Zhipeng Fan

Allogeneic Periodontal Ligament Stem Cell Therapy for Periodontitis in Swine

Periodontitis is one of the most widespread infectious diseases in humans. It is the main cause of tooth loss and associated with a number of systemic diseases. Until now, there is no appropriate method for functional periodontal tissue regeneration. Here, we establish a novel approach of using allogeneic periodontal ligament stem cells (PDLSCs) sheet to curing periodontitis in a miniature pig periodontitis model. Significant periodontal tissue regeneration was achieved in both the autologous and the allogeneic PDLSCs transplantation group at 12 weeks post‐PDLSCs transplantation. Based on clinical assessments, computed tomography (CT) scanning, and histological examination, there was no marked difference between the autologous and allogeneic PDLSCs transplantation groups. In addition, lack of immunological rejections in the animals that received the allogeneic PDLSCs transplantation was observed. Interestingly, we found that human PDLSCs fail to express human leukocyte antigen (HLA)‐II DR and costimulatory molecules. PDLSCs were not able to elicit T‐cell proliferation and inhibit T‐cell proliferation when stimulated with mismatched major histocompatibility complex molecules. Furthermore, we found that prostaglandin E2 (PGE2) plays a crucial role in PDLSCs‐mediated immunomodulation and periodontal tissue regeneration in vitro and in vivo. Our study demonstrated that PDLSCs possess low immunogenicity and marked immunosuppression via PGE2‐induced T‐cell anergy. We developed a standard technological procedure of using allogeneic PDLSCs to cure periodontitis in swine. STEM CELLS 2010;28:1829–1838

Allogeneic mesenchymal stem cell treatment alleviates experimental and clinical Sjögren syndrome

Sjögren syndrome (SS) is a systemic autoimmune disease characterized by dry mouth and eyes, and the cellular and molecular mechanisms for its pathogenesis are complex. Here we reveal, for the first time, that bone marrow mesenchymal stem cells in SS-like NOD/Ltj mice and human patients were defective in immunoregulatory functions. Importantly, treatment with mesenchymal stem cells (MSCs) suppressed autoimmunity and restored salivary gland secretory function in both mouse models and SS patients. MSC treatment directed T cells toward Treg and Th2, while suppressing Th17 and Tfh responses, and alleviated disease symptoms. Infused MSCs migrated toward the inflammatory regions in a stromal cell-derived factor-1-dependent manner, as neutralization of stromal cell-derived factor-1 ligand CXCR4 abolished the effectiveness of bone marrow mesenchymal stem cell treatment. Collectively, our study suggests that immunologic regulatory functions of MSCs play an important role in SS pathogenesis, and allogeneic MSC treatment may provide a novel, effective, and safe therapy for patients with SS.

Vitamin C Treatment Promotes Mesenchymal Stem Cell Sheet Formation and Tissue Regeneration by Elevating Telomerase Activity

Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell‐mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up‐regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc‐mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell‐based tissue regeneration. J. Cell. Physiol. 227: 3216–3224, 2012.

Functional Tooth Restoration by Allogeneic Mesenchymal Stem Cell-Based Bio-Root Regeneration in Swine

Our previous proof-of-concept study showed the feasibility of regenerating the dental stem cell-based bioengineered tooth root (bio-root) structure in a large animal model. Here, we used allogeneic dental mesenchymal stem cells to regenerate bio-root, and then installed a crown on the bio-root to restore tooth function. A root shape hydroxyapatite tricalcium phosphate scaffold containing dental pulp stem cells was covered by a Vc-induced periodontal ligament stem cell sheet and implanted into a newly generated jaw bone implant socket. Six months after implantation, a prefabricated porcelain crown was cemented to the implant and subjected to tooth function. Clinical, radiological, histological, ultrastructural, systemic immunological evaluations and mechanical properties were analyzed for dynamic changes in the bio-root structure. The regenerated bio-root exhibited characteristics of a normal tooth after 6 months of use, including dentinal tubule-like and functional periodontal ligament-like structures. No immunological response to the bio-roots was observed. We developed a standard stem cell procedure for bio-root regeneration to restore adult tooth function. This study is the first to successfully regenerate a functional bio-root structure for artificial crown restoration by using allogeneic dental stem cells and Vc-induced cell sheet, and assess the recipient immune response in a preclinical model.

Mesenchymal stem cells derived from inflamed periodontal ligaments exhibit impaired immunomodulation

AIM The purpose of this study was to investigate the immunomodulatory properties of periodontal ligament stem cells derived from inflamed periodontal ligament tissues. MATERIAL AND METHODS Periodontal ligament stem cells were identified and isolated from healthy or inflamed periodontal ligament tissues. Peripheral blood mononuclear cells were cocultured with inflamed or healthy periodontal ligament stem cells, and T-lymphocyte proliferation was determined by incubation with carboxyfluorescein succinimidyl ester. T-helper cells (Th1/Th2, Th17) and regulatory T cells were analysed by flow cytometry. Cytokine profiles in supernatants were tested with a cytometric bead array. RESULTS Compared to healthy cells, inflamed periodontal ligament stem cells showed significantly diminished inhibition of T-cell proliferation. In cocultures, stimulated peripheral blood mononuclear cells showed significantly less induction of CD4+CD25+FOXP3+ regulatory T cells and IL-10 secretion in the presence of inflamed compared with healthy periodontal ligament stem cells. Furthermore, suppression of Th17 differentiation and IL-17 production by inflamed periodontal ligament stem cells was significantly lesser than by healthy cells. CONCLUSION This study demonstrated that inflamed periodontal ligament stem cells had markedly dysfunctional immunomodulatory properties; this may contribute to an imbalanced immune response, acceleration of osteoclastogenesis and inflammatory alveolar bone loss in periodontitis.

Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane

In vivo recycling of nitrate (NO3−) and nitrite (NO2−) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate–nitrite–NO balance. More than 25% of the circulating NO3− is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO3− to NO2−, which enters circulation and leads to NO generation. The transporters for NO3− in salivary glands have not yet been identified. Here we report that sialin (SLC17A5), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO3−/H+ cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO3− or sialic acid (SA), but not by Br−, and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO3−-induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H+-dependent NO3− conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO3− secretion in saliva after intake of a NO3−-rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO3−/H+ transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.

Allogeneic Bone Marrow Mesenchymal Stem Cell Transplantation for Periodontal Regeneration

In this study, we investigated the possibility of using local administration of allogeneic bone marrow mesenchymal stem cells (BMMSCs) to induce tissue regeneration in periodontal defects in a rat model of periodontitis. BMMSCs isolated from rats were mixed with 0.9% NaCl solution and injected into periodontal defects. Control groups were 0.9% NaCl solution or left untreated. The clinical assessments, x-rays, and histological examinations were used to evaluate the effect. At 12 wks post-transplantation, quantitative analysis revealed average probing bone loss values of 1.2 ± 0.19, 1.6 ± 0.2, and 1.7 ± 0.14; the bone regeneration rate was 53%, 45%, and 44% in the BMMSC+NaCl group, NaCl group, and untreated group, respectively. The clinical assessments, x-rays, and histological examinations revealed significant periodontal tissue regeneration in the BMMSC injection group, compared with the control groups. The ELISA results showed that TNFα, IFNγ, and IL1β were 2,674.88 ± 102.77 pg/mL vs. 3,422.1 ± 51.98 pg/mL, 609.85 ± 25.5 pg/mL vs. 803.79 ± 33.85 pg/mL, and 1,038.46 ± 76.29 pg/mL vs. 1,175.26 ± 105.55 pg/mL in the BMMSC+NaCl group and NaCl group, respectively, indicating that BMMSC injection inhibited the inflammatory factors TNFα, IFNγ, and IL1β. Our results indicate that local administration of BMMSCs can repair defects due to periodontitis, exerting anti-inflammatory and immunomodulatory functions.

Active secretion and protective effect of salivary nitrate against stress in human volunteers and rats.

Up to 25% of the circulating nitrate in blood is actively taken up, concentrated, and secreted into saliva by the salivary glands. Salivary nitrate can be reduced to nitrite by the commensal bacteria in the oral cavity or stomach and then further converted to nitric oxide (NO) in vivo, which may play a role in gastric protection. However, whether salivary nitrate is actively secreted in human beings has not yet been determined. This study was designed to determine whether salivary nitrate is actively secreted in human beings as an acute stress response and what role salivary nitrate plays in stress-induced gastric injury. To observe salivary nitrate function under stress conditions, alteration of salivary nitrate and nitrite was analyzed among 22 healthy volunteers before and after a strong stress activity, jumping down from a platform at the height of 68 m. A series of stress indexes was analyzed to monitor the stress situation. We found that both the concentration and the total amount of nitrate in mixed saliva were significantly increased in the human volunteers immediately after the jump, with an additional increase 1h later (p<0.01). Saliva nitrite reached a maximum immediately after the jump and was maintained 1h later. To study the biological functions of salivary nitrate and nitrite in stress protection, we further carried out a water-immersion-restraint stress (WIRS) assay in male adult rats with bilateral parotid and submandibular duct ligature (BPSDL). Intragastric nitrate, nitrite, and NO; gastric mucosal blood flow; and gastric ulcer index (UI) were monitored and nitrate was administrated in drinking water to compensate for nitrate secretion in BPSDL animals. Significantly decreased levels of intragastric nitrate, nitrite, and NO and gastric mucosal blood flow were measured in BPSDL rats during the WIRS assay compared to sham control rats (p<0.05). Recovery was observed in the BPSDL rats upon nitrate administration. The WIRS-induced UI was significantly higher in the BPSDL animals compared to controls, and nitrate administration rescued the WIRS-induced gastric injury in BPSDL rats. In conclusion, this study suggests that stress promotes salivary nitrate secretion and nitrite formation, which may play important roles in gastric protection against stress-induced injury via the nitrate-dependent NO pathway.

MicroRNAome and Expression Profile of Developing Tooth Germ in Miniature Pigs

MicroRNAs (miRNAs) play important roles in the regulation of rodent tooth development, but little is known about their role in tooth development in large mammals. We identified 637 unique miRNA sequences in a large-scale screen for miRNA expression profiles in the developing lower deciduous molars of miniature pigs (Sus scrofa) using Illumina Solexa deep sequencing. These candidate miRNAs and another 105 known Sus scrofa miRNAs were included in the custom-designed microarray and used to analyze the miRNA expression profile in the bud, cap, early bell, and late bell stages of tooth development. Microarray analysis revealed 166 transcripts that were differentially expressed in the four stages. Bioinformatic analysis identified 18 key miRNAs, including let-7f, miR-128, miR-200b, and miR-200c, that might play key roles in tooth development. Taken together, our results not only identified the specific microRNAome and expression profile in developing lower deciduous molars of the miniature pig, but they also provided useful information for investigating the molecular mechanism of tooth development in the miniature pig.

Mesenchymal stromal cell-based treatment of jaw osteoradionecrosis in Swine.

Jaw osteoradionecrosis (ORN) is a common and serious complication of radiation therapy for head and neck cancers. Bone marrow mesenchymal stromal cells (BMMSCs) are multipotent postnatal stem cells and have been widely used in clinical therapies. In the present study, we generated the mandibular ORN model in swine using a combination of single-dose 25-Gy irradiation and tooth extraction. A typical ORN phenotype, including loss of bone regeneration capacity and collagen collapse with the obliteration of vessels, gradually appeared after irradiation. After autologous BMMSC transplantation, new bone and vessels were regenerated, and the advanced mandibular ORN was treated successfully. In summary, we developed a swine model of jaw ORN, and our results indicate that autologous BMMSC transplantation may be a promising therapeutic approach for ORN.