ISOLATION AND IDENTIFICATION OF CARTILAGE PROGENITOR CELLS AND INFLUENCE OF INTERLEUKIN 1β ON ITS CHONDROGENESIS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHOUJianxin ¹ , YANGXiaofei ² , LIYang ² , DINGPeng ² , JIANGYiqiu ² ,  GUIJianchao ²

1. Department of Orthopaedics, the First People's Hospital of Wujiang District of Suzhou City, Suzhou Jiangsu, 215200, P. R. China;
2. Department of Orthopaedics, Nanjing Hospital Affiliated to Nanjing Medical University;

Corresponding author：

GUIJianchao, Email: gui1997@126.com

Keywords：

Cartilage progenitor cells; Inflammatory factor; Interleukin 1β; Differentiation; Cartilage repair

DOI：

10.7507/1002-1892.20150187

Video：

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Objective To isolate and identify the cartilage progenitor cells (CPCs) from normal cartilage, and to explore the influence of interleukin 1β (IL-1β) in different concentrations on its chondrogenesis. Methods CPCs were isolated from normal cartilage of adult New Zealand white rabbit with the fibronectin adhesion assay;the cell phenotype was identified;and the cloning and differentiation of CPCs were observed. CPCs were incubated with H-DMEM in group A, with chondrogenic induced medium in group B, with chondrogenic induced medium+0.1 ng/mL IL-1β in group C and chondrogenic induced medium+1.0 ng/mL IL-1β in group D for 3 weeks. The histology, biochemistry, and real-time fluorescence quantitative PCR were performed to observe the effect of IL-1β on the chondrgenic differentiation. Results The CPCs from normal cartilage expressed positively stem cell phenotype, which have similar ability of cloning and differentiation to stem cells. The cell pellets in groups C and D were significantly smaller than those in group B, and cell showed hypertrophic morphology change. There were more expressions of collagen type Ⅱ and collagen type X in group B than in group A, in group B than in groups C and D, and in group C than group D with Safranin O staining. The biochemistry results showed that collagen type Ⅱ content, glycosaminoglycan (GAG) content, and the ratio of GAG/DNA were significantly lower in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05);but the DNA content was significantly higher in groups C and D than in group B (P<0.05), and no significant difference between groups C and D (P>0.05). The real-time fluorescence quantitative PCR results showed that the relative mRNA expressions of collagen type Ⅱ, collagen type X, and Sox-9 were significantly lower in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05), but the relative mRNA expressions of Runx-2 and matrix metalloproteinase 13 were significantly higher in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05). Conclusion There are CPCs having the character of stem cells in normal cartilage, and they have the capability of cloning and potential differentiation. IL-1β can inhibit the chondrogenesis of CPCs, and possibly promote the osteogenic differentiation.

Citation： ZHOUJianxin, YANGXiaofei, LIYang, DINGPeng, JIANGYiqiu, GUIJianchao. ISOLATION AND IDENTIFICATION OF CARTILAGE PROGENITOR CELLS AND INFLUENCE OF INTERLEUKIN 1β ON ITS CHONDROGENESIS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(7): 863-869. doi: 10.7507/1002-1892.20150187 Copy

1.	Marlovits S,Zeller P,Singer P,et al.Cartilage repair:generations of autologous chondrocyte transplantation.Eur J Radiol,2006,57(1):24-31.
2.	Ahmed TA,Hincke MT.Strategies for articular cartilage lesion repair and functional restoration.Tissue engineering Part B,Reviews,2010,16(3):305-329.
3.	Buckwalter JA.Articular cartilage:injuries and potential for healing.J Orthop Sports Phys Ther,1998,28(4):192-202.
4.	Solheim E,Øyen J,Hegna J,et al.Microfracture treatment of single or multiple articular cartilage defects of the knee:a 5-year median follow-up of 110 patients.Knee Surg Sports Traumatol Arthrosc,2010,18(4):504-508.
5.	Frank RM,Van Thiel GS,Slabaugh MA,et al.Clinical outcomes after microfracture of the glenohumeral joint.Am J Sports Med,2010,38(4):772-781.
6.	Gulotta LV,Rudzki JR,Kovacevic D,et al.Chondrocyte death and cartilage degradation after autologous osteochondral transplantation surgery in a rabbit model.Am J Sports Med,2009,37(7):1324-1333.
7.	Pangopoulos A,van Niekerk L,Triantafillopoulos I.Autologous chondrocyte implantation for knee cartilage injuries:moderate functional outcome and performance in patients with high-impact activities.Orthopedics,2012,35(1):e6-14.
8.	Cournil-Henrionnet C,Huselstein C,Wang Y,et al.Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion.Biorheology,2008,45(3-4):513-526.
9.	Schnabel M,Marlovits S,Eckhoff G,et al.Dedifferentiation-associated changes in morphology and gene expression in primary human articular chondrocytes in cell culture.Osteoarthritis Cartilage,2002,10(1):62-70.
10.	Dowthwaite GP,Bishop JC,Redman SN,et al.The surface of articular cartilage contains a progenitor cell population.J Cell Sci,2004,117(Pt 6):889-897.
11.	Kobayashi S,Takebe T,Zheng YW,et al.Presence of cartilage stem/progenitor cells in adult mice auricular perichondrium.PLoS One,2011,6(10):e26393.
12.	Hattori S,Oxford C,Reddi AH.Identification of superficial zone articular chondrocyte stem/progenitor cells.Biochem Biophys Res Commun,2007,358(1):99-103.
13.	Koelling S,Miosge N.Sex differences of chondrogenic progenitor cells in late stages of osteoarthritis.Arthritis Rheum,2010,62(4):1077-1087.
14.	Koelling S,Kruegel J,Irmer M,et al.Migratory chondrogenic progenitor cells from repair tissue during the later stages of human osteoarthritis.Cell Stem Cell,2009,4(4):324-335.
15.	Ousema PH,Moutos FT,Estes BT,et al.The inhibition by interleukin 1 of MSC chondrogenesis and the development of biomechanical properties in biomimetic 3D woven PCL scaffolds.Biomaterials,2012,33(35):8967-8974.
16.	Lima EG,Tan AR,Tai T,et al.Genipin enhances the mechanical properties of tissue-engineered cartilage and protects against inflammatory degradation when used as a medium supplement.J Biomed Mater Res A,2009,91(3):692-700.
17.	Estes BT,Fermor B,Guilak F.The influence of interleukin-1 and mechanical stimulation on human adipose derived adult stem cells undergoing chondrogenesis.Trans Orthop Res Soc,2004,29:765.
18.	Heldens GT,Blaney Davidson EN,Vitters EL,et al.Catabolic factors and osteoarthritis-conditioned medium inhibit chondrogenesis of human mesenchymal stem cells.Tissue Eng Part A,2012,18(1-2):45-54.
19.	Wehling N,Palmer GD,Pilapil C,et al.Interleukin-1beta and tumor necrosis factor alpha inhibit chondrogenesis by human mesenchymal stem cells through NF-kappaB-dependent pathways.Arthritis Rheum,2009,60(3):801-812.
20.	Joos H,Wildner A,Hogrefe C,et al.Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage.Arthritis Res Ther,2013,15(5):R119.
21.	Yin Z,Yang X,Jiang Y,et al.Platelet-rich plasma combined with agarose as a bioactive scaffold to enhance cartilage repair:an in vitro study.J Biomater Appl,2014,28(7):1039-1050.
22.	Gerter R,Kruegel J,Miosge N.New insights into cartilage repair-the role of migratory progenitor cells in osteoarthritis.Matrix Biol,2012,31(3):206-213.
23.	Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.Cytotherapy,2006,8(4):315-317.
24.	Williams R,Khan IM,Richardson K,et al.Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage.PLoS One,2010,5(10):e13246.
25.	Baptista LS,Silva KR,Pedrosa CS,et al.Bioengineered cartilage in a scaffold-free method by human cartilage-derived progenitor cells:a comparison with human adipose-derived mesenchymal stromal cells.Artif Organs,2013,37(12):1068-1075.
26.	McCarthy HE,Bara JJ,Brakspear K,et al.The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse.Vet J,2012,192(3):345-351.
27.	Tyler JA.Articular cartilage cultured with catabolin (pig interleukin 1) synthesizes a decreased number of normal proteoglycan molecules.Biochem J,1985,227(3):869-878.
28.	Siddiqui MA.The efficacy and tolerability of newer biologics in rheumatoid arthritis:best current evidence.Curr Opin Rheumatol,2007,19(3):308-313.
29.	Murakami S,Lefebvre V,de Crombrugghe B.Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha.J Biol Chem,2000,275(5):3687-3692.
30.	Dowthwaite GP,Bishop JC,Redman SN,et al.The surface of articular cartilage contains a progenitor cell population.J Cell Sci,2004,117(Pt 6):889-897.

1. Marlovits S,Zeller P,Singer P,et al.Cartilage repair:generations of autologous chondrocyte transplantation.Eur J Radiol,2006,57(1):24-31.
2. Ahmed TA,Hincke MT.Strategies for articular cartilage lesion repair and functional restoration.Tissue engineering Part B,Reviews,2010,16(3):305-329.
3. Buckwalter JA.Articular cartilage:injuries and potential for healing.J Orthop Sports Phys Ther,1998,28(4):192-202.
4. Solheim E,Øyen J,Hegna J,et al.Microfracture treatment of single or multiple articular cartilage defects of the knee:a 5-year median follow-up of 110 patients.Knee Surg Sports Traumatol Arthrosc,2010,18(4):504-508.
5. Frank RM,Van Thiel GS,Slabaugh MA,et al.Clinical outcomes after microfracture of the glenohumeral joint.Am J Sports Med,2010,38(4):772-781.
6. Gulotta LV,Rudzki JR,Kovacevic D,et al.Chondrocyte death and cartilage degradation after autologous osteochondral transplantation surgery in a rabbit model.Am J Sports Med,2009,37(7):1324-1333.
7. Pangopoulos A,van Niekerk L,Triantafillopoulos I.Autologous chondrocyte implantation for knee cartilage injuries:moderate functional outcome and performance in patients with high-impact activities.Orthopedics,2012,35(1):e6-14.
8. Cournil-Henrionnet C,Huselstein C,Wang Y,et al.Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion.Biorheology,2008,45(3-4):513-526.
9. Schnabel M,Marlovits S,Eckhoff G,et al.Dedifferentiation-associated changes in morphology and gene expression in primary human articular chondrocytes in cell culture.Osteoarthritis Cartilage,2002,10(1):62-70.
10. Dowthwaite GP,Bishop JC,Redman SN,et al.The surface of articular cartilage contains a progenitor cell population.J Cell Sci,2004,117(Pt 6):889-897.
11. Kobayashi S,Takebe T,Zheng YW,et al.Presence of cartilage stem/progenitor cells in adult mice auricular perichondrium.PLoS One,2011,6(10):e26393.
12. Hattori S,Oxford C,Reddi AH.Identification of superficial zone articular chondrocyte stem/progenitor cells.Biochem Biophys Res Commun,2007,358(1):99-103.
13. Koelling S,Miosge N.Sex differences of chondrogenic progenitor cells in late stages of osteoarthritis.Arthritis Rheum,2010,62(4):1077-1087.
14. Koelling S,Kruegel J,Irmer M,et al.Migratory chondrogenic progenitor cells from repair tissue during the later stages of human osteoarthritis.Cell Stem Cell,2009,4(4):324-335.
15. Ousema PH,Moutos FT,Estes BT,et al.The inhibition by interleukin 1 of MSC chondrogenesis and the development of biomechanical properties in biomimetic 3D woven PCL scaffolds.Biomaterials,2012,33(35):8967-8974.
16. Lima EG,Tan AR,Tai T,et al.Genipin enhances the mechanical properties of tissue-engineered cartilage and protects against inflammatory degradation when used as a medium supplement.J Biomed Mater Res A,2009,91(3):692-700.
17. Estes BT,Fermor B,Guilak F.The influence of interleukin-1 and mechanical stimulation on human adipose derived adult stem cells undergoing chondrogenesis.Trans Orthop Res Soc,2004,29:765.
18. Heldens GT,Blaney Davidson EN,Vitters EL,et al.Catabolic factors and osteoarthritis-conditioned medium inhibit chondrogenesis of human mesenchymal stem cells.Tissue Eng Part A,2012,18(1-2):45-54.
19. Wehling N,Palmer GD,Pilapil C,et al.Interleukin-1beta and tumor necrosis factor alpha inhibit chondrogenesis by human mesenchymal stem cells through NF-kappaB-dependent pathways.Arthritis Rheum,2009,60(3):801-812.
20. Joos H,Wildner A,Hogrefe C,et al.Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage.Arthritis Res Ther,2013,15(5):R119.
21. Yin Z,Yang X,Jiang Y,et al.Platelet-rich plasma combined with agarose as a bioactive scaffold to enhance cartilage repair:an in vitro study.J Biomater Appl,2014,28(7):1039-1050.
22. Gerter R,Kruegel J,Miosge N.New insights into cartilage repair-the role of migratory progenitor cells in osteoarthritis.Matrix Biol,2012,31(3):206-213.
23. Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.Cytotherapy,2006,8(4):315-317.
24. Williams R,Khan IM,Richardson K,et al.Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage.PLoS One,2010,5(10):e13246.
25. Baptista LS,Silva KR,Pedrosa CS,et al.Bioengineered cartilage in a scaffold-free method by human cartilage-derived progenitor cells:a comparison with human adipose-derived mesenchymal stromal cells.Artif Organs,2013,37(12):1068-1075.
26. McCarthy HE,Bara JJ,Brakspear K,et al.The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse.Vet J,2012,192(3):345-351.
27. Tyler JA.Articular cartilage cultured with catabolin (pig interleukin 1) synthesizes a decreased number of normal proteoglycan molecules.Biochem J,1985,227(3):869-878.
28. Siddiqui MA.The efficacy and tolerability of newer biologics in rheumatoid arthritis:best current evidence.Curr Opin Rheumatol,2007,19(3):308-313.
29. Murakami S,Lefebvre V,de Crombrugghe B.Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha.J Biol Chem,2000,275(5):3687-3692.
30. Dowthwaite GP,Bishop JC,Redman SN,et al.The surface of articular cartilage contains a progenitor cell population.J Cell Sci,2004,117(Pt 6):889-897.

Chinese Journal of Reparative and Reconstructive Surgery

ISOLATION AND IDENTIFICATION OF CARTILAGE PROGENITOR CELLS AND INFLUENCE OF INTERLEUKIN 1β ON ITS CHONDROGENESIS

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