalence (over 40% in the United States) and can be associated with other systemic ailments, for instance, arthritis, osteoporosis, metabolic syndrome, cancer, respiratory diseases, chronic kidney disease, and Alzheimer's disease. The common treatments for periodontitis are classified into invasive (surgical) and noninvasive (antibiotic therapy, scaling, and root planning) methods; however, these therapies have not reflected enough effectiveness for related patients. New documents inform the beneficial effects of plant-based compounds in healing various disorders, like periodontitis. In conjunction with this subject, it has been revealed that crocin, as an active component of saffron, regulates the balance between osteoclasts and osteoblasts and has a stroking role in the accumulation of the most common collagen in teeth and bone (type 1 collagen). Besides, this carotenoid compound possesses anti-inflammatory and anti-oxidative effects, which can be associated with the therapeutic processes of crocin in this oral disease. Hence, this narrative review study was performed to reflect the reparative/regenerative aspects of crocin agonist periodontitis.

Flavonoid extract from propolis alleviates periodontitis by boosting periodontium regeneration and inflammation resolution via regulating TLR4/MyD88/NF-κB and RANK/NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, propolis has been used for treating oral diseases for centuries, widely. Flavonoid extract is the main active ingredient in propolis, which has attracted extensive attention in recent years. AIM OF THE STUDY: The objective and novelty of the current study aims to identify the mechanism of total flavonoid extract of propolis (TFP) for the treatment of periodontitis, and evaluate the therapeutic effect of TFP-loaded liquid crystal hydrogel (TFP-LLC) in rats with periodontitis. METHODS: In this study, we used lipopolysaccharide-stimulated periodontal ligament stem cells (PDLSCs) to construct in vitro inflammation model, and investigated the anti-inflammatory effect of TFP by expression levels of inflammatory factors. Osteogenic differentiation was assessed using alkaline phosphatase activity and alizarin red staining. Meanwhile, the expression of toll like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), nuclear factor-kappa B (NF-κB), receptor activator of NF-κB (RANK) etc, were quantitated to investigate the therapeutic mechanism of TFP. Finally, we constructed TFP-LLC using a self-emulsification method and administered it to rats with periodontitis via periodontal pocket injection to evaluate the therapeutic effects. The therapeutic index, microcomputed tomography (Micro-CT), H&E staining, TRAP staining, and Masson staining were used for this evaluation. RESULTS: TFP reduced the expression of TLR4, MyD88, NF-κB and inflammatory factor in lipopolysaccharide-stimulated PDLSCs. Meanwhile, TFP simultaneously regulating alkaline phosphatase, RANK, runt-associated transcription factor-2 and matrix metalloproteinase production to accelerate osteogenic differentiation and collagen secretion. In addition, TFP-LLC can stably anchor to the periodontal lesion site and sustainably release TFP. After four weeks of treatment with TFP-LLC, we observed a decrease in the levels of NF-κB and interleukin-1ß (IL-1ß) in the periodontal tissues of rats, as well as a significant reduction in inflammation in HE staining. Similarly, Micro CT results showed that TFP-LLC could significantly inhibit alveolar bone resorption, increase bone mineral density (BMD) and reduce trabecular bone space (Tb.Sp) in rats with periodontitis. CONCLUSION: Collectively, we have firstly verified the therapeutic effects and mechanisms of TFP in PDLSCs for periodontitis treatment. Our results indicate that TFP perform anti-inflammatory and tissue repair activities through TLR4/MyD88/NF-κB and RANK/NF-κB pathways in PDLSCs. Meanwhile, for the first time, we employed LLC delivery system to load TFP for periodontitis treatment. The results showed that TFP-LLC could be effectively retained in the periodontal pocket and exerted a crucial role in inflammation resolution and periodontal tissue regeneration.

Effects of nano-micelles curcumin-based photodynamic therapy on expression of RUNX2 as an indicator of bone regeneration in orthodontic tooth movement.

OBJECTIVES: The aim was to evaluate the impact of nano-micelles curcumin (NMCur) based photodynamic therapy (PDT) during compressive force application on human PDL-derived fibroblasts (HPDFs) in vitro for up to 6 days on the expression of RUNX2 as an indicator of bone development and remodeling. MATERIALS AND METHODS: HPDFs viability during 2 g/cm2 compressive force application was investigated using membrane-impermeable DNA-binding stain propidium iodide (PI) in flow cytometry. Gene and protein expressions of RUNX2 were assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and flow cytometry, respectively, following NMCur-PDT at different concentrations of NMCur (25, 50, and 75 µM plus irradiation of 180 mW/cm2 diode laser at the wavelength of 450 ± 10 nm for 5 min) during the static compressive force of 2 g/cm2 on HPDFs via weight approach-based in-vitro loading model up to 6 days. One-way ANOVA and Tukey post hoc tests at a p-value equal to/or less than 0.05 were used to analyze the obtained data. RESULTS: After 6 days of application of compressive force, 99.21 ± 6.12% of HPDFs were PI negative and therefore considered alive, while only 0.89 ± 0.06% of the population were PI positive and considered dead. In comparison with controls (loaded HPDFs), expression of RUNX2 gene was dose-dependent and the highest expression (14.38-fold; P < 0.01) was observed at a concentration of 75 µM NMCur following 5 min of diode laser irradiation (i.e., 75 µM NMCur-PDT) during compressive force application on day 5. The greatest and lowest upregulations of RUNX2 protein were observed in 75 µM NMCur-PDT during compressive force application on HPDFs, on day 5 (3.19-fold; P < 0.01) and day 6 (2.09-fold; P < 0.05), respectively. CONCLUSION: NMCur-PDT during weight approach-based in-vitro loading model can promote orthodontic tooth movement by upregulating RUNX2 signaling pathway in HPDFs.

The molecular weight of hyaluronic acid influences metabolic activity and osteogenic differentiation of periodontal ligament cells.

OBJECTIVE: While HA is present naturally in periodontal tissues, its molecular weight can vary widely in vivo. The objective of this study was to directly compare the biological reactions of periodontal ligament cells to four distinct molecular weights of hyaluronic acid (HA). MATERIALS AND METHODS: Immortalized human periodontal ligament cells (PDL-hTERT) were cultured for 21 days in culture medium alone (control) or enriched with osteogenic supplements (OS group). Other 4 experimental groups were cultured in OS medium with the addition of HA with different molecular weights (HMW, MMW, LMW, and ULMW). The cell morphology was examined daily. WST1 assays were performed to evaluate metabolic activity. Von Kossa staining and calcium deposition assay were used to analyze osteogenic differentiation and mineralization. RESULTS: Cell morphology remained unaltered in all groups. Cells stimulated with OS alone or with the addition of hyaluronan showed all the typical microscopic appearance of osteogenic differentiation. Metabolic activity increased in all groups over time. Hyaluronan stimulated greater metabolic activity than the control group, with LMW HA and MMW HA showing the most significant increase. All groups showed mineral deposits and calcium deposition after 21 days of stimulation. CONCLUSION: Our results suggest that hyaluronan can promote metabolic activity and mineralization of PDL-hTERT cells, with LMW HA being the most effective. CLINICAL RELEVANCE: These results shed light on how the various molecular weight fractions of HA promote tissue regeneration and repair, as well as help to identify an optimal molecular weight range for this application in periodontal tissues.

A systematic review of the protein composition of whole saliva in subjects with healthy periodontium compared with chronic periodontitis.

CONTEXT: Periodontitis is a chronic multifactorial inflammatory disease linked to oral microbiota dysbiosis. This disease progresses to infection that stimulates a host immune/inflammatory response, with progressive destruction of the tooth-supporting structures. OBJECTIVE: This systematic review aims to present a robust critical evaluation of the evidence of salivary protein profiles for identifying oral diseases using proteomic approaches and summarize the use of these approaches to diagnose chronic periodontitis. DATA SOURCES: A systematic literature search was conducted from January 1st, 2010, to December 1st, 2022, based on PICO criteria following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and by searching the three databases Science Direct, Scopus, and Springer Link. STUDY SELECTION: According to the inclusion criteria, eight studies were identified to analyze the proteins identified by proteomics. RESULTS: The protein family S100 was identified as the most abundant in patients with chronic periodontitis. In this family, an increased abundance of S100A8 and S100A9 from individuals with the active disease was observed, which strongly relates to the inflammatory response. Moreover, the ratio S100A8/S100A9 and the metalloproteinase-8 in saliva could differentiate distinct periodontitis groups. The changes in protein profile after non-surgical periodontal therapy improved the health of the buccal area. The results of this systematic review identified a set of proteins that could be used as a complementary tool for periodontitis diagnosis using salivary proteins. CONCLUSION: Biomarkers in saliva can be used to monitor an early stage of periodontitis and the progression of the disease following therapy.

Effects of vitamin A in promoting proliferation and osteogenic differentiation of human periodontal ligament cells.

Human periodontal ligament cells (hPDLCs) are known as ideal seed cells for the regeneration of periodontal tissues. Several factors (i.e., vitamin D3, luteolin, and 6-bromoindirubin-3'-oxime) have been shown to promote osteogenic differentiation of hPDLCs. In this study, we aim to investigate the effect of vitamin A on cell proliferation, migration, and osteogenic differentiation of hPDLCs. hPDLCs were cultured in osteogenic induction medium supplemented with different concentrations of vitamin A. Cell proliferation and migration assays were conducted after 24, 48, and 72 h of incubation, whereas osteogenic differentiation and osteogenesis-related gene expression were assessed after 21 d only. Our results demonstrated that 1-µM vitamin A stimulation exerted the most potent promotion effect on cell proliferation, migration, and osteogenic differentiation of hPDLCs. It also induced significant upregulation of osteogenic differentiation-related genes and mitochondrial complexes II and IV in hPDLCs. Vitamin A may serve as a promising potential candidate for periodontal tissue regeneration.

Composite Graded Melt Electrowritten Scaffolds for Regeneration of the Periodontal Ligament-to-Bone Interface.

Periodontitis is a ubiquitous chronic inflammatory, bacteria-triggered oral disease affecting the adult population. If left untreated, periodontitis can lead to severe tissue destruction, eventually resulting in tooth loss. Despite previous efforts in clinically managing the disease, therapeutic strategies are still lacking. Herein, melt electrowriting (MEW) is utilized to develop a compositionally and structurally tailored graded scaffold for regeneration of the periodontal ligament-to-bone interface. The composite scaffolds, consisting of fibers of polycaprolactone (PCL) and fibers of PCL-containing magnesium phosphate (MgP) were fabricated using MEW. To maximize the bond between bone (MgP) and ligament (PCL) regions, we evaluated two different fiber architectures in the interface area. These were a crosshatch pattern at a 0/90° angle and a random pattern. MgP fibrous scaffolds were able to promote in vitro bone formation even in culture media devoid of osteogenic supplements. Mechanical properties after MgP incorporation resulted in an increase of the elastic modulus and yield stress of the scaffolds, and fiber orientation in the interfacial zone affected the interfacial toughness. Composite graded MEW scaffolds enhanced bone fill when they were implanted in an in vivo periodontal fenestration defect model in rats. The presence of an interfacial zone allows coordinated regeneration of multitissues, as indicated by higher expression of bone, ligament, and cementoblastic markers compared to empty defects. Collectively, MEW-fabricated scaffolds having compositionally and structurally tailored zones exhibit a good mimicry of the periodontal complex, with excellent regenerative capacity and great potential as a defect-specific treatment strategy.

Thyrotropin inhibits osteogenic differentiation of human periodontal ligament stem cells.

BACKGROUND AND OBJECTIVE: Periodontal ligament stem cells (PDLSCs) are derived from the periodontal ligament and have the characteristics of pluripotent differentiation, including osteogenesis, and are one of the important seed cells in oral tissue engineering. Thyrotropin (TSH) has been shown to regulate bone metabolism independently of thyroid hormone, including the fate of osteoblasts and osteoclasts, but whether it affects osteogenic differentiation of PDLSCs is unknown. MATERIALS AND METHODS: PDLSCs were isolated and cultured from human periodontal ligament and grown in osteogenic medium (containing sodium ß-glycerophosphate, ascorbic acid, and dexamethasone). Recombinant human TSH was added to the culture medium. Osteogenic differentiation of PDLSCs was assessed after 14 days by staining with alkaline phosphatase and alizarin red and by detection of osteogenic differentiation genes. Differentially expressed genes (DEGs) in PDLSCs under TSH were detected by high-throughput sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyzed the biological functions and signaling pathways involved in DEGs. RESULTS: We found that osteogenic differentiation of PDLSCs was significantly inhibited in the presence of TSH: including decreased calcium nodule formation, decreased alkaline phosphatase levels, and decreased collagen synthesis. Using high-throughput sequencing, we found changes in the expression of some osteogenesis-related genes, which may be the reason that TSH inhibits osteogenic differentiation of PDLSCs. CONCLUSION: Unless TSH is ≥10 mU/L, patients with subclinical hypothyroidism usually do not undergo thyroxine supplementation therapy. However, in this work, we found that elevated TSH inhibited the osteogenic differentiation of PDLSCs. Therefore, correction of TSH levels in patients with subclinical hypothyroidism may be beneficial to improve orthodontic, implant, and periodontitis outcomes in these patients.

In Vitro Effect of Photobiomodulation Therapy with 980 nm Diode Laser on Gene Expression of Key Regulators of Bone Remodeling by Human Periodontal Ligament Cells under Mild Orthodontic Forces.

This study investigated the effect of photobiomodulation (PBM) with 980 nm diode laser as monotherapy and in combination with compressive and tensile orthodontic forces on expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), sclerostin (SOST) and periostin (POSTN), by human periodontal ligament cells. Isolated cells were cultured and subjected to either tensile (10% elongation) or compressive forces (25 g cm-2 ) for 24 and 48 h. Subsequently, the cells received PBM (100 mW power, 3 or 6 J cm-2 energy density) immediately after load cycle. RT-PCR was applied to assess the genes expression. Data were analyzed by one-way ANOVA, followed by post hoc Tukey test (P ≤ 0.05). We found that PBM in combination with orthodontic forces led to upregulation of bone resorption genes (RANKL and SOST) at the pressure side and their downregulation at the tension side. The expression of osteogenic genes (OPG and POSTN) increased at the tension side and decreased at the pressure side. PBM alone did not affect gene expression. In conclusion, these findings suggest that this PBM protocol may be effective in enhancement of the gene expression in favor of bone remodeling acceleration that should be confirmed in future animal and human studies.

Melatonin promoted osteogenesis of human periodontal ligament cells by regulating mitochondrial functions through the translocase of the outer mitochondrial membrane 20.

BACKGROUND AND OBJECTIVE: Melatonin plays an important role in various beneficial functions, including promoting differentiation. However, effects on osteogenic differentiation, especially in human periodontal cells (hPDLCs), still remain inconclusive. Mitochondria are highly dynamic organelles that play an important role in various biological processes in cells, including energy metabolism and oxidative stress reaction. Furthermore, the translocase of the outer mitochondrial membrane 20 (TOM20) is responsible for recognizing and transporting precursor proteins. Thus, the objective of this study was to evaluate the functionality of melatonin on osteogenesis in human periodontal cells and to explore the involved mechanism of mitochondria. METHODS: The hPDLCs were extracted and identified by flow cytometry and multilineage differentiation. We divided hPDLCs into control group, osteogenic induction group, and osteogenesis with melatonin treatment group (100, 10, and 1 µM). Then we used a specific siRNA to achieve interference of TOM20. Alizarin red and Alkaline phosphatase staining and activity assays were performed to evaluate osteogenic differentiation. Osteogenesis-related genes and proteins were measured by qPCR and western blot. Mitochondrial functions were tested using ATP, NAD+/NADH, JC-1, and Seahorse Mito Stress Test kits. Finally, TOM20 and mitochondrial dynamics-related molecules expression were also assessed by qPCR and western blot. RESULTS: Our results showed that melatonin-treated hPDLCs had higher calcification and ALP activity as well as upregulated OCN and Runx2 expression at mRNA and protein levels, which was the most obvious in 1 µM melatonin-treated group. Meanwhile, melatonin supplement elevated intracellular ATP production and mitochondrial membrane potential by increasing mitochondrial oxidative metabolism, hence causing a lower NAD+ /NADH ratio. In addition, we also found that melatonin treatment raised TOM20 level and osteogenesis and mitochondrial functions were both suppressed after knocking down TOM20. CONCLUSION: We found that melatonin promoted osteogenesis of hPDLCs and 1 µM melatonin had the most remarkable effect. Melatonin treatment can reinforce mitochondrial functions by upregulating TOM20.

Channa Striata and Hyperbaric Oxygen Therapy Combination for Pressure Areas in Orthodontic Treatment

ABSTRACT Objective: To study the effect of using a combination of Channa Striata gel and hyperbaric oxygen therapy on pressure areas during orthodontic treatment. Material and Methods: The study was conducted using the ARRIVE Essential 10 guidelines. In this study, 35 3-4 months male guinea pigs (Cavia Cobaya) weighing 300-400 grams were used and divided into 5 groups (n=7). Decalcification was performed to dissolve the dental calcium and jawbone to cut the tissue properly. The decalcification was performed for 30 days. Then preparations were made with HE (Hematoxylin Eosin), observed using a microscope, and counted the number of osteoclasts and macrophages on a light microscope with 400 times magnification. The results of the preparations were analyzed using the SPSS program. Results: The Kruskal-Wallis test of macrophage cells and the ANOVA test of osteoclast cells showed significant results between all groups (p<0.05). Conclusion: The effect of hyperbaric oxygen therapy 2,4 ATA administered on days 8-14 and Channa Striata extract gel administered on days 3-14 can increase the number of macrophages in the periodontal ligament and osteoclasts in the alveolar bone in the pressure area during orthodontic tooth movement.

Botanicals and Oral Stem Cell Mediated Regeneration: A Paradigm Shift from Artificial to Biological Replacement.

Stem cells are a well-known autologous pluripotent cell source, having excellent potential to develop into specialized cells, such as brain, skin, and bone marrow cells. The oral cavity is reported to be a rich source of multiple types of oral stem cells, including the dental pulp, mucosal soft tissues, periodontal ligament, and apical papilla. Oral stem cells were useful for both the regeneration of soft tissue components in the dental pulp and mineralized structure regeneration, such as bone or dentin, and can be a viable substitute for traditionally used bone marrow stem cells. In recent years, several studies have reported that plant extracts or compounds promoted the proliferation, differentiation, and survival of different oral stem cells. This review is carried out by following the PRISMA guidelines and focusing mainly on the effects of bioactive compounds on oral stem cell-mediated dental, bone, and neural regeneration. It is observed that in recent years studies were mainly focused on the utilization of oral stem cell-mediated regeneration of bone or dental mesenchymal cells, however, the utility of bioactive compounds on oral stem cell-mediated regeneration requires additional assessment beyond in vitro and in vivo studies, and requires more randomized clinical trials and case studies.

An in vitro evaluation of ice apple water, Aloe vera, and propolis as a storage medium to preserve viability of human periodontal ligament fibroblasts.

Background: A number of media that create the best possible conditions to maintain periodontal ligament (PDL) cell viability after dental avulsion have been reported. Aim: The aim of this study is to evaluate ice apple water (IAW), Aloe vera, and propolis as a storage medium to preserve the viability of human PDL fibroblasts. Methods: An in vitro comparative type of study was performed on a PDL cell culture model. PDL fibroblasts obtained from the roots of healthy premolars were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with ice apple water (IAW), 7% propolis extract (PE), 30% Aloe vera extract (AVE), positive control DMEM supplemented with fetal bovine serum, negative control (NC) without any agent, and incubated at 37°C for 1 h, 3 h, and 24 h. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay after every test period. Optical density was measured at a wavelength of 490 nm. Statistical Analysis Used: The effects of the test storage media were evaluated by one-way analysis of variance test, followed by post hoc Tukey's multiple comparison test (P < 0.05). Results: Seven percent PE demonstrated the highest capacity of maintaining PDL cell viability at 1 h and 24 h. IAW showed a statistically significantly lower percentage of viable cells at all three test periods as compared to 7% PE. After 3 h, 30% AVE demonstrated maximum viable cells. Conclusions: Within the limitations of this study, propolis at a concentration of 7% was the most effective medium for maintaining PDL cell viability.

Human periodontal ligament stem cell encapsulation in alginate-fibrin-platelet lysate microbeads for dental and craniofacial regeneration.

OBJECTIVE: Tissue engineering is promising for dental and craniofacial regeneration. The objectives of this study were to develop a novel xeno-free alginate-fibrin-platelet lysate hydrogel with human periodontal ligament stem cells (hPDLSCs) for dental regeneration, and to investigate the proliferation and osteogenic differentiation of hPDLSCs using hPL as a cell culture nutrient supplement. METHODS: hPDLSCs were cultured with Dulbecco's modified eagle medium (DMEM), DMEM + 10% fetal bovine serum (FBS), and DMEM + hPL (1%, 2.5%, and 5%). hPDLSCs were encapsulated in alginate-fibrin microbeads (Alg+Fib), alginate-hPL microbeads (Alg+hPL), or alginate-fibrin-hPL microbeads (Alg+Fib+hPL). hPDLSCs encapsulated in alginate microbeads were induced with an osteogenic medium containing hPL or FBS. Quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) activity, ALP staining, and alizarin red (ARS) staining was investigated. RESULTS: hPDLSCs were released faster from Alg+Fib+hPL than from Alg+hPL. At 14 days, ALP activity was 44.1 ± 7.61 mU/mg for Alg+Fib+hPL group, higher than 28.07 ± 5.15 mU/mg of Alg+Fib (p<0.05) and 0.95 ± 0.2 mU/mg of control (p<0.01). At 7 days, osteogenic genes (ALP, RUNX2, COL1, and OPN) in Alg+Fib+hPL and Alg+Fib were 3-10 folds those of control. At 21 days, the hPDLSC-synthesized bone mineral amount in Alg+Fib+hPL and Alg+Fib was 7.5 folds and 4.3 folds that of control group, respectively. CONCLUSIONS: The 2.5% hPL was determined to be optimal for hPDLSCs. Adding hPL into alginate hydrogel improved the viability of the hPDLSCs encapsulated in the microbeads. The hPL-based medium enhanced the osteogenic differentiation of hPDLSCs in Alg+Fib+hPL construct, showing a promising xeno-free approach for delivering hPDLSCs to enhance dental, craniofacial and orthopedic regenerations.

Near Infrared Laser Photobiomodulation of Periodontal Ligament Stem Cells.

OBJECTIVE: To determine the effect of different energy densities of near infrared diode lasers with wavelengths of 810 or 940 nm on the proliferation and survival of periodontal ligament derived stem cells (PDLSCs). METHODS: After isolation and characterisation, PDLSCs were cultured in clear 96-well plates. Each well was irradiated by either 810 nm (L1) or 940 nm (L2) lasers, with energy densities of 0.5, 1.5 and 2.5 J/cm2 and an output power of 100 mW. A non-irradiated well was used as a control. Cellular viability was measured 24 hours after irradiation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and proliferation was measured 24, 48 and 72 hours after irradiation using trypan blue staining and counting. Propidium iodide (PI) staining was used to identify any pyknotic nuclei or nuclear fragmentation 72 hours after irradiation. RESULTS: An increase in viability was observed only in the group with the 940 nm laser irradiation at energy density of 2.5 J/cm2 (P < 0.001). The proliferation of cells was significantly increased in the group with 940 nm laser irradiation at energy density of 2.5 J/cm2 at all the time points examined in comparison to other groups (P < 0.001). PI staining showed no change in cell nuclei in any of the groups. CONCLUSION: Irradiation of PDLSCs with a 940 nm laser at an energy density of 2.5 J/cm2 could promote efficient cell proliferation.

Efficacy and safety of P11-4 for the treatment of periodontal defects in dogs.

OBJECTIVES: This study's aim was to investigate the safety and performance of a self-assembling peptide matrix (SAPM) P11-4 for the treatment of periodontal disease in a controlled pre-clinical study. MATERIALS AND METHODS: Acute buccal bony dehiscence defects (LxW: 5 × 3 mm) were surgically created on the distal root of four teeth on one mandible side of 7 beagle dogs followed by another identical surgery 8 weeks later on the contralateral side. SAPM P11-4 (with and without root conditioning with 24% EDTA (T1, T2)), Emdogain® (C) and a sham intervention (S) were randomly applied on the four defects at each time point. Four weeks after the second surgery and treatment, the animals were sacrificed, the mandibles measured by micro-computed tomography (µ-CT) and sections of the tissue were stained and evaluated histologically. RESULTS: Clinically and histologically, no safety concerns or pathological issues due to the treatments were observed in any of the study groups at any time point. All groups showed overall similar results after 4 and 12 weeks of healing regarding new cementum, functionality of newly formed periodontal ligament and recovery of height and volume of the new alveolar bone and mineral density. CONCLUSION: A controlled clinical study in humans should be performed in a next step as no adverse effects or safety issues, which might affect clinical usage of the product, were observed. CLINICAL RELEVANCE: The synthetic SAPM P11-4 may offer an alternative to the animal-derived product Emdogain® in the future.

Osteoinductive function of fucoidan on periodontal ligament stem cells: Role of PI3K/Akt and Wnt/ß-catenin signaling pathways.

BACKGROUND/OBJECTIVES: Fucoidan has been focused as a multifunctional therapeutic uses including bone health supplements. However, the critical molecular mechanisms of fucoidan for bone therapeutic agents have not been fully understood. We investigated the osteoinductive effect of fucoidan on periodontal ligament stem cells (PDLSCs) and how this polymer encouraged PDLSC osteogenesis. MATERIALS AND METHODS: Osteogenic induction of PDLSCs was processed by culturing cells with fucoidan treatment. Osteogenic differentiation of PDLSCs was verified by alkaline phosphatase (ALP) activity, matrix mineralization assay, intracellular calcium levels, and mRNA expression and protein levels of osteogenic markers. RESULTS: Fucoidan treatment showed higher osteogenic activity in the PDLSCs than the control groups. PDLSCs with fucoidan also presented increased levels of the phosphatidylinositol-3-kinase (PI3K) isoforms, p110α and p110γ compared to control cells. The phosphorylation of Akt, a PI3K downstream effector, was significantly increased at 90 min of fucoidan induction. Expression of ß-catenin, a coactivator of canonical Wnt pathways, was increased in PDLSCs with fucoidan. ß-catenin was found to link with PI3K activation during the fucoidan stimulation. When cells were blocked by PI3K inhibitor or ß-catenin-specific siRNA, fucoidan-induced osteogenic activity of PDLSCs was significantly attenuated. CONCLUSION: These findings suggest that the fucoidan stimulates osteogenic differentiation of PDLSCs via the PI3K/Akt and Wnt/ß-catenin pathways.

Effects of Photobiomodulation Therapy with Various Laser Wavelengths on Proliferation of Human Periodontal Ligament Mesenchymal Stem Cells.

Several methods have been proposed to enhance the regeneration and healing time in periodontal therapy. Photobiomodulation therapy (PBMT) is a recently suggested novel technique for this purpose. This study aimed to compare the efficacy of PBMT with various laser wavelengths and energy densities on proliferation of human periodontal ligament mesenchymal stem cells (PDLMSCs). The wells containing PDLMSCs were subjected to laser irradiation at 635, 660, 808 and 980 nm wavelengths with 1, 1.5, 2.5 and 4 J cm-2 energy densities. Cell proliferation and viability were evaluated after 1, 3 and 5 days with the methyl thiazolyl tetrazolium (MTT) assay and 4,6-diamidino-2-phenylindole (DAPI) staining. No significant difference was observed among the experimental and the control groups on day 1 (P > 0.05). On day 3, 808 nm laser at 4 J cm-2 energy density and 980 nm laser at all densities had significant differences with control group. On day 5, the control group had significant differences in cell proliferation with 808 nm laser at 2.5 and 4 J cm-2 energy densities, and 980 nm laser at all densities. PBMT with 635, 660, 808 and 980 nm wavelengths increased the proliferation of PDLMSCs but the maximum cell viability was prominent after irradiation by 980 nm laser with energy density of 4 J cm-2 on day 3.

Astaxanthin Inhibits Diabetes-Triggered Periodontal Destruction, Ameliorates Oxidative Complications in STZ-Injected Mice, and Recovers Nrf2-Dependent Antioxidant System.

Numerous studies highlight that astaxanthin (ASTX) ameliorates hyperglycemic condition and hyperglycemia-associated chronic complications. While periodontitis and periodontic tissue degradation are also triggered under chronic hyperglycemia, the roles of ASTX on diabetes-associated periodontal destruction and the related mechanisms therein are not yet fully understood. Here, we explored the impacts of supplemental ASTX on periodontal destruction and systemic complications in type I diabetic mice. To induce diabetes, C57BL/6 mice received a single intraperitoneal injection of streptozotocin (STZ; 150 mg/kg), and the hyperglycemic mice were orally administered with ASTX (12.5 mg/kg) (STZ+ASTX group) or vehicle only (STZ group) daily for 60 days. Supplemental ASTX did not improve hyperglycemic condition, but ameliorated excessive water and feed consumptions and lethality in STZ-induced diabetic mice. Compared with the non-diabetic and STZ+ASTX groups, the STZ group exhibited severe periodontal destruction. Oral gavage with ASTX inhibited osteoclastic formation and the expression of receptor activator of nuclear factor (NF)-κB ligand, 8-OHdG, γ-H2AX, cyclooxygenase 2, and interleukin-1ß in the periodontium of STZ-injected mice. Supplemental ASTX not only increased the levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and osteogenic transcription factors in the periodontium, but also recovered circulating lymphocytes and endogenous antioxidant enzyme activity in the blood of STZ-injected mice. Furthermore, the addition of ASTX blocked advanced glycation end products-induced oxidative stress and growth inhibition in human-derived periodontal ligament cells by upregulating the Nrf2 pathway. Together, our results suggest that ASTX does not directly improve hyperglycemia, but ameliorates hyperglycemia-triggered periodontal destruction and oxidative systemic complications in type I diabetes.