Proposing new standards for testing solubility of pulp preservation materials.

OBJECTIVES: Quality control testing of dental materials requires a standard to enable the generation of reproducible and comparable data. Currently there are no standards for testing materials used for vital pulp therapy. The aim of this study was to develop a new standard to evaluate solubility of pulp preservation materials. METHODS: The solubility of three materials used for vital pulp therapy: Biodentine, TheraCal and Activa was evaluated using two international standards for dental materials ISO 4049:2019 (S1) and ISO 6876:2012 (S2). For both standards, a modified methodology was evaluated. This included changing the volume of the solution used (S1M, S2M), using Dulbecco's modified eagle medium (DMEM) as an alternative to water (S1D, S2D) and periodic solution change for the ISO 4049 method (S1P, S1MP). Materials were characterised before and after completion of solubility test using scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis. RESULTS: The test materials exhibited different solubility values depending on the methodology used. Biodentine exhibited significantly lower solubility when lower volumes of solution were used when tested using both ISO methods (p ≤ 0.05). TheraCal and Activa showed negative solubility values after desiccation when tested using ISO 4049:2019. The Biodentine exhibited changes in its microstructure which was dependent on the method used to test solubility. CONCLUSIONS: The solubility values obtained were dependent on the method used. It is thus important to use methods that replicate the clinical environment for meaningful evaluations.

Differing responses of osteogenic cell lines to ß-glycerophosphate.

Ascorbic acid (Asc), dexamethasone (Dex) and ß-glycerophosphate (ß-Gly) are commonly used to promote osteogenic behaviour by osteoblasts in vitro. According to the literature, several osteosarcoma cells lines appear to respond differently to the latter with regards to proliferation kinetics and osteogenic gene transcription. Unsurprisingly, these differences lead to contrasting data between publications that necessitate preliminary studies to confirm the phenotype of the chosen osteosarcoma cell line in the presence of Asc, Dex and ß-Gly. The present study exposed Saos-2 cells to different combinations of Asc, Dex and ß-Gly for 14 days and compared the response with immortalised human mesenchymal stromal/stem cells (MSCs). Cell numbers, cytotoxicity, mineralised matrix deposition and cell proliferation were analysed to assess osteoblast-like behaviour in the presence of Asc, Dex and ß-Gly. Additionally, gene expression of runt-related transcription factor 2 (RUNX2); osteocalcin (OCN); alkaline phosphatase (ALP); phosphate regulating endopeptidase homolog X-linked (PHEX); marker of proliferation MKI67 and proliferating cell nuclear antigen (PCNA) was performed every two days during the 14-day cultures. It was found that proliferation of Saos-2 cells was significantly decreased by the presence of ß-Gly which contrasted with hMSCs where no change was observed. Furthermore, unlike hMSCs, Saos-2 cells demonstrated an upregulated expression of late osteoblastic markers, OCN and PHEX that suggested ß-Gly could affect later stages of osteogenic differentiation. In summary, it is important to consider that ß-Gly significantly affects key cell processes of Saos-2 when using it as an osteoblast-like cell model.

Exploring the neurogenic differentiation of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) have increasingly gained interest as a potential therapy for nerve regeneration in medicine and dentistry, however their neurogenic potential remains a matter of debate. This study aimed to characterize hDPSC neuronal differentiation in comparison with the human SH-SY5Y neuronal stem cell differentiation model. Both hDPSCs and SH-SY5Y could be differentiated to generate typical neuronal-like cells following sequential treatment with all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF), as evidenced by significant expression of neuronal proteins ßIII-tubulin (TUBB3) and neurofilament medium (NF-M). Both cell types also expressed multiple neural gene markers including growth-associated protein 43 (GAP43), enolase 2/neuron-specific enolase (ENO2/NSE), synapsin I (SYN1), nestin (NES), and peripherin (PRPH), and exhibited measurable voltage-activated Na+ and K+ currents. In hDPSCs, upregulation of acetylcholinesterase (ACHE), choline O-acetyltransferase (CHAT), sodium channel alpha subunit 9 (SCN9A), POU class 4 homeobox 1 (POU4F1/BRN3A) along with a downregulation of motor neuron and pancreas homeobox 1 (MNX1) indicated that differentiation was more guided toward a cholinergic sensory neuronal lineage. Furthermore, the Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 significantly impaired hDPSC neuronal differentiation and was associated with reduction of the ERK1/2 phosphorylation. In conclusion, this study demonstrates that extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) is necessary for sensory cholinergic neuronal differentiation of hDPSCs. hDPSC-derived cholinergic sensory neuronal-like cells represent a novel model and potential source for neuronal regeneration therapies.

Traditional Multiwell Plates and Petri Dishes Limit the Evaluation of the Effects of Ultrasound on Cells In Vitro.

Ultrasound accelerates healing in fractured bone; however, the mechanisms responsible are poorly understood. Experimental setups and ultrasound exposures vary or are not adequately characterized across studies, resulting in inter-study variation and difficulty in concluding biological effects. This study investigated experimental variability introduced through the cell culture platform used. Continuous wave ultrasound (45 kHz; 10, 25 or 75 mW/cm2, 5 min/d) was applied, using a Duoson device, to Saos-2 cells seeded in multiwell plates or Petri dishes. Pressure field and vibration quantification and finite-element modelling suggested formation of complex interference patterns, resulting in localized displacement and velocity gradients, more pronounced in multiwell plates. Cell experiments revealed lower metabolic activities in both culture platforms at higher ultrasound intensities and absence of mineralization in certain regions of multiwell plates but not in Petri dishes. Thus, the same transducer produced variable results in different cell culture platforms. Analysis on Petri dishes further revealed that higher intensities reduced vinculin expression and distorted cell morphology, while causing mitochondrial and endoplasmic reticulum damage and accumulation of cells in sub-G1 phase, leading to cell death. More defined experimental setups and reproducible ultrasound exposure systems are required to study the real effect of ultrasound on cells for development of effective ultrasound-based therapies not just limited to bone repair and regeneration.

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells.

Dental pulp stem cells (DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium (DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor (NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2 (MAP-2) and cytoskeletal marker ßIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and ßIII-tubulin were analysed by quantitative polymerase chain reaction (qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors (NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and ßIII-tubulin immunostaining. Furthermore, DPSCs-CM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls; however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

The anti-tumour activity of DNA methylation inhibitor 5-aza-2'-deoxycytidine is enhanced by the common analgesic paracetamol through induction of oxidative stress.

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.

Neurotrophic effects of dental pulp stem cells on trigeminal neuronal cells.

Evidence indicates that dental pulp stem cells (DPSC) secrete neurotrophic factors which play an important role in neurogenesis, neural maintenance and repair. In this study we investigated the trophic potential of DPSC-derived conditioned medium (CM) to protect and regenerate isolated primary trigeminal ganglion neuronal cells (TGNC). DPSC and TGNC were harvested by enzymatic digestion from Wister-Hann rats. CM was collected from 72 h serum-free DPSC cultures and neurotrophic factors; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF) were analysed by specific enzyme-linked immunosorbent assays (ELISAs). Primary co-cultures of DPSC and TGNC were established to evaluate the paracrine effects of DPSC. In comparison, NGF was used to evaluate its neurotrophic and neuritogenic effect on TGNC. Immunocytochemistry was performed to detect the neuronal-markers; neuronal nuclei (NeuN), microtubule-associated protein-2 (MAP-2) and ßIII-tubulin. Quantitative real time polymerase chain reaction (qRT-PCR) was used to analyse neuronal-associated gene expression of NeuN, MAP-2, ßIII-tubulin in addition to growth-associated protein-43 (GAP-43), Synapsin-I and thermo-sensitive transient receptor potential vanilloid channel-1 (TRPV1). DPSC-CM contained significant levels of NGF, BDNF, NT-3 and GDNF. DPSC and DPSC-CM significantly enhanced TGNC survival with extensive neurite outgrowth and branching as evaluated by immunocytochemistry of neuronal markers. DPSC-CM was more effective in stimulating TGNC survival than co-cultures or NGF treated culture. In comparison to controls, DPSC-CM significantly upregulated gene expression of several neuronal markers as well as TRPV1. This study demonstrated that DPSC-derived factors promoted survival and regeneration of isolated TGNC and may be considered as cell-free therapy for TG nerve repair.

A core curriculum in the biological and biomedical sciences for dentistry.

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 overarching 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 post-graduate setting. So, this document should be seen as a beginning and it will need regular review as BMS curricula in dentistry evolve.

Effect of Dentin Matrix Components on the Mineralization of Human Mesenchymal Stromal Cells.

IMPACT STATEMENT: This research has been conducted with the aim to contribute to the development of treatment modalities for the reconstruction of lost/damaged mineralized tissues. Currently, determining the most appropriate stromal cell population and signaling cues stands at the core of developing effective treatments. We provide new insights into the effect of innate inductive cues found in human dentin matrix components, on the osteogenic differentiation of various human stromal cell types. The effects of dentin extracellular matrix components on umbilical cord mesenchymal stromal cells have not been investigated before. The findings of this study could underpin translational research based on the development of techniques for mineralized tissue engineering and will be of great interest for the readership of Tissue Engineering Part A.

Dental Pulp Stem Cell Mechanoresponsiveness: Effects of Mechanical Stimuli on Dental Pulp Stem Cell Behavior.

Dental pulp is known to be an accessible and important source of multipotent mesenchymal progenitor cells termed dental pulp stem cells (DPSCs). DPSCs can differentiate into odontoblast-like cells and maintain pulp homeostasis by the formation of new dentin which protects the underlying pulp. DPSCs similar to other mesenchymal stem cells (MSCs) reside in a niche, a complex microenvironment consisting of an extracellular matrix, other local cell types and biochemical stimuli that influence the decision between stem cell (SC) self-renewal and differentiation. In addition to biochemical factors, mechanical factors are increasingly recognized as key regulators in DPSC behavior and function. Thus, microenvironments can significantly influence the role and differentiation of DPSCs through a combination of factors which are biochemical, biomechanical and biophysical in nature. Under in vitro conditions, it has been shown that DPSCs are sensitive to different types of force, such as uniaxial mechanical stretch, cyclic tensile strain, pulsating fluid flow, low-intensity pulsed ultrasound as well as being responsive to biomechanical cues presented in the form of micro- and nano-scale surface topographies. To understand how DPSCs sense and respond to the mechanics of their microenvironments, it is essential to determine how these cells convert mechanical and physical stimuli into function, including lineage specification. This review therefore covers some aspects of DPSC mechanoresponsivity with an emphasis on the factors that influence their behavior. An in-depth understanding of the physical environment that influence DPSC fate is necessary to improve the outcome of their therapeutic application for tissue regeneration.

Role of Piezo Channels in Ultrasound-stimulated Dental Stem Cells.

INTRODUCTION: Piezo1 and Piezo2 are mechanosensitive membrane ion channels. We hypothesized that Piezo proteins may play a role in transducing ultrasound-associated mechanical signals and activate downstream mitogen-activated protein kinase (MAPK) signaling processes in dental cells. In this study, the expression and role of Piezo channels were investigated in dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) after treatment with low-intensity pulsed ultrasound (LIPUS). METHODS: Cell proliferation was evaluated by bromodeoxyuridine incorporation. Western blots were used to analyze the proliferating cell nuclear antigen as well as the transcription factors c-fos and c-jun. Enzyme-linked immunosorbent assay and Western blotting were used to determine the activation of MAPK after LIPUS treatment. Ruthenium red (RR), a Piezo ion channel blocker, was applied to determine the functional role of Piezo proteins in LIPUS-stimulated cell proliferation and MAPK signaling. RESULTS: Western blotting showed the presence of Piezo1 and Piezo2 in both dental cell types. LIPUS treatment significantly increased the level of the Piezo proteins in DPSCs after 24 hours; however, no significant effects were observed in PDLSCs. Treatment with RR significantly inhibited LIPUS-stimulated DPSC proliferation but not PDLSC proliferation. Extracellular signal-related kinase (ERK) 1/2 MAPK was consistently activated in DPSCs over a 24-hour time period after LIPUS exposure, whereas phosphorylated c-Jun N-terminal kinase and p38 mitogen-activated protein kinase MAPK were mainly increased in PDLSCs. RR affected MAPK signaling in both dental cell types with its most prominent effects on ERK1/2/MAPK phosphorylation levels; the significant inhibition of LIPUS-induced stimulation of ERK1/2 activation in DPSCs by RR suggests that stimulation of DPSC proliferation by LIPUS involves Piezo-mediated regulation of ERK1/2 MAPK signaling. CONCLUSIONS: This study for the first time supports the role of Piezo ion channels in transducing the LIPUS response in dental stem cells.

Concise Review: Dental Pulp Stem Cells: A Novel Cell Therapy for Retinal and Central Nervous System Repair.

Dental pulp stem cells (DPSC) are neural crest-derived ecto-mesenchymal stem cells that can relatively easily and non-invasively be isolated from the dental pulp of extracted postnatal and adult teeth. Accumulating evidence suggests that DPSC have great promise as a cellular therapy for central nervous system (CNS) and retinal injury and disease. The mode of action by which DPSC confer therapeutic benefit may comprise multiple pathways, in particular, paracrine-mediated processes which involve a wide array of secreted trophic factors and is increasingly regarded as the principal predominant mechanism. In this concise review, we present the current evidence for the use of DPSC to repair CNS damage, including recent findings on retinal ganglion cell neuroprotection and regeneration in optic nerve injury and glaucoma. Stem Cells 2017;35:61-67.

Ultrasound Stimulation of Different Dental Stem Cell Populations: Role of Mitogen-activated Protein Kinase Signaling.

INTRODUCTION: Mesenchymal stem cells (MSCs) from dental tissues may respond to low-intensity pulsed ultrasound (LIPUS) treatment, potentially providing a therapeutic approach to promoting dental tissue regeneration. This work aimed to compare LIPUS effects on the proliferation and MAPK signaling in MSCs from rodent dental pulp stem cells (DPSCs) compared with MSCs from periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs). METHODS: Isolated MSCs were treated with 1-MHz LIPUS at an intensity of 250 or 750 mW/cm2 for 5 or 20 minutes. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) staining after 24 hours of culture following a single LIPUS treatment. Specific ELISAs were used to determine the total and activated p38, ERK1/2, and JNK MAPK signaling proteins up to 4 hours after treatment. Selective MAPK inhibitors PD98059 (ERK1/2), SB203580 (p38), and SP600125 (JNK) were used to determine the role of activation of the particular MAPK pathways. RESULTS: The proliferation of all MSC types was significantly increased after LIPUS treatment. LIPUS at a 750-mW/cm2 dose induced the greatest effects on DPSCs. BMSC proliferation was stimulated in equal measures by both intensities, whereas 250 mW/cm2 LIPUS exposure exerted maximum effects on PDLSCs. ERK1/2 was activated immediately in DPSCs after treatment. Concomitantly, DPSC proliferation was specifically modulated by ERK1/2 inhibition, whereas p38 and JNK inhibition exerted no effects. In BMSCs, JNK MAPK signaling was LIPUS activated, and the increase in proliferation was blocked by specific inhibition of the JNK pathway. In PDLSCs, JNK MAPK signaling was activated immediately after LIPUS, whereas p-p38 MAPK increased significantly in these cells 4 hours after exposure. Correspondingly, JNK and p38 inhibition modulated LIPUS-stimulated PDLSC proliferation. CONCLUSIONS: LIPUS promoted MSC proliferation in an intensity and cell-specific dependent manner via activation of distinct MAPK pathways.

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.

Isolation of adipose and bone marrow mesenchymal stem cells using CD29 and CD90 modifies their capacity for osteogenic and adipogenic differentiation.

Mesenchymal stem cells isolated from rats are frequently used for tissue engineering research. However, considerable differences have been identified between rat mesenchymal stem cells and those derived from humans, and no defined panel of markers currently exists for the isolation of these cells. The aim of this study was to examine the effects of cell sorting for CD29(+)/CD90(+) cells from rat adipose and bone marrow tissues on their differentiation and expression of stem cell-associated genes. Flow cytometry showed 66% and 78% CD29(+)/CD90(+) positivity within passage 1 of adipose and bone marrow cultures, respectively. CD29(+)/CD90(+) cells showed a reduction in both osteogenic and adipogenic differentiation when compared with unsorted cells, as determined by alizarin red and Oil Red-O staining, respectively. These findings could not entirely be explained by fluorescence-activated cell sorting-induced cell injury as sort recovery was only modestly affected in adipose-derived cells. Maintaining cells in fluorescence-activated cell sorting buffer did not affect adipose-derived cell viability, but a significant (p < 0.05) reduction was found in bone marrow-derived cell viability. Additionally, CD29(+)/CD90(+) selection was associated with a significant decrease in the expression of Lin28, Sox2, Nanog and CD73 in adipose-derived cell cultures, whereas differences in stem cell-associated gene expression were not observed in sorted bone marrow-derived cell cultures. In summary, this study demonstrated that fluorescence-activated cell sorting had differential effects on adipose-derived cells and bone marrow-derived cells, and both CD29(+)/CD90(+) cells displayed a significantly reduced capacity for osteogenic/adipogenic differentiation. In conclusion, we identify that maintaining heterogeneity within the mesenchymal stem cell population may be important for optimal differentiation.

Ultrasound field characterization and bioeffects in multiwell culture plates.

BACKGROUND: Ultrasound with frequencies in the kilohertz range has been demonstrated to promote biological effects and has been suggested as a non-invasive tool for tissue healing and repair. However, many challenges exist to characterize and develop kilohertz ultrasound for therapy. In particular there is a limited evidence-based guidance and standard procedure in the literature concerning the methodology of exposing biological cells to ultrasound in vitro. METHODS: This study characterized a 45-kHz low-frequency ultrasound at three different preset intensity levels (10, 25, and 75 mW/cm(2)) and compared this with the thermal and biological effects seen in a 6-well culture setup using murine odontoblast-like cells (MDPC-23). Ultrasound was produced from a commercially available ultrasound-therapy system, and measurements were recorded using a needle hydrophone in a water tank. The transducer was displaced horizontally and vertically from the hydrophone to plot the lateral spread of ultrasound energy. Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer. During ultrasound treatment, cell cultures were directly exposed to ultrasound by submerging the ultrasound transducer into the culture media. Four groups of cell culture samples were treated with ultrasound. Three with ultrasound at an intensity level of 10, 25, and 75 mW/cm(2), respectively, and the final group underwent a sham treatment with no ultrasound. Cell proliferation and viability were analyzed from each group 8 days after three ultrasound treatments, each separated by 48 h. RESULTS: The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells. Significant thermal variations were not detected in adjacent untreated wells. CONCLUSIONS: This study highlights the pitfalls of using multiwell plates when investigating the biological effect of kilohertz low-frequency ultrasound on adherent cell cultures.

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.

Comparative evaluation of methods for estimating retinal ganglion cell loss in retinal sections and wholemounts.

To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a(+), ßIII-tubulin(+) and Islet-1(+) RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG(+) and Brn3a(+) RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas ßIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of ßIII-tubulin(+) RGCs was greater than Brn3a+ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.