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Abstract
Study Design
In vitro experiment to determine the matrix synthesis of intervertebral disc (IVD) cell to various biologic inter-ventions and conditions.
Objectives
To elucidate biologic responses in terms of matrix synthesis of human IVD cells in vitro to various factors i.e. concentration of adenoviral vector and exogenous growth factor, duration of incubation, and type of culture methods.
Summary of Literature Review
Sophisticated method to delivery of growth factors, in continuous manner, is the genetic modi-fication of disc cells through gene transfer. Direct comparison of gene transfer and exogenous growth factor on matrix synthesis has not been reported.
Materials and Methods
IVD tissue was obtained from twenty three patients. Isolation and preparation of disc cells in monolayer (2 D) and alginate beads (3 D) culture were performed. Disc cells in 2 D and 3 D were treated with either A d/TGF- ß1 or exogenous TGF- ß1. Control cultures were treated with either saline or A d/luciferase. Matrix synthesis (newly synthesized proteoglycan) was measured in various conditions (concentration of adenoviral vector and exogenous growth factor, duration of incubation, and type of culture methods). Newly synthesized proteoglycan were analyzed using chromatography on Sephadex G- 25 in PD- 10 columns after S35- sulfate incorporation.
Results
A d/TGF- ß1 showed increase in proteoglycan synthesis (plateau at 75 MOI) in 3 D culture, (plateau at 25 MOI) in 2 D culture. In 3 D culture, A d/TGF- ß1showed significant increase in proteoglycan synthesis on day 1, 2, and 3 of incubation. In 2 D culture, A d/TGF- ß1showed significant increase in proteoglycan synthesis on day 2 of incubation with significant loss of anabolic effect on day 3. In 3 D culture, exogenous TGF- ß1showed increase in proteoglycan synthesis (plateau at 2 ng/ml) while in 2 D culture, there is no synthetic response to exogenous TGF- ß1.
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Fig. 1.
Content of newly synthesized proteoglycan as assayed by incorporation of 35S-sulfate. Human intervertebral disc cells in monolayer culture transduced by adenovirus-TGFß1 construct (10, 25, 50, 75, 100, 150, 300 MOI) showed increase in newly synthesized proteoglycan (p<0.05) with a plateau response with an MOI of 75 compared to those treated with normal saline.
Fig. 2.
Content of newly synthesized proteoglycan as assayed by incorporation of 35S-sulfate. Human intervertebral disc cells in monolayer culture treated by TGF-ß1 (2,10, 50 ng/ml) showed no increase in newly synthesized proteoglycan compared to those treated with normal saline.
Fig. 3.
Content of newly synthesized proteoglycan as assayed by incorporation of 35S-sulfate. Human intervertebral disc cells transduced by adenovirus-TGFß1 construct (10, 25, 50, 75, 100, 150, 300 MOI), cultured in 3 dimensional alginate beads, showed increase in newly synthesized proteoglycan with a plateau response with an MOI of 75 compared to those treated with normal saline (p<0.05).
Fig. 4.
Content of newly synthesized proteoglycan as assayed by incorporation of 35S-sulfate. Human intervertebral disc cells in 3 dimensional culture treated by TGF-ß1(2, 10, 50 ng/ml) showed significant increase in newly synthesized proteoglycan compared to those treated with normal saline (p<0.05).
Fig. 5.
Content of newly synthesized proteoglycan as assayed by incorporation of 35S-sulfate. Human intervertebral disc cells transduced by adenovirus-TGFß1 construct with an MOI of 75, cultured in 3 dimensional alginate beads, showed increase in newly synthesized proteoglycan in day 1, 2, and 3 without recognizable loss of anabolic effect. Disc cells culture in monolayer showed strong anabolic effect on day 2 with significant loss of anabolic effect on day 3.
Fig. 6.
Human intervertbral disc cells cultured in monolyaer and alginate beads with therapeutic gene transfer (TGF-ß1 gene) showed robust increase in proteoglycan synthesis compared to exogenous TGF-ß1 (p<0.05). Transduction with adenovirus-luciferase construct showed no difference in proteoglycan synthesis compared to normal saline control.
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