76 research outputs found
Evaluation of dental pulp stem cell heterogeneity and behaviour in 3D type I collagen gels
Dental pulp stem cells (DPSCs) are increasingly being advocated for regenerative medicine-based therapies. However, significant heterogeneity in the genotypic/phenotypic properties of DPSC subpopulations exists, influencing their therapeutic potentials. As most studies have established DPSC heterogeneity using 2D culture approaches, we investigated whether heterogeneous DPSC proliferative and contraction/remodelling capabilities were further evident within 3D type I collagen gels in vitro. DPSC subpopulations were isolated from human third molars and identified as high/low proliferative and multipotent/unipotent, following in vitro culture expansion and population doubling (PD) analysis. High proliferative/multipotent DPSCs, such as A3 (30 PDs and 80 PDs), and low proliferative/unipotent DPSCs, such as A1 (17 PDs), were cultured in collagen gels for 12 days, either attached or detached from the surrounding culture plastic. Collagen architecture and high proliferative/multipotent DPSC morphologies were visualised by Scanning Electron Microscopy and FITC-phalloidin/Fluorescence Microscopy. DPSC proliferation (cell counts), contraction (% diameter reductions), and remodelling (MMP-2/MMP-9 gelatin zymography) of collagen gels were also evaluated. Unexpectedly, no proliferation differences existed between DPSCs, A3 (30 PDs) and A1 (17 PDs), although A3 (80 PDs) responses were significantly reduced. Despite rapid detached collagen gel contraction with A3 (30 PDs), similar contraction rates were determined with A1 (17 PDs), although A3 (80 PDs) contraction was significantly impaired. Gel contraction correlated to distinct gelatinase profiles. A3 (30 PDs) possessed superior MMP-9 and comparable MMP-2 activities to A1 (17 PDs), whereas A3 (80 PDs) had significantly reduced MMP-2/MMP-9. High proliferative/multipotent DPSCs, A3 (30 PDs), further exhibited fibroblast-like morphologies becoming polygonal within attached gels, whilst losing cytoskeletal organization and fibroblastic morphologies in detached gels. This study demonstrates that heterogeneity exists in the gel contraction and MMP expression/activity capabilities of DPSCs, potentially reflecting differences in their abilities to degrade biomaterial scaffolds and regulate cellular functions in 3D environments and their regenerative properties overall. Thus, such findings enhance our understanding of the molecular and phenotypic characteristics associated with high proliferative/multipotent DPSCs
Isolation and characterisation of mesenchymal stem cells from rat bone marrow and the endosteal niche: A comparative study
Within bone, mesenchymal stromal cells (MSCs) exist within the bone marrow stroma (BM-MSC) and the endosteal niche, as cells lining compact bone (CB-MSCs). This study isolated and characterised heterogeneous MSC populations from each niche and subsequently investigated the effects of extensive cell expansion, analysing population doublings (PDs)/cellular senescence, colony-forming efficiencies (CFEs), MSC cell marker expression, and osteogenic/adipogenic differentiation. CB-MSCs and BM-MSCs demonstrated similar morphologies and PDs, reaching 100 PDs. Both populations exhibited consistent telomere lengths (12–17 kb), minimal senescence, and positive telomerase expression. CB-MSCs (PD15) had significantly lower CFEs than PD50. CB-MSCs and BM-MSCs both expressed MSC (CD73/CD90/CD105); embryonic (Nanog) and osteogenic markers (Runx2, osteocalcin) but no hematopoietic markers (CD45). CB-MSCs (PD15) strongly expressed Oct4 and p16INK4A. At early PDs, CB-MSCs possessed a strong osteogenic potency and low potency for adipogenesis, whilst BM-MSCs possessed greater overall bipotentiality for osteogenesis and adipogenesis. At PD50, CB-MSCs demonstrated reduced potency for both osteogenesis and adipogenesis, compared to BM-MSCs at equivalent PDs. This study demonstrates similarities in proliferative and mesenchymal cell characteristics between CB-MSCs and BM-MSCs, but contrasting multipotentiality. Such findings support further comparisons of human CB-MSCs and BM-MSCs, facilitating selection of optimal MSC populations for regenerative medicine purposes
Evaluation of the in vitro oral wound healing effects of pomegranate (Punica granatum) rind extract and punicalagin, in combination with Zn (II)
Pomegranate (Punica granatum) is a well-established folklore medicine, demonstrating benefits in treating numerous conditions partly due to its antimicrobial and anti-inflammatory properties. Such desirable medicinal capabilities are attributed to a high hydrolysable tannin content, especially punicalagin. However, few studies have evaluated the abilities of pomegranate to promote oral healing, during situations such as periodontal disease or trauma. Therefore, this study evaluated the antioxidant and in vitro gingival wound healing effects of pomegranate rind extract (PRE) and punicalagin, alone and in combination with Zn (II). In vitro antioxidant activities were studied using DPPH and ABTS assays, with total PRE phenolic content measured by Folin–Ciocalteu assay. PRE, punicalagin and Zn (II) combination effects on human gingival fibroblast viability/proliferation and migration were investigated by MTT assay and scratch wounds, respectively. Punicalagin demonstrated superior antioxidant capacities to PRE, although Zn (II) exerted no additional influences. PRE, punicalagin and Zn (II) reduced gingival fibroblast viability and migration at high concentrations, but retained viability at lower concentrations without Zn (II). Fibroblast speed and distance travelled during migration were also enhanced by punicalagin with Zn (II) at low concentrations. Therefore, punicalagin in combination with Zn (II) may promote certain anti-inflammatory and fibroblast responses to aid oral healin
Dental Pulp Stem Cell Heterogeneity: Finding Superior Quality “Needles” in a Dental Pulpal “Haystack” for Regenerative Medicine-Based Applications
Human dental pulp stem/stromal cells (hDPSCs) derived from the permanent secondary dentition are recognised to possess certain advantageous traits, which support their potential use as a viable source of mesenchymal stem/stromal cells (MSCs) for regenerative medicine-based applications. However, the well-established heterogeneous nature of hDPSC subpopulations, coupled with their limited numbers within dental pulp tissues, has impeded our understanding of hDPSC biology and the translation of sufficient quantities of these cells from laboratory research, through successful therapy development and clinical applications. This article reviews our current understanding of hDPSC biology and the evidence underpinning the molecular basis of their heterogeneity, which may be exploited to distinguish individual subpopulations with specific or superior characteristics for regenerative medicine applications. Pertinent unanswered questions which still remain, regarding the developmental origins, hierarchical organisation, and stem cell niche locations of hDPSC subpopulations and their roles in hDPSC heterogeneity and functions, will further be explored. Ultimately, a greater understanding of how key features, such as specific cell surface, senescence and other relevant genes, and protein and metabolic markers, delineate between hDPSC subpopulations with contrasting stemness, proliferative, multipotency, immunomodulatory, anti-inflammatory, and other relevant properties is required. Such knowledge advancements will undoubtedly lead to the development of novel screening, isolation, and purification strategies, permitting the routine and effective identification, enrichment, and expansion of more desirable hDPSC subpopulations for regenerative medicine-based applications. Furthermore, such innovative measures could lead to improved cell expansion, manufacture, and banking procedures, thereby supporting the translational development of hDPSC-based therapies in the future
Differential SOD2 and GSTZ1 profiles contribute to contrasting dental pulp stem cell susceptibilities to oxidative damage and premature senescence
Background
Dental pulp stem cells (DPSCs) are increasingly being advocated as viable cell sources for regenerative medicine-based therapies. However, significant heterogeneity in DPSC expansion and multi-potency capabilities are well-established, attributed to contrasting telomere profiles and susceptibilities to replicative senescence. As DPSCs possess negligible human telomerase (hTERT) expression, we examined whether intrinsic differences in the susceptibilities of DPSC sub-populations to oxidative stress-induced biomolecular damage and premature senescence further contributed to this heterogeneity, via differential enzymic antioxidant capabilities between DPSCs.
Methods
DPSCs were isolated from human third molars by differential fibronectin adhesion, and positive mesenchymal (CD73/CD90/CD105) and negative hematopoietic (CD45) stem cell marker expression confirmed. Isolated sub-populations were expanded in H2O2 (0–200 μM) and established as high or low proliferative DPSCs, based on population doublings (PDs) and senescence (telomere lengths, SA-β-galactosidase, p53/p16INK4a/p21waf1/hTERT) marker detection. The impact of DPSC expansion on mesenchymal, embryonic, and neural crest marker expression was assessed, as were the susceptibilities of high and low proliferative DPSCs to oxidative DNA and protein damage by immunocytochemistry. Expression profiles for superoxide dismutases (SODs), catalase, and glutathione-related antioxidants were further compared between DPSC sub-populations by qRT-PCR, Western blotting and activity assays.
Results
High proliferative DPSCs underwent > 80PDs in culture and resisted H2O2−induced senescence (50–76PDs). In contrast, low proliferative sub-populations exhibited accelerated senescence (4–32PDs), even in untreated controls (11-34PDs). While telomere lengths were largely unaffected, certain stem cell marker expression declined with H2O2 treatment and expansion. Elevated senescence susceptibilities in low proliferative DPSC (2–10PDs) were accompanied by increased oxidative damage, absent in high proliferative DPSCs until 45–60PDs. Increased SOD2/glutathione S-transferase ζ1 (GSTZ1) expression and SOD activities were identified in high proliferative DPSCs (10–25PDs), which declined during expansion. Low proliferative DPSCs (2–10PDs) exhibited inferior SOD, catalase and glutathione-related antioxidant expression/activities.
Conclusions
Significant variations exist in the susceptibilities of DPSC sub-populations to oxidative damage and premature senescence, contributed to by differential SOD2 and GSTZ1 profiles which maintain senescence-resistance/stemness properties in high proliferative DPSCs. Identification of superior antioxidant properties in high proliferative DPSCs enhances our understanding of DPSC biology and senescence, which may be exploited for selective sub-population screening/isolation from dental pulp tissues for regenerative medicine-based applications
Variation in human dental pulp stem cell ageing profiles reflect contrasting proliferative and regenerative capabilities
Background
Dental pulp stem cells (DPSCs) are increasingly being recognized as a viable cell source for regenerative medicine. Although significant variations in their ex vivo expansion are well-established, DPSC proliferative heterogeneity remains poorly understood, despite such characteristics influencing their regenerative and therapeutic potential. This study assessed clonal human DPSC regenerative potential and the impact of cellular senescence on these responses, to better understand DPSC functional behaviour.
Results
All DPSCs were negative for hTERT. Whilst one DPSC population reached >80 PDs before senescence, other populations only achieved 60 PDs).
Conclusions
This study has identified that proliferative and regenerative heterogeneity is related to contrasting telomere lengths and CD271 expression between DPSC populations. These characteristics may ultimately be used to selectively screen and isolate high proliferative capacity/multi-potent DPSCs for regenerative medicine exploitation
Synergistic activity of pomegranate rind extract and Zn (II) against Candida albicans under planktonic and biofilm conditions, and a mechanistic insight based upon intracellular ROS induction
Candida albicans (C. albicans) is an opportunistic pathogen, which causes superficial infection and can lead to mortal systemic infections, especially in immunocompromised patients. The incidence of C. albicans infections is increasing and there are a limited number of antifungal drugs used in treatment. Therefore, there is an urgent need for new and alternative antifungal drugs. Pomegranate rind extract (PRE) is known for its broad-spectrum antimicrobial activities, including against C. albicans and recently, PRE and Zn (II) have been shown to induce synergistic antimicrobial activity against various microbes. In this study, the inhibitory activities of PRE, Zn (II) and PRE in combination with Zn (II) were evaluated against C. albicans. Antifungal activities of PRE and Zn (II) were evaluated using conventional microdilution methods and the interaction between these compounds was assessed by in vitro checkerboard and time kill assays in planktonic cultures. The anti-biofilm activities of PRE, Zn (II) and PRE in combination with Zn (II) were assessed using confocal laser scanning microscopy, with quantitative analysis of biofilm biomass and mean thickness analysed using COMSTAT2 analysis. In addition, antimicrobial interactions between PRE and Zn (II) were assayed in terms reactive oxygen species (ROS) production by C. albicans. PRE and Zn (II) showed a potent antifungal activity against C. albicans, with MIC values of 4 mg/mL and 1.8 mg/mL, respectively. PRE and Zn (II) in combination exerted a synergistic antifungal effect, as confirmed by the checkerboard and time kill assays. PRE, Zn (II) and PRE and Zn (II) in combination gave rise to significant reductions in biofilm biomass, although only PRE caused a significant reduction in mean biofilm thickness. The PRE and Zn (II) in combination caused the highest levels of ROS production by C. albicans, in both planktonic and biofilm forms. The induction of excess ROS accumulation in C. albicans may help explain the synergistic activity of PRE and Zn (II) in combination against C. albicans in both planktonic and biofilm forms. Moreover, the data support the potential of the PRE and Zn (II) combination as a novel potential anti-Candida therapeutic system
Synergistic in vitro antimicrobial activity of pomegranate rind extract and zinc (II) against Micrococcus luteus under planktonic and biofilm conditions
Infectious diseases caused by microbial biofilms are a major clinical problem, and new antimicrobial agents that can inhibit biofilm formation and eradicate pre-formed biofilms are urgently needed. Pomegranate extracts are a well-established folkloric medicine and have been used in the treatment of infectious diseases since ancient times, whilst the addition of metal ions, including zinc (II), has enhanced the antimicrobial activity of pomegranate. Micrococcus luteus is generally a non-pathogenic skin commensal bacterium, although it can act as an opportunistic pathogen and cause serious infections, particularly involving catheterization and comorbidities. The aims of this study were to evaluate the holistic activity of pomegranate rind extract (PRE), Zn (II), and PRE/Zn (II) individually and in combination against M. luteus under both planktonic and biofilm conditions. Antimicrobial activity was detected in vitro using the broth dilution method, and synergistic activity was determined using checkerboard and time-kill assays. Effects on biofilm formation and eradication were determined by crystal violet and BacLightTM Live/Dead staining. PRE and Zn (II) exerted antimicrobial activity against M. luteus under both planktonic and biofilm conditions. After 4 h, potent synergistic bactericidal activity was also found when PRE and Zn (II) were co-administered under planktonic conditions (log reductions: PRE 1.83 ± 0.24, Zn (II) 3.4 ± 0.08, and PRE/Zn (II) 6.88 ± 1.02; p < 0.0001). In addition, greater heterogeneity was induced in the structure of M. luteus biofilm using the PRE/Zn (II) combination compared to when PRE and Zn (II) were applied individually. The activity of PRE and the PRE/Zn (II) combination could offer a novel antimicrobial therapy for the treatment of disease-associated infections caused by M. luteus and potentially other bacteria
Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: Consequences for bone repair during periodontal disease
A prime pathogenic agent associated with periodontitis is lipopolysaccharide (LPS) derived from Porphyromonas gingivalis. This study investigated the effects of P. gingivalis LPS on osteoblasts, which are responsible for alveolar bone repair. Bone cells were obtained from explants of rat alveolar bone chips and cultured with 0-200 ng ml-1 of P. gingivalis LPS. Porphyromonas gingivalis LPS significantly increased cell proliferation and inhibited osteoblast differentiation, as judged by reduced alkaline phosphatase activity. Analysis of biglycan mRNA and protein levels indicated that P. gingivalis LPS significantly delayed the normally high expression of biglycan during the early stages of culture, which are associated with cell proliferation and early differentiation of progenitor cells. In the presence of P. gingivalis LPS, decorin expression by the alveolar bone cells was reduced during periods of culture relating to collagen fibrillogenesis and mineral deposition. Analysis of glycosaminoglycan chains conjugated to these proteoglycans suggested that in the presence of P. gingivalis LPS, dermatan sulfate persisted within the matrix. This study suggests that P. gingivalis LPS influences the expression and processing of decorin and biglycan in the matrix, altering alveolar bone cell activity and osteoblast phenotype development. The consequences of this altered expression in relation to hindering bone repair as part of the cycle of events during periodontal disease are discussed. © 2008 Eur J Oral Sci
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