scholarly journals New Application of Osteogenic Differentiation from HiPS Stem Cells for Evaluating the Osteogenic Potential of Nanomaterials in Dentistry

Author(s):  
Giulia Tetè ◽  
Paolo Capparè ◽  
Enrico Gherlone

Objective: HiPS stem cells are commonly used for the study of medical disorders. The laboratory in which this study was conducted uses these cells for examining the treatment and cure of neurodegenerative diseases. Bone regeneration poses the greatest challenge for an oral surgeon both in terms of increased implant osseointegration and reducing bone healing times. The aim of this study was to validate the protocol in the literature to produce and then test in vitro osteoblasts with different nanomaterials to simulate bone regeneration. Method: hiPS clones (#2, #4, and #8) were differentiated into an osteoblast cell culture tested for alizarin red staining and for alkaline phosphatase testing at 14, 21 and 28 days, after the cells were plated. Results: The cells showed diffuse positivity under alizarin red staining and the alkaline phosphatase (ALP)-test, showing small formations of calcium clusters. Conclusion: Despite the limitations of our study, it is a starting point for further protocols, laying a solid foundation for research in the field of bone regeneration through the use of stem cells.

2020 ◽  
Author(s):  
Yi Zhao ◽  
Qiaoli Zhai ◽  
Hong Liu ◽  
Xun Xi ◽  
Shuai Chen ◽  
...  

Abstract BackgroundPeriodontal disease is a common disease that compromises the integrity of tooth-supporting tissues. Bone regeneration is the ultimate goal of periodontal therapies, in which osteogenic differentiation of human periodontal ligament stem cells plays a critical role. The tripartite motif (TRIM)16 is downregulated in periodontal tissues of patients with periodontitis and involved in osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs).However, the role of TRIM16 in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is largely unknown.MethodshPDLSCs were isolated and identified by immunophenotype assays using flow cytometry. Overexpression plasmids and specific short-hairpin RNAs (shRNAs) were constructed to manipulate the expression of target molecules. Alkaline phosphatase (ALP) staining, alizarin red staining (ARS) and enzyme‐linked immunosorbent assays (ELISA) were used to evaluate osteogenic potential capacity. Reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis were performed to determine the expression of osteogenic-related markers and activation of relevant signaling pathways. Co-immunoprecipitation assays were performed to confirm the interactions between proteins and the ubiquitination of RUNX2. A LC-MS/MS analysis was performed to explore the different expression proteins in present of TRIM16.ResultsTRIM16 significantly promoted alkaline phosphatase activity and mineralized nodule formation, and positively regulated the osteogenic differentiation of hPDLSCs by enhancing protein expression of RUNX2, COL1A1 and OCN. Mechanistically, TRIM16 serves as a pivotal factor that stabilizes RUNX2 protein levels by decreasing CHIP-mediated K48-linked ubiquitination degradation of the RUNX2 protein. Besides, TRIM16 significantly increased expression of COL1A1 via activation of p38MAPK/RUNX2.ConclusionThis study identified a novel mechanism of TRIM16 in regulating stability of the RUNX2 protein, which may promote the osteogenic differentiation of hPDLSCs. TRIM16 may be a potential target of stem cell based-bone regeneration for periodontal therapies.


2020 ◽  
Author(s):  
Yejia Yu ◽  
Mengyu Li ◽  
Yuqiong Zhou ◽  
Yueqi Shi ◽  
Wenjie Zhang ◽  
...  

Abstract Background: Dentigerous cyst (DC) is a bone destructive disease and remains a challenge for clinicians. Marsupialization enables bone to regenerate with capsules maintaining, making it a preferred therapeutic means for DC adjacent to vital anatomical structures. Given that capsules of DC derive from odontogenic epithelium remnants at embryonic stage, we investigated whether there were mesenchymal stem cells (MSCs) located in DC capsules and the role that they played in the bone regeneration after marsupialization.Methods: Samples obtained before and after marsupialization were used for histological detection and cell culture. The stemness of cells isolated from fresh tissues were analyzed by morphology, surface marker and multi-differentiation assays. Comparison of proliferation ability between Am-DCSCs and Bm-DCSCs were evaluated by Cell Counting Kit-8 (CCK-8), fibroblast colony-forming units (CFU-F) and 5’‐ethynyl‐2’‐deoxyuridine (EdU) assay. Their osteogenic capacity in vitro was detected by Alkaline phosphatase (ALP) and Alizarin Red staining (ARS), combined with Real-time polymerase chain reaction (RT-PCR) and immunofluorescence (IF) staining. Subcutaneous ectopic osteogenesis as well as cranial bone defect model in nude mice were performed to detect their bone regeneration and bone defect repair ability.Results: Bone tissue and strong ALP activity were detected in the capsule of DC after marsupialization. Two types of MSCs were isolated from fibrous capsules of DC both before (Bm-DCSCs) and after (Am-DCSCs) marsupialization. These fibroblast-like, colony forming cells expressed MSC markers (CD44+, CD90+, CD31-, CD34-, CD45-), and they could differentiate into osteoblast-, adipocyte- and chondrocyte-like cells under induction. Notably, Am-DCSCs performed better in cell proliferation and self-renewal. Moreover, Am-DCSCs showed greater osteogenic capacity both in vitro and in vivo compared with Bm-DCSCs. Conclusions: There are MSCs residing in capsules of DC, and the cell viability as well as osteogenic capacity of them are largely enhanced after marsupialization. Our findings suggested that MSCs might play a crucial role in the healing process of DC after marsupialization, thus providing new insight into the treatment for DC by promoting the osteogenic differentiation of MSCs inside capsules.


2008 ◽  
Vol 20 (1) ◽  
pp. 223
Author(s):  
A. Lima ◽  
E. Monaco ◽  
S. Wilson ◽  
D. Kim ◽  
C. Feltrin ◽  
...  

The quantity and accessibility of subcutaneous adipose tissue in humans make it an attractive alternative to bone marrow as a source of adult stem cells for therapeutic purposes. However, before such a cell source substitution can be proposed, the properties of stem cells derived from adipose (ADSCs) and bone marrow (MSCs) and their differentiated progeny must be compared in an animal model that adequately simulates the structure and physiology of humans. The objective of this work was to induce adult porcine stem cells isolated from subcutaneous adipose tissue and bone marrow to differentiate in vitro along the osteoblastic lineage and to compare their morphological, phenotypic, and genotypic properties. MSCs and ADSCs were isolated respectively from femurs and subcutaneous adipose tissue of adult pigs and cultured in vitro using DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin G-streptomycin, and 5.6 mg L–1 amphotericin B. After 3 passages, cells were differentiated along the osteogenic lineage using lineage-specific inducing medium. Osteogenic medium contained 100 nm dexamethasone, 10 mm β-glycerophosphate, and 0.005 mm ascorbic acid-2-phosphate. Osteogenic cultures were incubated for 4 weeks in 95% air and 5% CO2 at 39�C. Spent medium was replaced with fresh medium every 3 days. Histological staining with alkaline phosphatase, Von Kossa, and alizarin red S were performed at 0, 2, 4, 7, 14, 21, and 28 days of differentiation (dd). At the same time points, RNA was extracted. qPCR was performed on COL1A1, BGLAP, SPARC, and SPP1. As internal control, the geometrical mean of GTF2H, NUBP, and PPP2C was used. Relative mRNA abundance between cell types was calculated using 1/efficiencydCT. The osteogenic differentiation of both MSCs and ADScs was confirmed by the organization of the cells in nodules and by alkaline phosphatase-, Von Kossa-, and alizarin red S-positive staining. The percent relative abundance of the 4 genes in both cell types was COL1A1 (ca. 50) > SPARC (ca. 45) > SPP1 (ca. 5) > BGLAP ( < 0.1). Cell types showed similar mRNA abundance for COL1A1 and SPARC while SPP1 and BGLAP were, respectively, 10- and 19-fold higher in MSCs than in ADSCs. All of the genes had the same pattern among tissues during differentiation except for SPP1, which showed a >10-fold increase at 14 v. 0 dd only for MSCs. Adipose-derived stem cells demonstrated a clear osteogenic differentiation and similar expression and pattern of the two osteogenic genes most abundant in MSCs (COL1A1 and SPARC). However, the higher abundance of SPP1 and BGLAP and the different behavior of SPP1 in MSCs suggest a different transcription profile between the two cell types. From these preliminary results, adipose tissue can be a practical alternative source for stem cells in future human clinical applications.


2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yejia Yu ◽  
Mengyu Li ◽  
Yuqiong Zhou ◽  
Yueqi Shi ◽  
Wenjie Zhang ◽  
...  

Abstract Background Dentigerous cyst (DC) is a bone destructive disease and remains a challenge for clinicians. Marsupialization enables the bone to regenerate with capsule maintaining, making it a preferred therapeutic means for DC adjacent to vital anatomical structures. Given that capsules of DC are derived from odontogenic epithelium remnants at the embryonic stage, we investigated whether there were mesenchymal stem cells (MSCs) located in DC capsules and the role that they played in the bone regeneration after marsupialization. Methods Samples obtained before and after marsupialization were used for histological detection and cell culture. The stemness of cells isolated from fresh tissues was analyzed by morphology, surface marker, and multi-differentiation assays. Comparison of proliferation ability between MSCs isolated from DC capsules before (Bm-DCSCs) and after (Am-DCSCs) marsupialization was evaluated by Cell Counting Kit-8 (CCK-8), fibroblast colony-forming units (CFU-F), and 5′-ethynyl-2′-deoxyuridine (EdU) assay. Their osteogenic capacity in vitro was detected by alkaline phosphatase (ALP) and Alizarin Red staining (ARS), combined with real-time polymerase chain reaction (RT-PCR) and immunofluorescence (IF) staining. Subcutaneous ectopic osteogenesis as well as cranial bone defect model in nude mice was performed to detect their bone regeneration and bone defect repairability. Results Bone tissue and strong ALP activity were detected in the capsule of DC after marsupialization. Two types of MSCs were isolated from fibrous capsules of DC both before (Bm-DCSCs) and after (Am-DCSCs) marsupialization. These fibroblast-like, colony-forming cells expressed MSC markers (CD44+, CD90+, CD31−, CD34−, CD45−), and they could differentiate into osteoblast-, adipocyte-, and chondrocyte-like cells under induction. Notably, Am-DCSCs performed better in cell proliferation and self-renewal. Moreover, Am-DCSCs showed a greater osteogenic capacity both in vitro and in vivo compared with Bm-DCSCs. Conclusions There are MSCs residing in capsules of DC, and the cell viability as well as the osteogenic capacity of them is largely enhanced after marsupialization. Our findings suggested that MSCs might play a crucial role in the healing process of DC after marsupialization, thus providing new insight into the treatment for DC by promoting the osteogenic differentiation of MSCs inside capsules.


2014 ◽  
Vol 1621 ◽  
pp. 71-79 ◽  
Author(s):  
Pelagie Favi ◽  
Madhu Dhar ◽  
Nancy Neilsen ◽  
Roberto Benson

ABSTRACTAdvanced biomaterials that mimic the structure and function of native tissues and permit stem cells to adhere and differentiate is of paramount importance in the development of stem cell therapies for bone defects. Successful bone repair approaches may include an osteoconductive scaffold that permits excellent cell adhesion and proliferation, and cells with an osteogenic potential. The objective of this study was to evaluate the cell proliferation, viability and osteocyte differentiation of equine-derived bone marrow mesenchymal stem cells (EqMSCs) when seeded onto biocompatible and biodegradable calcium-deficient hydroxyapatite (CdHA) tubular-shaped bacterial cellulose scaffolds (BC-TS) of various sizes. The biocompatible gel-like BC-TS was synthesized using the bacterium Gluconacetobacter sucrofermentans under static culture in oxygen-permeable silicone tubes. The BC-TS scaffolds were modified using a periodate oxidation to yield biodegradable scaffolds. Additionally, CdHA was deposited in the scaffolds to mimic native bone tissues. The morphological properties of the resulting BC-TS and its composites were characterized using scanning electron microscopy. The ability of the BC-TS and its composites to support and maintain EqMSCs growth, proliferation and osteogenic differentiation in vitro was also assessed. BC-TS and its composites exhibited aligned nanofibril structures. MTS assay demonstrated increasing proliferation and viability with time (days 1, 2 and 3). Cell-scaffold constructs were cultured for 8 days under osteogenic conditions and the resulting osteocytes were positive for alizarin red. In summary, biocompatible and biodegradable CdHA BC-TS composites support the proliferation, viability and osteogenic differentiation of EqMSCs cultured onto its surface in vitro, allowing for future potential use for tissue engineering therapies.


2020 ◽  
Author(s):  
Yejia Yu ◽  
Mengyu Li ◽  
Yuqiong Zhou ◽  
Yueqi Shi ◽  
Wenjie Zhang ◽  
...  

Abstract Background: Dentigerous cyst (DC) is a bone destructive disease and remains a challenge for clinicians. Marsupialization enables bone to regenerate with capsules maintaining, making it a preferred therapeutic means for DC adjacent to vital anatomical structures. Given that capsules of DC derive from odontogenic epithelium remnants at embryonic stage, we investigated whether there were mesenchymal stem cells (MSCs) located in DC capsules and the role that they played in the bone regeneration after marsupialization. Methods: Samples obtained before and after marsupialization were used for histological detection and cell culture. The stemness of cells isolated from fresh tissues were analyzed by morphology, surface marker and multi-differentiation assays. Comparison of proliferation ability between Am-DCSCs and Bm-DCSCs were evaluated by Cell Counting Kit-8 (CCK-8), fibroblast colony-forming units (CFU-F) and 5’‐ethynyl‐2’‐deoxyuridine (EdU) assay. Their osteogenic capacity in vitro was detected by Alkaline phosphatase (ALP) and Alizarin Red staining (ARS), combined with Real-time polymerase chain reaction (RT-PCR) and immunofluorescence (IF) staining. Subcutaneous ectopic osteogenesis as well as cranial bone defect model in nude mice were performed to detect their bone regeneration and bone defect repair ability. Results: Bone tissue and strong ALP activity were detected in the capsule of DC after marsupialization. Two types of MSCs were isolated from fibrous capsules of DC both before (Bm-DCSCs) and after (Am-DCSCs) marsupialization. These fibroblast-like, colony forming cells expressed MSC markers (CD44+, CD90+, CD31-, CD34-, CD45-), and they could differentiate into osteoblast-, adipocyte- and chondrocyte-like cells under induction. Notably, Am-DCSCs performed better in cell proliferation and self-renewal. Moreover, Am-DCSCs showed greater osteogenic capacity both in vitro and in vivo compared with Bm-DCSCs. Conclusions: There are MSCs residing in capsules of DC, and the cell viability as well as osteogenic capacity of them are largely enhanced after marsupialization. Our findings suggested that MSCs might play a crucial role in the healing process of DC after marsupialization, thus providing new insight into the treatment for DC by promoting the osteogenic differentiation of MSCs inside capsules.


2020 ◽  
Vol 3 (3) ◽  
pp. 267-278
Author(s):  
Alan Jesus ◽  
Adriano Jesus ◽  
Flávia Lima ◽  
Luiz Freitas ◽  
Cássio Meira ◽  
...  

Autogenous bone grafting is needed in some bone tissue defects; however, it causes secondary surgical wounds and morbidity. Tissue bioengineering may be an alternative approach for bone regeneration. Here we investigated the osteogenic potential of dental pulp stem cells from deciduous teeth (DPSC) in association with a Ricinus bone compound (RBC) in a model of bone defect. The influence of the biomaterial RBC on the proliferation and osteogenic differentiation of DPSC was assessed in vitro by MTT metabolism and alizarin red staining, respectively. The morphologic analysis was performed using the optic and scanning electron (SEM) microscopies. For the in vivo study, 54 Wistar rats submitted to calvarial defects were filled with RBC or RBC+DPSC. A control group had the defects filled only with blood clots. Analyses were performed 15, 30 and 60 days after treatment using digital radiography, optical microscopy, SEM and chemical analysis by electron dispersive spectroscopy. The Ricinus bone compound (RBC) did not inhibit the osteogenic differentiation in vitro. No spontaneous regeneration was observed in the control group. The area of the calvarial defect of the RBC+DPSC group showed greater radiopacity on day 15. The RBC presented no reabsorption, was biocompatible and showed osteointegration, working as a mechanical filling. Only sparse ossification areas were found and those were larger and more developed on the RBC+DPSC group when compared to animals treated only with RBC. RBC in association with DPSC is a promising combination for applications in bone regeneration.  


2021 ◽  
Author(s):  
Tianli Wu ◽  
Zhihao Yao ◽  
Gang Tao ◽  
Fangzhi Lou ◽  
Hui Tang ◽  
...  

Abstract Objective: Although it has been demonstrated that adipose-derived stem cells (ASCs) from osteoporosis mice (OP-ASCs) exhibit impaired osteogenic differentiation potential, the molecular mechanism has not yet been elucidated. We found that Fzd6 was decreased in OP-ASCs compared with ASCs. This study investigates the effects and underlying mechanisms of Fzd6 in the osteogenic potential of OP-ASCs. Methods: Fzd6 expression in ASCs and OP-ASCs was measured by PCR gene chip. Fzd6 overexpression and silencing lentiviruses were used to evaluate the role of Fzd6 in the osteogenic differentiation of OP-ASCs. Real-time PCR (qPCR) and western blotting (WB) was performed to detect the expression of Fzd6 and bone-related molecules, including runt-related transcription factor 2 (Runx2) and osteopontin (Opn). Alizarin red staining and Alkaline phosphatase (ALP) staining was performed following osteogenic induction. Microscopic CT (Micro-CT), hematoxylin and eosin staining (H&E) staining, and Masson staining were used to assess the role of Fzd6 in osteogenic differentiation of osteoporosis (OP) mice in vivo.Results: Expression of Fzd6 was decreased significantly in OP-ASCs. Fzd6 silencing down-regulated the osteogenic ability of OP-ASCs in vitro. Overexpression of Fzd6 rescued the impaired osteogenic capacity in OP-ASCs in vitro. We obtained similar results in vivo.Conclusions: Fzd6 plays an important role in regulating the osteogenic ability of OP-ASCs both in vivo and in vitro. Overexpression of Fzd6 associated with the Wnt signaling pathway promotes the osteogenic ability of OP-ASCs, which provides new insights for the prevention and treatment of OP.


2020 ◽  
Vol 72 (5) ◽  
pp. 715-730 ◽  
Author(s):  
Yamuna Mohanram ◽  
Jingying Zhang ◽  
Eleftherios Tsiridis ◽  
Xuebin B. Yang

Abstract 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.


Author(s):  
H. A. Zhernasechanka ◽  
Ya. I. Isaikina ◽  
T. V. Filipovich ◽  
E. G. Liakh

Mesenchymal stem cells (MSC) represent a perspective resource for cell biotechnology. However the question of chondrogenic and osteogenic capacity of MSC of different origin remains under study.The aim of this study was to analyze the osteo-chondrogenic differentiation potential of MSC obtained from the bone marrow and placenta. The results of our studies have indicated that bone marrow-derived and placenta-derived MSC showed a chondrogenic potential in vitro after a chondrogenic induction with specific differentiation media. But for bone marrowderived MSC, the chondrogenic program was realized by expression of collagens (Coll2, Coll10), while in placenta-derived MSC cultures we found a progressive increase in COMP and Ver expression, so bone marrow-derived MSC is more preferable for use in cartilage tissue engineering. Regarding the results on alkaline phosphatase and alizarin red staining, bone marrowderived MSC showed a more significant osteogenic potential compared to placenta-derived MSC. Bone marrow-derived MSC in the composition of fibrin gel after osteogenic induction on the 14th day exhibited the activity of alkaline phosphatase, calcium depositions inside the cells and extracellular matrix, the increase in Sp7 and DMP expression.


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