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

The DNA demethylating agent 5-aza-2′-deoxycytidine (DAC, decitabine) has anti-cancer therapeutic potential, but its clinical efficacy is hindered by DNA damage-related side effects and its use in solid tumours is debated. Here we describe how paracetamol augments the effects of DAC on cancer cell proliferation and differentiation, without enhancing DNA damage. Firstly, DAC specifically upregulates cyclooxygenase-2-prostaglandin E2 pathway, inadvertently providing cancer cells with survival potential, while the addition of paracetamol offsets this effect. Secondly, in the presence of paracetamol, DAC treatment leads to glutathione depletion and finally to accumulation of ROS and/or mitochondrial superoxide, both of which have the potential to restrict tumour growth. The benefits of combined treatment are demonstrated here in head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukaemia cell lines, further corroborated in a HNSCC xenograft mouse model and through mining of publicly available DAC and paracetamol responses. The sensitizing effect of paracetamol supplementation is specific to DAC but not its analogue 5-azacitidine. In summary, the addition of paracetamol could allow for DAC dose reduction, widening its clinical usability and providing a strong rationale for consideration in cancer therapy., Highlights • Paracetamol works in synergy with DAC (decitabine) to reduce cancer cell viability. • The synergistic effect is specific for decitabine and not observed for 5-azacitidine. • Paracetamol allows for DAC dose reduction without inducing DNA damage. • Paracetamol counteracts DAC-triggered activation of COX-2-PGE2 pathway. • In the presence of paracetamol DAC induces mimicry of paracetamol overdose leading to oxidative stress.

Introduction The biomedical sciences (BMS) are a central part of the dental curriculum that underpins teaching and clinical practice in all areas of dentistry. Although some specialist groups have proposed curricula in their particular topic areas, there is currently no over-arching view of what should be included in a BMS curriculum for undergraduate dental programmes. To address this, the Association for Dental Education in Europe (ADEE) convened a Special Interest Group (SIG) with representatives from across Europe to develop a consensus BMS curriculum for dental programmes. Curriculum This paper summarises the outcome of the deliberations of this SIG, and details a consensus view from the SIG of what a BMS curriculum should include. Conclusions Given the broad nature of BMS applied to dentistry, this curriculum framework is advisory and seeks to provide programme planners with an indicative list of topics which can be mapped to specific learning objectives within their own curricula. As dentistry becomes increasingly specialised these will change, or some elements of the undergraduate curriculum may move to the postgraduate setting. So, this document should be seen as a beginning and it will need regular review as BMS curricula in dentistry evolve.

The DNA demethylating agent 5-aza-2’-deoxycytidine (DAC, decitabine) has anti-cancer therapeutic potential, but its clinical efficacy is hindered by DNA damage-related side effects. Here we describe how paracetamol augments the effects of DAC on cancer cell proliferation and differentiation, without enhancing DNA damage. Firstly, DAC specifically upregulates cyclooxygenase-2-prostaglandin E2 pathway, inadvertently increasing cancer cell survival, while the addition of paracetamol offsets this effect. Secondly, combined treatment leads to glutathione depletion and ROS accumulation with oxidative stress further enhanced by DAC suppressing anti-oxidant and thioredoxin responses. The benefits of combined treatment are demonstrated here in head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukaemia cell lines, further corroborated in a HNSCC xenograft mouse model and through mining of publicly available DAC and paracetamol responses. In summary, the addition of paracetamol could allow for DAC dose reduction, widening its clinical usability and providing a strong rationale for consideration in cancer therapy.

The dental pulp is a highly innervated connective tissue, rich in sensory nerves and supplemented with autonomic innervation and other neural elements, including glial and neural progenitor cells. It is therefore not surprising that application of almost all physiological stimuli to the human pulp results in the sensation of pain. Pulpal innervation also functions beyond pain sensation to modulate physiological functions such as blood flow regulation, inflammation and regeneration. In this chapter we will describe in detail aspects of pulpal neurogenesis and innervation and explain the neuronal basis for inflammation, healing and repair. The mechanisms of pain transduction and nociception at both molecular and cellular levels will be described, with particular emphasis on ion channels and their modulation during injury and inflammation; this will enable a better understanding of inflammatory and neuropathic tooth pain mechanisms and aid accurate diagnosis and treatment. Dental pulp stem cells, their neurogenic potential and application in neuronal repair and regeneration (in the dental pulp and beyond), will also be discussed with reference to emerging research and developments in this area.

BACKGROUND AIMS: Glaucoma is a leading cause of irreversible blindness involving loss of retinal ganglion cells (RGC). Mesenchymal stromal cells (MSC) have shown promise as a paracrine-mediated therapy for compromised neurons. It is, however, unknown whether dental pulp stem cells (DPSC) are effective as a cellular therapy in glaucoma and how their hypothesized influence compares with other more widely researched MSC sources. The present study aimed to compare the efficacy of adipose-derived stem cells, bone marrow-derived MSC (BMSC) and DPSC in preventing the loss of RGC and visual function when transplanted into the vitreous of glaucomatous rodent eyes. METHODS: Thirty-five days after raised intraocular pressure (IOP) and intravitreal stem cell transplantation, Brn3a(+) RGC numbers, retinal nerve fibre layer thickness (RNFL) and RGC function were evaluated by immunohistochemistry, optical coherence tomography and electroretinography, respectively. RESULTS: Control glaucomatous eyes that were sham-treated with heat-killed DPSC had a significant loss of RGC numbers, RNFL thickness and function compared with intact eyes. BMSC and, to a greater extent, DPSC provided significant protection from RGC loss and RNFL thinning and preserved RGC function. DISCUSSION: The study supports the use of DPSC as a neuroprotective cellular therapy in retinal degenerative disease such as glaucoma.

Retinal ganglion cells (RGCs) are responsible for propagating signals derived from visual stimuli in the eye to the brain, along their axons within the optic nerve to the superior colliculus, lateral geniculate nucleus and visual cortex of the brain. Damage to the optic nerve either through trauma, such as head injury, or degenerative disease, such as glaucoma causes irreversible loss of function through degeneration of non-regenerating RGC axons and death of irreplaceable RGCs, ultimately leading to blindness (Berry et al., 2008). The degeneration of RGCs and their axons is due to the loss of the necessary source of retrogradely transported neurotrophic factors (NTFs) being hindered by axonal injury. NTFs are survival factors for neurons and play a pivotal part in axon regeneration. Stem cells particularly mesenchymal stem cells (MSCs) have been shown to possess a natural intrinsic capacity for paracrine support, releasing multiple signalling molecules including NTFs. By transplanting MSCs into the vitreous, they are positioned adjacent to the injured retina to provide paracrine-mediated therapy for the retinal neuronal cells (Johnson et al., 2010a; Mead et al., 2013). Additionally, MSCs may be pre-differentiated into supportive glial-like cells, such as Schwann cells, which could further increase their potential for paracrine support of injured neurons (Martens et al., 2013). Thus, MSCs have received considerable attention as a new cellular therapy for both traumatic and degenerative eye disease, acting as an alternative source of NTFs, protecting injured RGCs and promoting regeneration of their axons

One of the principal aims of cell-based therapies is to deliver personalised medicine for the repair and regeneration of tissues lost to accidents or disease. To achieve this aim sub-zero temperatures (−196°) are applied that halt biological activity, thus preserving the cells for future clinical applications. The idea of banking stem cells as a means of ‘biological insurance’ has seen a recent rise in popularity that is at least in part due to increased media attention and a greater public awareness of regenerative medicine. Consequently, several companies now exist offering individuals the opportunity to store their own multipotent cells, with the aim of future therapeutic application to restore or regenerate a multitude of tissues throughout the body.

Introduction Ultrasound is a potential therapeutic tool for dental tissue repair, but its biological effects on odontoblasts have not been well characterized. In this study, the effects of low-intensity low-frequency ultrasound on the viability, proliferation, and differentiation of odontoblast-like cells were investigated. Methods Cell viability and proliferation were assessed after the treatment of adherent clonal MDPC-23 odontoblast-like cells with a 25-mW/cm 2 45-kHz ultrasound. An in vitro scratch wound healing assay was used to investigate the ultrasound effects on cell migration. Long-term cultures were used to study odontogenic differentiation and extracellular mineralization. Results Ultrasound exposure for up to 30 minutes did not significantly affect odontoblast-like cell viability but significantly increased cell numbers after 2 days in culture. Ultrasound did not influence the scratch wound closure rate in the absence or presence of the mitogen inhibitor mitomycin C, indicating that ultrasound did not influence cellular migration. Single and consecutive exposures to ultrasound resulted in the enhancement of in vitro mineralization after 14 days in culture with an osteogenic differentiation medium. This coincided with the up-regulation of gene expression of collagen type I, osteoadherin, dentine matrix protein 1, and osteocalcin as well as the expression of cell markers alkaline phosphatase and nestin. Conclusions These findings indicate that low-frequency ultrasound is able to influence proliferation and differentiation of odontoblast-like cells and may potentially be considered as a therapeutic tool for dental pulp and dentine repair.

Objectives: This study investigated the perception that dental students have regarding the relevance of oral biology (OB) to dental education and dentistry in general. Moreover, this study analysed students’ attitude towards OB learning approaches and resources. Methods: A questionnaire based on a Likert scale was used to survey pre-clinical/second (BDS2)- and final/fifth (BDS5)-year dental students at the School of Dentistry of the University of Birmingham (United Kingdom). In comparison, a small group of postgraduate specialist registrars were surveyed to evaluate the attitudes of practising dentists. Results: The results show that all study groups expressed a high level of perceived relevance of OB to dentistry. Students’ perception of OB for dental education, clinical training and practice also scored high. More than 40% of undergraduate students and about 55% of the postgraduates indicated a perceived change in their attitude towards OB with time characterised by increased appreciation of the subject. Lectures were considered as the most important teaching approach, whereas ‘group poster projects’ ranked lowest. Of the different study resources, lecture handouts received the overall highest importance score. Conclusions: The results indicate that dental students considered OB relevant for dental education and dentistry and suggest a positive attitude towards the subject. This study also suggested that dental students prefer teacher-centred/led teaching rather than student-directed learning of OB. The article addresses the role of OB and science-related research projects within the dental curriculum and discusses that close integration of basic sciences with dental education may enrich dental education and overall learning experience.

The functional loss that occurs after retinal/optic nerve injury is permanent and can arise through trauma or neurodegenerative conditions such as glaucoma. Neurotrophic factors (NTFs) promote survival of injured retinal ganglion cells (RGCs) and regeneration of their axons, suggesting their clinical utility to prevent further damage and restore lost function. Delivery of optimal concentrations of NTFs to RGCs is difficult to achieve by injection but single implants of stem cells which naturally secrete multiple NTFs for sustained periods better addresses this problem. This review discusses a relatively new source of adult stem cells, the dental pulp stem cells, and compares their efficacy and feasibility with other stem cells, such as the well-studied bone marrow-derived mesenchymal stem cells (BMSCs), in the context of cellular therapy for the retina.

Background Low-intensity ultrasound is considered an effective non-invasive therapy to stimulate hard tissue repair, in particular to accelerate delayed non-union bone fracture healing. More recently, ultrasound has been proposed as a therapeutic tool to repair and regenerate dental tissues. Our recent work suggested that low-frequency kilohertz-range ultrasound is able to interact with dental pulp cells which could have potential to stimulate dentine reparative processes and hence promote the viability and longevity of teeth. Methods In this study, the biophysical characteristics of low-frequency ultrasound transmission through teeth towards the dental pulp were explored. We conducted cell culture studies using an odontoblast-like/dental pulp cell line, MDPC-23. Half of the samples underwent ultrasound exposure while the other half underwent ‘sham treatment’ where the transducer was submerged into the medium but no ultrasound was generated. Ultrasound was applied directly to the cell cultures using a therapeutic ultrasound device at a frequency of 45 kHz with intensity settings of 10, 25 and 75 mW/cm2 for 5 min. Following ultrasound treatment, the odontoblast-like cells were detached from the culture using a 0.25% Trypsin/EDTA solution, and viable cell numbers were counted. Two-dimensional tooth models based on μ-CT 2D images of the teeth were analyzed using COMSOL as the finite element analysis platform. This was used to confirm experimental results and to demonstrate the potential theory that with the correct combination of frequency and intensity, a tooth can be repaired using small doses of ultrasound. Frequencies in the 30 kHz–1 MHz range were analyzed. For each frequency, pressure/intensity plots provided information on how the intensity changes at each point throughout the propagation path. Spatial peak temporal average (SPTA) intensity was calculated and related to existing optimal spatial average temporal average (SATA) intensity deemed effective for cell proliferation during tooth repair. Results The results demonstrate that odontoblast MDPC-23 cell numbers were significantly increased following three consecutive ultrasound treatments over a 7-day culture period as compared with sham controls underscoring the anabolic effects of ultrasound on these cells. Data show a distinct increase in cell number compared to the sham data after ultrasound treatment for intensities of 10 and 25 mW/cm2 (p < 0.05 and p < 0.01, respectively). Using finite element analysis, we demonstrated that ultrasound does indeed propagate through the mineralized layers of the teeth and into the pulp chamber where it forms a ‘therapeutic’ force field to interact with the living dental pulp cells. This allowed us to observe the pressure/intensity of the wave as it propagates throughout the tooth. A selection of time-dependent snapshots of the pressure/intensity reveal that the lower frequency waves propagate to the pulp and remain within the chamber for a while, which is ideal for cell excitation. Input frequencies and pressures of 30 kHz (70 Pa) and 45 kHz (31 kPa), respectively, with an average SPTA of up to 120 mW/cm2 in the pulp seem to be optimal and agree with the SATA intensities reported experimentally. Conclusions Our data suggest that ultrasound can be harnessed to propagate to the dental pulp region where it can interact with the living cells to promote dentine repair. Further research is required to analyze the precise physical and biological interactions of low-frequency ultrasound with the dental pulp to develop a novel non-invasive tool for dental tissue regeneration.

SUMMARY Conflicting data have been published on whether low-dose methotrexate (MTX) treatment of rheumatoid arthritis (RA) is able to slow down radiological joint damage, i.e. retard the destruction of articular cartilage and (subchondral) bone. We studied the effects of MTX on proteoglycan (PG) turnover and interleukin-1 (IL-1)- and RA mononuclear cell (RA-MNC)-mduced cartilage damage in human articular cartilage tissue cultures, and the effects of MTX on basal and RA-MNC-influenced proliferation and differentiation of osteoblasts in cultures of human bone-derived osteoblasts. MTX exerted no direct effect on cartilage nor did MTX influence IL-1- or RA-MNC-induce d cartilage damage, despite strong suppression of basal as well as mitogen- and antigen-induced RA-MNC proliferation. MTX induced strong inhibition of osteoblast proliferation, but did not significantly interfere with osteoblast differentiation (i.e. alkaline phosphatase activity). RA-MNC-enhanced proliferation and differentiation of osteoblasts were abolished by MTX. These results suggest that if MTX is able to induce retardation of radiological progression in RA, this is not based on an initial direct effect of MTX on cartilage as measured by PG turnover, nor on an initial inhibition of IL-1- or RA-MNC-induce d cartilage damage. However, longstanding MTX-induced inhibition of RA-MNC proliferation may lead to reduction of the catabolic activity involved in cartilage destruction. On the other hand, long-term inhibition of osteoblast proliferation may eventually lead to decreased bone formation and osteopenia. Whether this will turn out to be a problem of clinical importance in the treatment of RA has to be established.

This study investigated the effects of different frequencies of low intensity ultrasound on osteoblast migration using an in vitro scratch-wound healing assay. Mouse calvarial-derived MC3T3-E1 osteoblasts in culture were exposed to continuous 45 kHz ultrasound (25 mW/cm(2)) or pulsed 1 MHz ultrasound (250 mW/cm(2)) for 30 min followed by 2 days' culture. Ultrasound treatment with either kHz or MHz output similarly and significantly increased cell numbers after 2 days in culture compared with untreated control cultures. In the scratch-wound healing assay the presence of the cell proliferation inhibitor mitomycin C (MMC) did not influence scratch-wound closure in control cultures indicating that cell migration was responsible for the in vitro wound healing. Application of ultrasound significantly stimulated wound closure. MMC did not affect kHz-stimulated in vitro wound healing; however, MMC reduced in part the scratch-wound closure rate in MHz-treated cultures suggesting that enhanced cell proliferation as well as migration was involved in the healing promoted by MHz ultrasound. In conclusion, both continuous kHz and pulsed MHz ultrasound promoted osteoblastic migration; however, subtle differences were apparent in the manner the different ultrasound regimens enhanced in vitro scratch-wound healing.

Dental disease affects human health and the quality of life of millions worldwide. Tooth decay (caries) and diseases of the dental pulp result in loss of tooth vitality and function requiring invasive treatment to restore the tooth to health. "Therapeutic" low intensity pulsed ultrasound has been shown to accelerate bone fracture healing indicating that ultrasound may be used as a tool to facilitate hard tissue regeneration. We have shown recently that low frequency ultrasound is able to exert biological effects on odontoblast-like cells. In this paper, we postulate that low frequency, low intensity ultrasound may stimulate endogenous coronal tooth repair by stimulating dentine formation from existing odontoblasts or by activating dental pulp stem cells to differentiate into new reparative dentine-producing cells. Ultrasound therapy promoting dentine formation and repair may also have the potential benefit of alleviating dentine hypersensitivity by inducing occlusion of dentinal tubules. It is envisaged that therapeutic ultrasound may be used in future to facilitate dental tissue engineering and stem cell therapy applications for dental tissue regeneration. Further research is warranted in this clinically important area and we envisage that novel strategies in dental therapy will be realised that may ultimately lead to the development of novel non-invasive, multifunctional ultrasound devices for dental diagnostics, repair and regeneration.

Summary For many years articular cartilage has been the focus of research aimed at improving understanding of and treatment for osteoarthritis. Although much is known about the tissue, research has had little success in elucidating the pathogenesis of generalised osteoarthritis. A new hypothesis is required. Substantial changes in many tissues, including bone, muscle, ligaments, and joint capsule, as well as cartilage, are increasingly recognised in this disease, and not all these changes are localised to the affected joints. There is also a well established link with obesity. These observations, the common origins of the mesenchymal cells that maintain these tissues, and the possible role of neuroendocrine factors that can regulate bone mass, result in the hypothesis that systemic factors that include altered lipid metabolism could explain the diversity of physiological changes in generalised osteoarthritis. If proven, this hypothesis could have important implications for a new approach to pharmacological intervention in the early stages of the disease.

Purpose.: To investigate the potential therapeutic benefit of intravitreally implanted dental pulp stem cells (DPSCs) on axotomized adult rat retinal ganglion cells (RGCs) using in vitro and in vivo neural injury models. Methods.: Conditioned media collected from cultured rat DPSCs and bone marrow–derived mesenchymal stem cells (BMSCs) were assayed for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) secretion using ELISA. DPSCs or BMSCs were cocultured with retinal cells, with or without Fc-TrK inhibitors, in a Transwell system, and the number of surviving βIII-tubulin+ retinal cells and length/number of βIII-tubulin+ neurites were quantified. For the in vivo study, DPSCs or BMSCs were transplanted into the vitreous body of the eye after a surgically induced optic nerve crush injury. At 7, 14, and 21 days postlesion (dpl), optical coherence tomography (OCT) was used to measure the retinal nerve fiber layer thickness as a measure of axonal atrophy. At 21 dpl, numbers of Brn-3a+ RGCs in parasagittal retinal sections and growth-associated protein-43+ axons in longitudinal optic nerve sections were quantified as measures of RGC survival and axon regeneration, respectively. Results.: Both DPSCs and BMSCs secreted NGF, BDNF, and NT-3, with DPSCs secreting significantly higher titers of NGF and BDNF than BMSCs. DPSCs, and to a lesser extent BMSCs, promoted statistically significant survival and neuritogenesis/axogenesis of βIII-tubulin+ retinal cells in vitro and in vivo where the effects were abolished after TrK receptor blockade. Conclusions.: Intravitreal transplants of DPSCs promoted significant neurotrophin-mediated RGC survival and axon regeneration after optic nerve injury.

Background The development of ultrasound for use in dental tissues is hampered by the complex, multilayered nature of the teeth. The purpose of this preliminary study was to obtain the phase and group velocities associated with several directions of ultrasonic wave propagation in relation to the tooth structure, which would then lead to the determination of the elastic constants in dental hard tissue. Knowledge of these elastic constants can be used to feed back into numerical models (such as finite element) in order to simulate/predict ultrasonic wave propagation and behavior in the teeth. This will help to optimize ultrasonic protocols as potential noninvasive therapeutic tools for novel dental regenerative therapies. Methods An extracted human second molar was used to determine time-of-flight information from A-scan signatures obtained at various angles of inclination and rotation using a scanning acoustic microscope at 10 MHz. Phase and group velocities and associated slowness curves were calculated in order to determine the independent elastic constants in the human teeth. Results Results show that as the tooth was inclined at three azimuthal angles (Θin = 0°, 15°, and 30°) and rotated from Φin = 0° to 360° in order to cover the whole perimeter of the tooth, slowness curves constructed from the computed phase and group velocities versus angle of rotation confirm the inhomogeneous and anisotropic nature of the tooth as indicated by the nonuniform appearance of uneven circular shape patterns of the measurements when compared to those produced in a control isotropic fused quartz sample. Conclusions This study demonstrates that phase and group velocities of ultrasound as determined by acoustic microscopy change and are dependent on the direction of the tooth structure. Thus, these results confirm that the tooth is indeed a multilayered anisotropic structure underscoring that there is no single elastic constant sufficient to represent the complex structure of the tooth. The findings underline the importance to take into account these crucial characteristics in order to develop and optimize therapeutic as well as diagnostic applications of ultrasound in dental tissue repair, and further studies are warranted to analyze ultrasound transmission at various frequencies and intensities in different teeth to develop appropriate models for ultrasound biophysical behavior in dental tissues.