Comparing the Osteogenic Potential and Bone Regeneration Capacities of Dedifferentiated Fat Cells and Adipose-Derived Stem Cells In Vitro and In Vivo: Application of DFAT Cells Isolated by a Mesh Method

Background: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). Method: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. Results: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture. Conclusion: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies.

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Role of Fzd6 in Regulating the Osteogenic Differentiation of Adipose-derived Stem Cells in Osteoporosis

10.21203/rs.3.rs-328417/v1 ◽

2021 ◽

Author(s):

Tianli Wu ◽

Zhihao Yao ◽

Gang Tao ◽

Fangzhi Lou ◽

Hui Tang ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Wnt Signaling Pathway ◽

Osteogenic Potential ◽

Adipose Derived Stem Cells ◽

Alizarin Red ◽

Differentiation Potential

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.

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188 The Effect of Vitamin B12 on the Differentiation of Adipose-Derived Stem Cells into Osteoblasts

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab188 ◽

2018 ◽

Vol 30 (1) ◽

pp. 234

Author(s):

T. A. Bane ◽

J. C. Bertels ◽

K. M. Polkoff ◽

M. Rubessa ◽

M. B. Wheeler

Keyword(s):

Stem Cells ◽

Vitamin B12 ◽

Difficult Problem ◽

Negative Control ◽

Nodule Formation ◽

Adipose Derived Stem Cells ◽

Osteogenic Medium ◽

Alizarin Red ◽

Α Level

Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate if the osteogenic medium infused with vitamin B12 influences the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin B12 has been shown to have a stimulatory effect on osteoclastogenesis in vitro (Vaes et al. 2009 Calcified Tissue Int. 84, 413-422). Our hypothesis was that the presence of vitamin B12 in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). The ASC were divided in 8 different treatments: 8 concentrations of vitamin B12 in the osteogenic medium (0.1, 0.2, 1, 2, 10, and 20 μM) plus 2 control treatments (osteogenic medium without vitamin B12 and a negative control, DMEM). The medium was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red and Von Kossa stains. In each well, we counted the nodules and then divided them in 2 categories: formed and forming nodules. Data was analysed using the generalized linear model (GLM) procedure in SPSS (IBM/SPSS, Armonk, NY, USA). Bonferroni’s post hoc test was used to perform statistical multiple comparison. The α-level was set at 0.01. The results showed that the concentration of 20 μM vitamin B12 was detrimental for nodule formation. Table 1 illustrates the number of formed and forming nodules in addition to their standard deviation. There was no positive effect on nodule formation when different concentrations of vitamin B12 were added to the osteogenic medium. More experiments need to be conducted to determine if vitamin B12 can act synergistically with other vitamins to produce a significant role in ASC differentiation into osteoblasts. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with vitamin B12 incorporated into their matrix. Table 1.The average number of formed and forming osteoblast nodules compared between treatment groups (SD in parentheses)

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Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells

Indian Journal of Plastic Surgery ◽

10.1055/s-0039-1699220 ◽

2008 ◽

Vol 41 (01) ◽

pp. 08-14 ◽

Cited By ~ 1

Author(s):

Arash Zaminy ◽

Iraj Ragerdi Kashani ◽

Mohammad Barbarestani ◽

Azim Hedayatpour ◽

Reza Mahmoudi ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Culture Media ◽

Adipose Derived Stem Cells ◽

Osteogenic Medium ◽

Adult Rats ◽

Alizarin Red ◽

Alp Activity ◽

Proliferation And Differentiation

ABSTRACT Background: Osteogenesis driven by adipose-derived stem cells (ADSCs) is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM) could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs.Materials and Methods: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM). After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and flow cytometry, respectively.Results: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level) also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased.Conclusion: These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.

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197 THE EFFECT OF ZINC ON THE DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INTO OSTEOBLASTS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab197 ◽

2017 ◽

Vol 29 (1) ◽

pp. 207 ◽

Cited By ~ 1

Author(s):

J. C. Bertels ◽

M. Rubessa ◽

S.R. Schreiber ◽

M. B. Wheeler

Keyword(s):

Stem Cells ◽

Bone Formation ◽

Negative Control ◽

Nodule Formation ◽

Adipose Derived Stem Cells ◽

Osteogenic Medium ◽

Alizarin Red ◽

The Media ◽

Positive Effect

The aim of this project was to evaluate the effects of zinc in osteogenic media and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Zinc has a stimulatory effect on bone formation and mineralization in vivo and vitro (Seo et al. 2010 Nutr. Res. Pract. 4, 356–361). Our hypothesis was that the presence of zinc in the osteogenic media would positively influence both the speed of formation and the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided in 8 different treatments: 6 different concentrations of zinc in the osteogenic medium (8, 4, 0.8, 0.4, 0.08, and 0.04 mM) plus 2 control treatments (osteogenic medium without zinc and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 4 times. At the end of the culture period, cells were stained with Alizarin Red S. In each well, we counted the nodules and divided them in 2 categories: formed and forming nodules. The second evaluation that we did was to evaluate the diameter of the largest nodules (2/well) in each group. Data were analysed by ANOVA using the Generalized Linear Model procedure (SPSS, IBM Corp., Armonk, NY, USA). Bonferroni’s post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that the doses of zinc of both 4 and 8 mM were toxic to the whole cell populations in this treatment, which was indicated by cell death, whereas the concentrations of 0.8 and 0.4 mM were not cytotoxic but no nodules formed. Here we report the results that are greater than zero in Table 1. There is a positive effect on nodule formation when the zinc is added to the media. It is clear that the total number of nodules is different between the 0.08 mM zinc group and the control (P < 0.003). When we evaluated nodule diameter we found a direct correlation between the zinc concentration and the diameter of the nodules: 292.7 (±136.6) v. 366.8 (±218.7) v. 423.7 (±267.7) µm for the control, 0.04 mM zinc, and 0.08 mM zinc, respectively. The largest nodule was found in the 0.08 mM zinc treatment at 886.6 µm. These results confirmed the positive effect of this mineral on bone formation. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with zinc incorporated into their matrix. Table 1. The average number (SD in parentheses) of formed and forming osteoblast nodules compared between treatment groups

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Osteogenic potential of human adipose derived stem cells (hASCs) seeded on titanium trabecular spinal cages

Scientific Reports ◽

10.1038/s41598-020-75385-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Laura Caliogna ◽

Valentina Bina ◽

Laura Botta ◽

Francesco Maria Benazzo ◽

Marta Medetti ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Bone Structure ◽

Bone Matrix ◽

In Vitro Study ◽

In Vivo Studies ◽

Osteogenic Potential ◽

Osteogenic Medium

Abstract Spine degenerative conditions are becoming increasingly prevalent, affecting about 5.7% of the population in Europe, resulting in a significant reduction of life’s quality. Up to now, many materials have been used in manufacturing cage implants, used as graft substitutes, to achieve immediate and long-term spinal fixation. Particularly, titanium and its alloys are emerging as valuable candidates to develop new types of cages. The aim of this in vitro study was to evaluate the adhesion, proliferation and osteogenic differentiation of adipose derived mesenchymal stem cells (ASCs) seeded on trabecular titanium cages. ASCs adhered, proliferated and produced an abundant extracellular matrix during the 3 weeks of culture. In the presence of osteogenic medium, ASCs differentiated into osteoblast-like cells: the expression of typical bone genes, as well as the alkaline phosphatase activity, was statistically higher than in controls. Furthermore, the dispersive spectrometry microanalysis showed a marked increase of calcium level in cells grown in osteogenic medium. Plus, our preliminary data about osteoinduction suggest that this titanium implant has the potential to induce the ASCs to produce a secretome able to trigger a shift in the ASCs phenotype, possibly towards the osteogenic differentiation, as illustrated by the qRT-PCR and ALP biochemical assay results. The trabecular porous organization of these cages is rather similar to the cancellous bone structure, thus allowing the bone matrix to colonize it efficiently; for these reasons we can conclude that the architecture of this cage may play a role in modulating the osteoinductive capabilities of the implant, thus encouraging its engagement in in vivo studies for the treatment of spinal deformities and diseases.

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186 The effects of different concentrations of MgSO4 in osteogenic differentiation

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab186 ◽

2019 ◽

Vol 31 (1) ◽

pp. 217

Author(s):

L. R. Padoveze ◽

M. Rubessa ◽

C. E. Ambrosio ◽

M. B. Wheeler

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

In Vitro Study ◽

Control Group ◽

Nodule Formation ◽

Osteogenic Medium ◽

Alizarin Red ◽

Physiological Processes

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. Magnesium-based materials are biocompatible in vivo, and it is possible to determine the degradation period according to the necessities (Farraro et al. 2014 J. Biomech. 47, 1979-1986). Magnesium (Mg) is part of many physiological processes, and it promotes the osteogenesis of mesenchymal stem cells (Díaz-Tocados et al. 2017 Sci. Rep. 7, 7839.). Moreover, Mg up-regulates important genes associated with the osteogenic differentiation (Yoshizawa et al. 2014 Acta Biomater. 10, 2834-2842). The aim of this study was to evaluate the effect of different Mg concentrations in the osteogenic medium on the number of nodules of bone. Swine adipose stem cells (ASC) were previously isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). In this in vitro study, ASC were cultured during 4 weeks in osteogenic medium with addition of 0.1, 0.2, 1, 2, 10, or 20mM MgSO4. The medium was changed twice a week. Alizarin Red and Von Kossa staining were performed to evaluate the formation of nodules by mineralization of extracellular matrix (ECM), evidenced by dark red nodules and calcium deposit. The experiment was replicated 3 times in triplicate. Data were analysed using the generalized linear model (GLM) procedure, and Bonferroni’s post hoc test was used to perform statistical multiple comparison (SPSS Inc./IBM Corp., Chicago, IL, USA). The results showed enhanced nodule formation with 2mM Mg in the osteogenic medium (35.6v. 15.3, respectively for 2mM and Control). This result confirms the ability of magnesium to act in bone formation. There was no statistical difference among the different groups when we evaluated the Von Kossa staining results, indicating that the quality of the new formations was comparable to that of the control group even in an elevated nodule formation. In conclusion, a higher concentration of magnesium can improve nodule formation into osteogenic differentiation in vitro; the 2mM concentration showed the best nodule formation compared with the other groups. These results showed the value of magnesium in bone physiology.

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Stamina-Enhancing Effects of Human Adipose-Derived Stem Cells

Cell Transplantation ◽

10.1177/09636897211035409 ◽

2021 ◽

Vol 30 ◽

pp. 096368972110354

Author(s):

Eun-Jung Yoon ◽

Hye Rim Seong ◽

Jangbeen Kyung ◽

Dajeong Kim ◽

Sangryong Park ◽

...

Keyword(s):

Physical Activity ◽

Stem Cells ◽

Locomotor Activity ◽

Tissue Injury ◽

Male Rats ◽

Adipose Derived Stem Cells ◽

Sprague Dawley ◽

Serum Triglycerides

Stamina-enhancing effects of human adipose derived stem cells (hADSCs) were investigated in young Sprague-Dawley rats. Ten-day-old male rats were transplanted intravenously (IV) or intracerebroventricularly (ICV) with hADSCs (1 × 106 cells/rat), and physical activity was measured by locomotor activity and rota-rod performance at post-natal day (PND) 14, 20, 30, and 40, as well as a forced swimming test at PND 41. hADSCs injection increased the moving time in locomotor activity, the latency in rota-rod performance, and the maximum swimming time. For the improvement of physical activity, ICV transplantation was superior to IV injection. In biochemical analyses, ICV transplantation of hADSCs markedly reduced serum creatine phosphokinase, lactate dehydrogenase, alanine transaminase, and muscular lipid peroxidation, the markers for muscular and hepatic injuries, despite the reduction in muscular glycogen and serum triglycerides as energy sources. Notably, hADSCs secreted brain-derived neurotrophic factor (BDNF) and nerve growth factor in vitro, and increased the level of BDNF in the brain and muscles in vivo. The results indicate that hADSCs enhance physical activity including stamina not only by attenuating tissue injury, but also by strengthening the muscles via production of BDNF.

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High glucose induces early senescence in adipose-derived stem cells by accelerating p16 and mTOR

Biomedical Research and Therapy ◽

10.15419/bmrat.v6i6.548 ◽

2019 ◽

Vol 6 (6) ◽

pp. 3213-3221

Author(s):

Hieu Liem Pham ◽

Phuc Van Pham

Keyword(s):

Stem Cells ◽

High Glucose ◽

Glucose Concentration ◽

Culture Medium ◽

Diabetic Patients ◽

Adipose Derived Stem Cells ◽

Differentiation Potential ◽

Normal Glucose

Introduction: The senescence of stem cells is the primary reason that causes aging of stem cell-containing tissues. Some hypotheses have suggested that high glucose concentration in diabetic patients is the main factor that causes senescence of cells in those patients. This study aimed to evaluate the effects of high glucose concentrations on the senescence of adipose-derived stem cells (ADSCs). Methods: ADSCs were isolated and expanded from human adipose tissues. They were characterized and confirmed as mesenchymal stem cells (MSCs) by expression of surface markers, their shape, and in vitro differentiation potential. They were then cultured in 3 different media- that contained 17.5 mM, 35 mM, or 55 mM of D-glucose. The senescent status of ADSCs was recorded by the expression of the enzyme beta-galactosidase, cell proliferation, and doubling time. Real-time RT-PCR was used to evaluate the expression of p16, p21, p53 and mTOR. Results: The results showed that high glucose concentrations (35 mM and 55 mM) in the culture medium induced senescence of human ADSCs. The ADSCs could progress to the senescent status quicker than those cultured in the lower glucose-containing medium (17.5 mM). The senescent state was related to the up-regulation of p16 and mTOR genes. Conclusion: These results suggest that high glucose in culture medium can trigger the expression of p16 and mTOR genes which cause early senescence in ADSCs. Therefore, ADSCs should be cultured in low glucose culture medium, or normal glucose concentration, to extend their life in vitro as well as in vivo.

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