The effect of bone morphogenetic protein-2 on rat intervertebral disc cells in vitro.
METHODS
An in vitro experiment to determine the molecular and cellular effect of recombinant human bone morphogenetic protein-2 on cultured rat intervertebral disc cells was performed.


OBJECTIVE
To determine the effect of recombinant human bone morphogenetic protein-2 on cell proliferation, production of sulfated-glycosaminoglycan, and the expression of genes specific for chondrocytes (Type II collagen, aggrecan, and Sox9) in cultured rat intervertebral disc cells.


BACKGROUND
Intervertebral disc degeneration is associated with cellular and biochemical changes, which include decreased synthesis of cartilage specific gene products such as Type II collagen and aggrecan. Although bone morphogenetic protein-2 is known to induce chondrogenesis during new bone formation, the effects on intervertebral disc cells have not been characterized.


METHODS
Cells were isolated from the anulus fibrosus and transition zones of lumbar discs from Sprague-Dawley rats. The cells were grown in monolayer and treated with recombinant human bone morphogenetic protein-2 (0, 10, 100, 1000 ng/mL) in Dulbecco's Modified Eagle Medium/F-12 with 1% fetal bovine serum (day 0). On days 2, 4, and 7 after recombinant human bone morphogenetic protein-2 treatment, sulfated-glycosaminoglycan content in the media was quantified using 1,9-dimethylmethylene blue staining. The results were normalized according to culture duration and cell number. On day 7, mRNA was extracted for reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction to quantitate mRNAs of Type I collagen, Type II collagen, aggrecan, Sox9, osteocalcin, and glyceraldehyde phosphate dehydrogenase. Cell number was determined with a hemocytometer.


RESULTS
Recombinant human bone morphogenetic protein-2 at 100 and 1000 ng/mL yielded a 17% and 42% increase in cell number on day 4, and a 59% and 79% on day 7, respectively. Recombinant human bone morphogenetic protein-2 at 10 ng/mL had no effect on cell number. Sulfated-glycosaminoglycan increase was greatest at day 7, increasing by 1.3-, 2.1-, and 3.6-fold with recombinant human bone morphogenetic protein-2 treatments of 10, 100, and 1000 ng/mL, respectively. Increases in mRNA levels of Type II collagen, aggrecan, Sox9, and osteocalcin were observed with recombinant human bone morphogenetic protein-2 concentrations of 100 and 1000 ng/mL on day 7 as determined by reverse transcriptase-polymerase chain reaction. No detectable increase in mRNA level of Type I collagen was observed with any levels of recombinant human bone morphogenetic protein-2. Real-time polymerase chain reaction showed the greatest effect at 1000 ng/mL recombinant human bone morphogenetic protein-2, leading to an 11.5-fold increase in aggrecan, a 4.6-fold increase in Type II collagen, a 5.3-fold increase in Sox9, and a 1.9-fold increase in osteocalcin mRNA above untreated controls at day 7.


CONCLUSIONS
The results of this study show that recombinant human bone morphogenetic protein-2 enhances disc matrix production and chondrocytic phenotype of intervertebral disc cells. Recombinant human bone morphogenetic protein-2 increases cell proliferation and sulfated-glycosaminoglycan (proteoglycan) synthesis. It increases mRNA of Type II collagen, aggrecan, and Sox9 genes (chondrocyte specific genes), and osteocalcin, but not Type I collagen or glyceraldehyde phosphate dehydrogenase.
Tim Yoon
S
S
Emory University School of Medicine, Atlanta, GA 30033, USA. tim_yoon@emoryhealthcare.org
Su Kim
Keun
K
Li
Jun
J
Soo Park
Jin
J
Akamaru
Tomoyuki
T
Elmer
William A
WA
Hutton
William C
WC
D053674:Aggrecans; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics; D024043:Collagen Type II / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D016326:Extracellular Matrix Proteins; D005786:Gene Expression Regulation / Q000187:drug effects; D006609:High Mobility Group Proteins / Q000235:genetics; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D037181:Lectins, C-Type; D015675:Osteocalcin / Q000235:genetics; D011509:Proteoglycans / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D055758:SOX9 Transcription Factor; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics; D016212:Transforming Growth Factor beta
C508321:Acan protein, rat; D053674:Aggrecans; C525718:BMP2 protein, human; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D006609:High Mobility Group Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D012333:RNA, Messenger; D011994:Recombinant Proteins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D015675:Osteocalcin