Bone tissue engineering using 3D silk scaffolds and human dental pulp stromal cells epigenetic reprogrammed with the selective histone deacetylase inhibitor MI192.

Epigenetics plays a critical role in regulating mesenchymal stem cells' (MSCs) fate for tissue repair and regeneration. There is increasing evidence that the inhibition of histone deacetylase (HDAC) isoform 3 can enhance MSC osteogenesis. This study investigated the potential of using a selective HDAC2 and 3 inhibitor, MI192, to promote human dental pulp stromal cells (hDPSCs) bone-like tissue formation in vitro and in vivo within porous Bombyx Mori silk scaffolds. Both 2 and 5 wt% silk scaffolds were fabricated and characterised. The 5 wt% scaffolds possess thicker internal lamellae, reduced scaffold swelling and degradation rates, whilst increased compressive modulus in comparison to the 2 wt% silk scaffold. MI192 pre-treatment of hDPSCs on 5 wt% silk scaffold significantly enhanced hDPSCs alkaline phosphatase activity (ALP). The expression of osteoblast-related genes (RUNX2, ALP, Col1a, OCN) was significantly upregulated in the MI192 pre-treated cells. Histological analysis confirmed that the MI192 pre-treated hDPSCs-silk scaffold constructs promoted bone extracellular matrix (ALP, Col1a, OCN) deposition and mineralisation compared to the untreated group. Following 6 weeks of subcutaneous implantation in nude mice, the MI192 pre-treated hDPSCs-silk scaffold constructs enhanced the vascularisation and extracellular matrix mineralisation compared to untreated control. In conclusion, these findings demonstrate the potential of using epigenetic reprogramming and silk scaffolds to promote hDPSCs bone formation efficacy, which provides evidence for clinical translation of this technology for bone augmentation.

Enhancing osteogenic potential of hDPSCs by resveratrol through reducing oxidative stress via the Sirt1/Nrf2 pathway.

CONTEXT: The osteogenic potential of the human dental pulp stromal cells (hDPSCs) was reduced in the state of oxidative stress. Resveratrol (RSV) possesses numerous biological properties, including osteogenic potential, growth-promoting and antioxidant activities. OBJECTIVE: This study investigates the osteogenic potential of RSV by activating the Sirt1/Nrf2 pathway on oxidatively stressed hDPSCs and old mice. MATERIALS AND METHODS: The hDPSCs were subjected to reactive oxygen species (ROS) fluorescence staining, cell proliferation assay, ROS activity assay, superoxide dismutase (SOD) enzyme activity, the glutathione (GSH) concentration assay, alkaline phosphatase staining, real-time polymerase chain reaction (RT-PCR) and Sirt1 immunofluorescence labelling to assess the antioxidant stress and osteogenic ability of RSV. Forty female Kunming mice were divided into Old, Old-RSV, Young and Young-RSV groups to assess the repair of calvarial defects of 0.2 mL RSV of 20 mg/kg/d for seven days by injecting intraperitoneally at 4 weeks after surgery using micro-computed tomography, nonlinear optical microscope and immunohistochemical analysis. RESULTS: RSV abates oxidative stress by alleviating the proliferation, mitigating the ROS activity, increasing the SOD enzyme activity and ameliorating the GSH concentration (RSV IC50 in hDPSCs is 67.65 ± 9.86). The antioxidative stress and osteogenic capabilities of RSV were confirmed by the up-regulated gene expression of SOD1, xCT, RUNX2 and OCN, as well as Sirt1/Nrf2. The collagen, bone matrix formation and Sirt1 expression, are significantly increased after RSV treatment in mice. DISCUSSION AND CONCLUSIONS: For elderly or patients with oxidative stress physiological states such as hypertension, heart disease, diabetes, etc., RSV may potentially improve bone augmentation surgery in regenerative medicine.

The Selective Histone Deacetylase Inhibitor MI192 Enhances the Osteogenic Differentiation Efficacy of Human Dental Pulp Stromal Cells.

The use of human dental pulp stromal cells (hDPSCs) has gained increasing attention as an alternative stem cell source for bone tissue engineering. The modification of the cells' epigenetics has been found to play an important role in regulating differentiation, with the inhibition of histone deacetylases 3 (HDAC3) being linked to increased osteogenic differentiation. This study aimed to induce epigenetic reprogramming using the HDAC2 and 3 selective inhibitor, MI192 to promote hDPSCs osteogenic capacity for bone regeneration. MI192 treatment caused a time-dose-dependent change in hDPSC morphology and reduction in viability. Additionally, MI192 successfully augmented hDPSC epigenetic functionality, which resulted in increased histone acetylation and cell cycle arrest at the G2/M phase. MI192 pre-treatment exhibited a dose-dependent effect on hDPSCs alkaline phosphatase activity. Quantitative PCR and In-Cell Western further demonstrated that MI192 pre-treatment significantly upregulated hDPSCs osteoblast-related gene and protein expression (alkaline phosphatase, bone morphogenic protein 2, type I collagen and osteocalcin) during osteogenic differentiation. Importantly, MI192 pre-treatment significantly increased hDPSCs extracellular matrix collagen production and mineralisation. As such, for the first time, our findings show that epigenetic reprogramming with the HDAC2 and 3 selective inhibitor MI192 accelerates the osteogenic differentiation of hDPSCs, demonstrating the considerable utility of this MSCs engineering approach for bone augmentation strategies.

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.

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.

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.

The effect of mechanical loading on osteogenesis of human dental pulp stromal cells in a novel in vitro model.

Tooth loss often results in alveolar bone resorption because of lack of mechanical stimulation. Thus, the mechanism of mechanical loading on stem cell osteogenesis is crucial for alveolar bone regeneration. We have investigated the effect of mechanical loading on osteogenesis in human dental pulp stromal cells (hDPSCs) in a novel in vitro model. Briefly, 1 × 10(7) hDPSCs were seeded into 1 ml 3% agarose gel in a 48-well-plate. A loading tube was then placed in the middle of the gel to mimic tooth-chewing movement (1 Hz, 3 × 30 min per day, n = 3). A non-loading group was used as a control. At various time points, the distribution of live/dead cells within the gel was confirmed by fluorescence markers and confocal microscopy. The correlation and interaction between the factors (e.g. force, time, depth and distance) were statistically analysed. The samples were processed for histology and immunohistochemistry. After 1-3 weeks of culture in the in-house-designed in vitro bioreactor, fluorescence imaging confirmed that additional mechanical loading increased the viable cell numbers over time as compared with the control. Cells of various phenotypes formed different patterns away from the reaction tube. The cells in the middle part of the gel showed enhanced alkaline phosphatase staining at week 1 but reduced staining at weeks 2 and 3. Additional loading enhanced Sirius Red and type I collagen staining compared with the control. We have thus successfully developed a novel in-house-designed in vitro bioreactor mimicking the biting force to enhance hDPSC osteogenesis in an agarose scaffold and to promote bone formation and/or prevent bone resorption.

Bone tissue engineering by using a combination of polymer/Bioglass composites with human adipose-derived stem cells.

Translational research in bone tissue engineering is essential for "bench to bedside" patient benefit. However, the ideal combination of stem cells and biomaterial scaffolds for bone repair/regeneration is still unclear. The aim of this study is to investigate the osteogenic capacity of a combination of poly(DL-lactic acid) (PDLLA) porous foams containing 5 wt% and 40 wt% of Bioglass particles with human adipose-derived stem cells (ADSCs) in vitro and in vivo. Live/dead fluorescent markers, confocal microscopy and scanning electron microscopy showed that PDLLA/Bioglass porous scaffolds supported ADSC attachment, growth and osteogenic differentiation, as confirmed by enhanced alkaline phosphatase (ALP) activity. Higher Bioglass content of the PDLLA foams increased ALP activity compared with the PDLLA only group. Extracellular matrix deposition after 8 weeks in the in vitro cultures was evident by Alcian blue/Sirius red staining. In vivo bone formation was assessed by using scaffold/ADSC constructs in diffusion chambers transplanted intraperitoneally into nude mice and recovered after 8 weeks. Histological and immunohistochemical assays indicated significant new bone formation in the 40 wt% and 5 wt% Bioglass constructs compared with the PDLLA only group. Thus, the combination of a well-developed biodegradable bioactive porous PDLLA/Bioglass composite scaffold with a high-potential stem cell source (human ADSCs) could be a promising approach for bone regeneration in a clinical setting.

Informing future cartilage repair strategies: a comparative study of three different human cell types for cartilage tissue engineering.

A major clinical need exists for cartilage repair and regeneration. Despite many different strategies having been pursued, the identification of an optimised cell type and of pre-treatment conditions remains a challenge. This study compares the cartilage-like tissue generated by human bone marrow stromal cells (HBMSCs) and human neonatal and adult chondrocytes cultured on three-dimensional (3D) scaffolds under various conditions in vitro and in vivo with the aim of informing future cartilage repair strategies based upon tissue-engineering approaches. After 3 weeks in vitro culture, all three cell types showed cartilage-like tissue formation on 3D poly (lactide-co-glycolide) acid scaffolds only when cultured in chondrogenic medium. After 6 weeks of chondro-induction, neonatal chondrocyte constructs revealed the most cartilage-like tissue formation with a prominent superficial zone-like layer, a middle zone-like structure and the thinnest fibrous capsule. HBMSC constructs had the thickest fibrous capsule formation. Under basal culture conditions, neonatal articular chondrocytes failed to form any tissue, whereas HBMSCs and adult chondrocytes showed thick fibrous capsule formation at 6 weeks. After in vivo implantation, all groups generated more compact tissues compared with in vitro constructs. Pre-culturing in chondrogenic media for 1 week before implantation reduced fibrous tissue formation in all cell constructs at week 3. After 6 weeks, only the adult chondrocyte group pre-cultured in chondrogenic media was able to maintain a more chondrogenic/less fibrocartilaginous phenotype. Thus, pre-culture under chondrogenic conditions is required to maintain a long-term chondrogenic phenotype, with adult chondrocytes being a more promising cell source than HBMSCs for articular cartilage tissue engineering.

The effect of epigenetic reprogramming using MI192 HDAC inhibitor on enhancing the osteogenesis of human adipose-derived stem cells in vitro.

The ability to control stem cell function is the key to stem cell-based therapy and living tissue regeneration. In natural conditions, histone deacetylases (HDAC) are regarded as the important defining epigenetic reprogramming for stem cell differentiation. To date, human adipose-derived stem cells (hADSCs) have been widely utilised for bone tissue engineering applications. The present study aimed to examine the effect of a novel HDAC2&3-selective inhibitor, MI192, on hADSCs epigenetic reprogramming for regulating its osteogenic potential in vitro. The results confirmed that MI192 treatment reduced the hADSCs viability in a time and dose-dependent manner. The optimal concentration and pre-treatment time of MI192 for hADSCs osteogenic induction was 30 µM and 2 days representatively. A quantitative biochemical assay confirmed that the pre-treatment with MI192 (30 µM) for 2 days significantly enhanced hADSCs alkaline phosphatase (ALP) specific activity (P<0.05) compared with that of the valproic acid (VPA) pre-treatment group. Real-time PCR analysis revealed that MI192 pre-treatment up-regulated hADSCs gene expressions of osteogenic markers (e.g., Runx2, Col1, and OCN) under the osteogenic induction. DNA flow cytometric analysis indicated that two days' pre-treatment with MI192 (30 µM) resulted in G2/M arrest in hADSCs and this G2/M arrest was reversible. Our results suggest that MI192 is capable of epigenetic reprogramming of hADSCs via HDAC inhibition for controlling the cell cycle, resulting in enhancing hADSCs osteogenic differentiation, which indicates the potential of using MI192 for promoting bone tissue regeneration.

GelMA Hydrogel Reinforced with 3D Printed PEGT/PBT Scaffolds for Supporting Epigenetically-Activated Human Bone Marrow Stromal Cells for Bone Repair.

Epigenetic approaches using the histone deacetylase 2 and 3 inhibitor-MI192 have been reported to accelerate stem cells to form mineralised tissues. Gelatine methacryloyl (GelMA) hydrogels provide a favourable microenvironment to facilitate cell delivery and support tissue formation. However, their application for bone repair is limited due to their low mechanical strength. This study aimed to investigate a GelMA hydrogel reinforced with a 3D printed scaffold to support MI192-induced human bone marrow stromal cells (hBMSCs) for bone formation. Cell culture: The GelMA (5 wt%) hydrogel supported the proliferation of MI192-pre-treated hBMSCs. MI192-pre-treated hBMSCs within the GelMA in osteogenic culture significantly increased alkaline phosphatase activity (p ≤ 0.001) compared to control. Histology: The MI192-pre-treated group enhanced osteoblast-related extracellular matrix deposition and mineralisation (p ≤ 0.001) compared to control. Mechanical testing: GelMA hydrogels reinforced with 3D printed poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) scaffolds exhibited a 1000-fold increase in the compressive modulus compared to the GelMA alone. MI192-pre-treated hBMSCs within the GelMA-PEGT/PBT constructs significantly enhanced extracellular matrix collagen production and mineralisation compared to control (p ≤ 0.001). These findings demonstrate that the GelMA-PEGT/PBT construct provides enhanced mechanical strength and facilitates the delivery of epigenetically-activated MSCs for bone augmentation strategies.

A long-lasting guided bone regeneration membrane from sequentially functionalised photoactive atelocollagen.

The fast degradation of collagen-based membranes in the biological environment remains a critical challenge, resulting in underperforming Guided Bone Regeneration (GBR) therapy leading to compromised clinical results. Photoactive atelocollagen (AC) systems functionalised with ethylenically unsaturated monomers, such as 4-vinylbenzyl chloride (4VBC), have been shown to generate mechanically competent materials for wound healing, inflammation control and drug delivery, whereby control of the molecular architecture of the AC network is key. Building on this platform, the sequential functionalisation with 4VBC and methacrylic anhydride (MA) was hypothesised to generate UV-cured AC hydrogels with reduced swelling ratio, increased proteolytic stability and barrier functionality for GBR therapy. The sequentially functionalised atelocollagen precursor (SAP) was characterised via TNBS and ninhydrin colourimetric assays, circular dichroism and UV-curing rheometry, which confirmed nearly complete consumption of collagen's primary amino groups, preserved triple helices and fast (< 180 s) gelation kinetics, respectively. Hydrogel's swelling ratio and compression modulus were adjusted depending on the aqueous environment used for UV-curing, whilst the sequential functionalisation of AC successfully generated hydrogels with superior proteolytic stability in vitro compared to both 4VBC-functionalised control and the commercial dental membrane Bio-Gide®. These in vitro results were confirmed in vivo via both subcutaneous implantation and a proof-of-concept study in a GBR calvarial model, indicating integrity of the hydrogel and barrier defect, as well as tissue formation following 1-month implantation in rats. STATEMENT OF SIGNIFICANCE: Collagen-based membranes remain a key component in Guided Bone Regeneration (GBR) therapy, but their properties, e.g. proteolytic stability and soft tissue barrier functionality, are still far from optimal. This is largely attributed to the complex molecular configuration of collagen, which makes chemical accessibility and structure-function relations challenging. Here, we fabricated a UV-cured hydrogel network of atelocollagen, whereby triple helices were sequentially functionalised with two distinct ethylenically unsaturated monomers. The effects of the sequential functionalisation and UV-curing on the macroscopic properties, degradation behaviour and GBR capability were investigated in vitro and in vivo. The results highlight the key role of the sequential functionalisation and provide important insights for the design of future, longer-lasting resorbable membranes for GBR therapy.

CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

Heterogeneity of bone marrow mesenchymal stromal cells (MSCs, frequently referred to as "mesenchymal stem cells") clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterized by CD317 expression (CD317pos (29.77 ± 3.00% of the total MSC population), comprising CD317dim (28.10 ± 4.60%) and CD317bright (1.67 ± 0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarization towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

Corrigendum: CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

[This corrects the article DOI: 10.3389/fimmu.2022.903796.].

Role of the store-operated Ca2+ channel in ATP-induced Ca2+ signalling in mesenchymal stem cells and regulation of cell functions.

It is well-known that extracellular ATP acts as an autocrine/paracrine signal to regulate cell functions by inducing intracellular Ca2+ signalling through its cognate receptors, namely, the ligand-gated ion channel P2X receptors that mediate Ca2+ influx and/or the Gq/11-coupled P2Y receptors that link to Ca2+ release from the ER. The reduction in ER Ca2+ can trigger further extracellular Ca2+ entry by activating the store-operated Ca2+ (SOC) channel. Mesenchymal stem cells (MSC) play an important role in the homeostasis of residing tissues and have promising applications in regenerative medicines. MSC can release ATP spontaneously or in response to diverse stimuli, and express multiple P2X and Gq/11-coupled P2Y receptors that participate in ATP-induced Ca2+ signalling and regulate cell function. There is increasing evidence to show the contribution of the SOC channel in ATP-induced Ca2+ signalling in MSC. In this mini-review, we discuss the current understanding of the expression of the SOC channel in MSC and its potential role in mediating ATP-induced Ca2+ signalling and regulation of MSC differentiation, proliferation and migration.

MI192 induced epigenetic reprogramming enhances the therapeutic efficacy of human bone marrows stromal cells for bone regeneration.

Human bone marrow stromal cells (hBMSCs) have been extensively utilised for bone tissue engineering applications. However, they are associated with limitations that hinder their clinical utility for bone regeneration. Cell fate can be modulated via altering their epigenetic functionality. Inhibiting histone deacetylase (HDAC) enzymes have been reported to promote osteogenic differentiation, with HDAC3 activity shown to be causatively associated with osteogenesis. Therefore, this study aimed to investigate the potential of using an HDAC2 & 3 selective inhibitor - MI192 to induce epigenetic reprogramming of hBMSCs and enhance its therapeutic efficacy for bone formation. Treatment with MI192 caused a time-dose dependant reduction in hBMSCs viability. MI192 was also found to substantially alter hBMSCs epigenetic function through reduced HDAC activity and increased histone acetylation. hBMSCs were pre-treated with MI192 (50 µM) for 48 h prior to osteogenic induction. MI192 pre-treatment significantly upregulated osteoblast-related gene/protein expression (Runx2, ALP, Col1a and OCN) and enhanced alkaline phosphatase specific activity (ALPSA) (1.43-fold) (P ≤ 0.001). Moreover, MI192 substantially increased hBMSCs extracellular matrix calcium deposition (1.4-fold) (P ≤ 0.001) and mineralisation when compared to the untreated control. In 3D microtissue culture, MI192 significantly promoted hBMSCs osteoblast-related gene expression and ALPSA (> 2.41-fold) (P ≤ 0.001). Importantly, MI192 substantially enhanced extracellular matrix deposition (ALP, Col1a, OCN) and mineralisation (1.67-fold) (P ≤ 0.001) within the bioassembled-microtissue (BMT) construct. Following 8-week intraperitoneal implantation within nude mice, MI192 treated hBMSCs exhibited enhanced extracellular matrix deposition and mineralisation (2.39-fold) (P ≤ 0.001) within the BMT when compared to the untreated BMT construct. Taken together, these results demonstrate that MI192 effectively altered hBMSCs epigenetic functionality and is capable of promoting hBMSCs osteogenic differentiation in vitro and in vivo, indicating the potential of using epigenetic reprogramming to enhance the therapeutic efficacy of hBMSCs for bone augmentation strategies.

Comparing bone tissue engineering efficacy of HDPSCs, HBMSCs on 3D biomimetic ABM-P-15 scaffolds in vitro and in vivo.

Human bone marrow mesenchymal stem cells (HBMSCs) has been the gold standard for bone regeneration. However, the low proliferation rate and long doubling time limited its clinical applications. This study aims to compare the bone tissue engineering efficacy of human dental pulp stem cells (HDPSCs) with HBMSCs in 2D, and 3D anorganic bone mineral (ABM) coated with a biomimetic collagen peptide (ABM-P-15) for improving bone-forming speed and efficacy in vitro and in vivo. The multipotential of both HDPSCs and HBMSCs have been compared in vitro. The bone formation of HDPSCs on ABM-P-15 was tested using in vivo model. The osteogenic potential of the cells was confirmed by alkaline phosphatase (ALP) and immunohistological staining for osteogenic markers. Enhanced ALP, collagen, lipid droplet, or glycosaminoglycans production were visible in HDPSCs and HBMSCs after osteogenic, adipogenic and chondrogenic induction. HDPSC showed stronger ALP staining compared to HBMSCs. Confocal images showed more viable HDPSCs on both ABM-P-15 and ABM scaffolds compared to HBMSCs on similar scaffolds. ABM-P-15 enhanced cell attachment/spreading/bridging formation on ABM-P-15 scaffolds and significantly increased quantitative ALP specific activities of the HDPSCs and HBMSCs. After 8 weeks in vivo implantation in diffusion chamber model, the HDPSCs on ABM-P-15 scaffolds showed extensive high organised collagenous matrix formation that was positive for COL-I and OCN compared to ABM alone. In conclusion, the HDPSCs have a higher proliferation rate and better osteogenic capacity, which indicated the potential of combining HDPSCs with ABM-P-15 scaffolds for improving bone regeneration speed and efficacy.

Harnessing the HDAC-histone deacetylase enzymes, inhibitors and how these can be utilised in tissue engineering.

There are large knowledge gaps regarding how to control stem cells growth and differentiation. The limitations of currently available technologies, such as growth factors and/or gene therapies has led to the search of alternatives. We explore here how a cell's epigenome influences determination of cell type, and potential applications in tissue engineering. A prevalent epigenetic modification is the acetylation of DNA core histone proteins. Acetylation levels heavily influence gene transcription. Histone deacetylase (HDAC) enzymes can remove these acetyl groups, leading to the formation of a condensed and more transcriptionally silenced chromatin. Histone deacetylase inhibitors (HDACis) can inhibit these enzymes, resulting in the increased acetylation of histones, thereby affecting gene expression. There is strong evidence to suggest that HDACis can be utilised in stem cell therapies and tissue engineering, potentially providing novel tools to control stem cell fate. This review introduces the structure/function of HDAC enzymes and their links to different tissue types (specifically bone, cardiac, neural tissues), including the history, current status and future perspectives of using HDACis for stem cell research and tissue engineering, with particular attention paid to how different HDAC isoforms may be integral to this field.

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.

Chemical probing of single cancer cells with gold nanoaggregates by surface-enhanced Raman scattering.

By using near-infrared surface-enhanced Raman scattering (SERS) with 60 nm gold nanoparticles (Au-NPs) to probe the chemical composition inside single human osteosarcoma cells we have shown that the SERS intensity may increase by a factor of 3-6 times in different parts of the cells depending on the density of gold nanoaggregates within the probed volume after the cell is dehydrated. The cellular points of low-density gold nanoaggregates exhibit more significant increase of SERS signal levels, the cellular macrochemicals such as nucleic acids show conformational changes, and new components can be probed after the cell is completely dried. A comparative study between viable and apoptotic cells indicates that most of the Au-NPs that enter the living cell reside in the cytoplasm and around the nucleus, whereas glyoxal-induced apoptotic cells show relatively uniform distribution of Au-NPs and, interestingly, the presence of DNA fragments is detected throughout the cell, including the cell surface.