Tissue Eng Regen Med.  2019 Feb;16(1):69-80. 10.1007/s13770-018-0164-4.

Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure

• Shahmoradi SR
• Kabir Salmani M
• Soleimanpour HR
• Tavakoli A
• Hosaini K
• Haghighipour N
• Bonakdar S

• KMID: 2441257
• DOI: http://doi.org/10.1007/s13770-018-0164-4

Abstract

BACKGROUND
Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro.
METHODS
In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively.
RESULTS
Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively.
CONCLUSION
The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.

Keyword

Bone marrow mesenchymal stem cells; Chondrogenic differentiation; Hydrostatic pressure; Demineralized bone matrix scaffold

MeSH Terms

Aggrecans
Alcian Blue
Bone Marrow
Bone Matrix*
Cartilage
Cartilage, Articular
Collagen Type I
Collagen Type II
Eosine Yellowish-(YS)
Extracellular Matrix
Fluorescent Antibody Technique
Gene Expression
Hematoxylin
Humans*
Hydrostatic Pressure*
In Vitro Techniques
Mesenchymal Stromal Cells*
Osteocalcin
Real-Time Polymerase Chain Reaction
RNA, Messenger
Aggrecans
Alcian Blue
Collagen Type I
Collagen Type II
Eosine Yellowish-(YS)
Hematoxylin
Osteocalcin
RNA, Messenger