CYLD inhibits osteoclastogenesis to ameliorate alveolar bone loss in mice with periodontitis.

Periodontitis is a chronic immune inflammatory disease that can lead to the destruction and loss of the tooth-supporting apparatus. During this process, the balance between bone absorption mediated by osteoclasts and bone formation mediated by osteoblasts is damaged. Consistent with previous studies, we observed that depletion of cylindromatosis (CYLD) resulted in an osteoporotic bone phenotype. However, the effect of CYLD deficiency on periodontitis is undetermined. Here, we investigated whether CYLD affects periodontal tissue homeostasis in experimental periodontitis in Cyld knockout (KO) mice, and we explored the underlying mechanisms. Interestingly, we discovered significant alveolar bone density loss and severely reduced alveolar bone height in Cyld KO mice with experimentally induced periodontitis. We observed increased osteoclast number and activity in both the femurs and alveolar bones, accompanied by the downregulation of osteogenesis genes and upregulation of osteoclastogenesis genes of alveolar bones in ligatured Cyld KO mice. Taken together, our findings demonstrate that the deletion of CYLD in mice plays a vital role in the pathogenesis of periodontal bone loss and suggest that CYLD might exert an ameliorative effect on periodontal inflammatory responses.

Cleaning and Disinfecting Oval-Shaped Root Canals: Ex Vivo Evaluation of Three Rotary Instrumentation Systems with Passive Ultrasonic Irrigation.

Background and Objectives: Successful root canal treatment depends on the thorough removal of biofilms through chemomechanical preparation. This study aimed to investigate and compare the cleaning and disinfecting efficiency of oval-shaped root canals using XP-endo Shaper (XPS), ProTaper Next (PTN), and HyFlex CM (HCM) in combination with passive ultrasonic irrigation (PUI). Materials and Methods: Ninety extracted teeth were contaminated and randomly divided into three groups: XPS, PTN, and HCM. Each group was assigned to three subgroups: subgroup A (sterile saline), subgroup B (3% sodium hypochlorite and 17% ethylenediaminetetraacetic acid), and subgroup C (3% sodium hypochlorite, 17% ethylenediaminetetraacetic acid, and PUI). Bacterial sampling was conducted both from baseline samples and samples after chemomechanical preparation. Scanning electron microscopy (SEM) was used to evaluate the residue bacterial biofilms, hard tissue debris, and smear layers on the buccolingual walls of oval-shaped root canals. Results: When combined with sterile saline, XPS demonstrated a higher reduction of bacterial counts and was more effective in eradicating Enterococcus faecalis in the middle third of the canals compared to the other instruments (p < 0.05). Additionally, when used with antimicrobial irrigants, XPS was more effective in disinfecting the coronal third of the canals than the other instruments (p < 0.05). Furthermore, XPS reduced hard tissue debris more effectively in the middle third of canals than in the apical third (p < 0.05). Conclusions: XPS outperforms PTN and HCM in disinfecting oval-shaped root canals. Despite the fact that combining XPS and PUI improves cleaning and disinfecting, removing hard tissue debris from the critical apical area remains challenging.

Downregulation of Prolactin-Induced Protein Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells.

BACKGROUND Periodontal ligament stem cells (PDLSCs) are promising seed cells for bone tissue engineering and periodontal regeneration applications. However, the mechanism underlying the osteogenic differentiation process remains largely unknown. Previous reports showed that prolactin-induced protein (PIP) was upregulated after PDLSCs osteogenic induction. However, few studies have reported on the function of PIP in osteogenic differentiation. The purpose of the present study was to investigate the effect of PIP on osteogenic differentiation of PDLSCs. MATERIAL AND METHODS The expression pattern of PIP during PDLSCs osteogenic differentiation was detected and the effect of each component in the osteogenic induction medium on PIP was also tested by qRT-PCR. Then, the PIP knockdown cells were established using lentivirus. The knockdown efficiency was measured and the proliferation, apoptosis, and osteogenic differentiation ability were examined to determine the functional role of PIP on PDLSCs. RESULTS QRT-PCR showed that PIP was sustainedly upregulated during the osteogenic induction process and the phenomenon was mainly caused by the stimulation of dexamethasone in the induction medium. CCK-8 and flow cytometer showed that knocking down PIP had no influence on proliferation and apoptosis of PDLSCs. ALP staining and activity, Alizarin Red staining, and western blot analysis demonstrated PIP knockdown enhanced the osteogenic differentiation and mineralization of PDLSCs. CONCLUSIONS PIP was upregulated after osteogenic induction; however, PIP knockdown promoted PDLSCs osteogenic differentiation. PIP might be a by-product of osteogenic induction, and downregulating of PIP might be a new target in bone tissue engineering applications.

Epigenetic modifier trichostatin A enhanced osteogenic differentiation of mesenchymal stem cells by inhibiting NF-κB (p65) DNA binding and promoted periodontal repair in rats.

We wished to evaluate whether epigenetic modifiers have a beneficial effect on treating experimental periodontitis and mechanisms for regulating the cell fate of mesenchymal stem cells (MSCs) in inflammatory microenvironments. We isolated MSCs from healthy and inflamed gingival tissues to investigate whether trichostatin A (TSA) could improve osteogenic differentiation and resolve inflammation in vitro. The tissue regenerative potentials were evaluated when treated with a temperature-dependent, chitosan-scaffold-encapsulated TSA, in a rat model of periodontitis. After induction with the conditioned medium, TSA treatment increased the osteogenic differentiation potential of inflamed MSCs and healthy MSCs. In addition, interleukin-6 and interleukin-8 levels in supernatants were significantly decreased after TSA treatment. Moreover, TSA promoted osteogenic differentiation by inhibiting nuclear factor-κB (p65) DNA binding in MSCs. In rats with experimental periodontitis, 7 weeks after local injections of chitosan-scaffold-encapsulated TSA, histology and microcomputed tomography showed a significant increase in alveolar bone volume and less inflammatory infiltration compared with vehicle-treated rats. The concentrations of interferon-γ and interleukin-6 were significantly decreased in the gingival crevicular fluid after TSA treatment. This study demonstrated that TSA had anti-inflammatory properties and could promote periodontal tissue repair, which indicated that epigenetic modifiers hold promise as a potential therapeutic option for periodontal tissue repair.

Demethylation of IGFBP5 by Histone Demethylase KDM6B Promotes Mesenchymal Stem Cell-Mediated Periodontal Tissue Regeneration by Enhancing Osteogenic Differentiation and Anti-Inflammation Potentials.

Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered a promising method for periodontitis treatment. The molecular mechanism underlying directed differentiation and anti-inflammatory actions remains unclear, thus limiting potential MSC application. We previously found that insulin-like growth factor binding protein 5 (IGFBP5) is highly expressed in dental tissue-derived MSCs compared with in non-dental tissue-derived MSCs. IGFBP5 is mainly involved in regulating biological activity of insulin-like growth factors, and its functions in human MSCs and tissue regeneration are unclear. In this study, we performed gain- and loss-of-function assays to test whether IGFBP5 could regulate the osteogenic differentiation and anti-inflammatory potential in MSCs. We found that IGFBP5 expression was upregulated upon osteogenic induction, and that IGFBP5 enhanced osteogenic differentiation in MSCs. We further showed that IGFBP5 prompted the anti-inflammation effect of MSCs via negative regulation of NFκB signaling. Depletion of the histone demethylase lysine (K)-specific demethylase 6B (KDM6B) downregulated IGFBP5 expression by increasing histone K27 methylation in the IGFBP5 promoter. Moreover, IGFBP5 expression in periodontal tissues was downregulated in individuals with periodontitis compared with in healthy people, and IGFBP5 enhanced MSC-mediated periodontal tissue regeneration and alleviated local inflammation in a swine model of periodontitis. In conclusion, our present results reveal a new function for IGFBP5, provide insight into the mechanism underlying the directed differentiation and anti-inflammation capacities of MSCs, and identify a potential target mediator for improving tissue regeneration.

Identification of differential microRNA expression during tooth morphogenesis in the heterodont dentition of miniature pigs, SusScrofa.

BACKGROUND: It has been found that microRNAs (miRNAs) play important roles in the regulation of tooth development, and most likely increase the complexity of the genetic network, thus lead to greater complexity of teeth. But there has been no research about the key microRNAs associated with tooth morphogenesis based on miRNAs expression profiles. Compared to mice, the pig model has plentiful types of teeth, which is similar with the human dental pattern. Therefore, we used miniature pigs as large-animal models to investigate differentially expressed miRNAs expression during tooth morphogenesis in the early developmental stages of tooth germ. RESULTS: A custom-designed miRNA microarray with 742 miRNA gene probes was used to analyze the expression profiles of four types of teeth at three stages of tooth development. Of the 591 detectable miRNA transcripts, 212 miRNAs were continuously expressed in all types of tooth germ, but the numbers of miRNA transcript among the four different types of teeth at each embryonic stage were statistically significant differences (p < 0.01). The hierarchical clustering and principal component analysis results suggest that the miRNA expression was globally altered by types and temporal changes. By clustering analysis, we predicted 11 unique miRNA sequences that belong to mir-103 and mir-107, mir-133a and mir-133b, and mir-127 isomiR families. The results of real-time reverse-transcriptase PCR and in situ hybridization experiments revealed that five representative miRNAs may play important roles during different developmental stages of the incisor, canine, biscuspid, and molar, respectively. CONCLUSIONS: The present study indicated that these five miRNAs, including ssc-miR-103 and ssc-miR-107, ssc-miR-133a and ssc-miR-133b, and ssc-miR-127, may play key regulatory roles in different types of teeth during different stages and thus may play critical roles in tooth morphogenesis during early development in miniature pigs.

Construction of a cDNA library for miniature pig mandibular deciduous molars.

BACKGROUND: The miniature pig provides an excellent experimental model for tooth morphogenesis because its diphyodont and heterodont dentition resembles that of humans. However, little information is available on the process of tooth development or the exact molecular mechanisms controlling tooth development in miniature pigs or humans. Thus, the analysis of gene expression related to each stage of tooth development is very important. RESULTS: In our study, after serial sections were made, the development of the crown of the miniature pigs' mandibular deciduous molar could be divided into five main phases: dental lamina stage (E33-E35), bud stage (E35-E40), cap stage (E40-E50), early bell stage (E50-E60), and late bell stage (E60-E65). Total RNA was isolated from the tooth germ of miniature pig embryos at E35, E45, E50, and E60, and a cDNA library was constructed. Then, we identified cDNA sequences on a large scale screen for cDNA profiles in the developing mandibular deciduous molars (E35, E45, E50, and E60) of miniature pigs using Illumina Solexa deep sequencing. Microarray assay was used to detect the expression of genes. Lastly, through Unigene sequence analysis and cDNA expression pattern analysis at E45 and E60, we found that 12 up-regulated and 15 down-regulated genes during the four periods are highly conserved genes homologous with known Homo sapiens genes. Furthermore, there were 6 down-regulated and 2 up-regulated genes in the miniature pig that were highly homologous to Homo sapiens genes compared with those in the mouse. CONCLUSION: Our results not only identify the specific transcriptome and cDNA profile in developing mandibular deciduous molars of the miniature pig, but also provide useful information for investigating the molecular mechanism of tooth development in the miniature pig.

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.

Periodontal ligament stem cells regulate B lymphocyte function via programmed cell death protein 1.

Periodontal ligament stem cells (PDLSCs) have provided novel cell sources for tooth and periodontal tissue regeneration. Allogeneic PDLSCs can reconstruct periodontal ligament tissue that has been damaged by periodontal diseases and regulate T-cell immunity. However, the effect of PDLSCs on B cells remains unknown. Here, we treated periodontitis in a miniature pig model using allogeneic PDLSCs and showed a reduction in humoral immunity in the animals. When cocultured with normal B cells, human PDLSCs (hPDLSCs) had similar effects as bone marrow mesenchymal stem cells in suppressing B cell proliferation, differentiation, and migration, while intriguingly, hPDLSCs increased B cell viability by secreting interleukin-6. Mechanistically, hPDLSCs suppressed B cell activation through cell-to-cell contact mostly mediated by programmed cell death protein 1 and programmed cell death 1 ligand 1. Our data revealed a previously unrecognized function of PDLSCs in regulating humoral immune responses, which may represent a novel therapeutic strategy for immune-related disorders.

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.

Design and Analysis of a Microgripper with Three-Stage Amplification Mechanism for Micromanipulation.

This paper proposes a novel microgripper with two working modes. The microgripper is designed with symmetric structure and each part is actuated by one piezoelectric actuator, respectively. To achieve desired output displacement, each part of the microgripper is designed with three-stage amplification mechanism to amplify the displacement of the PZT actuator. According to the size of the microobjects, the grasping operation can be completed by one finger moving or two fingers moving simultaneously. Then, the theoretical analysis is carried out to calculated the key characteristics, including amplification, input stiffness and frequency. Finite element analysis (FEA) is conducted to optimize the structural parameters and investigate the performance of the microgripper. Finally, a prototype is machined by wire electro-discharge machining (WEDM) method and experiments are carried out to verify the performance of the microgripper. The results indicate that the amplification is 10.41 and the motion stroke of one jaw is 118.34 µm when the input voltage is 100 V. The first natural frequency is 746.56 Hz. By picking and placing the wires with different diameters and slices with different thickness, the grasping stability is verified.

3D ring artifacts removal algorithm combined low-rank tensor decomposition with spatial-sequential total variation regularization and its application in phase-contrast microtomography.

PURPOSE: High-resolution synchrotron radiation X-ray phase contrast microtomography (PC-µCT) images often suffer from severe ring artifacts, which are mainly caused by undesirable responses of detector elements. In the medical imaging field, the existence of ring artifacts can lead to degraded visual quality and can directly affect diagnosis accuracy. Thus, removing or at least effectively reducing ring artifacts is indispensable. METHOD: The existing ring artifacts removal algorithms mainly focus on two-dimensional (matrix-based) priors, and these algorithms fail to consider correlations hidden in sequential computed tomography (CT) images. This paper proposed a novel three-dimensional (tensor-based) ring artifacts removal algorithm for synchrotron radiation X-ray PC-µCT images. In the sinogram domain, ring artifacts manifest as vertical stripe artifacts. From an image decomposition perspective, a degraded sinogram can be decomposed into a stripe artifacts component and an underlying clean sinogram component. The proposed algorithm is designed to detect and remove stripe artifacts from a degraded sinogram by fully identifying the characteristics of the two components. Specifically, for the stripe artifacts component, tensor Tucker decomposition is used to describe its low-rank character. For the underlying clean sinogram component, spatial-sequential total variation regularization is adopted to enhance the piecewise smoothness. Moreover, the Frobenius norm term is further used to model Gaussian noise. An efficient augmented Lagrange multiplier method is designed to solve the proposed optimization model. RESULTS: The proposed method is evaluated utilizing both simulations and real data containing different ring artifacts patterns. In the simulations, the human chest CT images are used for evaluating the proposed method. We compare the peak signal-to-noise ratio (PSNR), structural similarity (SSIM), and mean absolute error (MAE) results of our algorithm with the Naghia's method, the RRRTV method, the wavelet-FFT method, and the SDRSD-GIF method. The proposed method was also evaluated on real data from rat liver samples and rat tooth samples. Our proposed method outperforms the competing methods in terms of both qualitative and quantitative evaluation results. Additionally, the 3D visualization results were presented to make the ring artifacts removal effect more intuitive. CONCLUSION: The experimental results on simulations and real data clearly demonstrated that the proposed algorithm can significantly improve the quality of PC-µCT images compared with the existing popular algorithms, and it has great potential to promote the application of high-resolution imaging for visualizing biological tissues.

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.

Fabrication of pH-responsive TA-keratin bio-composited hydrogels encapsulated with photoluminescent GO quantum dots for improved bacterial inhibition and healing efficacy in wound care management: In vivo wound evaluations.

Wounds origins serious complications of lives of human beings which may leads to death. The important issue for the problem is infection during wound care management which delays wound healing process. These kinds of infections may be caused by the overuse or misuse of antibiotics, antidotes, usage of new drugs, not properly sterilized surgical instruments, not appropriate for pH level and imperfect wound dressing etc. during or after surgery. Hence in this report, antimicrobial action of pH responsive TA/KA composited hydrogel crosslinked with GO-QDs (TA/KA-GOQDs) using citric acid as cross-linker has been reported by demonstrating in-vitro and in-vivo studies for wound care management. The prepared samples of GOQDs, TA/KA hydrogel and TA/KA-GOQDs were characterized using FT-IR, XRD, SEM and TEM techniques. pH responsive hydrogel property of TA/KA was evaluated by swelling studies. In-vitro antibacterial studies was carried out by direct contact test method. Further, the prepared samples were tested in a wound healing model of rate with the wound of size 1.5 cm2 for in-vivo studies. After 16 days of treatment, the prepared samples for wound healing causes 100% wound areas closure. Histological observations were made by MT and HE staining process which proves keratinocytes proliferation by biocompatible and biocomposited TA/KA-GOQDs. The pH responsive TA/KA-GOQDs proved as efficient wound healing agent by faster keratinocytes proliferation within a compact period.

Azithromycin Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells after Stimulation with TNF-α.

BACKGROUND AND OBJECTIVE: This study investigated the effects and underlying mechanisms of azithromycin (AZM) treatment on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) after their stimulation with TNF-α in vitro. Methods. PDLSCs were isolated from periodontal ligaments from extracted teeth, and MTS assay was used to evaluate whether AZM and TNF-α had toxic effects on PDLSCs viability and proliferation. After stimulating PDLSCs with TNF-α and AZM, we analyzed alkaline phosphatase staining, alkaline phosphatase activity, and alizarin red staining to detect osteogenic differentiation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to detect the mRNA expression of osteogenic-related genes, including RUNX2, OCN, and BSP. Western blotting was used to measure the NF-κB signaling pathway proteins p65, phosphorylated p65, IκB-α, phosphorylated IκB-α, and ß-catenin as well as the apoptosis-related proteins caspase-8 and caspase-3. Annexin V assay was used to detect PDLSCs apoptosis. RESULTS: TNF-α stimulation of PDLSCs decreased alkaline phosphatase and alizarin red staining, alkaline phosphatase activity, and mRNA expression of RUNX2, OCN, and BSP in osteogenic-conditioned medium. AZM enhanced the osteogenic differentiation of PDLSCs that were stimulated with TNF-α. Western blot analysis showed that ß-catenin, phosphorated p65, and phosphorylated IκB-α protein expression decreased in PDLSCs treated with AZM. In addition, pretreatment of PDLSCs with AZM (10 µg/ml, 20 µg/ml) prevented TNF-α-induced apoptosis by decreasing caspase-8 and caspase-3 expression. CONCLUSIONS: Our results showed that AZM promotes PDLSCs osteogenic differentiation in an inflammatory microenvironment by inhibiting the WNT and NF-κB signaling pathways and by suppressing TNF-α-induced apoptosis. This suggests that AZM has potential as a clinical therapeutic for periodontitis.

Depletion of MEIS2 inhibits osteogenic differentiation potential of human dental stem cells.

Dental mesenchymal stem cells (MSCs) are a reliable and promising cell source for the regeneration of tooth,bone and other tissues . However, the molecular mechanisms underlying their differentiation are still largely unknown, which restricts their further wide application. Here, we investigate regulatory function of homeobox gene MEIS2 in the osteogenic differentiation potential of MSCs using stem cells from apical papilla (SCAPs) and dental pulp stem cells (DPSCs) by loss-of-function experiments. Our findings demonstrated that knockdown of MEIS2 in SCAPs and DPSCs decreased alkaline phosphatase (ALP) activity and mineralization, and inhibited the mRNA expression of ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Besides, depletion of MEIS2 resulted in reduced expression of the key osteogenesis-related transcription factor, osterix (OSX) but not in the expression of runt-related transcription factor 2 (RUNX2). Furthermore, MEIS2 expression significantly increased during osteogenic induction and was strongly upregulated by BMP4 stimulation. Taken together, these results indicated that MEIS2 played an essential role in maintaining osteogenic differentiation potential of dental tissue- derived MSCs. These findings will provide new insights into the mechanisms underlying directed differentiation of MSCs, and identify a potential target gene in dental tissues derived MSCs for promoting the tissue regeneration.

Osteoinductive activity of ErhBMP-2 after anterior cervical diskectomy and fusion with a ß-TCP interbody cage in a goat model.

Bone morphogenetic protein (BMP)-2 induces bone and cartilage tissue formation. Large amounts of BMP-2 are difficult to purify or to produce in vitro using eukaryotic cells. The goal of the present study was to assess the clinical use of Escherichia coli-derived recombinant human BMP-2 (ErhBMP-2) on bone fusion after cervical and lumbar spine surgery in a goat model, compared with the standard autogenous iliac bone grafting. Thirty-six goats were randomized to 3 groups: (A) autogenous iliac bone grafting, (B) cervical interbody fusion cage containing ß-tricalcium phosphate (ß-TCP), or (C) cervical interbody fusion cage containing ß-TCP+ErhBMP-2 (2.5 mg). Cervical bone repair was evaluated using radiographs and computed tomography scans at 0, 3, and 6 months. Histological analyses were performed on cervical samples. Two goats died from infection. The differences in intervertebral height among the groups were not significant 3 months postoperatively but became significant after 6 months between groups A vs B and C (P=.04); there was no difference between groups B and C at 6 months. Adding ErhBMP-2 significantly increased cervical fusion at 6 months (P=.04). Histological examinations showed that ß-TCP+ErhBMP-2 increased new bone area, material degradation rate, and depth of tissue penetration and decreased residual material area, all in a time-dependent manner. Escherichia coli-derived rhBMP-2 combined with an enhanced fusion cage containing ß-TCP induced bone formation in a goat model. Furthermore, its ability to promote bone fusion was similar to autogenous iliac bone grafting.

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.

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.

Human dental pulp stem cell is a promising autologous seed cell for bone tissue engineering.

BACKGROUND: The seed cell is a core problem in bone tissue engineering research. Recent research indicates that human dental pulp stem cells (hDPSCs) can differentiate into osteoblasts in vitro, which suggests that they may become a new kind of seed cells for bone tissue engineering. The aim of this study was to evaluate the osteogenic differentiation of hDPSCs in vitro and bone-like tissue formation when transplanted with three-dimensional gelatin scaffolds in vivo, and hDPSCs may become appropriate seed cells for bone tissue engineering. METHODS: We have utilized enzymatic digestion to obtain hDPSCs from dental pulp tissue extracted during orthodontic treatment. After culturing and expansion to three passages, the cells were seeded in 6-well plates or on three-dimensional gelatin scaffolds and cultured in osteogenic medium. After 14 days in culture, the three-dimensional gelatin scaffolds were implanted subcutaneously in nude mice for 4 weeks. In 6-well plate culture, osteogenesis was assessed by alkaline phosphatase staining, Von Kossa staining, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the osteogenesis-specific genes type I collagen (COL I), bone sialoprotein (BSP), osteocalcin (OCN), RUNX2, and osterix (OSX). In three-dimensional gelatin scaffold culture, X-rays, hematoxylin/eosin staining, and immunohistochemical staining were used to examine bone formation. RESULTS: In vitro studies revealed that hDPSCs do possess osteogenic differentiation potential. In vivo studies revealed that hDPSCs seeded on gelatin scaffolds can form bone structures in heterotopic sites of nude mice. CONCLUSIONS: These findings suggested that hDPSCs may be valuable as seed cells for bone tissue engineering. As a special stem cell source, hDPSCs may blaze a new path for bone tissue engineering.