An animal model study of osteochondral defect repair by human adipose stem cells and pomegranate fruit hydroalchoholic extract

Document Type : Original Research Article


1 Department of Anatomical Sciences, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Anatomical Sciences, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Genetic and Molecular Biology, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran


Objective: Articular cartilage damages do not repair spontaneously. Tissue engineering is a promising approach to repair cartilage damage. Transforming growth factor-beta (TGF-β) members are the known induction factors in chondrogenic differentiation. However, hypertrophy of the chondrocytes resulting from mesenchymal stem cells (MSCs) induction by TGF-β is inevitable. Pomegranate fruit contains many ingredients which are useful in ensuring the health of organs. This study was designed to investigate the Pomegranate Fruit hydroalchoholic Extract (PFE) capability in human adipose derived stem cells (hASCs) differentiation into the chondrocytes on fibrin scaffold.
Materials and Methods: Pomegranate fruit hydroalchoholic extract (PFE) was prepared. hASCs were isolated, expanded, labeled, and seeded on the fibrin scaffold. The constructs were divided into three groups including TGF-β3, PFE, and control. The constructs were induced for 14 days, then, the MTT assay, Real-Time Polymerase Chain Reaction (PCR), and histochemistry assessments were run, and finally, the constructs were transplanted into the knee defect of rats. The gross and histological assessments of the transplants were done after 8 weeks.
Results: The viability rate, COL2A1, Aggrecan (ACAN) and COL10A1 genes expression levels, and histological criterion of the PFE samples were significantly higher than that of the control. The macroscopic grades and histological results of the PFE samples were close to that of the TGF-β3. The number of positive cells for COLІI protein were higher significantly in the PFE group than the control.
Conclusion: PFE was effective in the chondrogenic induction of hASCs. Further studies are needed to find out the events of the chondrogenic induction using PFE. 


Ahmed S, Wang N, Hafeez B, Cheruvu V,
Haqqi T. 2005. Punica granatum L. extract
inhibits IL-1β–Induced expression of
matrix metalloproteinases by inhibiting the
activation of MAP kinases and NF-κB in
human chondrocytes in vitro. J Nutr, 135:
Akpinar-Bayizit A, Ozcan T, Yilmaz-Ersan L.
2012. The therapeutic potential of
pomegranate and its products for prevention
of cancer. Asian Pac J Cancer, 2: 331-373.
Blom AB, Brockbank S, van Lent P, van
Beuningen H, Geurts J, Takahashi N,
Clement K. 2009. Involvement of the Wnt
signaling pathway in experimental and
human osteoarthritis: prominent role of
Wnt‐ induced signaling protein 1. Arthritis
Rheumatol, 60: 501-512.
Catelas I, Sese N, Wu B, Dunn J, Helgerson S,
Tawil B. 2006. Human mesenchymal stem
cell proliferation and osteogenic
differentiation in fibrin gels in vitro. Tissue
Eng, 12: 2385-2396.
Cerdá B, Soto C, Albaladejo M, Martinez P,
Sanchez-Gascon F, Tomás-Barberán F,
Espin J. 2006. Pomegranate juice
supplementation in chronic obstructive
pulmonary disease: a 5-week randomized,
double-blind, placebo-controlled trial. Eur J
Clin Nutr, 60: 245-253.
Chandra R, Bab K, Jadhav V, Jaime A, Silva
T. 2010. Origin, history and domestication
of pomegranate. Fruit Veg Cereal Sci,
Biotech, 2: 1-6.
Dahlin R, Kinard L, Lam J, Needham C, Lu S,
Kasper F, Mikos G. 2014. Articular
chondrocytes and mesenchymal stem cells
seeded on biodegradable scaffolds for the
repair of cartilage in a rat osteochondral
defect model. Biomaterials, 35: 7460-7469.
Davidson E, Scharstuhl A, Vitters E, Van der
Kraan P, Van Den Berg W. 2005. Reduced
transforming growth factor-beta signaling in
cartilage of old mice: role in impaired repair
capacity. Arthritis Res Ther, 7: 1338-1347.
Garbacki N, Angenot L, Bassleer C, Damas J,
Tits M. 2002. Effects of prodelphinidins
isolated from Ribes nigrum on chondrocyte
metabolism and COX activity. NaunynSchmiedeberg's Arch Pharmacol, 365: 434-
Goebel L, Orth P, Müller A, Zurakowski D,
Bücker A, Cucchiarini M, Madry H. 2012.
Articular defect repairing by hASCs and pomegranate
AJP, Vol. 13, No. 2, Mar-Apr 2023 187
Experimental scoring systems for
macroscopic articular cartilage repair
correlate with the MOCART score assessed
by a high-field MRI at 9.4 T–comparative
evaluation of five macroscopic scoring
systems in a large animal cartilage defect
model. Osteoarthr Cartil, 20: 1046-1055.
Hajhashemi V, Ghannadi A, Hajiloo M. 2010.
Analgesic and anti-inflammatory effects of
Rosa damascena hydroalcoholic extract and
its essential oil in animal models. Int J
Pharm Res, 9: 163-168.
Hashemibeni B, Valiani A, Esmaeli M, Kazemi
M, Aliakbari M, Iranpour FG. 2018.
Comparison of the efficacy of piascledine
and transforming growth factor β1 on
chondrogenic differentiation of human
adipose-derived stem cells in fibrin and
fibrin-alginate scaffolds. Iran J Basic Med
Sci, 21: 212-218.
Ho T, Cool S, Hui J, Hutmacher DW. 2010. The
influence of fibrin based hydrogels on the
chondrogenic differentiation of human bone
marrow stromal cells. Biomaterials, 31: 38-
Jung S, Rhie J, Kwon H, Jun Y, Seo J, Yoo G,
Oh J. 2010. In vivo cartilage formation using
chondrogenic-differentiated human
adipose-derived mesenchymal stem cells
mixed with fibrin glue. Craniofac Surg, 21:
Kang S, Choi B, Lee E, Kim S, Yi H, Park H,
Ku S. 2015. Inhibitory effect of dried
pomegranate concentration powder on
melanogenesis in B16F10 melanoma cells;
involvement of p38 and PKA signaling
pathways. Int J Mol Sci, 16: 24219-24242.
Katani M, Zolfaghari B, Soleimani M, Valiani
A, Hashemibeni B. 2018. The effect of
pomegranate extract on producing type II
collagen in differentiation of adiposederived stem cells in to chondrocytes. J Res
Med Sci, 35: 1540-1545.
Khalifé S, Zafarullah M. 2011. Molecular
targets of natural health products in arthritis.
Arthritis Res Ther, 13: 102-104.
Landers R, Hübner U, Schmelzeisen R,
Mülhaupt R. 2002. Rapid prototyping of
scaffolds derived from thermoreversible
hydrogels and tailored for applications in
tissue engineering. Biomaterials, 23: 4437-
Longtin R. 2003. The pomegranate: nature's
power fruit? J Natl Cancer Inst, 95: 346-348.
Miguel M, Neves M, Antunes M. 2010.
Pomegranate (Punica granatum L.): A
medicinal plant with myriad biological
properties-A short review. J Med Plant Res,
4: 2836-2847.
Monsefi M, Parvin F, Talaei-Khozani T. 2012.
Effects of pomegranate extracts on cartilage,
bone and mesenchymal cells of mouse
fetuses. Br J Nutr, 107: 683-690.
Mueller MB, Fischer M, Zellner J, Berner A,
Dienstknecht T, Prantl L, Angele P. 2010.
Hypertrophy in mesenchymal stem cell
chondrogenesis: effect of TGF-β isoforms
and chondrogenic conditioning. Cells
Tissues Organs, 192: 158-166.
Patel C, Dadhaniya P, Hingorani L, Soni M.
2008. Safety assessment of pomegranate
fruit extract: acute and subchronic toxicity
studies. Food Chem Toxicol, 46: 2728-
Risbud M, Sittinger M. 2002. Tissue
engineering: advances in in vitro cartilage
generation. Trends Biotechnol, 20: 351-356.
Seeram N, Nair M. 2002. Inhibition of lipid
peroxidation and structure− activity-related
studies of the dietary constituents
anthocyanins, anthocyanidins, and
catechins. J Agric Food Chem, 50: 5308-
Sekiya I, Vuoristo J, Larson B, Prockop D.
2002. In vitro cartilage formation by human
adult stem cells from bone marrow stroma
defines the sequence of cellular and
molecular events during chondrogenesis.
Proc Natl Acad Sci, 99: 4397-4402.
Shukla M, Gupta K, Rasheed Z, Khan K , Haqqi
T. 2008a. Bioavailable
constituents/metabolites of pomegranate
(Punica granatum L) preferentially inhibit
COX2 activity ex vivo and IL-1betainduced PGE 2 production in human
chondrocytes in vitro. J Inflamm, 5: 9-19.
Shukla M, Gupta K, Rasheed Z, Khan K , Haqqi
T. 2008b. Consumption of hydrolyzable
tannins-rich pomegranate extract suppresses
inflammation and joint damage in
rheumatoid arthritis. Nutr J, 24: 733-743.
Steinert A, Ghivizzani S, Rethwilm A, Tuan R,
Evans C, Nöth U. 2007. Major biological
obstacles for persistent cell-based
regeneration of articular cartilage. Arthritis
Res Ther, 9: 213-228.
Teimourinejad A, Hashemibeni B, Salehi H,
Mostafavi F, Kazemi M, Bahramian H.
2020. Chondrogenic activity of two herbal
Teimourinejad et al.
AJP, Vol. 13, No. 2, Mar-Apr 2023 188
products; pomegranate fruit extract and
avocado/soybean unsaponifiable. Res
Pharm Sci , 15: 358-366.
Toi M, Bando H, Ramachandran C, Melnick S,
Imai A, Fife R, Lansky E. 2003. Preliminary
studies on the anti-angiogenic potential of
pomegranate fractions in vitro and in vivo.
Angiogenesis, 6: 121-128.
Valcourt U, Gouttenoire J, Moustakas A,
Herbage D, Mallein-Gerin F. 2002.
Functions of TGF-β family type I receptors
and Smad proteins in the hypertrophic
maturation and osteoblastic differentiation
of chondrocytes. J Biol Chem, 5: 151-153
Xue D, Zheng Q, Zong C, Li Q, Li H, Qian S,
Pan Z. 2010. Osteochondral repair using
porous poly (lactide‐ co‐ glycolide)/nano‐
hydroxyapatite hybrid scaffolds with
undifferentiated mesenchymal stem cells in
a rat model. J Biomed Mater Res A, 94:
Yang S, Wu C, Shih T, Chen P, Lin F. 2008.
Three‐ dimensional culture of human
nucleus pulposus cells in fibrin clot:
comparisons on cellular proliferation and
matrix synthesis with cells in alginate. Artif
Organs, 32: 70-73.