A novel culture platform for fast proliferation of human annulus fibrosus cells.
Tissue engineering provides a promising approach to treat degenerative disc disease, which usually requires a large quantity of seed cells. A simple and reliable in vitro culture system to expand seed cells in a timely fashion is necessary to implement the application clinically. Here, we sought to establish a cost-effective culture system for expanding human annulus fibrosus cells using extracellular matrix (ECM) proteins as culture substrates. Cells were cultured onto a plastic surface coated with various types of ECMs, including fibronectin, vitronectin, collagen type I, gelatin and cell-free matrix deposited by human nucleus pulposus cells. AF cell morphology, growth, adhesion and phenotype (anabolic and catabolic markers) were assessed by microscopy, real-time RT-PCR, western blotting, zymography, immunofluorescence staining and biochemical assays. Fibronectin, collagen and gelatin promoted cell proliferation and adhesion in a dose-dependent manner. Fibronectin elevated mRNA expression of proteoglycan and enhanced glycosaminoglycan production. Both collagen and gelatin increased protein expression of type II collagen. Consistent with increased cell adhesion, collagen and fibronectin promoted formation of focal adhesion complexes in the cell-matrix junction, suggesting enhanced binding of the actin network with both ECM substrates. On the other hand, fibronectin, collagen and gelatin decreased expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 in media. Finally, a mixture of fibronectin (1.7 μg/mL) and collagen (1.3 μg/mL) was identified as the most promising in vitro culture substrate system in promoting proliferation and maintaining anabolic-catabolic balance. Our method provides a simple and cost-effective platform for tissue engineering applications in intervertebral disc research.
Department of Orthopaedic Surgery, University of Virginia, Cobb Hall, 135 Hospital Dr., Charlottesville, VA 22908.
Li
L
Xiao
Department of Orthopaedic Surgery, University of Virginia, Cobb Hall, 135 Hospital Dr., Charlottesville, VA 22908.|Department of Anesthesiology, China Medical University, No. 36 Sanhao Street, Heping District, Shenyang, Liaoning, China 110004.
Mengmeng
M
Ding
Department of Orthopaedic Surgery, University of Virginia, Cobb Hall, 135 Hospital Dr., Charlottesville, VA 22908.
Osama
O
Saadoon
Department of Orthopaedic Surgery, University of Virginia, Cobb Hall, 135 Hospital Dr., Charlottesville, VA 22908.
Eric
E
Vess
Department of Orthopaedic Surgery, University of Virginia, Cobb Hall, 135 Hospital Dr., Charlottesville, VA 22908.
Andrew
A
Fernandez
Department of Anesthesiology, China Medical University, No. 36 Sanhao Street, Heping District, Shenyang, Liaoning, China 110004.
Ping
P
Zhao
Department of Orthopaedic Surgery, University of Virginia, Cobb Hall, 135 Hospital Dr., Charlottesville, VA 22908.
Li
L
Jin
Department of Orthopaedic Surgery, University of Virginia, Cobb Hall, 135 Hospital Dr., Charlottesville, VA 22908.
Xudong
X
Li
D000070616:Annulus Fibrosus / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005353:Fibronectins / Q000494:pharmacology; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D005780:Gelatin / Q000494:pharmacology; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors; D016596:Vinculin / Q000378:metabolism
D005353:Fibronectins; D012333:RNA, Messenger; D016596:Vinculin; D005780:Gelatin; D003094:Collagen; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9
Cell proliferation; Extracellular matrix; In vitro culture; Intervertebral disc degeneration; Tissue engineering