Ben A. Scheven

Dentine as a bioactive extracellular matrix

As a mineralised connective tissue, dentine is well adapted to its functional role as a major structural component of the tooth. Although similar in composition to bone, dentine matrix is not remodelled physiologically and traditionally, has been regarded as a rather inert tissue. Nevertheless, dentine-pulp demonstrates strong regenerative potential which allows it to respond to disease and traumatic injury. Such responses are strongly influenced by cell-matrix interactions and modified by disease processes, including infection and inflammation. The identification of many bioactive molecules bound within dentine matrix has allowed their potential involvement in regenerative and other tissue responses to be better understood and new opportunities to be recognised for novel clinical therapies.

Intravitreally Transplanted Dental Pulp Stem Cells Promote Neuroprotection and Axon Regeneration of Retinal Ganglion Cells After Optic Nerve Injury

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.

Paracrine-Mediated Neuroprotection and Neuritogenesis of Axotomised Retinal Ganglion Cells by Human Dental Pulp Stem Cells: Comparison with Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells

We have investigated and compared the neurotrophic activity of human dental pulp stem cells (hDPSC), human bone marrow-derived mesenchymal stem cells (hBMSC) and human adipose-derived stem cells (hAMSC) on axotomised adult rat retinal ganglion cells (RGC) in vitro in order to evaluate their therapeutic potential for neurodegenerative conditions of RGC. Using the transwell system, RGC survival and length/number of neurites were quantified in coculture with stem cells in the presence or absence of specific Fc-receptor inhibitors to determine the role of NGF, BDNF, NT-3, VEGF, GDNF, PDGF-AA and PDGF-AB/BB in stem cell-mediated RGC neuroprotection and neuritogenesis. Conditioned media, collected from cultured hDPSC/hBMSC/hAMSC, were assayed for the secreted growth factors detailed above using ELISA. PCR array determined the hDPSC, hBMSC and hAMSC expression of genes encoding 84 growth factors and receptors. The results demonstrated that hDPSC promoted significantly more neuroprotection and neuritogenesis of axotomised RGC than either hBMSC or hAMSC, an effect that was neutralized after the addition of specific Fc-receptor inhibitors. hDPSC secreted greater levels of various growth factors including NGF, BDNF and VEGF compared with hBMSC/hAMSC. The PCR array confirmed these findings and identified VGF as a novel potentially therapeutic hDPSC-derived neurotrophic factor (NTF) with significant RGC neuroprotective properties after coculture with axotomised RGC. In conclusion, hDPSC promoted significant multi-factorial paracrine-mediated RGC survival and neurite outgrowth and may be considered a potent and advantageous cell therapy for retinal nerve repair.

Stem cell treatment of degenerative eye disease

Stem cell therapies are being explored extensively as treatments for degenerative eye disease, either for replacing lost neurons, restoring neural circuits or, based on more recent evidence, as paracrine-mediated therapies in which stem cell-derived trophic factors protect compromised endogenous retinal neurons from death and induce the growth of new connections. Retinal progenitor phenotypes induced from embryonic stem cells/induced pluripotent stem cells (ESCs/iPSCs) and endogenous retinal stem cells may replace lost photoreceptors and retinal pigment epithelial (RPE) cells and restore vision in the diseased eye, whereas treatment of injured retinal ganglion cells (RGCs) has so far been reliant on mesenchymal stem cells (MSC). Here, we review the properties of non-retinal-derived adult stem cells, in particular neural stem cells (NSCs), MSC derived from bone marrow (BMSC), adipose tissues (ADSC) and dental pulp (DPSC), together with ESC/iPSC and discuss and compare their potential advantages as therapies designed to provide trophic support, repair and replacement of retinal neurons, RPE and glia in degenerative retinal diseases. We conclude that ESCs/iPSCs have the potential to replace lost retinal cells, whereas MSC may be a useful source of paracrine factors that protect RGC and stimulate regeneration of their axons in the optic nerve in degenerate eye disease. NSC may have potential as both a source of replacement cells and also as mediators of paracrine treatment.

VEGF and odontoblast-like cells: Stimulation by low frequency ultrasound

OBJECTIVE Vascular endothelial growth factor (VEGF) has been implicated in the regulation of dental pulp and dentine repair. Therapeutic ultrasound was shown to be effective for fracture repair. We investigated whether low frequency ultrasound influences the production of VEGF by odontoblast-like cells. Moreover, we examined the direct effects of VEGF on odontoblast-like cell proliferation. DESIGN MDPC-23, an established odontoblast-like cell line, was exposed to increasing intensities of 30kHz ultrasound using an ultrasonic tip probe. RESULTS After 24h cell culture, WST-1 analysis of cell viability and number showed a dose-dependent decrease in the number of viable cells with increasing ultrasound power. However, the relative concentration of VEGF as analysed by ELISA and normalised to cell number was significantly increased in the culture supernatants indicating an ultrasound-induced stimulation of odontoblastic VEGF secretion. Analysis of VEGF gene expression by sqRT-PCR revealed the expression of the main VEGF isoforms in the MDPC-23 cells, i.e. VEGF(120) and VEGF(164) as well as to a minor extent VEGF(188). Low power ultrasound increased gene expression of all VEGF isoforms. Addition of recombinant VEGF to the cell cultures significantly stimulated cell proliferation. Gene expression of the VEGF receptors Flt1/VEGFR1 and KDR/VEGFR2 was detected in the MDPC-23, suggesting the possibility that VEGF may act on the odontoblast-like cells in an autocrine manner. CONCLUSIONS Our results indicate that ultrasound promoted VEGF expression and production by odontoblast-like cells and that VEGF may have autocrine effects on these cells. It is proposed that ultrasound may influence odontoblast activity and dentine repair by modulating production of endogenous growth factors in the dentine-pulp complex.

A comparison of the in vitro mineralisation and dentinogenic potential of mesenchymal stem cells derived from adipose tissue, bone marrow and dental pulp

Stem-cell-based therapies provide a biological basis for the regeneration of mineralised tissues. Stem cells isolated from adipose tissue (ADSCs), bone marrow (BMSCs) and dental pulp (DPSCs) have the capacity to form mineralised tissue. However, studies comparing the capacity of ADSCs with BMSCs and DPSCs for mineralised tissue engineering are lacking, and their ability to regenerate dental tissues has not been fully explored. Characterisation of the cells using fluorescence-activated cell sorting and semi-quantitative reverse transcription PCR for MSC markers indicated that they were immunophenotypically similar. Alizarin red (AR) staining and micro-computed tomography (µCT) analyses demonstrated that the osteogenic potential of DPSCs was significantly greater than that of BMSCs and ADSCs. Scanning electron microscopy and AR staining showed that the pattern of mineralisation in DPSC cultures differed from ADSCs and BMSCs, with DPSC cultures lacking defined mineralised nodules and instead forming a diffuse layer of low-density mineral. Dentine matrix components (DMCs) were used to promote dentinogenic differentiation. Their addition to cultures resulted in increased amounts of mineral deposited in all three cultures and significantly increased the density of mineral deposited in BMSC cultures, as determined by µCT analysis. Addition of DMCs also increased the relative gene expression levels of the dentinogenic markers dentine sialophosphoprotein and dentine matrix protein 1 in ADSC and BMSC cultures. In conclusion, DPSCs show the greatest potential to produce a comparatively high volume of mineralised matrix; however, both dentinogenesis and mineral volume was enhanced in ADSC and BMSC cultures by DMCs, suggesting that these cells show promise for regenerative dental therapies.

The effects of cryopreservation on cells isolated from adipose, bone marrow and dental pulp tissues☆

The effects of cryopreservation on mesenchymal stem cell (MSC) phenotype are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation (10% dimethyl-sulfoxide) on the morphology, viability, gene-expression and relative proportion of MSC surface-markers on cells derived from rat adipose, bone marrow and dental pulp. Cryopreservation significantly reduced the number of viable cells in bone marrow and dental pulp cell populations but had no observable effect on adipose cells. Flow cytometry analysis demonstrated significant increases in the relative expression of MSC surface-markers, CD90 and CD29/CD90 following cryopreservation. sqRT-PCR analysis of MSC gene-expression demonstrated increases in pluripotent markers for adipose and dental pulp, together with significant tissue-specific increases in CD44, CD73-CD105 following cryopreservation. Cells isolated from different tissue sources did not respond equally to cryopreservation with adipose tissue representing a more robust source of MSCs.

Low-intensity Low-frequency Ultrasound Promotes Proliferation and Differentiation of Odontoblast-like Cells

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.

Effects of Glial Cell Line-derived Neurotrophic Factor on Dental Pulp Cells

This study investigated the effects of glial cell line-derived neurotrophic factor (GDNF) on dental pulp cells (DPCs). Cultures of DPCs expressed GDNF as well as its receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium did not significantly affect DPC growth; however, GDNF dose-dependently increased viable cell number under serum-free culture conditions. Live/dead, lactate dehydrogenase (LDH), and caspases-3/-7 assays demonstrated that cell death occurred under serum-free conditions, and that GDNF significantly reduced the number of dead cells by inhibiting apoptotic cell death. GDNF also stimulated cell proliferation in serum-free conditions, as assessed by the BrdU incorporation assay. The effect of GDNF was abolished in the presence of inhibitors to GFRα1 and RET suggesting receptor-mediated events. This study also demonstrated that GDNF counteracted TNFα-induced DPC cytotoxicity, suggesting that GDNF may be cytoprotective under disease conditions. In conclusion, our findings indicate that GDNF promotes cell survival and proliferation of DPCs and suggest that GDNF may play a multifunctional role in the regulation of dental pulp homeostasis.

Mesenchymal stromal cell-mediated neuroprotection and functional preservation of retinal ganglion cells in a rodent model of glaucoma.

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.