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"INTRODUCTION
Lower back pain affects 80 % of adults and has estimated costs up to 1 – 2 % of GDP1,2. Current surgical treatments include spinal fusion, but outcomes are poor with reoperation required in 20% of patients3. An emerging and more regenerative approach is nucleus augmentation, which aims to restore healthy disc anatomy and biomechanical function through the delivery of a biomaterial into the nucleus of a degenerated intervertebral disc. Self-assembling peptides (PEP) can be used with glycosaminoglycans (GAG) to form a PEP-GAG hydrogel and tuned to improve the biocompatibility and mechanical properties for use as a nucleus augmentation material. This work focuses on assessing three different peptides and their viability for nucleus augmentation.
EXPERIMENTAL METHODS
All samples contain peptide (20 mg/ml) with or without chondroitin sulfate (136 mg/ml).
Contact, extract, and filter diffusion assays were used to assess PEP-GAG hydrogel and counterion cytotoxicity with L929 and BHK cells.
Small amplitude oscillatory shear rheology was used to assess mechanical properties after different modes of delivery.
Peptide samples were made at a range of concentrations in D2O to determine the critical concentration (c*) for self-assembly using 1H NMR.
CryoSEM and FIB-SEM images were obtained using FEI – Helios G4 CX Dual beam FIBSEM.
RESULTS AND DISCUSSION
Cytotoxicity testing of different PEP-GAG hydrogels showed some slight cytotoxicity in the indirect and extract assays but not in the direct contact assay with the three different peptides behaving similarly across the assays. A greater difference in sample cytotoxicity was seen when comparing trifluoroacetate (TFA), acetate and HCl counterions with HCl showing the least cytotoxicity compared to TFA and acetate.
As the end polar amino acids change from ser-ser to glu-ser to glu-glu the c* decreases as a result of the increased number and strength of intermolecular hydrogen bonding. When injected down different needle lengths, gauges and designs, there was a small reduction in gel stiffness (G’) when the needle design was changed, however this small change was not clinically relevant.
CryoSEM images showed that all three PEP-GAG hydrogels were able to form fibrous networks.
CONCLUSION
All three peptides investigated were able to form PEP-GAG hydrogels with low levels of cytotoxicity. Changing the end polar amino acid between glutamine and serine allows for tuning of the material properties. The mechanical properties as determined by rheology suggest that the PEP-GAG hydrogels are suitable for nucleus augmentation. The use of a minimally invasive clinical delivery device slightly reduces the gel stiffness but provides the advantage of reduced damage to the annulus fibrosus and simultaneous injection of the two components to allow better mixing in situ. The PEP-GAG hydrogels offer a promising new approach to repair the degenerated intervertebral disc to treat lower back pain.
REFERENCES
1. Kent, P.M. et al, Chiropr. Osteopat. 13:13, 2005.
2. Philips, C.J. et al, Expert. Rev. Pharmacoecon. Outcome Res. 6:591-601, 2006.
3. Gillet, P. J Spinal Disord Tech. 16:338-345. 2003.
ACKNOWLEDGMENTS
The authors would like to acknowledge Stuart Micklethwaite and LEMAS for their support obtaining cryoSEM and FIB-SEM images."
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