Therapeutic applications of dental pulp stem cells in regenerating dental,periodontal and oral-related structures

Dental pulp stem cells (DPSCs) have emerged as a promising tool with greatpotential for use in tissue regeneration and engineering. Some of the mainadvantages of these cells are their multifaceted differentiation capacity, along withtheir high proliferation rate, a relative simplicity of extraction and culture thatenables obtaining patient-specific cell lines for their use in autologous celltherapy. PubMed, Scopus and Google Scholar databases were searched forrelevant articles related to the use of DPSCs in regeneration of dentin-pulpcomplex (DPC), periodontal tissues, salivary gland and craniomaxillofacial bonedefects. Few studies were found regarding the use of DPSCs for regeneration ofDPC. Scaffold-based combined with DPSCs isolated from healthy pulps was thestrategy used for DPC regeneration. Studies involved subcutaneous implantationof scaffolds loaded with DPSCs pretreated with odontogenic media, or performedon human tooth root model as a root slice. Most of the studies were related toperiodontal tissue regeneration which mainly utilized DPSCs/secretome. Forperiodontal tissues, DPSCs or their secretome were isolated from healthy orinflamed pulps and they were used either for preclinical or clinical studies.Regarding salivary gland regeneration, the submandibular gland was the onlymodel used for the preclinical studies and DPSCs or their secretome were isolatedonly from healthy pulps and they were used in preclinical studies. Likewise,DPSCs have been studied for craniomaxillofacial bone defects in the form ofmandibular, calvarial and craniofacial bone defects where DPSCs were isolatedonly from healthy pulps for preclinical and clinical studies. From the previousresults, we can conclude that DPSCs is promising candidate for dental and oraltissue regeneration.

Application of platelet-rich plasma with stem cells in bone and periodontal tissue engineering

Presently, there is a high paucity of bone grafts in the United States and worldwide. Regenerating bone is of prime concern due to the current demand of bone grafts and the increasing number of diseases causing bone loss. Autogenous bone is the present gold standard of bone regeneration. However, disadvantages like donor site morbidity and its decreased availability limit its use. Even allografts and synthetic grafting materials have their own limitations. As certain specific stem cells can be directed to differentiate into an osteoblastic lineage in the presence of growth factors(GFs), it makes stem cells the ideal agents for bone regeneration.Furthermore, platelet-rich plasma(PRP), which can be easily isolated from whole blood, is often used for bone regeneration, wound healing and bone defect repair. When stem cells are combined with PRP in the presence of GFs, they are able to promote osteogenesis. This review provides in-depth knowledge regarding the use of stem cells and PRP in vitro, in vivo and their application in clinical studies in the future.

Local icariin application enhanced periodontal tissue regeneration and relieved local inflammation in a minipig model of periodontitis

Periodontitis is an inflammatory autoimmune disease. Treatment should alleviate inflammation, regulate the immune reaction and promote periodontal tissue regeneration. Icariin is the main active ingredient of Epimedii Folium, and it is a promising compound for the enhancement of mesenchymal stem cell function, promotion of bone formation, inhibition of bone resorption, alleviation of inflammation and regulation of immunity. The study investigated the effect of icariin on periodontal tissue regeneration in a minipig model of periodontitis. The minipig model of periodontitis was established. Icariin was injected locally. The periodontal clinical assessment index, a computed tomography(CT) scan, histopathology and enzyme-linked immune sorbent assay(ELISA)were used to evaluate the effects of icariin. Quantitative analysis results 12 weeks post-injection demonstrated that probing depth,gingival recession, attachment loss and alveolar bone regeneration values were(3.72 ± 1.18) mm vs.(6.56 ± 1.47) mm,(1.67 ± 0.59)mm vs.(2.38 ± 0.61) mm,(5.56 ± 1.29) mm vs.(8.61 ± 1.72) mm, and(25.65 ± 5.13) mm3 vs.(9.48 ± 1.78) mm3 in the icariin group and0.9% NaCl group, respectively. The clinical assessment, CT scan, and histopathology results demonstrated significant enhancement of periodontal tissue regeneration in the icariin group compared to the 0.9% NaCl group. The ELISA results suggested that the concentration of interleukin-1 beta(IL-1β) in the icariin group was downregulated compared to the 0.9% NaCl group, which indicates that local injection of icariin relieved local inflammation in a minipig model of periodontitis. Local injection of icariin promoted periodontal tissue regeneration and exerted anti-inflammatory and immunomodulatory function. These results support the application of icariin for the clinical treatment of periodontitis.

Mass acquisition of human periodontal ligament stem cells

The periodontal ligament(PDL)is an essential fibrous tissue for tooth retention in the alveolar bone socket.PDL tissue further functions to cushion occlusal force,maintain alveolar bone height,allow orthodontic tooth movement,and connect tooth roots with bone.Severe periodontitis,deep caries,and trauma cause irreversible damage to this tissue,eventually leading to tooth loss through the destruction of tooth retention.Many patients suffer from these diseases worldwide,and its prevalence increases with age.To address this issue,regenerative medicine for damaged PDL tissue as well as the surrounding tissues has been extensively investigated regarding the potential and effectiveness of stem cells,scaffolds,and cytokines as well as their combined applications.In particular,PDL stem cells(PDLSCs)have been well studied.In this review,I discuss comprehensive studies on PDLSCs performed in vivo and contemporary reports focusing on the acquisition of large numbers of PDLSCs for therapeutic applications because of the very small number of PDLSCs available in vivo.

The Use of Zein and Shuanghuangbu for Periodontal Tissue Engineering

Aim Tissue engineering is a promising area with a broad range of applications in the fields of regenerative medicine and human health. The emergence of periodontal tissue engineering for clinical treatment of periodontal disease has opened a new therapeutic avenue. The choice of scaffold is crucial. This study was conducted to prepare zein scaffold and explore the suitability of zein and Shuanghuangbu for periodontal tissue engineering.Methodology A zein scaffold was made using the solvent casting/particulate leaching method with sodium chloride （NaC1） particles as the porogen. The physical properties of the zein scaffold were evaluated by observing its shape and determining its pore structure and porosity. Cytotoxicity testing of the scaffold was carried out via in vitro cell culture experiments, including a liquid extraction experi- ment and the direct contact assay. Also, the Chinese medicine Shuanghuangbu, as a growth factor, was diluted by scaffold extract into different concentrations. This Shuanghuangbu-scaffold extract was then added to periodontal ligament cells （PDLCs） in order to determineits effect on cell proliferation. Results The zein scaffold displayed a sponge-like structure with a high porosity and sufficient thickness. The porosity and pore size of the zein scaffold can be controlled by changing the porogen particles dosage and size. The porosity was up to 64.1%-78.0%. The pores were well-distributed, interconnected, and porous. The toxicity of the zein scaffold was graded as 0-1. Furthermore, PDLCs displayed full stretching and vigorous growth under scanning electronic microscope （SEM）. Shuanghuangbu-scaffold extract could reinforce proliferation activity of PDLCs compared to the control group, especially at 100 μg.mL^-1 （P〈0.01）. Conclusion A zein scaffold with high porosity, open pore wall structure, and good biocompatibility is conducive to the growth of PDLCs. Zein could be used as scaffold to repair periodontal tissue defects. Also, Shuanghuangbuscaffold extract can enhance the proliferation activity of PDLCs. Altogether, these findings provide the basis for in vivo testing on animals.

A new approach to transfect NF-κB decoy oligodeoxynucleotides into the periodontal tissue using the ultrasound-microbubble method

The objective of this study is to investigate the effect of the ultrasound-microbubble technique in nuclear factor kappa B(NF-κB) decoy oligodeoxynucleotide(ODN) transfection in the gingival tissue in mice. The 6-FAM-labeled scrambled decoy ODN with microbubbles was applied to the periodontal tissue in 8-week-old male C57BL/6J mice by ultrasound radiation at low(LUM-Sc) and high(HUM-Sc) intensities to optimize the transfection condition of the ultrasound-microbubble method.Histological inspections were performed two hours after transfection to compare the expression with that in the sham-operated group without ultrasound radiation(A-Sc). Then, an NF-κB decoy was transfected into the periodontal tissue using the highintensity ultrasound-microbubble(HUM-NF) technique to examine the anti-inflammatory effects of the decoy ODN. Western blot analysis was performed to investigate the expression of interleukin(IL)-1β, IL-6 and intercellular adhesion molecule-1(ICAM-1)in the gingival tissues in the HUM-Sc, the HUM-NF and control groups. The fluorescence microscopy results showed that the fluorescent intensity in the periodontal tissues in the LUM-Sc and HUM-Sc groups was significantly higher than that in the A-Sc and the control groups. The fluorescent intensity in the HUM-Sc group, especially in the gingival connective tissue,was the highest of all groups. Western blot analysis indicated that the protein expression levels of IL-1β, IL-6 and ICAM-1 in the HUM-NF group were significantly lower than those in the HUM-Sc and the control groups. These findings suggest that the high-intensity ultrasound-microbubble technique is an effective tool for decoy transfection into the periodontal tissue.

Research progress of strontium in promoting periodontal tissue regeneration

Periodontal disease is a chronic infectious disease of the oral cavity.Its main clinical features are periodontal pocket formation and alveolar bone resorption.Scholars'research hotspot is to achieve periodontal tissue regeneration in patients.Studies have found that strontium has certain potential in promoting periodontal tissue regeneration.In recent years,scholars have been conducting research on strontium and periodontal tissue regeneration,with a view to opening a new path for periodontal disease treatment.This article reviews the research status of strontium and periodontal tissue regeneration.The review results show that strontium can induce the proliferation and differentiation of PDLSCs and promote the regeneration of lost bone tissue;it can inhibit osteoclast activity and induce osteoclast apoptosis through a variety of signaling pathways,thereby inhibiting bone resorption;Promote bone formation;Strontium also has the function of promoting early angiogenesis and suppressing immune inflammatory response.Because the current research on strontium and periodontal tissue regeneration is only focused on in vivo and in vitro experiments,there is no relevant clinical trial to apply strontium to periodontal tissue regeneration.Therefore,if strontium is used to promote periodontal tissue regeneration to achieve the treatment of periodontal disease,further research is needed.

Effectiveness of two regeneration materials in autogenous tooth transplantation in larger sockets:an experimental study in beagle dogs

Objective： To evaluate the effectiveness of regenerous tissue and bone substitute in autogenous tooth transplantation in the larger recipient socket. Methods：In 3 Beagle dogs, 18 incisors were transplanted to the recipient sockets, 2 mm wider mesio-distally. The regenerous tissue group, the bone substitute group and the control group contained 7, 7 and 4 teeth respectively. No additional material was used in control group. Clinical and radiographic examinations were done every month and were sacrificed 3 months later. Subsequently, decalcified sections were prepared for routine histological evaluation. Ordinal scores for root surface resorption were analyzed using the Kruskal-Wallis test. Results：All donor teeth survived. A statistically significant difference was found among all three treatment groups（P= 0.0001）. The proliferating tissue in space positively affected the periodontal healing without any resorption. Inflammatory resorption of the root surface and formation of new bone were observed in the bone substitute group. Surface resorptions of the roots were found in the control group. Conclusion：Proliferating tissues enhance the regeneration of periodontal tissues in larger recipient sockets and prevent root resorption. Sinbone HT is beneficial for the stabilization of the transplanted teeth in larger sockets.

Dimethyloxallyl glycine/nanosilicates-loaded osteogenic/angiogenic difunctional fibrous structure for functional periodontal tissue regeneration

The coupled process of osteogenesis-angiogenesis plays a crucial role in periodontal tissue regeneration.Although various cytokines or chemokines have been widely applied in periodontal in situ tissue engineering,most of them are macromolecular proteins with the drawbacks of short effective half-life,poor stability and high cost,which constrain their clinical translation.Our study aimed to develop a difunctional structure for periodontal tissue regeneration by incorporating an angiogenic small molecule,dimethyloxalylglycine(DMOG),and an osteoinductive inorganic nanomaterial,nanosilicate(nSi)into poly(lactic-co-glycolic acid)(PLGA)fibers by electrospinning.The physiochemical properties of DMOG/nSi-PLGA fibrous membranes were characterized.Thereafter,the effect of DMOG/nSi-PLGA membranes on periodontal tissue regeneration was evaluated by detecting osteogenic and angiogenic differentiation potential of periodontal ligament stem cells(PDLSCs)in vitro.Additionally,the fibrous membranes were transplanted into rat periodontal defects,and tissue regeneration was assessed with histological evaluation,micro-computed tomography(micro-CT),and immunohistochemical analysis.DMOG/nSi-PLGA membranes possessed preferable mechanical property and biocompatibility.PDLSCs seeded on the DMOG/nSi-PLGA membranes showed up-regulated expression of osteogenic and angiogenic markers,higher alkaline phosphatase(ALP)activity,and more tube formation in comparison with single application.Further,in vivo study showed that the DMOG/nSi-PLGA membranes promoted recruitment of CD90+/CD34stromal cells,induced angiogenesis and osteogenesis,and regenerated cementum-ligament-bone complex in periodontal defects.Consequently,the combination of DMOG and nSi exerted admirable effects on periodontal tissue regeneration.DMOG/nSi-PLGA fibrous membranes could enhance and orchestrate osteogenesis-angiogenesis,and may have the potential to be translated as an effective scaffold in periodontal tissue engineering.

Application of Induced Pluripotent Stem Cells Derived from Human Periodontal Ligament Cells in Bone Regeneration

The purpose of this study was to primarily culture human periodontal ligament cells(hPDLCs)and to reprogram hPDLCs with exogenous genes via a lentivirus-mediated transfection system.Then induced pluripotent stem cells derived from h PDLCs(hPDLC-iPSCs)were identified.Alizarin red staining was used to observe the formation of mineralized nodules and real-time Polymerase Chain Reaction(PCR)was used to detect the expression of osteogenic genes.For the in vivo experiment,nude mouse skull defect models were established and cell sheets were made to repair the bone defect.The reprogrammed cells were positive for alkaline phosphatase(ALP)staining and embryonic stem cells(ESCs)-specific proteins,and could form teratomas.After osteogenic induction,alizarin red staining showed that the number of mineralized nodules in the h PDLC-i PSCs group was more and the osteogenic related factors ALP,osteocalcin(OCN),Col-I and Runx2 were also expressed higher in hPDLC-iPSCs.The hPDLC-iPSC cell sheets were all successfully made.Histological analysis showed that the h PDLC-i PSC cell sheet got new bone formation.These results demonstrated that hPDLC-iPSCs were successfully generated from human periodontal ligament fibroblasts and hPDLC-iPSC cell sheets provided new options for bone tissue engineering.

Development of a multicellular 3D-bioprinted microtissue model of human periodontal ligament-alveolar bone biointerface:Towards a pre-clinical model of periodontal diseases and personalized periodontal tissue engineering

While periodontal (PD) disease is among principal causes of tooth loss worldwide, regulation of concomitant soft and mineralized PD tissues, and PD pathogenesis have not been completely clarified yet. Besides, relevant pre-clinical models and in vitro platforms have limitations in simulating human physiology. Here, we have harnessed three-dimensional bioprinting (3DBP) technology for developing a multi-cellular microtissue model resembling PD ligament-alveolar bone (PDL-AB) biointerface for the first time. 3DBP parameters were optimized;the physical, chemical, rheological, mechanical, and thermal properties of the constructs were assessed. Constructs containing gelatin methacryloyl (Gel-MA) and hydroxyapatite-magnetic iron oxide nanoparticles showed higher level of compressive strength when compared with that of Gel-MA constructs. Bioprinted self-supporting microtissue was cultured under flow in a microfluidic platform for >10 days without significant loss of shape fidelity. Confocal microscopy analysis indicated that encapsulated cells were homogenously distributed inside the matrix and preserved their viability for >7 days under microfluidic conditions. Immunofluorescence analysis showed the cohesion of stromal cell surface marker-1+ human PDL fibroblasts containing PDL layer with the osteocalcin+ human osteoblasts containing mineralized layer in time, demonstrating some permeability of the printed constructs to cell migration. Preliminary tetracycline interaction study indicated the uptake of model drug by the cells inside the 3D-microtissue. Also, the non-toxic levels of tetracycline were determined for the encapsulated cells. Thus, the effects of tetracyclines on PDL-AB have clinical significance for treating PD diseases. This 3D-bioprinted multi-cellular periodontal/osteoblastic microtissue model has potential as an in vitro platform for studying processes of the human PDL.

PREPARATION OF CHITOSAN/HYDROXYAPATITE GUIDED MEMBRANE USED FOR PERIODONTAL TISSUE REGENERATION

In an effort to develop biomaterials to meet guided tissue regeneration （GTR） standards for periodontal tissue recovery, a homogeneous and transparent chitosan （CS）/hydroxyapatite （HA） membrane with potential applications as GTR barrier in periodontal therapy has been prepared via in situ compositing. The membrane has been designed to have a smoothrough asymmetric structure that meets the demand for GTR. Component and morphology of the membrane are characterized by XRD and SEM. It can be indicated that HA was in situ synthesized uniformly in the CS membrane. Mechanical experiments of the membranes with various HA contents show that their tensile strengths are adequate for periodontal therapy. Biological properties of the membrane have been performed by cell toxicity assays, hemolysis tests and animal experiments. Results indicate that the membrane has good biocompatibility and inductive effect for cell growth. Therefore this membrane can be potentially applied as GTR barrier membrane for periodontal tissue regeneration.

Effects of secretive bone morphogenetic protein 2 induced by gene transfection on the biological changes of NIH3T3 cells

Background Bone morphogenetic proteins （BMPs）, which belong to the transforming growth factor beta superfamily, are powerful regulators of cartilage and bone formation. This study investigated the biological changes of NIH3T3 ceils incubated with secretive BMP2 that was induced by gene transfection through transwell.Methods Eukaryonic expression vector （pcDNA3. 1-B2 ） was transfered into NIH333 cells with Sofast^TM, a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expressions of BMP2 were determined by immunohistochemical stain and enzyme-linked immunosorbent assay. NIH333 ceils were co-cultured with hBMP2 gene transfecting ceils through transwell, and the ultrastructure, alkaline phosphatase activity and the expression of osteocalcin （the marker of osteogenetic differentiation） changes were observed.Results There were cytoplasmic and extracellular expressions of BMP2 in transfecting NIH3T3 ceils. The ultrastructural changes, the high activity of alkaline phosphatase and the positive stain of osteocalcin suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells.Conclusion Secretive BMP2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast ceils.