Tissue engineering of the intervertebral disc with cultured nucleus pulposus cells using atelocollagen scaffold and growth factors.
METHODS
In vitro experiment using rabbit nucleus pulposus (NP) cells seeded in atelocollagen scaffolds under the stimulation of growth factors.


OBJECTIVE
To demonstrate the effect of anabolic growth factors in rabbit NP cells cultured in atelocollagen type I and type II.


BACKGROUND
Atelocollagen provides intervertebral disc (IVD) cells for a biocompatible environment to produce extracellular matrix. IVD cells with exogenous transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) also render an increase in matrix synthesis. However, the effect of anabolic growth factors in NP cells cultured in atelocollagens was not elucidated before.


METHODS
Rabbit NP cell was harvested, enzymatically digested, and cultured. The NP cells were seeded to atelocollagen type I and type II scaffolds, and then cultures were exposed to TGF-β1 (10 ng/mL) and/or BMP-2 (100 ng/mL). DNA synthesis was measured using [4H]-thymidine incorporation. Newly synthesized proteoglycan was measured using [35S]-sulfate incorporation. Reverse transcription-polymerase chain reactions (RT-PCRs) for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin were performed.


RESULTS
Rabbit NP cells cultured in atelocollagen type I scaffold showed an increase (1.7 to 2.4-fold) in DNA synthesis in response to TGF-β1 and/or BMP-2 (P < 0.05), whereas NP cultures in atelocollagen type II demonstrated a 30% increase in DNA synthesis only with combination of both growth factors compared with control (P < 0.05). Rabbit NP cells in atelocollagen type II scaffold with TGF-β1 and combination of both growth factors exhibited robust 5.3- and 5.4-fold increases in proteoglycan synthesis (P < 0.05), whereas any cultures in atelocollagen type I failed to show any significant increase compared with control. Rabbit NP cells in atelocollagen type I and type II scaffolds with TGF-β1 and/or BMP-2 demonstrated the upregulation of aggrecan, collagen type I, and collagen type II mRNA expression compared with saline control (P < 0.05). The response in transcriptional level was more robust in atelocollagen type II than in type I. In any event, there is no recognizable expression of osteocalcin (P < 0.05).


CONCLUSIONS
NP cells in atelocollagens under the stimulation of TGF-β1 and BMP-2 exhibited anabolic responses in transcriptional and translational levels. Hence, such an approach can provide a suitable engineered tissue for IVD regeneration with potential for robust refurbishment of matrix.
Brain Korea 21, Medical Science Graduate School, Seoul, Korea.
Kwang-Il
KI
Lee
Seong-Hwan
SH
Moon
Hyang
H
Kim
Un-Hye
UH
Kwon
Ho-Joong
HJ
Kim
Si-Nae
SN
Park
Hwal
H
Suh
Hwan-Mo
HM
Lee
Hak-Sun
HS
Kim
Heoung-Jae
HJ
Chun
Il-Keun
IK
Kwon
Ju-Woong
JW
Jang
D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D002478:Cells, Cultured; D003094:Collagen; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007403:Intervertebral Disc / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D023822:Tissue Engineering; D054457:Tissue Scaffolds; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D055396:Bone Morphogenetic Protein 2; D036341:Intercellular Signaling Peptides and Proteins; D053773:Transforming Growth Factor beta1; C059821:atelocollagen; D003094:Collagen