Zili Wang1, William C Hutton, S Tim Yoon. 1. Emory Spine Center, Emory University School of Medicine and VA Medical Center, Atlanta, GA 30033, USA. zwang2@emory.edu
Abstract
STUDY DESIGN: An in vitro study using human intervertebral disc cells. OBJECTIVE: To evaluate the effect of link protein peptide (LPP) on the expression of disc extracellular matrix macromolecules sulfated glycosaminoglycan, aggrecan, and collagen II. To determine the effect of LPP on catabolic regulators and to compare LPP with both BMP2 and BMP7 in terms of osteoinductivity. SUMMARY OF BACKGROUND DATA: Previous studies have shown that N-terminal link-protein peptide (LPP) can induce synthesis of proteoglycans and collagen II in cartilaginous cell types. However, the effect of LPP on human disc cells remains to be investigated. Moreover, the effects of LPP on the catabolic regulators and the osteoinductive potential of LPP are unknown. METHODS: Human intervertebral disc cells were cultured in alginate beads and treated with LPP, truncated LPPs, and the reverse sequence of LPP (LPR). The levels of aggrecan and collagen II mRNAs were measured by real-time polymerase chain reaction. Sulfated glycosaminoglycan content was assayed using the dimethylmethylene blue method. The protein levels of collagen II and catabolic regulators were determined by enzyme-linked immunosorbent assays and Western blots. The relative osteoinductive potential of LPP was evaluated by comparing the effect of LPP, BMP2, and BMP7 on in vitro markers of osteoinductivity. RESULTS: LPP upregulates expression of aggrecan and collagen II at both mRNA and protein levels. The full length of LPP is required for this upregulation. Neither the reverse sequences of LPP nor the truncated LPPs were as effective as the full-length LPP. LPP selectively inhibits expression of the catabolic regulators interleukin (IL)-1β and MMP1 and has no effect on expression of the other catabolic regulators tumor necrosis factor α, ADAMTS1, ADAMTS4, ADAMTS5, MMP3, and MMP9. LPP has relatively little osteoinductive effect compared with BMP2 and BMP7. CONCLUSION: LPP could have value in stimulating the growth and regeneration of degenerated discs with less concern of creating unwanted bone. LEVEL OF EVIDENCE: N/A.
STUDY DESIGN: An in vitro study using human intervertebral disc cells. OBJECTIVE: To evaluate the effect of link protein peptide (LPP) on the expression of disc extracellular matrix macromolecules sulfated glycosaminoglycan, aggrecan, and collagen II. To determine the effect of LPP on catabolic regulators and to compare LPP with both BMP2 and BMP7 in terms of osteoinductivity. SUMMARY OF BACKGROUND DATA: Previous studies have shown that N-terminal link-protein peptide (LPP) can induce synthesis of proteoglycans and collagen II in cartilaginous cell types. However, the effect of LPP on human disc cells remains to be investigated. Moreover, the effects of LPP on the catabolic regulators and the osteoinductive potential of LPP are unknown. METHODS:Human intervertebral disc cells were cultured in alginate beads and treated with LPP, truncated LPPs, and the reverse sequence of LPP (LPR). The levels of aggrecan and collagen II mRNAs were measured by real-time polymerase chain reaction. Sulfated glycosaminoglycan content was assayed using the dimethylmethylene blue method. The protein levels of collagen II and catabolic regulators were determined by enzyme-linked immunosorbent assays and Western blots. The relative osteoinductive potential of LPP was evaluated by comparing the effect of LPP, BMP2, and BMP7 on in vitro markers of osteoinductivity. RESULTS:LPP upregulates expression of aggrecan and collagen II at both mRNA and protein levels. The full length of LPP is required for this upregulation. Neither the reverse sequences of LPP nor the truncated LPPs were as effective as the full-length LPP. LPP selectively inhibits expression of the catabolic regulators interleukin (IL)-1β and MMP1 and has no effect on expression of the other catabolic regulators tumornecrosis factor α, ADAMTS1, ADAMTS4, ADAMTS5, MMP3, and MMP9. LPP has relatively little osteoinductive effect compared with BMP2 and BMP7. CONCLUSION:LPP could have value in stimulating the growth and regeneration of degenerated discs with less concern of creating unwanted bone. LEVEL OF EVIDENCE: N/A.
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