Effects of basic fibroblast growth factor on chondrocyte phenotype and dedifferentiation in porous tantalum-chondrocyte composites in vitro
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Graduate School,Southern Medical University;Department of Joint Surgery, Tangshan second Hospital,Graduate School,Southern Medical University;Department of Anatomy, Basic Medical College, Hebei United University,Department of Orthopaedics,Affiliated Hospital,Hebei United University,Department of Orthopaedics,Affiliated Hospital,Hebei United University,Graduate School,Southern Medical University;Department of Hand Surgery, Tangshan second Hospita,Graduate School,Southern Medical University;Department of Orthopaedics, Affiliated Hospital, Hebei United University,Experimental Center,Hebei United University,Graduate School,Southern Medical University;Department of Orthopaedics, Affiliated Hospital, Hebei United University

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Supported by National Science and Technology Supporting Project of Science and Technology Ministry of China (2012BAE06B03).

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    Abstract:

    Objective To study the effect of basic fibroblast growth factor (bFGF) of different concentrations on phenotypes and dedifferentiation of rabbit chondrocytes in porous tantalum-chondrocyte composites in vitro, so as to provide theoretic basis for cartilage defect repair. Methods The articular chondrocytes from 3-week-old rabbit were cultured and identified by type Ⅱ collagen immunocytochemistry and Safranin O staining. The 3rd generation chondrocytes were implanted in the porous tantalum and was treated with bFGF of various concentrations. The bFGF-chondrocyte-porous tantalum composites (bFGF compostes) were then divided into 5 groups: group A (1 ng/mL bFGF composites), group B (10 ng/mL bFGF composites), group C (50 ng/mL bFGF composites), group D (chondrocyte-porous tantalum), and group E (pure chondrocyte). The proliferation of chondrocytes was measured by MTT and the cell morphology and growth were observed by scanning electron microscopy (SEM). Phenotypes and dedifferentiation (type Ⅰ, Ⅱ, Ⅸ, and Ⅹ collagen) of the chondrocytes were detected by immunocytochemical method. Type Ⅱ and Ⅹ collagen mRNA was tested by real-time PCR. Results Type Ⅱ collagen immunocytochemistry and Safranin O staining were positive, confirming that the cultured cells were chondrocytes. MTT results showed that chondrocyte proliferation in groups A, B, C, and D were significantly greater than that in group E (P<0.05), with the most significant proliferation promoting effect seen at 10 ng/mL; significant difference was also found between different groups (P<0.05). SEM observation: the chondrocytes grew adherently on the surface of the scaffolds and within scaffold pores, in the early stage the chondrocytes were spherical, after 24 h the chondrocytes stretched, lengthened, and extended pseudopodia gradually over time, and the chondrocytes connected with each other across the pores and covering the scaffold. Immunocytochemical staining showed that collagen Ⅱ and Ⅸ expressions were significantly stronger in 10 ng/mL bFGF-tantalum-chondrocytes group (group B) compared with the control group (group E, P<0.05), and there were significant differences between different groups (P<0.05); while collagen Ⅰ and Ⅹ expressions were significantly weaker compared with the control group (P<0.05). Expression of collagen type Ⅱ mRNA in the chondrocytes was significantly up-regulated in group A, B, C, and D compared with group E (P<0.05), and collagen type Ⅹ gene was significantly down-regulated (P<0.05). Conclusion bFGF can maintain chondrocyte phenotype in tantalum-chondrocyte composites, inhibit dedifferentiation and promote the secretion function of chondrocytes.

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History
  • Received:July 09,2014
  • Revised:November 24,2014
  • Adopted:January 21,2015
  • Online: March 26,2015
  • Published:
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