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Regeneration of dentin and preserving pulp vitality are essential targets for vital pulp therapy. Our study aimed to evaluate a novel biomimetic pulp capping agent with increased dentin regenerative activities. To produce demineralised dentin matrix (DDM) particles, human extracted teeth were ground and treated with ethylene diamine tetra-acetic acid solution. DDM particles were added to sodium alginate and this combination was dripped into a 5% calcium chloride to obtain DDM hydrogel (DDMH). The eluants of both DDMH and mineral trioxide aggregate (MTA) were tested using an MTT assay to detect their cytotoxic effect on dental pulp stem cells (DPSC). Collagen-I (COL-I) gene expression was analysed on DPSC exposed to different dilutions of pulp capping material eluants by real-time quantitative polymerase chain reaction. Acridine orange staining was used to monitor the cell growth over the tested materials. Agar diffusion assay was utilised to test the antibacterial effect of DDMH and MTA compared to controls. MTT assay revealed that neat eluates of DDMH promoted DPSC viability. However, neat eluates of MTA were cytotoxic on DPSC after 72 h of culture. Moreover, DPSC were capable of growth and attached to the surface of DDMH, while they showed a marked reduction in their number when cultured on the MTA surface for one week, as shown by the acridine orange stain. In DPSC cultured with DDMH eluates, the COL-I gene was overexpressed compared to those cultured with MTA eluants. DDMH had significant antimicrobial activity in comparison to MTA after 24 h incubation. This in vitro study showed that DDMH could be an alternative pulp capping agent for regenerative endodontics., (© 2024. The Author(s).)

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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ascorbic acid (Asc), dexamethasone (Dex) and β-glycerophosphate (β-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 β-Gly. The present study exposed Saos-2 cells to different combinations of Asc, Dex and β-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 β-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 β-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 β-Gly could affect later stages of osteogenic differentiation. In summary, it is important to consider that β-Gly significantly affects key cell processes of Saos-2 when using it as an osteoblast-like cell model., (© 2023. Springer Nature Limited.)

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 βIII-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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Al-Maswary et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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/cm 2 , 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., Competing Interests: Conflict of interest disclosure The authors report no potential competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: None

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 βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-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 βIII-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., Competing Interests: None

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 E 2 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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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 βIII-tubulin. Quantitative real time polymerase chain reaction (qRT-PCR) was used to analyse neuronal-associated gene expression of NeuN, MAP-2, βIII-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.

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.

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., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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., (© 2016 AlphaMed Press.)

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., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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.

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.

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.

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., (Copyright © 2015. Published by Elsevier B.V.)

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(+), βIII-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 βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-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.

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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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: 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/cm(2) 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/cm(2) (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/cm(2) 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.

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.

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.

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., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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., (© 2011 John Wiley & Sons A/S.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (AlP) activity showing a significant dose-dependent inhibition of relative AlP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated that GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFRα1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

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.

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.

In this study, the effects of low frequency ultrasound (US) were examined on odontoblasts, the primary cell responsible for dentinogenesis and dentine repair. An established odontoblast-like cell line, MDPC-23, was subjected to 30 kHz ultrasound at three different power settings. US induced a marginal level of cell death (3% to 4%) at lower amplitudes rising to 25% cell death at the highest power tested. The latter was reflected in a 30% decrease in cell attachment after 4 to 24 h of culture, while the number of adherent cells was reduced by approximately 10% to 15% in the lower power groups. Cell replication after 24 h, as measured by BrdU incorporation, showed no significant changes in the US-treated groups. Gene expression analyses demonstrated a moderate dose-dependent increase in the expression of GAPDH (glyseraldehyde-3-phosphate dehydrogenase)-normalised collagen type I, osteopontin (OPN), transforming growth factor-beta1 (TGFbeta1) and the heat shock protein (hsp) 70. The greatest change was found in the expression of the small hsp 25/27, which showed a two- to six-fold increase following US treatment. No significant effects were observed for alkaline phosphatase (ALP) and core-binding factor A1 (CBFA1/Runx2) expression levels. This is the first report describing US effects on odontoblasts. Further studies are warranted to elucidate US effects on odontoblast function and to evaluate US as a therapeutic application in dentine repair.

This investigation studied how the behaviour of isolated osteoblasts on standard tissue culture polystyrene compared with cells cultured on cut surfaces of dentin, a natural calcified material. Cellular attachment, viability and growth were monitored in parallel cultures of human osteosarcoma cell lines (MG63, HOS TE85, SaOS-2) and primary human osteoblast-like cells (HOBs). Culture plastic was either left untreated or roughened with abrasive paper of various grit sizes (4000-1200 grit) in order to obtain a level of roughness comparable to that of the dentin slices. Cell counting and intracellular BCECF staining showed that after an initial incubation of 2 h, the primary cells attached and spread out more quickly on the different substrates than the three cell lines. The primary cells also showed a stronger mitochondrial staining and viability on dentin. During subsequent culture morphological differences appeared with the cells on dentin displaying more cellular extensions. All three cell lines proliferated more slowly on dentin than on plastic. In contrast, the primary HOBs were not significantly affected in their growth by the different substrates. Total and specific alkaline phosphatase (AP) activity of the cell lines was not significantly affected by the different substrates after short-term adhesion, but it was increased for the primary cells on the dentin. However, after 2-3 days of culture, AP was decreased on the dentin slices for both the cell lines and primary HOBs. Plasma treatment of the roughened plastic did not alter cellular viability or AP activity, suggesting that grinding of the surface did not affect the property of the culture plastic to support cell attachment and growth. In conclusion, the results show that not only do osteoblastic cells behave differently on a natural calcified substrate surface than on standard culture plastic, but also that differences were evident between the various cell types, in particular the primary HOB versus the continuous cell lines., (Copyright 2002 Elsevier Science Ltd. All rights reserved.)

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.

A validated in vitro system was used to investigate the nature of osteoclast-inducing growth factors (OGF) present in fetal rat calvarial conditioned medium (RCCM). Evidence is presented here that macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, is an essential factor for the induction of osteoclast differentiation in this system. Specific polyclonal antibodies against MIP-1alpha significantly inhibited development of TRAP-positive osteoclast precursors and multinucleated osteoclasts induced by RCCM. Anti-MIP-1alpha antibody treatment was accompanied by an increase in the number of macrophage-like cells, suggesting that bone-derived MIP-1alpha is involved in the direction of preosteoclast formation with an inhibitory action on progenitor cell proliferation. Reverse-phase HPLC of RCCM resolved multiple fractions with OGF activity. OGF fractions separated at low acetonitrile (AcN) concentrations (

A "sequential culture step" system was devised to study osteoclast differentiation from newborn porcine bone marrow cells. Nonadherent cells were collected from cultures of bone marrow cells, and subsequently precultured at a low cell density in low-serum medium supplemented with L929-conditioned medium (L9-CM) derived M-CSF/CSF-1. After 4 d, adherent cells mainly composed of M-CSF-dependent macrophage/osteoclast progenitors, but devoid of stromal-like cells, were further cultured in medium supplemented with L9-CM and CM derived from serum-free cultures of fetal rat calvarial bones. This phase was characterized by a rapid induction of mono- and multinucleated (pre)osteoclast-like cells, positive for cytochemical TRAP activity, but negative for nonspecific esterase (NSE) staining. The presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] stimulated osteoclast generation, whereas calcitonin treatment significantly inhibited this process. The osteoclastic nature of the cells was confirmed by the occurrence of extensive, characteristic bone resorption on dentin slices, which was associated with release of type I collagen N-telopeptides from the bone matrix into the culture medium. The presence of a DNA synthesis inhibitor (HU) during the first 3 d of culture completely inhibited osteoclast formation, whereas HU treatment during the last phase did not affect production of multinucleated osteoclast-like cells. Likewise, a specific antibody directed against M-CSF during the first preculture period, completely abolished osteoclast formation. Adding the antibody during the last phase of the culture, however, strongly inhibited multinucleated osteoclast formation, accompanied by a significant increase in a mononuclear TRAP-positive, NSE-positive (osteoclast precursor) cell fraction. These results indicate that M-CSF is essential for progenitor proliferation as well as for (pre)osteoclast maturation and/ or fusion into multinucleated cells, but also suggest that additional soluble (bone-derived) factors are involved as cofactors in the differentiation process to committed mononuclear osteoclast precursors. The porcine marrow culture approach provides a suitable model system to investigate specific soluble osteoclast-inducing factors affecting different stages of osteoclast development.

DHEA, an adrenocortical steroid, and its sulfate derivative (DHEA-S), have been implicated in many biological functions, including the regulation of bone mass. In this study, we examined whether DHEA/DHEA-S are capable of directly affecting bone cell proliferation and differentiation, and compared this with the effects of, and interaction with, the established bone cell modulating steroid, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Two in vitro models of human osteoblastic cells were used, viz. MG63 osteosarcoma cell line and normal primary osteoblast-like cells (HOB). Our results show that DHEA and DHEA-S failed on their own to exert direct, independent significant effects on the growth and differentiation of human osteoblastic cells, but treating the cells in conjunction with 1,25(OH)2D3 resulted in enhancement of specific A1P activity. Moreover, 1,25(OH)2D3-induced osteocalcin production was potentiated by the adrenal steroids in both cell models. DHEA-S proved in general to be more potent than DHEA. In conclusion, this study shows that the effects of DHEA/DHEA-S on osteoblastic cell growth and differentiation are likely to be mediated via an effect on 1,25(OH)2D3-induced changes in bone cells, suggesting a distinctive role for these steroids in the regulation of bone metabolism.

A novel osteoclast generation and bone resorption assay system is described in which enhanced osteoclastic generation and bone resorption is induced in porcine bone marrow cell cultures cultured in low-serum medium supplemented with fibroblastic cell (L929) conditioned medium (CM). Numerous osteoclasts, which could be identified by TRAP staining and specific labelling with 121F antibody, were generated in a time-dependent and L929-CM concentration-dependent fashion. A specific antibody against murine M-CSF/CSF-1 abolished osteoclast formation indicating that M-CSF is the essential component of the L929-CM driven osteoclast generation. Culturing on devitalized bone slices resulted in extensive osteoclast-mediated resorption as visualized microscopically. After 16 days in culture, practically the entire bone slice surface was excavated by the osteoclastic cells. Bone resorption could be monitored with time using a novel enzyme-linked immunoassay measuring type I collagen N-telopeptides in culture supernatants. Release of collagen fragments from the slices was paralleled by osteoclastic secretion of TRAP. Salmon calcitonin significantly inhibited collagen fragment and TRAP release. 1,25-Dihydroxyvitamin D3 greatly promoted osteoclast generation and subsequent bone resorption, but its presence was not essential for this process to occur.

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)-induced 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-induced 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-induced 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 was designed to investigate whether methotrexate (MTX), used in the treatment of rheumatoid arthritis (RA), affects proliferation and differentiation of human osteoblasts in culture. The effects of MTX were assessed by analyzing markers of proliferation and differentiation of human trabecular bone-derived osteoblast-like cells cultured in the presence or absence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). Treatment of the osteoblastic cells with MTX resulted in a strong dose-dependent inhibition of cell proliferation with half maximal response at a dose of 30 nM. MTX did not interfere with cellular alkaline phosphatase (AP) activity, the number of cells expressing cytochemical AP, or basal osteocalcin production. Addition of 1,25(OH)2D3 to the cultures caused an enhanced AP expression and osteocalcin production coinciding with a decreased osteoblast proliferation. Coincubation of 1,25(OH)2D3 with MTX in doses > or = 100 nM further inhibited osteoblast growth and induced a significant stimulation of AP expression and activity, and production of osteocalcin above the values reached in the 1,25(OH)2D3 cultures. In conclusion, MTX proved to be a potent inhibitor of osteoblast proliferation but did not affect basal osteoblastic phenotypic expression. In the presence of the osteoblast differentiation-promoter, 1,25(OH)2D3, MTX further inhibited cell growth which was associated with enhanced AP activity and osteocalcin production. Thus, MTX may have profound effects on bone metabolism and remodeling by interfering with bone cell turnover.

Endogenous growth factors may be involved in the prevention of bone loss by estrogen and progestins in postmenopausal women. The present study was performed to compare the action of estrogen/progestins on bone-derived cells with the effects of exogenously added purified growth factors. Human osteoblast-like (HOB) cells were incubated with 17 beta-estradiol (E2), progesterone (P), dydrogesterone (DD), 20 alpha-dihydroxydydrogesterone (DHD), with and without the growth factors, insulin-like growth factors-I/-II (IGF-I/-II) or transforming growth factor-beta type 1 (TGF-beta 1) for 24 h under serum-free conditions. Cell growth and DNA synthesis were assessed by spectophotometrical analysis of total cell number and immunochemical detection of BrdU incorporation, respectively. Compared with the sex steroids, incubation of the cells with IGF-I or TGF-beta 1 resulted in at least a two-fold increase of total HOB cell numbers. No difference in stimulating HOB growth was observed between IGF-II and the female sex steroids E2 and P. Combining IGF-I/-II or TGF-beta 1 with either E2 or P did not result in a significantly further increase in the human osteoblast-like cell growth. In conclusion, the bone anabolic growth factors, IGF-I and TGF-beta 1, may be more important regulators of osteoblast proliferation than the female sex steroids. An interaction of estrogen/gestagens with the growth factors IGF-I/-II or TGF-beta 1 was not evident from the growth of human bone-forming cells in short-term cultures.

Estrogen/gestagen replacement therapy prevents excess bone loss in postmenopausal women. The mode of action by which these sex steroids exert their anabolic effects on bone has not been completely clarified yet. In this study, 17 beta-estradiol (E2), as well as progestins progesterone (P), dydrogesterone (DD), 20 alpha-dihydroxydydrogesterone (DHD), medroxyprogesterone acetate (MPA), and cyproterone acetate (CPA) were able to stimulate the mitogenesis and differentiation of normal adult human osteoblast-like (HOB) cells harvested from female trabecular bone explants. The different progestins exerted a more pronounced stimulatory effect on HOB proliferation than E2 did. The combination of E2 with P, DD, or DHD did not result in a statistically significant further increase of HOB proliferation, as compared with the progestins alone. In general, E2 showed a stronger differentiation-inducing effect than the progestins, as measured by histochemical staining of the HOB cells for alkaline phosphatase activity. Combining E2 and the progestins did not result in a further increase of the number of alkaline phosphatase positive cells, compared with E2 alone. The different progestins proved to be equally potent in stimulating HOB proliferation and differentiation. In conclusion, progestins as well as E2 exerted anabolic but differential effects on normal adult human osteoblasts in vitro.

Here we report that osteoblast-like cells derived from female and male adult human trabecular bone are able to directly respond to 17 beta-estradiol (E2) and progesterone (P). In short-term (1 day) cultures using serum-free and phenol red-free medium, both steroid hormones were found to stimulate DNA synthesis and growth of the human osteoblast-like cells. P was more potent in stimulating osteoblast growth compared to E2. On the other hand, E2 showed a stronger differentiation-inducing effect as determined by analysis of the number of cells displaying cytochemical alkaline phosphatase (AP) activity, a marker for the mature osteoblast phenotype. Combination of E2 and P resulted in a further increase in DNA synthesis, but did not further affect the number of cells expressing AP activity. In conclusion, female sex steroids may be involved in regulating bone mass in human adults via a direct anabolic action on the bone forming cells.

Recombinant GH and IGF-I/-II were studied for their capacity to directly influence the growth of human bone cells maintained under defined serum-free conditions. Normal human osteoblast-like cell (HOB) cultures were established from trabecular bone explants obtained from adult human femoral head samples. IGF-I and IGF-II as well as GH stimulated the growth of the HOB cultures in a dose-dependent manner. Growth stimulatory effects were also found using the human osteogenic sarcoma cell line, SaOS-2. IGF-I and -II were powerful enhancers of the SaOS-2 cell growth and their effects greatly exceeded GH effects on these cells. The role of endogenously produced IGFs was studied using a specific monoclonal antibody to IGF-I having a partial cross-reactivity with IGF-II (sm1.2B). The IGF-I stimulated HOB growth was completely neutralised by sm1.2B to the level of control+antibody which in general showed a slight stimulation compared to controls without the antibody. Interestingly, sm1.2B was not able to interfere with the action of GH on the HOB suggesting that GH effects may be attributed to an action independent of endogenous IGF-I/-II. Unlike the HOB, SaOS-2 cells were strongly inhibited by sm1.2B in control medium indicating an autocrine role of IGF-I/-II in osteosarcoma cell growth. Sm1.2B completely neutralised the stimulatory effects of IGF-I and IGF-II on the SaOS-2 cells. Moreover, GH effects on the osteogenic sarcoma cells were abolished by the anti-IGF antibody showing that GH was acting via endogenously produced IGFs.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence and biological activity of an Osteoclast Growth Factor (OGF) was investigated in serum-free medium conditioned by periostless fetal rat calvaria in culture. OGF activity was assessed using in vitro systems of fetal rat long bones and adult rat bone marrow cells. Rat calvaria conditioned medium (RCCM) increased the number of osteoclasts in the long bone cultures, partly due to stimulation of progenitor proliferation. RCCM did not exert a direct bone-resorbing activity (45Calcium release assay) on the pre-existing osteoclasts residing in the long bones, but stimulated bone resorption in long term cultures, apparently in an indirect manner by enhancing the number of osteoclasts. In cultures of bone marrow cells isolated from adult rats, RCCM markedly stimulated the formation of mononuclear cells which were positively stained for tartrate-resistant acid phosphatase (TRAP). The osteoclastic nature of the cells was confirmed by specific labeling with 125I-calcitonin. Formation of the TRAP-positive cells was significantly inhibited by salmon calcitonin. CM from fetal rat skin cultures did not display a significant OGF activity. Furthermore, unlike the bone marrow cells, peritoneal macrophages did not respond to RCCM and remained devoid of TRAP activity. Neutralization experiments with a specific antibody to GM-CSF indicated that OGF activity in the RCCM could not be ascribed to this hemopoietic growth factor. Secretion of OGF activity was mainly dependent on protein synthesis as addition of cycloheximide to the calvaria cultures significantly inhibited the secretion of OGF into the medium. G3000 HPLC fractionation of RCCM revealed two major OGF peaks with Mr 14,000 and 70,000. Two subsequent reverse-phase HPLC steps using the lower Mr OGF fraction led to a highly purified OGF fraction. The results of this study further provide evidence that bone tissue produces factor(s) which specifically govern the process of osteoclast development, thus providing information about one of the mechanisms controlling bone resorption.

Longitudinal growth was studied using an in vitro model system of intact rat long bones. Metatarsal bones from 18- and 19-day-old rat fetuses, entirely (18 days) or mainly (19 days) composed of chondrocytes, showed a steady rate of growth and radiolabelled thymidine incorporation for at least 7 days in serum-free media. Addition of recombinant human insulin-like growth factor-I to the culture media resulted in a direct stimulation of the longitudinal growth. Recombinant human growth hormone was also able to stimulate bone growth, although this was generally accomplished after a time lag of more than 2 days. A monoclonal antibody to IGF-I abolished both the IGF-I and GH-stimulated growth. However, the antibody had no effect on the growth of the bone explants in control, serum-free medium. Unlike the fetal long bones, bones from 2-day-old neonatal rats were arrested in their growth after 1-2 days in vitro. The neonatal bones responded to IGF-I and GH in a similar fashion as the fetal bones. Thus in this study in vitro evidence of a direct effect of GH on long bone growth via stimulating local production of IGF by the growth plate chondrocytes is presented. Furthermore, endogenous growth factors, others than IGFs, appear to play a crucial role in the regulation of fetal long bone growth.