EXPERIMENTAL STUDY ON OSTEOGENESIS OF SYNOVIUM-DERIVED MESENCHYMAL STEM CELLS IN VITRO AND IN VIVO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHENGWeiwei ,  YANGMin , WUCheng , SUXianlin

Department of Orthopedics and Traumatology, Yijishan Hospital, Wannan Medical College, Wuhu Anhui, 241001, P. R. China;

Corresponding?author：

YANGMin, Email: pkuyang@hotmail.com

Keywords：

Synovium-derived mesenchymal stem cells; Osteogenic induction; Alizarin red; Alkaline phosphatase; New bone formation; Rat

DOI：

10.7507/1002-1892.20160021

Video：

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Objective To investigate the osteogenic differentiation potential and the biological features of synovium-derived mesenchymal stem cells (SMSCs) in vitro and to observe the osteogenic capability of the composite scaffolds constructed with SMSCs and hydroxylapatite/chitosan/poly L-latic acid (HA/CS/PLLA) in vivo. Method SMSCs were separated and cultured with adherent method and enzymatic digestion method. Specific phenotypes of SMSCs were detected by flow cytometry after purification. Then, SMSCs were identified by oil red O staining, alkaline phosphatase (ALP) staining, and alizarin red staining after adipogenic and osteogenic induction, respectively. In vitro experiments:the expressions of osteogenic related genes[osteocalcin (OCN), collagen type I, ALP, and Runx-2] were detected by real-time fluorescent quantitative PCR at 1, 7, 14, 21, and 28 days after osteogenic induction; ALP activities were also determined by ELISA at 1, 3, 5, 7, 9, and 11 days after osteogenic induction; meanwhile, extracellular matrix calcium mineralization was detected by alizarin red S method at 7, 14, 21, and 28 days after osteogenic induction; the normal SMSCs were harvested as control group. In vivo experiments:Twenty-four Sprague Dawley (SD) rats were randomly divided into experimental group (n=12) and control group (n=12) . The 3rd passage SMSCs were seeded on HA/CS/PLLA to construct composite scaffolds, after adhesion for 72 hours in vitro, the composite scaffolds were implanted into the right thigh muscle of 12 SD rats as experimental group; HA/CS/PLLA was implanted into the right thigh muscle of the other 12 SD rats as control group. At 4 and 8 weeks after implantation, the scaffolds were harvested for X-ray film and histological examination to observe ectopic bone formation. Results The positive rates of CD147, CD90, CD105, and CD44 were more than 95%, while the positive rates of CD117, CD34, CD14, and CD45 were less than 10%. Oil red O staining demonstrated red lipid droplets in the cytoplasm, and alizarin red staining showed flaky red calcifications, and cytoplasm was dyed brown by the ALP staining. The mRNA expressions of collagen type I, ALP, and Runx-2 were significantly increased at 7 days after osteogenic induction, and OCN mRNA expression was significantly increased at 14 days after osteogenic induction; ALP activity was significantly higher at 5, 7, 9, 11 days after osteogenic induction in the SMSC-induced group than control group and reached a maximum at 7 days (P<0.05) . Calcium mineralization was significantly enhanced at 14 days after osteogenic induction, and gradually increased with time (P<0.05) ; moreover, it was significantly higher in the SMSC-induced group than control group (P<0.05) . X-ray and histological examination demonstrated that the new bone tissues formed in 2 groups, but bone formation content of the experimental group was significantly more than that of the control group at 4 and 8 weeks after implantation (P<0.05) . Conclusions SMSCs can be induced into osteoblasts both in vitro and in vivo, so SMSCs might be a promising seed cells for bone tissue engineering.

Citation： ZHENGWeiwei, YANGMin, WUCheng, SUXianlin. EXPERIMENTAL STUDY ON OSTEOGENESIS OF SYNOVIUM-DERIVED MESENCHYMAL STEM CELLS IN VITRO AND IN VIVO. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(1): 102-109. doi: 10.7507/1002-1892.20160021 Copy

1.	Sakaguchi Y, Sekiya I, Yagishita K, et al. Comparison of human stem cells derived from various mesenchymal tissues. Arthritis Rheum, 2005, 52(8) :2521-2529.
2.	Yoshimura H, Muneta T, Nimura A, et al. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res, 2007, 327(3) :449-462.
3.	Liu H, Wei X, Ding X, et al. Comparison of cellular responses of mesenchymal stem cells derived from bone marrow and synovium on combined silk scaffolds. J Biomed Mater Res A, 2015, 103(1) :115-125.
4.	Zhang CY, Zhang CL, Wang JF, et al. Fabrication and in vitro investigation of nanohydroxyapatite, chitosan, poly ternary biocomposite. J Appol Polym Sci, 2013, 127(3) :2152-2159.
5.	Vandenabeele F, De Bari C, Moreels M, et al. Morphological and immunocytochemical characterization of cultured fibroblast-like cells derived from adult human synovial membrane. Arch Histol Cytol, 2003, 66(2) :145-153.
6.	汪洋,王友,芮云峰,等.豬滑膜源性MSCs體外多向分化潛能的研究.中國修復重建外科雜志, 2009, 23(6) :737-741.
7.	Coiffier G, Albert JD. Gout and and calcium pyrophosphate crystal arthropathies:pathophysiology. Rev Prat, 2015, 65(5) :666-667, 669-670.
8.	Ryu K, Iriuchishima T, Oshida M, et al. The prevalence of and factors related to calcium pyrophosphate dihydrate crystal deposition in the knee joint. Osteoarthritis Cartilage, 2014, 22(7) :975-979.
9.	Crawford A, Frazer A, Lippitt JM, et al. A case of chondromatosis indicates a synovial stem cell aetiology. Rheumatology, 2006, 45(12) :1529-1533.
10.	Li J, Campbell DD, Bal GK, et al. Can arthroscopically harvested synovial stem cells be preferentially sorted using stage-specific embryonic antigen 4 antibody for cartilage, bone, and adipose regeneration? Arthroscopy, 2014, 30(3) :352-361.
11.	Lee WJ, Hah YS, Ock SA, et al. Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs. Exp Cell Res, 2015, 333(2) :273-288.
12.	陸鈺,王鑫,甘朝兵,等.多孔納米羥基磷灰石/殼聚糖支架材料復合成骨細胞的異位成骨研究.生物骨科材料與臨床研究, 2012, 9(1) :21-25.
13.	Ripamonti U, Ramoshebi LN, Matsaba T, et al. Bone induction by BMSCs/OPs and related family members in primates. J Bone Joint Surg (Am), 2001, 83-A Suppl 1(Pt2) :S116-127.
14.	Hofmann S, Hagenmüller H, Koch AM, et al. Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials, 2007, 8(6) :1152-1162.
15.	Csaki C, Matis U, Mobasheri A, et al. Chondrogenesis,osteogenesis and adipogenesis of canine mesenchymal stem cells:a biochemical,morphological and ultrastructural study. Histochem Cell Biol, 2007, 128(6) :507-520.
16.	Niemeyer P, Kornacker M, Mehlhorn A, et al. Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro. Tissue Eng, 2007, 13(1) :111-121.
17.	Mukherjee A, Wilson EM, Rotwein P. Selective signaling by Akt2 promotes bone morphogenetic protein 2-mediated osteoblast differentiation. Mol Cell Biol, 2010, 30(4) :1018-1027.
18.	張路,趙文志.不同生物支架材料修復骨缺損的性能與評價.中國組織工程研究與臨床康復, 2012, 51(14) :9679-9682.
19.	LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem Rev, 2008, 108(11) :4742-4753.
20.	Di Bella C, Farlie P, Penington AJ. Bone regeneration in a rabbit critical-sized skull defect using autologous adipose-derived cells. Tissue Eng Part A, 2008, 14(4) :483-490.
21.	Yoon E, Dhar S, Chun DE, et al. In vivo osteogenic potential of human adipose-derived stem cells/poly lactide-co-glycolic acid constructs for bone regeneration in a rat critical-sized calvarial defect model. Tissue Eng, 2007, 13(3) :619-627.
22.	El Marsafy S, Larghero J. Mesenchymal Stem Cells:key actors in tumor niche. Curr Stem Cell Res Ther, 2015, 10(6) :523-529.

1. Sakaguchi Y, Sekiya I, Yagishita K, et al. Comparison of human stem cells derived from various mesenchymal tissues. Arthritis Rheum, 2005, 52(8) :2521-2529.
2. Yoshimura H, Muneta T, Nimura A, et al. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res, 2007, 327(3) :449-462.
3. Liu H, Wei X, Ding X, et al. Comparison of cellular responses of mesenchymal stem cells derived from bone marrow and synovium on combined silk scaffolds. J Biomed Mater Res A, 2015, 103(1) :115-125.
4. Zhang CY, Zhang CL, Wang JF, et al. Fabrication and in vitro investigation of nanohydroxyapatite, chitosan, poly ternary biocomposite. J Appol Polym Sci, 2013, 127(3) :2152-2159.
5. Vandenabeele F, De Bari C, Moreels M, et al. Morphological and immunocytochemical characterization of cultured fibroblast-like cells derived from adult human synovial membrane. Arch Histol Cytol, 2003, 66(2) :145-153.
6. 汪洋,王友,芮云峰,等.豬滑膜源性MSCs體外多向分化潛能的研究.中國修復重建外科雜志, 2009, 23(6) :737-741.
7. Coiffier G, Albert JD. Gout and and calcium pyrophosphate crystal arthropathies:pathophysiology. Rev Prat, 2015, 65(5) :666-667, 669-670.
8. Ryu K, Iriuchishima T, Oshida M, et al. The prevalence of and factors related to calcium pyrophosphate dihydrate crystal deposition in the knee joint. Osteoarthritis Cartilage, 2014, 22(7) :975-979.
9. Crawford A, Frazer A, Lippitt JM, et al. A case of chondromatosis indicates a synovial stem cell aetiology. Rheumatology, 2006, 45(12) :1529-1533.
10. Li J, Campbell DD, Bal GK, et al. Can arthroscopically harvested synovial stem cells be preferentially sorted using stage-specific embryonic antigen 4 antibody for cartilage, bone, and adipose regeneration? Arthroscopy, 2014, 30(3) :352-361.
11. Lee WJ, Hah YS, Ock SA, et al. Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs. Exp Cell Res, 2015, 333(2) :273-288.
12. 陸鈺,王鑫,甘朝兵,等.多孔納米羥基磷灰石/殼聚糖支架材料復合成骨細胞的異位成骨研究.生物骨科材料與臨床研究, 2012, 9(1) :21-25.
13. Ripamonti U, Ramoshebi LN, Matsaba T, et al. Bone induction by BMSCs/OPs and related family members in primates. J Bone Joint Surg (Am), 2001, 83-A Suppl 1(Pt2) :S116-127.
14. Hofmann S, Hagenmüller H, Koch AM, et al. Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials, 2007, 8(6) :1152-1162.
15. Csaki C, Matis U, Mobasheri A, et al. Chondrogenesis,osteogenesis and adipogenesis of canine mesenchymal stem cells:a biochemical,morphological and ultrastructural study. Histochem Cell Biol, 2007, 128(6) :507-520.
16. Niemeyer P, Kornacker M, Mehlhorn A, et al. Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro. Tissue Eng, 2007, 13(1) :111-121.
17. Mukherjee A, Wilson EM, Rotwein P. Selective signaling by Akt2 promotes bone morphogenetic protein 2-mediated osteoblast differentiation. Mol Cell Biol, 2010, 30(4) :1018-1027.
18. 張路,趙文志.不同生物支架材料修復骨缺損的性能與評價.中國組織工程研究與臨床康復, 2012, 51(14) :9679-9682.
19. LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem Rev, 2008, 108(11) :4742-4753.
20. Di Bella C, Farlie P, Penington AJ. Bone regeneration in a rabbit critical-sized skull defect using autologous adipose-derived cells. Tissue Eng Part A, 2008, 14(4) :483-490.
21. Yoon E, Dhar S, Chun DE, et al. In vivo osteogenic potential of human adipose-derived stem cells/poly lactide-co-glycolic acid constructs for bone regeneration in a rat critical-sized calvarial defect model. Tissue Eng, 2007, 13(3) :619-627.
22. El Marsafy S, Larghero J. Mesenchymal Stem Cells:key actors in tumor niche. Curr Stem Cell Res Ther, 2015, 10(6) :523-529.

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Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON OSTEOGENESIS OF SYNOVIUM-DERIVED MESENCHYMAL STEM CELLS IN VITRO AND IN VIVO

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