Self-Assembling Peptides for Cartilage Regeneration

Loss of glycosaminoglycans (GAGs) in osteoarthritic (OA) cartilage contributes to a decrease in mechanical properties and function in vitro, and is considered to be a major contributor to disease progression. The aims of this investigation were to test the hypothesis that a combination of self-assembling peptides (SAPs) and chondroitin sulfate (glycosaminoglycan; GAG) would restore the biomechanical properties of GAG depleted porcine condylar cartilage, ideally to a level intrinsic to native porcine condylar cartilage. The SAPs investigated were members of the P11 series which have been designed to spontaneously self-assemble into three-dimensional fibrilar hydrogels, in response to physiological conditions. Initial studies were carried out to determine which of three peptides (P11-4, P11-8 and P11-12) demonstrated high β-sheet percentage, long-woven fibrilar networks and high stiffness; when mixed with chondroitin sulfate at two different GAG molar ratios (1:16 and 1:64) in physiological conditions, using FTIR analysis, transmission electron microscopy and rheology. The β-sheet percentage, dimensions of fibrils and stiffness were dependent upon the peptide, GAG molar ratio and Na2+ salt concentration. P11-4 and P11-8: GAG mixtures had high β-sheet percentage ranging from 50.6-91 % and 81.7-92 %, respectively. Fibril lengths of the P11-4 and P11-8: GAG mixtures were in the range 498- 3518 nm and the elastic shear modulus (G’) ranged from 4,479-10,720 Pa and 7,722-26,854 Pa, respectively. P11-4 and P11-8: GAG mixtures were selected for further investigation. In order to produce a GAG depleted cartilage model, porcine femoral condylar cartilage was subjected to three different methods of GAG depletion (1) coating the surface with chondroitinase ABC (2) injecting chondroitinase ABC into the cartilage (3) washing the condyles in sodium dodecyl sulfate (SDS). GAG depletion was successfully achieved following two 24 hour washes in 0.1 % (w/v) SDS and buffer washes. Histological analysis of safranin O stained sections revealed an absence of GAGs. Quantification of GAGs using the dimethylemethylene blue assay revealed that 75 % of GAGs had been removed. In order to assess the effects of peptide: GAG mixtures on the biomechanical properties of the GAG depleted porcine condylar cartilage a biomechanical test method was developed. A series of indentation tests using different loads, followed by finite element analysis of the data were performed on native and GAG depleted porcine condylar cartilage; to identify a suitable load for detection of a significant difference in the deformation, equilibrium elastic modulus and permeability between the native and GAG depleted porcine condylar cartilages. A load of 0.31 N was identified as the most appropriate. GAG depleted porcine condylar cartilage was injected with P11-4 and P11-8 alone, P11-4 and P11-8 : GAG mixtures at a molar ratio of 1:64 and chondroitin sulfate alone. The average percentage deformation of the medial condylar cartilage samples injected with P11-4 alone and P11-4: GAG mixture was 15.5 % and 8.7 % and for P11-8 alone and P11-8: GAG mixture was 11.4 % and 9.1 % respectively; compared to 6.3 % for the native cartilage and 12.6 % for the GAG depleted cartilage. The average equilibrium elastic modulus of the medial cartilage samples injected with P11-4 alone and P11-4: GAG mixture was 0.16 MPa and 0.43 MPa and for P11-8 alone and P11-8: GAG, 0.23 MPa and 0.35 MPa, respectively; compared to 0.49 MPa for the native cartilage and 0.21 MPa for the GAG depleted cartilage. Statistical analysis (ANOVA) showed that a mixture of P11-4: GAG, but not P11-8: GAG restored both the percentage deformation and equilibrium elastic modulus of the GAG depleted cartilage to levels that were not significantly different to the native cartilage. This study has shown that the use of P11-4 in combination with chondroitin sulfate has future potential for development as a minimally invasive treatment for early stage osteoarthritis.