Link-N Peptide: A Stepping Stone towards Biological Repair of Intervertebral Disk Degeneration
Authors: Gawri R. , Antoniou J. , Ouellet J. , Roughley P. , Steffen T. , Haglund L. A. , Mwale F. 
Published: June 2012

Introduction

Back pain is a fairly common problem which affects a large portion of the population across all ages and has an impact on quality of life. Intervertebral disk (IVD) degeneration is the single most common implicated cause of back pain. Presently, there is no medical treatment or therapeutic agent to address this problem and surgery is the only offered option. Link-N peptide represents the 16 amino acid sequence from the N-terminus of the link protein that stabilizes the proteoglycan aggregates present in cartilage and disk. Link-N peptide is released from the link protein as a result of proteolysis, and has been suggested to play a role in matrix homeostasis by promoting new matrix synthesis. We evaluated its regenerative potential in intact human IVD.

Materials and Methods

Lumbar IVDs were obtained through organ donations via Transplant Quebec. Disks from seven individuals, five disks per spine, were harvested. Cells were isolated from nucleus pulposus (NP) and inner annulus fibrosus regions of the disks. Single cells were beaded in 1.2% alginate and cultured in DMEM containing 1 g/L glucose and 10% FBS. Alginate beads were exposed to 10–10000 ng/mL Link-N peptide for 48 hours. Intact disks were prepared for organ culture by parallel cuts through the adjacent vertebral bodies close to the end plates, and the remaining bone and the calcified part of the cartilage endplates were removed using a high-speed bone burr. Disks were maintained and cultured with no external load applied in DMEM containing 1 g/L glucose and supplemented with 1% FBS. Link-N was conjugated with 5-TAMRA dye then injected into the center of the disk. The distribution of Link-N in the medium and within the disk was studied to determine whether Link-N is retained in the disk. Disks from adjacent levels were matched for the degree of degeneration and were injected in their NP region with 50µCi 35SO4 along with 0.1 mg or 1 mg of Link-N in 100 µL of medium per disk and harvested after 48 hours. Sustained effect of Link-N was evaluated by injecting the disk with Link-N and injecting 35SO4 1 week later. Proteoglycan synthesis was evaluated by measuring 35SO4 incorporation.

Results

When human lumbar disk cells from NP and iAF regions beaded in alginate were exposed to Link-N peptide for 48 hours, proteoglycan synthesis was observed to increase in a dose-dependent manner with the maximal response at 1000 ng/mL Link-N. Fluorescently labelled Link-N peptide was injected into the disks to determine if Link-N is retained in the disks matrix or freely diffuses throughout the tissue and equilibrates with surrounding medium. Samples were taken continually from the surrounding medium and from the disk tissue at the termination of the experiment. Fluorescent-Link-N was detectable in the medium at 24 hours and reached equilibrium after 48 hours. The fluorescent peptide was found in the NP and NP/iAF junction but not in the remaining AF. Thus loss of Link-N appears to occur by diffusion through the endplates (Fig. 1). Cell viability was maintained in the NP, at >96%, after injection of 1 mg of Link-N/disk. Disks injected with Link-N showed increased proteoglycan synthesis in the NP and iAF compared to adjacent level control disks matched for grade of degeneration. To evaluate the duration of the effect, disks were injected with 35SO4 1 week after the injection of Link-N. Proteoglycan synthesis remained elevated in Link-N injected disks compared to adjacent level control disks suggesting a sustained effect.

Conclusion

Link-N peptide has previously been shown to promote matrix protein synthesis by bovine disk cells in monolayer and pellet cultures. In this work, we show that Link-N can promote proteoglycan synthesis not only in human disk cells cultured in 3D constructs, but also in intact adult human disks where the cells are in their native environment. Recently, an increase in disk height measured by MRI was shown in an in vivo rabbit model, where degenerated disks were injected with Link-N. If a similar restoration of disk function could be achieved in the human disks, then Link-N could be a promising candidate for biologically induced disk repair, and could provide an alternative to surgical intervention for early stage disk degeneration. Link-N has a significant cost advantage over growth factors, such as BMP7, TGFβ and, GDF5. Based on prior in vivo studies in the rabbit, Link-N is over 100 times less expensive than recombinant growth factors that have a similar repair response. Thus, Link-N peptide injection could be both effective and cost-efficient therapy for retarding the ongoing degenerative process in early stage disk disease and help relieve back pain. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared