Waruna Lakmal Dissanayaka

Coculture of Dental Pulp Stem Cells with Endothelial Cells Enhances Osteo-/Odontogenic and Angiogenic Potential In Vitro

INTRODUCTION Dental pulp stem cells (DPSCs) have received much attention as a promising population of stem cells in regenerative endodontics. Securing a good blood supply during regeneration is a challenging task because of the constricted apical canal opening, which allows only a limited blood supply. The aim of this study was to investigate any potential synergistic effects of dental pulp stem cells and endothelial cells (ECs) on osteo-/odontogenic and angiogenic differentiation in vitro. METHODS Different ratios of DPSCs and ECs were cultured in direct contact using optimized medium for coculture. The 70% confluent cocultures were incubated in the osteo-/odontogenic differentiation medium for up to 3 weeks. Alkaline phosphatase (ALP) activity, the expression levels of ALP, bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) genes, and alizarin red staining for mineralization at different time points were analyzed. The tubular network formation on Matrigel and the gene expression levels of CD117, VEGF, CD34, and Flk-1 were used as assays to analyze angiogenesis. RESULTS The quantification of ALP in DPSC:EC cocultures revealed a greater ALP activity compared with DPSC-alone cultures. At all the time points, 1:1 cultures showed a significantly greater ALP activity than that of DPSC-alone cultures. Alizarin red staining and quantification revealed a much greater amount of calcification in the 1:1 and 1:5 cocultures compared with other cultures (P < .01). The expression levels of ALP, BSP, and DSPP genes further confirmed the greater osteo-/odontogenic differentiation in cocultures compared with those of DPSC-alone cultures. Matrigel assay showed that the addition of DPSCs stabilized preexisting vessel-like structures formed by ECs and increased the longevity of them. CONCLUSIONS Direct coculture of DPSCs and ECs enhances the in vitro differentiation toward osteo-/odontogenic and angiogenic phenotypes.

Regeneration of dental pulp tissue in immature teeth with apical periodontitis using platelet-rich plasma and dental pulp cells

AIM To investigate the new tissues growing into the pulp space of immature dog teeth that were infected, disinfected and filled with blood clot (BC), dental pulp cells (DPCs), platelet-rich plasma (PRP) or a combination of DPCs and PRP in immature dog teeth with apical periodontitis. METHODOLOGY Fifty-six immature roots from mandibular premolars of four beagles were divided into four experimental groups (n = 40) and two control groups. After the induction of apical periodontitis, the root canals of experimental groups were disinfected with NaOCl irrigation and a tri-antibiotic paste medication. The canals were then filled with different materials according to the experimental group: BC group, DPCs group, PRP group or DPCs + PRP group. Access cavities were sealed with MTA and composite. Radiographs were taken after 90 days, and the jaws including the teeth were processed for histologic analysis. The data were statistically analysed using chi-square evaluation and Students t-test. RESULTS Radiographic analyses demonstrated no significant difference between experimental groups in periradicular bone healing (P > 0.05), whilst those groups that used DPCs produced a significantly greater root thickening (P < 0.01). The histologic evaluation showed that the groups with PRP formed more tissues in the canals (P = 0.01). The groups with DPCs had substantially more mineralized tissue formation in the canal than those without DPCs, especially in the apical third. In DPCs + PRP group, bone-like tissue grew into the canal space from the periapical tissue. CONCLUSIONS A combination of DPCs + PRP increased vital tissue regeneration within the root canals of immature teeth associated with apical periodontitis.

Transplantation of Dental Pulp Stem Cells and Platelet-rich Plasma for Pulp Regeneration

INTRODUCTION The loss of dental pulp may weaken teeth, rendering them susceptible to reinfection, fracture, and subsequent tooth loss. Therefore, regeneration of pulp is considered an ideal treatment to preserve teeth. The aim of this study was to explore the capacity of dental pulp stem cells (DPSCs) and platelet-rich plasma (PRP) to regenerate dental pulp in canine mature permanent teeth. METHODS Pulpectomy with apical foramen enlarged to a #80 file was performed in 16 upper premolars of 4 beagle dogs. Four experimental groups were randomly established: (1) the blood clot group, (2) the autologous DPSCs group, (3) the PRP group, and (4) the DP + PRP group (a mixture of DPSCs and PRP). Four lower premolars without any further treatment after pulpectomy were used as the control group. All teeth were sealed with mineral trioxide aggregate and composite. Twelve weeks after transplantation, the teeth were subjected to radiographic and histologic examination. RESULTS Twenty-four of 32 experimental root canals gained newly formed tissues. All canals with an introduction of a blood clot showed histologic evidence of vital tissue formation. Cementum-like and periodontal ligament-like tissues along the internal root canal walls were typical structures in most cases. There is no significant difference between groups with or without autologous DPSC transplantation (exact chi-square test, P < .05). CONCLUSIONS New vital tissues can be regenerated in permanent canine teeth after pulpectomy and enlargement of the apical foramen. Histologically, transplantation of DPSCs and/or PRP into root canals showed no enhancement in new tissue formation compared with inducement of a blood clot into the root canals alone.

Characterization of Dental Pulp Stem Cells Isolated from Canine Premolars

INTRODUCTION Dogs are commonly used animal models for regenerative endodontics research. Although several studies have used stem cells isolated from dog teeth to investigate the dentin/pulp regeneration in vivo, less attention has been paid for the characterization of these cells. Therefore, this study aimed to characterize the dental pulp stem cells isolated from dog teeth (cDPSCs) in order to further define the dog as an animal model for regenerative endodontics. METHODS Stem cells were isolated from freshly extracted premolars of 10-month-old Beagles. The isolated cells were investigated for their stem cell properties by analysis of their clonogenic and growth characteristics; expression of mesenchymal stem cell markers; and evaluation of their osteo/odontogenic, adipogenic, and neurogenic potential. RESULTS A colony formation assay showed the existence of a clonogenic cell population in cDPSCs isolated. The growth curves revealed a higher proliferation rate of cDPSCs compared with hBMMSCs. cDPSCs expressed mesenchymal stem cell markers STRO-1, CD146, and Nanog. However, they were negative for CD73, CD105, and CD45. cDPSCs were able to differentiate into odontoblast-like cells as shown by increased alkaline phosphatase activity, dentin sialoprotein expression, and formation of mineralized nodules. cDPSCs also showed the neurogenic and adipogenic differentiation potential at a lower level compared with those of hDPSCs and hBMMSCs. CONCLUSIONS The results of this study confirmed the stem cell properties of cDPSCs at a comparable level to those of hDPSCs and hBMMSCs. Overall, the data presented in this study provide evidence in supportive of using cDPSCs and dogs as an animal model in dental tissue engineering via stem cell-based approaches.

Scaffold-free Prevascularized Microtissue Spheroids for Pulp Regeneration

Creating an optimal microenvironment that mimics the extracellular matrix (ECM) of natural pulp and securing an adequate blood supply for the survival of cell transplants are major hurdles that need to be overcome in dental pulp regeneration. However, many currently available scaffolds fail to mimic essential functions of natural ECM. The present study investigated a novel approach involving the use of scaffold-free microtissue spheroids of dental pulp stem cells (DPSCs) prevascularized by human umbilical vein endothelial cells (HUVECs) in pulp regeneration. In vitro-fabricated microtissue spheroids were inserted into the canal space of tooth-root slices and were implanted subcutaneously into immunodeficient mice. Histological examination revealed that, after four-week implantation, tooth-root slices containing microtissue spheroids resulted in well-vascularized and cellular pulp-like tissues, compared with empty tooth-root slices, which were filled with only subcutaneous fat tissue. Immunohistochemical staining indicated that the tissue found in the tooth-root slices was of human origin, as characterized by the expression of human mitochondria, and contained odontoblast-like cells organized along the dentin, as assessed by immunostaining for nestin and dentin sialoprotein (DSP). Vascular structures formed by HUVECs in vitro were successfully anastomosed with the host vasculature upon transplantation in vivo, as shown by immunostaining for human CD31. Collectively, these findings demonstrate that prevascularized, scaffold-free, microtissue spheroids can successfully regenerate vascular dental pulp-like tissue and also highlight the significance of the microtissue microenvironment as an optimal environment for successful pulp-regeneration strategies.

In Vitro Analysis of Scaffold-free Prevascularized Microtissue Spheroids Containing Human Dental Pulp Cells and Endothelial Cells

INTRODUCTION Scaffolds often fail to mimic essential functions of the physiologic extracellular matrix (ECM) that regulates cell-cell communication in tissue microenvironments. The development of scaffold-free microtissues containing stem cell-derived ECM may serve as a successful alternative to the use of artificial scaffolds. The current study aimed to fabricate 3-dimensional microtissue spheroids of dental pulp cells (DPCs) prevascularized by human umbilical vein endothelial cells (HUVECs) and to characterize these scaffold-free spheroids for the in vitro formation of pulplike tissue constructs. METHODS Three-dimensional microtissue spheroids of DPC alone and DPC-HUVEC co-cultures were fabricated using agarose micro-molds. Cellular organization within the spheroids and cell viability (live/dead assay) were assessed at days 1, 7, and 14. Microtissue spheroids were allowed to self-assemble into macrotissues, induced for odontogenic differentiation (21 days), and examined for expression levels of osteo/odontogenic markers: alkaline phosphatase, bone sialoprotein and RUNX2 (Real-time PCR), mineralization (von-Kossa), and prevascularisation (immunohistochemistry for CD31). RESULTS The DPC microtissue microenvironment supported HUVEC survival and capillary network formation in the absence of a scaffolding material and external angiogenic stimulation. Immunohistochemical staining for CD31 showed the capillary network formed by HUVECs did sustain-for a prolonged period-even after the microtissues transformed into a macrotissue. Induced, prevascularized macrotissues showed enhanced differentiation capacity compared with DPC alone macrotissues, as shown by higher osteo/odontogenic gene expression levels and mineralization. CONCLUSIONS These findings provide insight into the complex intercellular cross talk occurring between DPCs and HUVECs in the context of angiogenesis and pulp regeneration and highlight the significance of developing a favorable 3-dimensional microenvironment that can, in turn, contribute toward successful pulp regeneration strategies.

Prevalence and distribution of developmental enamel defects in children with cerebral palsy in Beijing, China

AIM To investigate the prevalence and distribution of developmental enamel defects in children with cerebral palsy (CP) in Beijing, China. DESIGN A total of 135 children aged 1.5-6 years with moderate or severe congenital CP diagnosed in Beijing Boai Hospital from year 2005 to 2009 were recruited. The children underwent dental examination at the hospital dental clinic. RESULTS Enamel defects (opacity and/or hypoplasia) were found in 44 (32.6%) out of 135 CP children. Enamel hypoplasia was found in 35 (25.9%) of the CP children, opacity alone was found in 5 (3.7%) of the CP children, and mixed defects (opacity and hypoplasia) was found in 4 (3.0%) of the CP children. Most of the enamel defects were located symmetrically in the primary incisors and first molars. 42.4% of children with enamel defects were born prematurely (<37 weeks) where as only 23.2% of them were born at normal gestational age. No statistically significant difference in the prevalence of enamel defects was found in relation to birth weight (P > 0.05). CONCLUSIONS A high prevalence of developmental enamel defects was found among the children with CP. The prevalence of defects varied with the tooth type and was associated with gestational age of the children.

Storage media enhance osteoclastogenic potential of human periodontal ligament cells via RANKL-independent signaling

BACKGROUND Hanks balanced salt solution (HBSS) and milk have gained wide acceptance as storage media for avulsed tooth. However, the effect of the media and storage time on the periodontal ligament (PDL) cells involvement in the development of root resorption is still unclear. The purpose of this study was to evaluate whether precultured PDL cells in HBSS, milk, or modified Eagles medium alpha (α-MEM) would affect osteoclastogenesis. MATERIALS AND METHODS PDL cells were precultured in HBSS, milk, or α-MEM for 1 h or 6 h before being co-cultured with RAW 264.7 cells for an additional 3 days for mRNA analysis and 11 days for osteoclastogenesis assay. RESULTS Cyclooxygenase-2 (COX-2) mRNA was detected immediately in PDL cells precultured in the three storage media. The expression was up-regulated markedly in all co-cultures when compared with RAW cells alone. As a result of the co-culture, interleukin-1β (IL-1β) expression was detectable in both PDL and RAW cells. TRAP+ multinucleated, osteoclast-like cells developed in all co-cultures; the number of TRAP+ cells was highest (P < 0.05) in the co-cultures that PDL cells precultured in milk for 6 h. The mRNA level of receptor activator of nuclear factor-kappa B ligand (RANKL) was not detected in PDL cells. Osteoprotegerin (OPG) mRNA expression reduced with increased preculture time, but the difference was not significant (P > 0.05). CONCLUSIONS PDL cells kept in the three storage media led to TRAP+ multinucleated, osteoclast-like cells formation via RANKL-independent signaling. The ability to induce osteoclastogenesis may be considered as one of the factors to evaluate the ability of storage medium to maintain PDL viability after tooth avulsion.

In vivo periodontal tissue regeneration by periodontal ligament stem cells and endothelial cells in three-dimensional cell sheet constructs

BACKGROUND AND OBJECTIVE Chronic periodontitis causes damage to tooth-supporting tissues, resulting in tooth loss in adults. Recently, cell-sheet-based approaches have been studied to overcome the limitations of conventional cytotherapeutic procedures for periodontal regeneration. The purpose of the present study was to investigate the regenerative potential of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs) in three-dimensional (3D) cell sheet constructs for periodontal regeneration in vivo. MATERIAL AND METHODS PDLSCs, HUVECs or co-cultures of both cells were seeded onto temperature-responsive culture dishes, and intact cell sheets were fabricated. Cell sheets were wrapped around the prepared human roots in three different combinations and implanted subcutaneously into immunodeficient mice. RESULTS Histological evaluation revealed that after 2, 4 and 8 wk of implantation, periodontal ligament-like tissue arrangements were observed around the implanted roots in experimental groups compared with controls. Vascular lumens were also observed in periodontal compartments of HUVEC-containing groups. Periodontal ligament regeneration, cementogenesis and osteogenesis were evident in the experimental groups at both weeks 4 and 8, as shown by immunostaining for periostin and bone sialoprotein. Human cells in the transplanted cell sheets were stained by immunohistochemistry for the presence of human mitochondria. CONCLUSIONS The 3D cell sheet-based approach may be potentially beneficial and is thus encouraged for future regenerative periodontal therapy.

Stimulation of EphB2/ephrin-B1 signalling by tumour necrosis factor alpha in human dental pulp stem cells.

The aim of this study was to investigate whether in vitro stimulation of dental pulp stem cells (DPSCs) by tumour necrosis factor alpha (TNF‐α) would induce secretion of EphB2/ephrin‐B1 signalling.