A possible approach to stimulate proteoglycan and collagen synthesis for treating intervertebral disc degeneration (IDD) is introduction of growth factors. The objective of Part I of this study was to screen the effect of human recombinant bone morphogenetic protein (BMP)-2 and BMP-12 on nucleus pulposus (NP) cells and to investigate the effect of Ad/BMP-12 on NP and anulus fibrosus (AF) cells. Cells were isolated from degenerated human discs and cultured in monolayer. RhBMP-2 (25, 50, 100, 200, 300, ng/ml) and rhBMP-12 (25, 50, 100 ng/ml) stimulated NP cells in serumless media (1% ITS) for 2 days. Ad/BMP-12 (50, 100, 150 MOI) transduced NP and AF cells, then pellets (150,000 cells/pellet) were formed and incubated in serumless media (1% ITS) for 6 days. Proteoglycan, collagen, and non-collagenous protein synthesis were measured. RhBMP-2 had a more substantial effect on upregulating matrix synthesis than rhBMP-12. Ad/BMP-12 significantly increased matrix synthesis. Total DNA content was increased pellets stimulated by Ad/BMP-12 when compared to control. The increase in matrix synthesis was attributed to both an increase in cell number and in matrix synthesis per cell.
Intervertebral disc (IVD) is the largest avascular organ in the body, it has been suggested that lack of nutrition may be one of the cause of IDD. The goals of the second part of this study were to develop a rabbit disc organ culture method to study the effect of FBS concentration in disc metabolism and to attempt gene therapy in the organ culture. Twenty-seven rabbit lumber intervertebral discs were harvested and cultured for 2 weeks in serumless media (1% ITS) or F-12/DMEM (5%, 10%, 15% FBS). NP and AF wet weight, dry weight, % hydration, glycosaminoglycans (GAG), DNA, and lactate content were measured. In 25 discs, 6x106 PFU of Ad/lacZ or Ad/Luciferase was injected into the NP 2 days after the culture to determine the effect of gene transfer. Histology and viability staining of 10 discs were used to show cell morphology and viability. Due to low cell viability, we did not observe successful gene transfer in the organ culture.
NP and AF lactate content and AF DNA content were significantly higher in the 15% FBS group than serumless group.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-11192003-172535 |
| Date | 02 February 2004 |
| Creators | Teng, Pang-ning |
| Contributors | Rebecca K Studer, James D Kang, Lars G Gilbertson, Richard R Koepsel |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-11192003-172535/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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