scholarly journals Electrical Stimulation of Adipose-Derived Stem Cells in 3D Nanofibrillar Cellulose Increases Their Osteogenic Potential

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1696
Author(s):  
Mesude Bicer ◽  
Jonathan Sheard ◽  
Donata Iandolo ◽  
Samuel Y. Boateng ◽  
Graeme S. Cottrell ◽  
...  
Keyword(s):  
Stem Cells ◽  
Electrical Stimulation ◽  
Osteoporotic Fractures ◽  
Bone Grafts ◽  
Osteogenic Potential ◽  
Ageing Population ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Nanofibrillar Cellulose ◽  
Biophysical Cues

Due to the ageing population, there is a steadily increasing incidence of osteoporosis and osteoporotic fractures. As conventional pharmacological therapy options for osteoporosis are often associated with severe side effects, bone grafts are still considered the clinical gold standard. However, the availability of viable, autologous bone grafts is limited making alternative cell-based strategies a promising therapeutic alternative. Adipose-derived stem cells (ASCs) are a readily available population of mesenchymal stem/stromal cells (MSCs) that can be isolated within minimally invasive surgery. This ease of availability and their ability to undergo osteogenic differentiation makes ASCs promising candidates for cell-based therapies for bone fractures. Recent studies have suggested that both exposure to electrical fields and cultivation in 3D can positively affect osteogenic potential of MSCs. To elucidate the osteoinductive potential of a combination of these biophysical cues on ASCs, cells were embedded within anionic nanofibrillar cellulose (aNFC) hydrogels and exposed to electrical stimulation (ES) for up to 21 days. ES was applied to ASCs in 2D and 3D at a voltage of 0.1 V/cm with a duration of 0.04 ms, and a frequency of 10 Hz for 30 min per day. Exposure of ASCs to ES in 3D resulted in high alkaline phosphatase (ALP) activity and in an increased mineralisation evidenced by Alizarin Red S staining. Moreover, ES in 3D aNFC led to an increased expression of the osteogenic markers osteopontin and osteocalcin and a rearrangement and alignment of the actin cytoskeleton. Taken together, our data suggest that a combination of ES with 3D cell culture can increase the osteogenic potential of ASCs. Thus, exposure of ASCs to these biophysical cues might improve the clinical outcomes of regenerative therapies in treatment of osteoporotic fractures.

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

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.

scholarly journals JKAMP Inhibits the Osteogenic Capacity of Adipose-derived Stem Cells in Diabetic Osteoporosis by Modulating the Wnt Signaling Pathway Through Intragenic DNA Methylation

2020 ◽  
Author(s):  
Shuanglin Peng ◽  
Sirong Shi ◽  
Gang Tao ◽  
Yanjing Li ◽  
Dexuan Xiao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Signaling Pathway ◽  
Underlying Mechanism ◽  
Osteogenic Potential ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Diabetic Osteoporosis

Abstract Background: Diabetic osteoporosis (DOP) is a systemic metabolic bone disease caused by diabetes mellitus (DM). Adipose-derived stem cells (ASCs) play an important role in bone regeneration. Our previous study confirmed that ASCs from DOP mice (DOP-ASCs) have a lower osteogenesis potential compared with control ASCs (CON-ASCs). However, the cause of this poor osteogenesis has not been elucidated. Therefore, this study investigated the underlying mechanism of the decline in the osteogenic potential of DOP-ASCs from the perspective of epigenetics and explored methods to enhance their osteogenic capacity. Methods: The expression level of JNK1-associated membrane protein (JKAMP) and degree of DNA methylation in CON-ASCs and DOP-ASCs were measured by mRNA expression profiling and MeDIP sequencing, respectively. JKAMP small interfering RNA (siRNA) and a Jkamp overexpression plasmid were used to assess the role of JKAMP in osteogenic differentiation of CON-ASCs and DOP-ASCs. Immunofluorescence, qPCR, and western blotting were used to measure changes in expression of Wnt signaling pathway-related genes and osteogenesis-related molecules after osteogenesis induction. Alizarin red and ALP staining was used to confirm the osteogenic potential of stem cells. Bisulfite-specific PCR (BSP) was used to detect JKAMP methylation degree. Results: Expression of JKAMP and osteogenesis-related molecules (RUNX2 and OPN) in DOP-ASCs was decreased significantly in comparison with CON-ASCs. JKAMP silencing inhibited the Wnt signaling pathway and reduced the osteogenic ability of CON-ASCs. Overexpression of JKAMP in DOP-ASCs rescued the impaired osteogenic capacity caused by DOP. Moreover, JKAMP in DOP-ASCs contained intragenic DNA hypermethylated regions related to the downregulation of JKAMP expression. Conclusions: Intragenic DNA methylation inhibits the Wnt signaling pathway by suppressing expression of JKAMP and the osteogenic ability of DOP-ASCs. This study shows an epigenetic explanation for the reduced osteogenic ability of DOP-ASCs, and provides a potential therapeutic target to prevent and treat osteoporosis.

scholarly journals 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

2021 ◽  
Vol 22 (22) ◽  
pp. 12392
Author(s):  
Kiyofumi Takabatake ◽  
Masakazu Matsubara ◽  
Eiki Yamachika ◽  
Yuki Fujita ◽  
Yuki Arimura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Potential ◽  
Fat Cells ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Dedifferentiated Fat Cells ◽  
Dfat Cells

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.

scholarly journals JKAMP inhibits the osteogenic capacity of adipose-derived stem cells in diabetic osteoporosis by modulating the Wnt signaling pathway through intragenic DNA methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shuanglin Peng ◽  
Sirong Shi ◽  
Gang Tao ◽  
Yanjing Li ◽  
Dexuan Xiao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Signaling Pathway ◽  
Underlying Mechanism ◽  
Osteogenic Potential ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Diabetic Osteoporosis

Abstract Background Diabetic osteoporosis (DOP) is a systemic metabolic bone disease caused by diabetes mellitus (DM). Adipose-derived stem cells (ASCs) play an important role in bone regeneration. Our previous study confirmed that ASCs from DOP mice (DOP-ASCs) have a lower osteogenesis potential compared with control ASCs (CON-ASCs). However, the cause of this poor osteogenesis has not been elucidated. Therefore, this study investigated the underlying mechanism of the decline in the osteogenic potential of DOP-ASCs from the perspective of epigenetics and explored methods to enhance their osteogenic capacity. Methods The expression level of JNK1-associated membrane protein (JKAMP) and degree of DNA methylation in CON-ASCs and DOP-ASCs were measured by mRNA expression profiling and MeDIP sequencing, respectively. JKAMP small interfering RNA (siRNA) and a Jkamp overexpression plasmid were used to assess the role of JKAMP in osteogenic differentiation of CON-ASCs and DOP-ASCs. Immunofluorescence, qPCR, and western blotting were used to measure changes in expression of Wnt signaling pathway-related genes and osteogenesis-related molecules after osteogenesis induction. Alizarin red and ALP staining was used to confirm the osteogenic potential of stem cells. Bisulfite-specific PCR (BSP) was used to detect JKAMP methylation degree. Results Expression of JKAMP and osteogenesis-related molecules (RUNX2 and OPN) in DOP-ASCs was decreased significantly in comparison with CON-ASCs. JKAMP silencing inhibited the Wnt signaling pathway and reduced the osteogenic ability of CON-ASCs. Overexpression of JKAMP in DOP-ASCs rescued the impaired osteogenic capacity caused by DOP. Moreover, JKAMP in DOP-ASCs contained intragenic DNA hypermethylated regions related to the downregulation of JKAMP expression. Conclusions Intragenic DNA methylation inhibits the osteogenic ability of DOP-ASCs by suppressing expression of JKAMP and the Wnt signaling pathway. This study shows an epigenetic explanation for the reduced osteogenic ability of DOP-ASCs and provides a potential therapeutic target to prevent and treat osteoporosis.

Differential osteogenic potential of human adipose-derived stem cells co-cultured with human osteoblasts on polymeric microfiber scaffolds

2015 ◽  
Vol 104 (2) ◽  
pp. 377-387 ◽  
Author(s):  
Ismail Rozila ◽  
Pedram Azari ◽  
Sha'ban Munirah ◽  
Wan Kamarul Zaman Wan Safwani ◽  
Seng Neon Gan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Potential ◽  
Adipose Derived Stem Cells ◽  
Human Osteoblasts

Osteogenic Potential of Autogenous Bone Grafts Harvested with Four Different Surgical Techniques

2011 ◽  
Vol 90 (12) ◽  
pp. 1428-1433 ◽  
Author(s):  
R.J. Miron ◽  
E. Hedbom ◽  
N. Saulacic ◽  
Y. Zhang ◽  
A. Sculean ◽  
...  
Keyword(s):  
Cell Attachment ◽  
Surgical Techniques ◽  
Bone Grafts ◽  
Osteogenic Potential ◽  
Autogenous Bone ◽  
Mrna Levels ◽  
Alizarin Red ◽  
Osteogenic Response ◽  
Autogenous Bone Grafts

The osteogenic potential of autogenous bone grafts is superior to that of allografts and xenografts because of their ability to release osteoinductive growth factors and provide a natural osteoconductive surface for cell attachment and growth. In this in vitro study, autogenous bone particles were harvested by four commonly used techniques and compared for their ability to promote an osteogenic response. Primary osteoblasts were isolated and seeded on autogenous bone grafts prepared from the mandibles of miniature pigs with a bone mill, piezo-surgery, bone scraper, and bone drill (bone slurry). The osteoblast cultures were compared for their ability to promote cell attachment, proliferation, and differentiation. After 4 and 8 hrs, significantly higher cell numbers were associated with bone mill and bone scraper samples compared with those acquired by bone slurry and piezo-surgery. Similar patterns were consistently observed up to 5 days. Furthermore, osteoblasts seeded on bone mill and scraper samples expressed significantly elevated mRNA levels of collagen, osteocalcin, and osterix at 3 and 14 days and produced more mineralized tissue as assessed by alizarin red staining. These results suggest that the larger bone graft particles produced by bone mill and bone scraper techniques have a higher osteogenic potential than bone slurry and piezo-surgery.

Assessment of the osteogenic potential of alendronate on isolated adipose-derived stem cells: An ex-vivo and in-vivo study

2015 ◽  
Vol 10 (1) ◽  
pp. 32
Author(s):  
MarwaM Ellithy ◽  
MohamedS Ayoub ◽  
EffatA Abbas ◽  
MohamedA Abd El Hamid ◽  
HouryM Baghdadi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
In Vivo Study ◽  
Osteogenic Potential ◽  
Adipose Derived Stem Cells

Heparin‐Conjugated Decellularized Bone Particles Promote Enhanced Osteogenic Signaling of PDGF‐BB to Adipose‐Derived Stem Cells in Tissue Engineered Bone Grafts

2019 ◽  
Vol 8 (10) ◽  
pp. 1801565 ◽  
Author(s):  
Alexandra N. Rindone ◽  
Bartlomiej Kachniarz ◽  
Chukwuebuka C. Achebe ◽  
Ryan C. Riddle ◽  
Aine N. O'Sullivan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Grafts ◽  
Adipose Derived Stem Cells ◽  
Bone Particles ◽  
Osteogenic Signaling

scholarly journals Chondrogenic and osteogenic potential of adipose derived stem cells from RA and OA patients

2011 ◽  
Vol 70 (Suppl 2) ◽  
pp. A25-A25 ◽  
Author(s):  
U. Skalska ◽  
T. Burakowski ◽  
I. Janicka ◽  
A. Kornatka ◽  
P. Maldyk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Potential ◽  
Adipose Derived Stem Cells

280 OSTEOGENIC ACTIVITY OF IN HOUSE-PRODUCED PORCINE BMP2 ON ADIPOSE-DERIVED STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 288 ◽  
Author(s):  
A. C. M. Ercolin ◽  
M. Mkrtschjan ◽  
M. Bionaz ◽  
T. Jensen ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Acetic Acid ◽  
Bone Regeneration ◽  
Treatment Time ◽  
Nodule Formation ◽  
Adipose Derived Stem Cells ◽  
Accurate Analysis ◽  
Alizarin Red ◽  
Coding Sequence ◽  
In Cells

In our laboratory, we extensively study the possibility of using adipose-derived stem cells (ASC) for maxillofacial bone regeneration. This includes also the tissue repair of large critical-size osteotomies requiring the use of tridimensional scaffolds. Bone regeneration in scaffolds can be greatly enhanced by the use of specific growth factors such as BMP2. In the present study, we compared the activity of commercially available human BMP2 (hBMP2) with in house-produced porcine BMP2 (pBMP2). The latter was synthesised using the BMP2 coding sequence from mRNA obtained from porcine ASC cell cultures. The coding sequence of the mature protein was cloned into a pET-21 plasmid and produced in E. coli as inclusion bodies. The activity of pBMP2 and hBMP2 was tested on ASC isolated from male pigs at passage 4 and at approximately 80% confluence in 48-well plates. Cells were treated in triplicate with hBMP2 or pBMP2 at 0.5, 5, 50, 500, or 1000 ng mL–1, adipogenic medium (AM), osteogenic medium (OM), or normal DMEM medium supplemented with acetic acid (used to resuspend BMP2 as the control) for 5 or 17 days. Cells were harvested for Alizarin Red S (AR) quantification and expression of osteogenic genes. For the AR analysis, cells were fixed with formalin and treated with AR. The AR was then extracted by acetic acid and neutralized with ammonium hydroxide before spectrophotometer reading at an absorbance of 420 nm. Data were analysed using GLM of SAS (SAS Institute Inc., Cary, NC, USA) with treatment, time, concentration, and all interactions as main effects. Using an inverted robotic stage microscope, images of the entire well for each replicate were taken every 2 to 3 days. Images revealed formation of osteogenic nodules in OM and characteristic large cells filled with lipid droplets in AM. No evident nodule formation was observed in the other treated cells at any time point. The AR was higher than control in both hBMP2 and pBMP2 at 0.5, 50, and 1000 ng mL–1 but not at 5 and 500 ng mL–1. There was no overall difference between hBMP2 and pBMP2 but the former had the highest AR value at 5 days in cells treated with 0.5 ng mL–1 and pBMP2 at 17 days with 1000 ng mL–1. Interestingly, both had higher values compared to OM, particularly at 5 days. We also observed an increase of AR due to time in cells treated with acetic acid (control). Overall, the data appear to indicate an increase in calcium accumulation in cells treated with both hBMP2 and pBMP2, with an early increase in the former and a late and larger increase in the latter. This might indicate a larger but slower activity of pBMP2 compared with hBMP2. The lack of formation of osteogenic nodules by both BMP2 might indicate an insufficiency of BMP2 to induce osteogenesis in porcine ASC. This last observation, together with the lack of increased AR accumulation compared with control at the 5 and 50 ng mL–1 doses, suggests the need for a more accurate analysis of BMP2 activity by measuring expression of BMP2-related genes. Finally, the data provide preliminary support for the equivalency of activity of pBMP2 and hBMP2 for in vivo bone regeneration.

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