An in-vivo Intraoral Defect Model for Assessing the Use of P11-4 Self-Assembling Peptide in Periodontal Regeneration.

Periodontal disease is one of the most common diseases worldwide. It has a significant impact on oral health and subsequently the individual's quality of life. However, optimal regeneration of periodontal tissues, using current treatments, has yet to be achieved. Peptide self-assembly has provided a step-change in nanobiotechnology and regenerative medicine fields. Our aim was to investigate the effects of a self-assembling peptide (SAP; P11-4) on periodontal regeneration in a preclinical model. Twenty-six bilateral maxillary critical-sized periodontal defects were created surgically in 13 rats. Defects on one side of the mouth were filled with P11-4 hydrogel; the contra-lateral defect was untreated (control). Rats were sacrificed immediately post-surgery (time 0) and after 2 and 4 weeks. Retrieved maxillae were processed for histological, immunohistochemical, and histomorphometric assessments. The results of histological analysis showed greater organization of periodontal fibers in defects treated with P11-4, at both time points, when compared to untreated defects. Histomorphometry showed that treated defects had both a significant increase in functional periodontal ligament length and a reduction in epithelial down growth after 4 weeks. At 2 weeks, treated defects showed a significant increase in expression of osteocalcin and osteoprotegerin as judged by immunohistochemistry. Also, a significantly higher osteoprotegerin/RANKL ratio was shown in treated defects. In conclusion, the results demonstrated enhanced regeneration of periodontal tissues when SAP P11-4 was used to fill periodontal defects in rats. The findings of this study suggest that SAP P11-4 is a promising novel candidate for periodontal regenerative therapy. Further investigations are required for optimization before clinical use.

Biomimetic peptide enriched nonwoven scaffolds promote calcium phosphate mineralisation.

Cell-free translational strategies are needed to accelerate the repair of mineralised tissues, particularly large bone defects, using minimally invasive approaches. Regenerative bone scaffolds should ideally mimic aspects of the tissue's ECM over multiple length scales and enable surgical handling and fixation during implantation in vivo. Leveraging the knowledge gained with bioactive self-assembling peptides (SAPs) and SAP-enriched electrospun fibres, we presented a cell free approach for promoting mineralisation via apatite deposition and crystal growth, in vitro, of SAP-enriched nonwoven scaffolds. The nonwoven scaffold was made by electrospinning poly(ε-caprolactone) (PCL) in the presence of either peptide P11-4 (Ac-QQRFEWEFEQQ-Am) or P11-8 (Ac QQRFOWOFEQQ-Am), in light of the polymer's fibre forming capability and its hydrolytic degradability as well as the well-known apatite nucleating capability of SAPs. The 11-residue family of peptides (P11-X) has the ability to self-assemble into ß-sheet ordered structures at the nano-scale and to generate hydrogels at the macroscopic scale, some of which are capable of promoting biomineralisation due to their apatite-nucleating capability. Both variants of SAP-enriched nonwoven used in this study were proven to be biocompatible with murine fibroblasts and supported nucleation and growth of apatite minerals in simulated body fluid (SBF) in vitro. The fibrous nonwoven provided a structurally robust scaffold, with the capability to control SAP release behaviour. Up to 75% of P11-4 and 45% of P11-8 were retained in the fibres after 7 day incubation in aqueous solution at pH 7.4. The encapsulation of SAP in a nonwoven system with apatite-forming as well as localised and long-term SAP delivery capabilities is appealing as a potential means of achieving cost-effective bone repair therapy for critical size defects.

Developing a Tooth in situ Organ Culture Model for Dental and Periodontal Regeneration Research.

In this study we have realized the need for an organ culture tooth in situ model to simulate the tooth structure especially the tooth attachment apparatus. The importance of such a model is to open avenues for investigating regeneration of the complex tooth and tooth attachment tissues and to reduce the need for experimental animals in investigating dental materials and treatments in the future. The aim of this study was to develop a porcine tooth in situ organ culture model and a novel bioreactor suitable for future studies of periodontal regeneration, including application of appropriate physiological loading. The Objectives of this study was to establish tissue viability, maintenance of tissue structure, and model sterility after 1 and 4 days of culture. To model diffusion characteristics within the organ culture system and design and develop a bioreactor that allows tooth loading and simulation of the chewing cycle. Methods: Twenty-one porcine first molars were dissected aseptically in situ within their bony sockets. Twelve were used to optimize sterility and determine tissue viability. The remainder were used in a 4-day organ culture study in basal medium. Sterility was determined for medium samples and swabs taken from all tissue components, using standard aerobic and anaerobic microbiological cultures. Tissue viability was determined at days 1 and 4 using an XTT assay and Glucose consumption assays. Maintenance of structure was confirmed using histology and histomorphometric analysis. Diffusion characteristics were investigated using micro-CT combined with finite element modeling. A suitable bioreactor was designed to permit longer term culture with application of mechanical loading to the tooth in situ. Result: XTT and Glucose consumption assays confirmed viability throughout the culture period for all tissues investigated. Histological and histomorphometric analysis confirmed maintenance of tissue structure. Clear microbiological cultures indicated maintenance of sterility within the organ culture system. The novel bioreactor showed no evidence of medium contamination after 4 days of culture. Finite element modeling indicated nutrient availability to the periodontium. Conclusion: A whole tooth in situ organ culture system was successfully maintained over 4 days in vitro.

A biomimetic self-assembling peptide promotes bone regeneration in vivo: A rat cranial defect study.

Rationally designed, pH sensitive self-assembling ß-peptides (SAPs) which are capable of reversibly switching between fluid and gel phases in response to environmental triggers are potentially useful injectable scaffolds for skeletal tissue engineering applications. SAP P11-4 (CH3COQQRFEWEFEQQNH2) has been shown to nucleate hydroxyapatite mineral de novo and has been used in dental enamel regeneration. We hypothesised that addition of mesenchymal stromal cells (MSCs) would enhance the in vivo effects of P11-4 in promoting skeletal tissue repair. Cranial defects were created in athymic rats and filled with either Bio-Oss® (anorganic bone chips) or P11-4 ±â€¯human dental pulp stromal cells (HDPSCs). Unfilled defects served as controls. After 4 weeks, only those defects filled with P11-4 alone showed significantly increased bone regeneration (almost complete healing), compared to unfilled control defects, as judged using quantitative micro-CT, histology and immunohistochemistry. In silico modelling indicated that fibril formation may be essential for any mineral nucleation activity. Taken together, these data suggest that self-assembling peptides are a suitable scaffold for regeneration of bone tissue in a one step, cell-free therapeutic approach.

The Unfolded Protein Response in Amelogenesis and Enamel Pathologies.

During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other "professional" secretory cells, ameloblasts employ the unfolded protein response (UPR) to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum)/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI) and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed.

Amelogenesis Imperfecta; Genes, Proteins, and Pathways.

Amelogenesis imperfecta (AI) is the name given to a heterogeneous group of conditions characterized by inherited developmental enamel defects. AI enamel is abnormally thin, soft, fragile, pitted and/or badly discolored, with poor function and aesthetics, causing patients problems such as early tooth loss, severe embarrassment, eating difficulties, and pain. It was first described separately from diseases of dentine nearly 80 years ago, but the underlying genetic and mechanistic basis of the condition is only now coming to light. Mutations in the gene AMELX, encoding an extracellular matrix protein secreted by ameloblasts during enamel formation, were first identified as a cause of AI in 1991. Since then, mutations in at least eighteen genes have been shown to cause AI presenting in isolation of other health problems, with many more implicated in syndromic AI. Some of the encoded proteins have well documented roles in amelogenesis, acting as enamel matrix proteins or the proteases that degrade them, cell adhesion molecules or regulators of calcium homeostasis. However, for others, function is less clear and further research is needed to understand the pathways and processes essential for the development of healthy enamel. Here, we review the genes and mutations underlying AI presenting in isolation of other health problems, the proteins they encode and knowledge of their roles in amelogenesis, combining evidence from human phenotypes, inheritance patterns, mouse models, and in vitro studies. An LOVD resource (http://dna2.leeds.ac.uk/LOVD/) containing all published gene mutations for AI presenting in isolation of other health problems is described. We use this resource to identify trends in the genes and mutations reported to cause AI in the 270 families for which molecular diagnoses have been reported by 23rd May 2017. Finally we discuss the potential value of the translation of AI genetics to clinical care with improved patient pathways and speculate on the possibility of novel treatments and prevention strategies for AI.

Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin.

Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.

A Fourth KLK4 Mutation Is Associated with Enamel Hypomineralisation and Structural Abnormalities.

"Amelogenesis imperfecta" (AI) describes a group of genetic conditions that result in defects in tooth enamel formation. Mutations in many genes are known to cause AI, including the gene encoding the serine protease, kallikrein related peptidase 4 (KLK4), expressed during the maturation stage of amelogenesis. In this study we report the fourth KLK4 mutation to be identified in autosomal recessively-inherited hypomaturation type AI, c.632delT, p.(L211Rfs*37) (NM_004917.4, NP_004908.4). This homozygous variant was identified in five Pakistani AI families and is predicted to result in a transcript with a premature stop codon that escapes nonsense mediated decay. However, the protein may misfold, as three of six disulphide bonds would be disrupted, and may be degraded or non-functional as a result. Primary teeth were obtained from one affected individual. The enamel phenotype was characterized using high-resolution computerized X-ray tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and microhardness testing (MH). Enamel from the affected individual (referred to as KLK4 enamel) was hypomineralised in comparison with matched control enamel. Furthermore, KLK4 inner enamel was hypomineralised compared with KLK4 outer enamel. SEM showed a clear structural demarcation between KLK4 inner and outer enamel, although enamel structure was similar to control tissue overall. EDX showed that KLK4 inner enamel contained less calcium and phosphorus and more nitrogen than control inner enamel and KLK4 outer enamel. MH testing showed that KLK4 inner enamel was significantly softer than KLK4 outer enamel (p < 0.001). However, the hardness of control inner enamel was not significantly different to that of control outer enamel. Overall, these findings suggest that the KLK4 c.632delT mutation may be a common cause of autosomal recessive AI in the Pakistani population. The phenotype data obtained mirror findings in the Klk4-/- mouse and suggest that KLK4 is required for the hardening and mineralization of the inner enamel layer but is less essential for hardening and mineralization of the outer enamel layer.

Defects in the acid phosphatase ACPT cause recessive hypoplastic amelogenesis imperfecta.

We identified two homozygous missense variants (c.428C>T, p.(T143M) and c.746C>T, p.(P249L)) in ACPT, the gene encoding acid phosphatase, testicular, which segregates with hypoplastic amelogenesis imperfecta in two unrelated families. ACPT is reported to play a role in odontoblast differentiation and mineralisation by supplying phosphate during dentine formation. Analysis by computerised tomography and scanning electron microscopy of a primary molar tooth from an individual homozygous for the c.746C>T variant revealed an enamel layer that was hypoplastic, but mineralised with prismatic architecture. These findings implicate variants in ACPT as a cause of early failure of amelogenesis during the secretory phase.

Amelogenesis imperfecta caused by N-terminal enamelin point mutations in mice and men is driven by endoplasmic reticulum stress.

'Amelogenesis imperfecta' (AI) describes a group of inherited diseases of dental enamel that have major clinical impact. Here, we identify the aetiology driving AI in mice carrying a p.S55I mutation in enamelin; one of the most commonly mutated proteins underlying AI in humans. Our data indicate that the mutation inhibits the ameloblast secretory pathway leading to ER stress and an activated unfolded protein response (UPR). Initially, with the support of the UPR acting in pro-survival mode, Enamp.S55I heterozygous mice secreted structurally normal enamel. However, enamel secreted thereafter was structurally abnormal; presumably due to the UPR modulating ameloblast behaviour and function in an attempt to relieve ER stress. Homozygous mutant mice failed to produce enamel. We also identified a novel heterozygous ENAMp.L31R mutation causing AI in humans. We hypothesize that ER stress is the aetiological factor in this case of human AI as it shared the characteristic phenotype described above for the Enamp.S55I mouse. We previously demonstrated that AI in mice carrying the Amelxp.Y64H mutation is a proteinopathy. The current data indicate that AI in Enamp.S55I mice is also a proteinopathy, and based on comparative phenotypic analysis, we suggest that human AI resulting from the ENAMp.L31R mutation is another proteinopathic disease. Identifying a common aetiology for AI resulting from mutations in two different genes opens the way for developing pharmaceutical interventions designed to relieve ER stress or modulate the UPR during enamel development to ameliorate the clinical phenotype.

Rapid cell separation with minimal manipulation for autologous cell therapies.

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of >98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities.

Remineralization of natural early caries lesions in vitro by P11 -4 monitored with photothermal radiometry and luminescence.

AIM: The efficacy of self-assembling peptide P11 -4 to regenerate enamel in natural early caries lesions was evaluated over 50 days by photothermal radiometry and luminescence using The Canary System (CS) and The Canary Lab (CL). METHODS: Baseline readings for sound and carious sites on smooth surfaces of extracted teeth were obtained by scanning with CS and CL. Teeth were then randomly assigned to a treatment group (TG, treated with P11 -4), a placebo group (PG, same vehicle as treatment group without P11 -4), or a control group (CG, no treatment). All the teeth were then placed in artificial saliva to facilitate natural remineralization, and the sites were rescanned with CS and CL at 7, 14, 30, and 50 days. RESULTS: For carious sites in TG, mean canary numbers (CN) derived from CS decreased significantly (P<.01) from 44±3.8 at baseline to 24±4.9 at day 50; the mean CN for the TG derived from CL also decreased significantly (P<.05), from 65 at baseline to 45 at day 50. In contrast, no significant changes in CN were observed for carious sites in the CG or PG using either CS or CL. CONCLUSIONS: P11 -4 promoted the regeneration of early caries.

Deletion of amelotin exons 3-6 is associated with amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective dental enamel formation. Amelotin (AMTN) is a secreted protein thought to act as a promoter of matrix mineralization in the final stage of enamel development, and is strongly expressed, almost exclusively, in maturation stage ameloblasts. Amtn overexpression and Amtn knockout mouse models have defective enamel with no other associated phenotypes, highlighting AMTN as an excellent candidate gene for human AI. However, no AMTN mutations have yet been associated with human AI. Using whole exome sequencing, we identified an 8,678 bp heterozygous genomic deletion encompassing exons 3-6 of AMTN in a Costa Rican family segregating dominant hypomineralised AI. The deletion corresponds to an in-frame deletion of 92 amino acids, shortening the protein from 209 to 117 residues. Exfoliated primary teeth from an affected family member had enamel that was of a lower mineral density compared to control enamel and exhibited structural defects at least some of which appeared to be associated with organic material as evidenced using elemental analysis. This study demonstrates for the first time that AMTN mutations cause non-syndromic human AI and explores the human phenotype, comparing it with that of mice with disrupted Amtn function.

In Vitro Engineering of High Modulus Cartilage-Like Constructs.

To date, the outcomes of cartilage repair have been inconsistent and have frequently yielded mechanically inferior fibrocartilage, thereby increasing the chances of damage recurrence. Implantation of constructs with biochemical composition and mechanical properties comparable to natural cartilage could be advantageous for long-term repair. This study attempted to create such constructs, in vitro, using tissue engineering principles. Bovine synoviocytes were seeded on nonwoven polyethylene terephthalate fiber scaffolds and cultured in chondrogenic medium for 4 weeks, after which uniaxial compressive loading was applied using an in-house bioreactor for 1 h per day, at a frequency of 1 Hz, for a further 84 days. The initial loading conditions, determined from the mechanical properties of the immature constructs after 4 weeks in chondrogenic culture, were strains ranging between 13% and 23%. After 56 days (sustained at 84 days) of loading, the constructs were stained homogenously with Alcian blue and for type-II collagen. Dynamic compressive moduli were comparable to the high end values for native cartilage and proportional to Alcian blue staining intensity. We suggest that these high moduli values were attributable to the bioreactor setup, which caused the loading regime to change as the constructs developed, that is, the applied stress and strain increased with construct thickness and stiffness, providing continued sufficient cell stimulation as further matrix was deposited. Constructs containing cartilage-like matrix with response to load similar to that of native cartilage could produce long-term effective cartilage repair when implanted.

Detection of early stage changes associated with adipogenesis using Raman spectroscopy under aseptic conditions.

There is growing interest in the development of methods capable of non-invasive characterization of stem cells prior to their use in cell-based therapies. Raman spectroscopy has previously been used to detect biochemical changes commensurate with the osteogenic, cardiogenic, and neurogenic differentiation of stem cells. The aim of this study was to characterize the adipogenic differentiation of live adipose derived stem cells (ASCs) under aseptic conditions. ASCs were cultured in adipogenic or basal culture medium for 14 days in customized culture flasks containing quartz windows. Raman spectra were acquired every 3 days. Principal component analysis (PCA) was used to identify spectral changes in the cultures over time. Adipogenic differentiation was confirmed using quantitative reverse transcription polymerase chain reaction for the marker genes PPARγ and ADIPOQ and Oil red O staining performed. PCA demonstrated that lipid associated spectral features varied throughout ASC differentiation with the earliest detection of the lipid associated peak at 1,438 cm(-1) after 3 days of induction. After 7 days of culture there were clear differences between the spectra acquired from ASCs in adipogenic or basal culture medium. No changes were observed in the spectra acquired from undifferentiated ASCs. Significant up-regulation in the expression of both PPARγ and ADIPOQ genes (P < 0.001) was observed after 14 days of differentiation as was prominent Oil red O staining. However, the Raman sampling process resulted in weaker gene expression compared with ASCs that had not undergone Raman analysis. This study demonstrated that Raman spectroscopy can be used to detect biochemical changes associated with adipogenic differentiation in a non-invasive and aseptic manner and that this can be achieved as early as three days into the differentiation process.

Aseptic Raman spectroscopy can detect changes associated with the culture of human dental pulp stromal cells in osteoinductive culture.

There is an unmet need for the non-invasive characterisation of stem cells to facilitate the translation of cell-based therapies. Raman spectroscopy has proven utility in stem cell characterisation but as yet no method has been reported capable of taking repeated Raman measurements of living cells aseptically over time. The aim of this study was to determine if Raman spectroscopy could be used to monitor changes in a well characterised cell population (human dental pulp stromal cells (DPSCs)) by taking repeated Raman measurements from the same cell populations in osteoinductive culture over time and under aseptic conditions. DPSCs were isolated from extracted premolar teeth from 3 consenting donors. Following in vitro expansion, DPSCs were maintained for 28 days in osteo-inductive medium. Raman spectra were acquired from the cells at days 0, 3, 7, 10, 14 and 28. Principal component analysis (PCA) was carried out to assess if there was any temporal spectral variation. At day 28, osteoinduction was confirmed using alizarin red staining and qRT-PCR for alkaline phosphatase and osteocalcin. Alizarin red staining was positive in all samples at day 28 and significant increases in alkaline phosphatase (p < 0.001) and osteocalcin (p < 0.05) gene expression were also observed compared with day 0. PCA of the Raman data demonstrated trends in PC1 from days 0-10, influenced by protein associated features and PC2 from days 10-28, influenced by DNA/RNA associated features. We conclude that spectroscopy can be used to monitor changes in Raman signature with time associated with the osteoinduction of DPSCs using repeated measurements via an aseptic methodology.

Investigating the Vascularization of Tissue-Engineered Bone Constructs Using Dental Pulp Cells and 45S5 Bioglass® Scaffolds.

Identification of a suitable cell source combined with an appropriate 3D scaffold is an essential prerequisite for successful engineering of skeletal tissues. Both osteogenesis and angiogenesis are key processes for bone regeneration. This study investigated the vascularization potential of a novel combination of human dental pulp stromal cells (HDPSCs) with 45S5 Bioglass® scaffolds for tissue-engineered mineral constructs in vivo and in vitro. 45S5 Bioglass scaffolds were produced by the foam replication technique with the standard composition of 45 wt% SiO2, 24.5 wt% Na2O, 24.5 wt% CaO, and 6 wt% P2O5. HDPSCs were cultured in monolayers and on porous 45S5 Bioglass scaffolds under angiogenic and osteogenic conditions for 2-4 weeks. HDPSCs expressed endothelial gene markers (CD34, CD31/PECAM1, and VEGFR2) under both conditions in the monolayer. A combination of HDPSCs with 45S5 Bioglass enhanced the expression of these gene markers. Positive immunostaining for CD31/PECAM1 and VEGFR2 and negative staining for CD34 supported the gene expression data, while histology revealed evidence of endothelial cell-like morphology within the constructs. More organized tubular structures, resembling microvessels, were seen in the constructs after 8 weeks of implantation in vivo. In conclusion, this study suggests that the combination of HDPSCs with 45S5 Bioglass scaffolds offers a promising strategy for regenerating vascularized bone grafts.

Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.