Emerging roles of platelet concentrates and platelet-derived extracellular vesicles in regenerative periodontology and implant dentistry.

Platelet concentrates (PCs) are easily obtained from autogenous whole blood after centrifugation and have evolved through three generations of development to include platelet-rich plasma, platelet-rich fibrin, and concentrated growth factor. Currently, PCs are widely used for sinus floor elevation, alveolar ridge preservation, periodontal bone defects, guided bone regeneration, and treatment of gingival recession. More recently, PCs have been leveraged for tissue regeneration to promote oral soft and hard tissue regeneration in implant dentistry and regenerative periodontology. PCs are ideal for this purpose because they have a high concentration of platelets, growth factors, and cytokines. Platelets have been shown to release extracellular vesicles (P-EVs), which are thought to be essential for PC-induced tissue regeneration. This study reviewed the clinical application of PCs and P-EVs for implant surgery and periodontal tissue regeneration.

Intrinsic Surface Effects of Tantalum and Titanium on Integrin α5ß1/ ERK1/2 Pathway-Mediated Osteogenic Differentiation in Rat Bone Mesenchymal Stromal Cells.

BACKGROUND/AIMS: Accumulating evidence demonstrates the superior osteoinductivity of tantalum (Ta) to that of titanium (Ti); however, the mechanisms underlying these differences are unclear. Thus, the objective of the present study was to examine the effects of Ta and Ti surfaces on osteogenesis using rat bone mesenchymal stromal cells (rBMSCs) as a model. METHODS: Ta and Ti substrates were polished to a mirror finish to minimize the influences of structural factors, and the intrinsic surface effects of the two materials on the integrin α5ß1/mitogen-activated protein kinases 3 and 1 (ERK1/2) cascade-mediated osteogenesis of rBMSCs were evaluated. Alkaline phosphatase (ALP) activity, Alizarin Red staining, real-time polymerase chain reaction, and western blot assays of critical osteogenic markers were conducted to evaluate the effects of the two substrates on cell osteogenesis. Moreover, the role of the integrin α5ß1/ERK1/2 pathway on the osteoinductive performance of Ta and Ti was assessed by up- and down-regulation of integrin α5 and ß1 with RNA interference, as well as through ERK1/2 inhibition with U0126. RESULTS: Osteogenesis of rBMSCs seeded on the Ta surface was superior to that of cells seeded on the Ti surface in terms of ALP activity, extracellular matrix calcification, and the expression of integrin α5, integrin ß1, ERK1/2, Runt-related transcription factor 2, osteocalcin, collagen type I, and ALP at both the mRNA and protein levels. Moreover, down-regulation of integrin α5 or integrin ß1, or ERK1/2 inhibition severely impaired the osteoblastic differentiation on the Ta surface. By contrast, over-expression of integrin α5 or integrin ß1 improved osteogenesis on the Ti substrates, while subsequent ERK1/2 inhibition abrogated this effect. CONCLUSION: The integrin α5ß1/ERK1/2 pathway plays a crucial role in regulating rBMSCs osteogenic differentiation; thus, the greater ability of a Ta surface to trigger integrin α5ß1/ERK1/2 signaling may explain its better osteoinductivity. The different effects of Ta and Ti surfaces on rBMSC osteogenesis are considered to be related to the conductive behaviors between integrin α5ß1 and the oxides spontaneously formed on the two metals. These results should facilitate the development of engineering strategies with Ta and Ti surfaces for improved osteogenesis in endosteal implants.

RNA-Seq investigation and in vivo study the effect of strontium ranelate on ovariectomized rat via the involvement of ROCK1.

Strontium ranelate (SrR) is an anti-osteoporosis drug with excellent osteogenic and angiogenic capacity. In this study, we aimed to investigate the osteogenesis and angiogenesis effects of SrR and the underlying mechanism involved. RNA-Seq was conducted to examine the effects of SrR on gene expression in ovariectomy rat bone marrow mesenchymal stem cells (OVX-rBMSCs). To validate the different expressed gene in vitro, the effects of gene interference and overexpression in osteogenic induction environment of OVX-rBMSCs and in primary osteoblasts were studied. RNA-Seq showed that ROCK1 significantly increased after SrR treatment in OVX-rBMSCs, and further validated by real-time PCR and western blotting. Overexpression of ROCK1 promoted osteogenic differentiation of OVX-rBMSCs and induced cell viability and inhibited apoptosis of primary osteoblasts, which was reversed by inhibition of ROCK1 by RNA interference or ROCK1 inhibitor (Y-27632) after SrR treatment. Furthermore, the SrR was loaded on nano-structured hydroxyapatite (nano-HAp) particulates to promote osteogenesis and angiogenesis in repairing of the femoral condyle bone defect using ovariectomy rat model. Taken together, ROCK1 is one of the targets that SrR promotes the osteogenic differentiation of OVX-rBMSCs and cell viability of primary osteoblasts, the nano-HAp particles could act as carriers for SrR to repair bone defects.

Effects of Sr-HT-Gahnite on osteogenesis and angiogenesis by adipose derived stem cells for critical-sized calvarial defect repair.

Tissue engineering strategies to construct vascularized bone grafts are now attracting much attention. Strontium-hardystonite-Gahnite (Sr-HT-Gahnite) is a strong, highly porous, and biocompatible calcium silicate based bio-ceramic that contains strontium and zinc ions. Adipose derived stem cells (ASCs) have been demonstrated to have the ability in promoting osteogenesis and angiogenesis. In this study, the effects of Sr-HT-Gahnite on cell morphology, cell proliferation, and osteogenic differentiation of ASCs were systematically investigated. The cell proliferation, migration and angiogenic differentiation of human umbilical vein endothelial cell (HUVECs) were studied. Beta-tricalcium phosphate/hydroxyapatite (TCP/HA) bioceramic scaffolds were set as the control biomaterial. Both bio-ceramics exhibited no adverse influence on cell viability. The Sr-HT-Gahnite scaffolds promoted cell attachment and alkaline phosphatase (ALP) activity of ASCs. The Sr-HT-Gahnite dissolution products enhanced ALP activity, matrix mineralization, and angiogenic differentiation of ASCs. They could also improve cell proliferation, migration, and angiogenic differentiation of HUVECs. Levels of in vivo bone formation with Sr-HT Gahnite were significantly higher than that for TCP/HA. The combination of Sr-HT-Gahnite and ASCs promoted both osteogenesis and angiogenesis in vivo study, compared to Sr-HT-Gahnite and TCP/HA bio-ceramics when administered alone, suggesting Sr-HT-Gahnite can act as a carrier for ASCs for construction of vascularized tissue-engineered bone.

Molecular, Cellular and Pharmaceutical Aspects of Autologous Grafts for Peri-implant Hard and Soft Tissue Defects.

The lack of supporting hard and soft tissues always prevents the rehabilitation with dental implants. Among various hard and soft tissue augmentation procedures, autologous grafts have been considered to be the gold standard. Autologous mesenchymal stem cells (MSCs) from bone marrow, dental tissue and adipose tissue have been described as promising alternatives for bone regeneration in the field of dental implantation. Mucosal cells, gingival fibroblasts and dental progenitor cells (DPS) can enhance peri-implant soft tissue augmentation and regenerate periodontal tissues around dental implants. Obtained from patients, platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are enriched in autologous platelets, which contain a great deal of growth factors and cytokines that are conducive to the regeneration of both hand and soft tissues around dental implants. Pharmaceutical treatments for osteoporosis and diabetes should be locally applied with implant procedures to restrict the resorption of autologous bone grafts and reduction of bone volume. Although autografts hold great potentials for dental implants, new approaches should also be explored with minimally invasion donor sites methods such as tissue engineering combined with autologous three factors and bio-3D printing involving selfassembling cell aggregates.

Maxillary sinus floor elevation with a tissue-engineered bone composite of deciduous tooth stem cells and calcium phosphate cement in goats.

The study aimed to assess the effect of maxillary sinus floor elevation with tissue-engineered bone constructed from deciduous tooth stem cells (DTSCs) and calcium phosphate cement (CPC). The stem cells from goat deciduous teeth (SGDs) were isolated and transfected by means of the adenovirus with an enhanced green fluorescent protein gene (AdEGFP). As many as 18 bilateral maxillary sinuses of nine goats were randomly allocated into three groups (n = 6/group): group A (SGDs-CPC compound), group B (CPC alone) and group C (autogenous bone obtained from an iliac crest). All the samples were evaluated by computed tomography (CT), histology and histomorphometric analysis. Furthermore, the fate of implanted SGDs was traced using an immunohistochemical staining method in the decalcified samples. SGDs might be differentiated into osteoblasts in an osteogenic medium. In the present study, three-dimensional CT analysis showed that the volume of newly formed bone in group A was greater than that in the other two groups. After a healing period of 3 months, sequential analyses of triad-colour fluorescence labelling, histology and histomorphology indicated that the SGDs-CPC compound primarily promoted bone formation and mineralization at 2 and 3 months after the operation. Moreover, the areas of new bone formation in elevated sinuses were 41.82 ± 6.24% in the SGDs-CPC group, which was significantly higher than the 30.11 ± 8.05% in the CPC-alone group or the 23.07 ± 10.21% in the autogenous bone group. Immunohistochemical staining revealed that GFP and OCN were both expressed in the new bone tissue for the samples with eGFP, which suggested that the implanted SGDs might have contributed to new bone formation on the elevated sinus floor. SGDs can promote new bone formation and maturation in the goat maxillary sinus, and the tissue-engineered bone composite of SGDs and CPC might be a potential substitute for existing maxillary sinus floor elevation methods. Copyright © 2014 John Wiley & Sons, Ltd.

Dose-dependent Effects of Strontium Ranelate on Ovariectomy Rat Bone Marrow Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells.

In clinic, strontium ranelate (SrR) is a useful drug to treat osteoporosis by orally taken method, but some side effect appeared in recent years. The aim of this study is to evaluate the effectiveness and safety of SrR on cells by direct application, to study the possibility of local application of this drug. Qualitative ALP staining, quantitative ALP activity assay, alizarin red staining, realtime PCR and westernblot assay were used to evaluate the osteogenesis ability of SrR under normal or osteogenic induction environment of ovariectomy bone marrow mesenchymal stem cells (OVX-BMSCs). The angiogenesis ability of SrR was studied by immunofluorescence staining of CD31 and vWF of OVX-BMSCs under angiogenesis induction environment, transwell, tubeformation and realtime PCR assay of HUVECs. Signaling pathway of PI3K/AKT/mTOR was also studied. The result demonstrated that SrR could enhance proliferation and osteogenic differentiation of OVX-BMSCs. The osteogenesis effect of SrR has been proved by the better performed of ALP activity, alizarin red staining and the remarkable up-regulation of ALP, Col-I, Runx2, OCN, BMP-2, BSP, OPG of the OVX-BMSCs, and reduction of RANKL. In addition, SrR promotes angiogenesis differentiation of both OVX-BMSCs and HUVECs. Higher intensity of immunostaining of CD31 and vWF, better result of transwell and tubeformation assay could be observed in SrR treated group, and increasing mRNA levels of VEGF and Ang-1 in the OVX-BMSCs, VEGF in HUVECs were learnt. Signaling pathway assay showed that PI3K/AKT/mTOR signaling pathway was involved in this SrR triggered angiogenesis procedure. The thrombosis marker ET-1, PAI-1 and t-PA were up-regulated, but no significant differences for low concentration (<0.5mM). The concentration between 0.25-0.5mM may be more appropriate for local application, and locally application of SrR could be considered as a promising way for bone regeneration.

Occlusal force characteristics of masseteric muscles after intramuscular injection of botulinum toxin A(BTX - A)for treatment of temporomandibular disorder.

Our aim was to evaluate the occlusal force and therapeutic efficacy of the masseteric muscles after intramuscular injection of botulinum toxin A (BTX-A) for the treatment of patients with concurrent temporomandibular disorders (TMD) and bruxism. Thirty patients with TMD associated with bruxism were randomised into three groups (n=10 in each group), and treated by bilateral intramuscular injection of BTX-A into the masseter, placebo, or control. We used an occlusal force analysis system to collect several measures of occlusal force such as duration of biting and closing, the maximum occlusal force, and the distribution of occlusal force. The occlusal force in the intercuspid position was reduced in all three groups. There was a significant difference between the BTX-A and placebo groups (F(df=1)=8.08, p=0.01) but not between the control group and the other two(F(df=1)=4.34, p=0.047). The duration of occlusion was significantly increased in the BTX-A group after 3 months' treatment (t=4.07, p=0.003). The asymmetrical distribution of occlusal force was reduced in all three groups, but not significantly so (Levene's test F(df=2)=0.25, p=0.78,ANOVA F(df=2)=0.50, p=0.61). Treatment of TMD with BTX-A is effective in reducing the occlusal force, but psychological intervention plays an important part in treatment.

Bioinformatics Analysis of the Effects of Tobacco Smoke on Gene Expression.

This study was designed to explore the effects of tobacco smoke on gene expression through bioinformatics analyses. Gene expression profile GSE17913 was downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) in buccal mucosa tissues between 39 active smokers and 40 never smokers were identified. Gene Ontology Specifically, the DEG distribution in the pathway of Metabolism of xenobiotics by cytochrome P450 was shown in Fig 2[corrected] were performed, followed by protein-protein interaction (PPI) network, transcriptional regulatory network as well as miRNA-target regulatory network construction. In total, 88 up-regulated DEGs and 106 down-regulated DEGs were identified. Among these DEGs, cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and CYP1B1 were enriched in the Metabolism of xenobiotics by cytochrome P450 pathway. In the PPI network, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), and CYP1A1 were hub genes. In the transcriptional regulatory network, transcription factors of MYC associated factor X (MAX) and upstream transcription factor 1 (USF1) regulated many overlapped DEGs. In addition, protein tyrosine phosphatase, receptor type, D (PTPRD) was regulated by multiple miRNAs in the miRNA-DEG regulatory network. CYP1A1, CYP1B1, YWHAZ and PTPRD, and TF of MAX and USF1 may have the potential to be used as biomarkers and therapeutic targets in tobacco smoke-related pathological changes.

Induction of Osteogenic Differentiation of Human Adipose-Derived Stem Cells by a Novel Self-Supporting Graphene Hydrogel Film and the Possible Underlying Mechanism.

Graphene and its derivatives have received increasing attention from scientists in the field of biomedical sciences because of their unique physical properties, which are responsible for their interesting biological functions. With a range of extraordinary properties such as high surface area, high mechanical strength, and ease of functionalization, graphene is considered highly promising for application in bone tissue engineering. Here, we examined the effect of using a self-supporting graphene hydrogel (SGH) film to induce the osteogenic differentiation of human adipose-derived stem cells (hADSCs). In comparison to conventional graphene and carbon fiber films, the SGH film had higher mechanical strength and flexibility. Moreover, we found that the SGH film was nontoxic and biocompatible. Of particular interest is the fact that the film alone could stimulate the osteogenic differentiation of hADSCs, independent of additional chemical inducers. Such effects are stronger for the SGH film than for graphene or carbon fiber films, although the induction capacity of the SGH film is not as high as that of the osteogenic-induced medium. The excellent osteoinductivity of the SGH film is closely related to its remarkable physical properties that include specific nanostructures, surface morphology, strong cell adherence, reasonable surface hydrophilicity, and high protein absorption.

The concentrations of IL-8 and IL-6 in gingival crevicular fluid during nickel-chromium alloy porcelain crown restoration.

We explored gum irritation and cytotoxicity caused by nickel-chromium (Ni-Cr) alloy porcelain by interleukin-8 (IL-8), interleukin-6 (IL-6) and gingival crevicular fluid (GCF) volumes at different time points peri-crown restoration. This prospective study was conducted in 60 young adults. The total amount and concentrations of IL-8 and IL-6 per site, GCF volumes, and blood neutrophil counts were performed prior to and at 1 week, 3 months, and 6 months after Ni-Cr alloy-porcelain crown restoration. Thirty male and 30 female subjects, aged 20-35 years old were enrolled. The total amount and concentrations of IL-8 and IL-6 per site, GCF volumes increased after nickel-chromium (Ni-Cr) alloy-porcelain crown restoration, and reached its peak at the third month as the GCF volume increased by 52.20 %, the total amount and concentrations of IL-8 increased by 112.11 and 22.75 %; the total amount and concentrations of IL-6 increased by 77.66 and 17.17 % when compared to baseline. In particular, the increase of IL-8 concentration was found in female patients at 3 months after restoration; while the neutrophil count of the peripheral blood did not change significantly. The increase in the total amount and the concentrations of IL-8 and IL-6 and GCF volume may be related to the cytotoxicity induced by Ni-Cr alloy. The significant increase of IL-8 concentration in females indicates that more attention should be given to women during Ni-Cr alloy porcelain crown restoration.

Engineering of bone using porous calcium phosphate cement and bone marrow stromal cells for maxillary sinus augmentation with simultaneous implant placement in goats.

The aim of this study was to explore the effects of maxillary sinus floor elevation and simultaneous dental implantation with a tissue-engineered bone complex of calcium phosphate cement (CPC) scaffolds combined with bone marrow stromal cells (BMSCs). A large animal goat model is used with the tissue engineering method. Eighteen bilateral maxillary sinus of nine goats were randomly allocated into three groups; the CPC/BMSC complex (n=6) was used to elevate maxillary sinus floor with a simultaneous implant placement; the effects were compared with those treated with CPC alone (n=6) or autogenous bone (n=6). After a healing period of 3 months, sequential triad-color fluorescence labeling, micro-CT, as well as histological and histomorphometric analyses indicated that the tissue-engineered BMSC/CPC complex could promote earlier bone formation and mineralization, and maximally maintain the volume and height of the augmented maxillary sinus. By comparison, CPC-alone or autogenous bone achieved less bone formation and later mineralization. Besides, the average bone-implant contact value reflecting the osseointegration was 35.63%±9.42% in the BMSCs/CPC group, significantly higher than 22.47%±4.28% in the CPC-alone group or 28.26%±8.03% in the autogenous bone group. In conclusion, CPC serves as a potential substrate for BMSCs for the maxillary sinus floor augmentation and simultaneous implantation. The tissue-engineered bone might enhance the stability of implants and thus be of great significance to achieve improved quality to restore the oral function in clinic.

[Ectopic osteogenesis of SGDs in goat's muscle pouch].

PURPOSE: To investigate ectopic osteogenesis of composites SGDs with porous calcium phosphate cement(pCPC) in goat's muscle pouch. METHODS: SGDs were cultivated with modified tissue culture techniques, then were induced into osteoblasts in the third passage, the osteogenic-induced SGDs were combined with pCPC and transplanted into the goat left dorsal muscle pouch, the pCPC without cells was transplanted into the right dorsal muscle pouch as negative controls. The transplants were harvested at 2-,4-,6-,8- week and prepared for histological examination. The morphologic quantitative analysis was made by SPSS 16.0 software package. RESULTS: Bone formation was not detected in pCPC without cells by histological examination. 2,4,6,8 weeks after transplantation in SGDs-pCPC group, the percentages of bone formation were (1.24±0.25)%,(1.59±0.23)%,(4.12±0.39)% and (5.68±0.58)%,respectively.There was no significant difference in bone formation at 2 and 4 weeks after transplantation (P>0.05). At 8- week, the percentages of bone formation were higher than that at 6- week in SGDs-pCPC group, and both significantly higher than that at 2- and 4- week(P<0.05). CONCLUSION: SGDs combined with pCPC have the ability of ectopic osteogenesis.