|Year : 2020 | Volume
| Issue : 1 | Page : 54-59
Cartilage tissue formation from human adipose-derived stem cells via herbal component (Avocado/soybean unsaponifiables) in scaffold-free culture system
Arefeh Basiri1, Batool Hashemibeni2, Mohammad Kazemi3, Ali Valiani4, Maryam Aliakbari4, Nazem Ghasemi4
1 Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran; Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Dental Research Center, Department of Anatomical Sciences, School of Medicine, Dental Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
4 Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Web Publication||21-Jan-2020|
Dr. Nazem Ghasemi
Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Background: The use of stem cells, growth factors, and scaffolds to repair damaged tissues is a new idea in tissue engineering. The aim of the present study is the investigation of Avocado/soybean (A/S) effects on chondrogenic differentiation of human adipose-derived stem cells (hADSCs) in micromass culture to access cartilage tissue with high quality.
Materials and Methods: In this an experimental study After hADSCs characterization, chondrogenic differentiation was induced using transforming growth factor beta 1 (TGF-β1) (10 ng/ml) and different concentrations (5, 10, and 20 μg/ml) of A/S in micromass culture. The efficiency of A/S on specific gene expression (types I, II, and X collagens, SOX9, and aggrecan) was evaluated using quantitative polymerase chain reaction. In addition, histological study was done using hematoxylin and eosin and toluidine blue staining all data were analyzed using one-way analysis of variance (ANOVA) and P ≤ 0.05 was considered to be statistically significant.
Results: The results of this study indicated that A/S can promote chondrogenic differentiation in a dose-dependent manner. In particular, 5 ng/ml A/S showed the highest expression of type II collagen, SOX9, and aggrecan which are effective and important markers in chondrogenic differentiation. In addition, the expression of types I and X collagens which are hypertrophic and fibrous factors in chondrogenesis is lower in present of 5 ng/ml A/S compared with TGF-β1 group (P ≤ 0.05). Moreover, the sulfated glycosaminoglycans in the extracellular matrix and the presence of chondrocytes within lacuna were more prominent in 5 ng/ml A/S group than other groups.
Conclusion: It can be concluded that A/S similar to TGF-β1 is able to facilitate the chondrogenic differentiation of hADSCs and do not have adverse effects of TGF-β1. Thus, TGF-β1 can be replaced by A/S in the field of tissue engineering.
Keywords: Adult stem cells, cell culture techniques, tissue engineering
|How to cite this article:|
Basiri A, Hashemibeni B, Kazemi M, Valiani A, Aliakbari M, Ghasemi N. Cartilage tissue formation from human adipose-derived stem cells via herbal component (Avocado/soybean unsaponifiables) in scaffold-free culture system. Dent Res J 2020;17:54-9
|How to cite this URL:|
Basiri A, Hashemibeni B, Kazemi M, Valiani A, Aliakbari M, Ghasemi N. Cartilage tissue formation from human adipose-derived stem cells via herbal component (Avocado/soybean unsaponifiables) in scaffold-free culture system. Dent Res J [serial online] 2020 [cited 2020 Feb 26];17:54-9. Available from: http://www.drjjournal.net/text.asp?2020/17/1/54/276236
| Introduction|| |
Tissue engineering using stem cells, growth factors, and scaffolds with aimed to improving the biological functions of damaged tissues is a new therapeutic method for tissue regenerative. Due to cartilage tissue have not blood supply and the recovery period of cartilage lesion is long, so tissue engineering can be a suitable method for cartilage repair.
Transforming growth factor beta (TGF-β) family is one the most factors which used for in vitro chondrogenic differentiation. It has been reported that TGF-β family members via specific receptors and with intracellular signaling are able to promote cartilage-specific gene expression.,,
In spite of this, our previous study confirms that the expression of type X collagen as a hypertrophic marker is high in cartilage which produced using TGF-β1. On the other hand, a previous study revealed that TGF-β1 can induce type I collagen expression through intracellular signaling effects. Moreover, TGF-β1when used in vivo has other side effects such as osteophyte formation and synovial membrane inflammation. Thus, the TGF-β replacing with other agents with fewer side effects is a necessity.
Avocado/soybean (A/S) (ratio 1:2) which is an herbal component used to decrease symptoms of cartilage defects. The main action mechanism of this agent is not clear; nevertheless, several potential pathways have been assumed. Among these mechanisms, increase of TGF-β expression, accumulation of extracellular matrix, and collagenase reduction, fibrinolysis, and matrix metalloproteinase activity inhibition may be significant. It is important to know that A/S is able to alter the cross-linking of collagen fibers and facilitate the wound-healing process. It has been reported that arthrocen (an A/S unsaponifiable) has a potential to reduce joint inflammation and pain associated with end-stage osteoarthritis. In a similar experiment, A/S (300 mg/day) was given to 4822 patients with symptomatic knee osteoarthritis as a routine medication. The results of this study revealed that a large number of patients who received AS for 6 months showed gradual decrease of joint pain, improvement in functional ability, and a considerable diminution in nonsteroidal anti-inflammatory drugs intake.
With respect to the wide beneficial effects A/S, in the present study, we evaluated the effects of several doses of A/S cartilage formation from human adipose-derived stem cells (hADSCs) in micromass culture system.
| Materials and Methods|| |
Human adipose-derived stem cells isolation and culture
This is an experimental study which was done at Isfahan University of Medical Sciences. All materials (except those specified) which used in this study were purchased from Sigma-Aldrich. Moreover, all procedures were approved by the Ethics Committee of Isfahan University of Medical Sciences. To hADSCs isolation, all following steps were done according to the previous study. Human abdominal fat was obtained from lipoaspirate samples; the samples were washed with phosphate-buffer saline (PBS) and then treated with 0.075% type I collagenase. Enzyme activity was neutralized after 30 min, the samples were centrifuged at 1400 rpm for 10 min, and finally, the cellular pellet was resuspended in Dulbecco's modified Eagle medium (DMEM/low glucose) solution and was cultured in standard condition.
Cell surface marker characterization
To identify specific markers of stem cell, hADSCs (within third passages) incubated with respective fluorochrome-conjugated antibodies against CD14, CD44, CD105, and CD90 which were conjugated with FITC (BioLegend Company) for 30 min. After incubation, the cells were washed with PBS and stained with DAPI. Finally, the mean percent of fluorescent cells were analyzed by a fluorescence microscope (Olympus BX51, Tokyo City, Japan).
Micromass culture system and chondrogenic differentiation
The induction of hADSCs into chondrocyte was carried out according to the previous study. Briefly, hADSCs within the third passages at a density of 2.5 × 105 cells in 12.5 μl medium were placed in a 24-well plate in a 37°C humidified incubator with a 5% CO2 environment for ½ h to form cell aggregates. In the following, chondrogenic differentiation media (1 ml) consisting of DMEM-high glucose (Gibco), penicillin and streptomycin 1% (Gibco), dexamethasone 10-7M, ascorbate-2-phosphate 50 μg/ml, bovine serum albumin 0.5 mg/ml, linoleic acid 5 μg/ml, 10 μg/ml insulin, 5.5 μg/l transferring, 5 μg/l selenium (ITS), with adding TGF-β1 10 ng/ml (Group 1), A/S 5 μg/ml (Group 2), A/S 10 μg/ml (Group 3), A/S 20 μg/ml (Group 4), TGF-β1/A/S 10 μg/ml (Group 5), and without any growth factor (group control) were carefully added around the cell aggregates. The medium was substituted every 3–4 days for 14 days. Meanwhile, the mean expression of cartilage-specific genes in stem cells was evaluated before induction of chondrogenesis and was considered as stem group.
Three samples in each group were fixed with formaldehyde and then dehydrated in ascending ethanol. In the following, they were clarified with xylol, embedded in paraffin, sectioned into 4-μm thickness, and finally stained using hematoxylin and eosin and toluidine blue. The comparison of glycosaminoglycans secretion in the extracellular matrix and the morphology of chondrocytes within lacuna were studied using a light microscope (Nikon, Japan).
Real-time polymerase chain reaction
To determine the cartilage-specific genes expression, RNA extraction (three samples per group) was done using TRIzol reagents (Invitrogen). In the following, RNA was reverse transcribed using Revert AidFirst Strand cDNA Synthesis Kit (Fermentase) with oligo (dT) primers, and then real-time polymerase chain reaction (PCR) was performed using SYBR Green PCR Master Mix (Fermentase) and the Step One Plus™ quantitative real-time PCR detection system (Applied Biosystems). Primers were designed for each gene using the Allele ID software (Primer Biosoft), which generated the following sequences: Collagen II (Forward: CTGGTGATGATGGTGAAG, Reverse: CCTGGATAACCTCTGTGA), collagen X (Forward: AGAATCCATCTGAGAATATGC, Reverse: CCTCTTACTGCTATACCTTTAC), SOX9 (Forward: TTCAGCAGCCAATAAGTG, Reverse: GTGGAATGTCTTGAAGGTTA), aggrecan (Forward: GTGGGACTGAAGTTCTTG, Reverse: GTTGTCATGGTCTGAAGTT). In addition, GAPDH primer was used as an internal control within these sequences: Forward: AAGCTCATTTCCTGGTATG, Reverse: CTTCTTCTTGTGCTCTTG.
Finally, all data were analyzed using one-way analysis of variance (ANOVA). In addition, Data are presented as mean ± standard error of the mean and P ≤ 0.05 was considered to be statistically significant.
| Results|| |
Human adipose-derived stem cells characterization
As shown in [Figure 1]a and [Figure 1]b, hADSCS before micromass formation exhibited fibroblast-like morphology; however, after micromass formation, these cells were aggregated and numerous cellular mass was formed. Moreover, immunocytochemistry results indicated that the high percent of hADSCs was CD105 and CD90 positive (98.27 ± 0.17 and 99.2 ± 0.11, respectively) but was CD14 and CD45 negative [Figure 1]c [Figure 1]d [Figure 1]e [Figure 1]f.
|Figure 1: Phase contrast images of cell morphology. Human adipose-derived stem cells within third passage (a), human adipose-derived stem cells micromass formation before chondrogenesis (b) and CD marker expression in human adipose-derived stem cells (c-f) (a and b, ×40).|
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Histological analysis of samples
Morphological study of the samples revealed that in 5 μg A/S group, the sulfated glycosaminoglycans in the extracellular matrix were higher than other groups [Figure 2]. In addition, the presence of chondrocytes within lacuna was more prominent in this group [Figure 3].
|Figure 2: Toluidine blue staining of the samples in end stage of chondrogenesis. The presence of sulfated glycosaminoglycans in the extracellular matrix was higher in 5 μg Avocado/soybean group than other groups (×40 and ×100).|
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|Figure 3: The samples in all groups. The presence of chondrocytes within lacuna was more prominentin 5 μg Avocado/soybean group than other groups (H and E, ×100).|
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Real-time polymerase chain reaction analysis
The results of real-time PCR indicated that A/S administered for 2 weeks significantly increased cartilage-specific genes expression of hADSCs in a dose-dependent manner. As seen in [Figure 4], in 5 ng/ml A/S administered group, the expression of aggrecan and collagen II genes was upregulated in comparison with TGF-β, stem, and control groups (P ≤ 0.05), but the expression of collagen I and collagen X was lower than TGF-β group (P ≥ 0.05). Therefore, 5 ng/ml A/S applied for 2 weeks was determined to be the optimal treatment for highest cartilage-specific genes expression.
|Figure 4: The results of real-time polymerase chain reaction for cartilage-specific genes expression. The expression of aggrecan, collagen II, and SOX9 genes was similar in transforming growth factor beta and avocado/soybean 5 ng/ml treated group and was increased significantly compared to stem and control groups (P < 0.05). In addition, the expression of collagen I and collagen X was lower in avocado/soybean 5 ng/ml treated group than transforming growth factor beta group.|
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| Discussion|| |
Age-related disorders including inflammatory, chronic, and acute joints lesions are principal cause of disability in the elderly. Of these, arthritis is the most common type of degenerative disorder which can lead to severe disability. It has been shown, for example, that stem cell transplantation is able to inhibit cartilage degeneration progression and capable to promote cartilage regeneration via paracrine effects including antioxidative and anti-inflammatory effects as well as chondroprotective effects.,,, It is important to know that stem cell transplants to cartilage repair may be to create teratomas;, thus, in vitro predifferentiation of these cells by the use of specific agent such as TGF-β family is suggested. It has been reported that TGF-β family members are able to promote chondrogenic differentiation and endochondral ossification., Although these multifunctional growth factors by intracellular signaling through specific receptors involved in several biological and pathological processes of the body, the exact roles of them in maintaining stem cells and tissue homeostasis have not been well documented.
According to our current published data, TGF-β3 is able to promote chondrogenic differentiation of hADSCs via increased chondrogenic genes expression in these cells. On the other hand, in similar experiment, TGF-β3 affect significantly on cellular proliferation, but TGF-β1 promotes chondrocyte differentiation. Of interest, our recent study showed that TGF-β1is able to express a high level of collagen type X which is a hypertrophic marker during chondrogenic differentiation. Unfortunately, due to the high number of side effects of TGF-β isoforms, the use of other effective agent on chondrogenesis is recommended.
A/S which is a plant extract has several effects on cell differentiation, cell viability, and proliferation. In addition, A/S also is able to reduce reactive oxygen species via inhibits cholesterol absorption and biosynthesis. Meanwhile, this agent in clinical level can reduce pain and stiffness and so is able to improve joint function. Overall, A/S is able to modify osteoarthritis pathogenesis through inhibiting some molecules and pathways, prohibit cartilage degeneration via inhibiting the produce and function of matrix metalloproteinases, inhibit fibrinolysis through stimulating the expression of plasminogen activator inhibitor, promote cartilage repair via stimulating collagen and aggrecan synthesis, decrease cholesterol absorption and endogenous cholesterol biosynthesis, and diminish pain and joint stiffness.
Here, in the presence of different dosage of A/S, we have differentiated hADECs into chondrocytes, and then, the expression of cartilage-specific genes was evaluated.
The real-time PCR results demonstrated that during chondrogenic differentiation, A/S a dose-dependent manner has potentially beneficial effects on cartilage-specific genes expression. As seen in [Figure 2], 5 ng/ml A/S has the highest effect on aggrecan, collagen II, and SOX9 genes expression. In addition, the expression of collagen I and collagen X genes is at low level which is another beneficial effect. These results is consistent with the recent study by others, which have also shown that, AS is able to reduces the expression of type X collagen better than other agents such as kartogenin in the stem cells.
Although, the TGF-β1/(A/S) administration can increase cartilage-specific gene expression, this composition is not able to reduce the expression of hypertrophic (collagen X) and fibrocartilage (collagen I) markers. Therefore, to in vitro chondrogenic differentiation, 5 ng/ml A/S is an effective and beneficial agent compared to other dosage.
| Conclusion|| |
It can be concluded that A/S is able to promote chondrogenic differentiation of hADSCs by the effect on gene expression via intracellular signaling pathway. Thus, this agent with 5 ng/ml dosage may be an ideal agent for in vitro chondrogenic differentiation of stem cells to application in cartilage lesions.
We are grateful to the Isfahan University of Medical Sciences for their financial support.
Financial support and sponsorship
We are grateful to the Isfahan University of Medical Sciences for their financial support (Grant No. 394247).
Conflicts of interest
The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.
| References|| |
Howard D, Buttery LD, Shakesheff KM, Roberts SJ. Tissue engineering: Strategies, stem cells and scaffolds. J Anat 2008;213:66-72.
Longobardi L, O'Rear L, Aakula S, Johnstone B, Shimer K, Chytil A, et al.
Effect of IGF-I in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling. J Bone Miner Res 2006;21:626-36.
Tuli R, Tuli S, Nandi S, Huang X, Manner PA, Hozack WJ, et al.
Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk. J Biol Chem 2003;278:41227-36.
Nakamura K, Shirai T, Morishita S, Uchida S, Saeki-Miura K, Makishima F, et al.
P38 mitogen-activated protein kinase functionally contributes to chondrogenesis induced by growth/differentiation factor-5 in ATDC5 cells. Exp Cell Res 1999;250:351-63.
Mardani M, Kabiri A, Esfandiari E, Esmaeili A, Pourazar A, Ansar M, et al.
The effect of platelet rich plasma on chondrogenic differentiation of human adipose derived stem cells in transwell culture. Iran J Basic Med Sci 2013;16:1163-9.
Pan X, Chen Z, Huang R, Yao Y, Ma G. Transforming growth factor β1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts. PLoS One 2013;8:e60335.
van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB. Differential effects of local application of BMP-2 or TGF-beta 1 on both articular cartilage composition and osteophyte formation. Osteoarthritis Cartilage 1998;6:306-17.
Boumediene K, Felisaz N, Bogdanowicz P, Galera P, Guillou GB, Pujol JP, et al.
Avocado/soya unsaponifiables enhance the expression of transforming growth factor beta1 and beta2 in cultured articular chondrocytes. Arthritis Rheum 1999;42:148-56.
Basiri A, Hashemibeni B, Mardani M, Esfandiari E. Ps-19: The effect of avocado/soybean unsaponifiables (Piascledine) on viability of human articular chondrocytes and adipose derived stem cells. Cell J (Yakhteh) 2014;16 Suppl 1:345.
Christiansen BA, Bhatti S, Goudarzi R, Emami S. Management of osteoarthritis with avocado/Soybean unsaponifiables. Cartilage 2015;6:30-44.
Werman MJ, Mokady S, Nimni ME, Neeman I. The effect of various avocado oils on skin collagen metabolism. Connect Tissue Res 1991;26:1-0.
Goudarzi R, Reid A, McDougall JJ. Evaluation of the novel avocado/soybean unsaponifiable arthrocen to alter joint pain and inflammation in a rat model of osteoarthritis. PLoS One 2018;13:e0191906.
Głuszko P, Stasiek M. Symptom-modifying effects of oral avocado/soybean unsaponifiables in routine treatment of knee osteoarthritis in Poland. An open, prospective observational study of patients adherent to a 6-month treatment. Reumatologia 2016;54:217-26.
Ghasemi N, Razavi S, Mardani M, Esfandiari E, Salehi H, Zarkesh Esfahani SH, et al.
Transplantation of human adipose-derived stem cells enhances remyelination in lysolecithin-induced focal demyelination of rat spinal cord. Mol Biotechnol 2014;56:470-8.
Mardani M, Roshankhah S, Hashemibeni B, Salahshoor M, Naghsh E, Esfandiari E, et al.
Induction of chondrogenic differentiation of human adipose-derived stem cells by low frequency electric field. Adv Biomed Res 2016;5:97.
] [Full text]
Cinelli M, Guiducci S, Del Rosso A, Pignone A, Del Rosso M, Fibbi G, et al.
Piascledine modulates the production of VEGF and TIMP-1 and reduces the invasiveness of rheumatoid arthritis synoviocytes. Scand J Rheumatol 2006;35:346-50.
Mobasheri A, Richardson S, Mobasheri R, Shakibaei M, Hoyland JA. Hypoxia inducible factor-1 and facilitative glucose transporters GLUT1 and GLUT3: Putative molecular components of the oxygen and glucose sensing apparatus in articular chondrocytes. Histol Histopathol 2005;20:1327-38.
Murphy JM, Fink DJ, Hunziker EB, Barry FP. Stem cell therapy in a caprine model of osteoarthritis. Arthritis Rheum 2003;48:3464-74.
Desando G, Cavallo C, Sartoni F, Martini L, Parrilli A, Veronesi F, et al.
Intra-articular delivery of adipose derived stromal cells attenuates osteoarthritis progression in an experimental rabbit model. Arthritis Res Ther 2013;15:R22.
Chen L, Tredget EE, Wu PY, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One 2008;3:e1886.
Kuroda K, Kabata T, Hayashi K, Maeda T, Kajino Y, Iwai S, et al.
The paracrine effect of adipose-derived stem cells inhibits osteoarthritis progression. BMC Musculoskelet Disord 2015;16:236.
Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro
. Nat Biotechnol 2000;18:399-404.
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al.
Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145-7.
Leonard CM, Fuld HM, Frenz DA, Downie SA, Massagué J, Newman SA, et al.
Role of transforming growth factor-beta in chondrogenic pattern formation in the embryonic limb: Stimulation of mesenchymal condensation and fibronectin gene expression by exogenenous TGF-beta and evidence for endogenous TGF-beta-like activity. Dev Biol 1991;145:99-109.
Seo HS, Serra R. Deletion of tgfbr2 in prx1-cre expressing mesenchyme results in defects in development of the long bones and joints. Dev Biol 2007;310:304-16.
Xu X, Zheng L, Yuan Q, Zhen G, Crane JL, Zhou X, et al.
Transforming growth factor-β in stem cells and tissue homeostasis. Bone Res 2018;6:2.
Zamani S, Hashemibeni B, Esfandiari E, Kabiri A, Rabbani H, Abutorabi R, et al.
Assessment of TGF-β3 on production of aggrecan by human articular chondrocytes in pellet culture system. Adv Biomed Res 2014;3:54.
James AW, Xu Y, Lee JK, Wang R, Longaker MT. Differential effects of TGF-beta1 and TGF-beta3 on chondrogenesis in posterofrontal cranial suture-derived mesenchymal cells in vitro
. Plast Reconstr Surg 2009;123:31-43.
Christiansen BA, Bhatti S, Goudarzi R, Emami S. Management of osteoarthritis with avocado/Soybean unsaponifiables. Cartilage 2015;6:30-44.
Izadi MA, Valiani A, Bahramian H, Esfandiari E, Hashemibeni B. Which factor is better for cartilage tissue engineering from human adipose-derived stem cells? Kartogenin or avocado soybean unsaponifiable. Pharmacophore 2018;9:140-8.
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