Osmotic Pressure of Biomimetic Aggrecan from “Grafting “To Synthesis

Abstract

IntroductionThe proteoglycan content of the nucleus pulposus (NP) decreases linearly with degeneration.1 This loss of proteoglycan is attributed to both a reduction of the synthesis of aggrecan by the NP cells and an increase degradation by MMP's and proteases, which attack the aggrecan protein core, resulting in aggrecan fragmentation.2-5 We envision a treatment strategy for restoration of the NP, by the addition of an enzymatically-resistant, biomimetic aggrecan (BioAGG) to the NP. This BioAGG, which “grafts” chondroitin sulfate (CS) bristles “to” a synthetic polymer core resistant to enzymatic degradation, will have osmotic pressure as a key functional property for application in the NP. We hypothesize that osmotic pressure can be impacted by both the number and density of the CS bristles incorporated, which will influence the fixed charge density and configurational entropy of this biomimetic macromolecule. Materials and MethodsTo synthesize BioAGG, linear CS molecules (bristles) with primary amine end groups were “grafted to” functional groups along poly-acrylic acid (PAA) chains. Reactions were performed with two PAA molecules at different lengths (250 kDa and 10 kDa) and varying ratios (mol/mol) of CS molecules to AA functional groups (CS; AA). Reaction kinetics was monitored using fluorescamine assay. Resulting macromolecules were purified via membrane dialysis. Polymer samples were dialyzed for 96 hours against DI water (50K MWCO) and lyophilized. Their chemical structure was confirmed with 1H NMR (300 MHz UnityInova NMR Spectrometer, 30 mg/mL samples in D2O).Using a custom membrane osmometer, osmotic pressures of the synthesized molecules were measured and compared to CS alone and an unreacted mixture of PAA and CS. All solutions were tested at the same fixed charge density based on calculations of fixed charge density. ResultsThree different molecules were fabricated using two molecular weights of the PAA core (250 kDa and 10 kDa). For PAA 250-CS samples, we varied the bristle density by controlling the molar ratio of CS to acrylic acid monomer units (CS: AA). PAA250-CS-high with “high” bristle density was synthesized with CS: AA at 1:15, and PAA250-CS-low with “low” bristle density was synthesized with CS:AA at 1:30. The molar ratio of CS: AA for PAA10-CS was kept constant at 1:10. Our theoretical estimation of the molecular weight of BioAGG samples has been done from conjugation data for CS, as determined using a fluorescamine assay, which is sensitive to the reduction in primary amines (PA) on CS. Results of these calculations are seen in Table 1.Table 1 Table 1MoleculeMolecular Wt (kDa)Number of BristlesBristle Spacing (nm)PAA250-CS-high≈2000≈854 to 8PAA250-CS-low≈1100≈4010 to 15PAA250-CS-high≈250≈93 to 5Chemical structures of the synthesized molecules were confirmed via 1H-NMR showing characteristic peaks for CS and corresponding polymer backbones for all purified compounds. No degradation of CS bristles after the synthesis and purification was observed. At the highest concentrations examined (25 mg/mL PAA250: CS) no gelation was observed. Osmotic pressure result of the PAA250-CS-high was 560 kPA about 8× higher than 70 kPA for CS alone (Fig. 1). Interestingly, when the same concentrations of PAA250 and CS are just mixed in solution (PAA250 + CS mixture), the osmotic pressure is close to that of CS alone (∼70 kPA). The same PAA250 with a lower CS bristle density, (PAA250-CS-low) is about 20% lower than the high-density version (410 kPA). A lower molecular weight with high bristle density (PAA10-CS) has an intermediate osmotic pressure between the PAA250-CS and CS alone (140 kPA). ConclusionBioAGG molecules were synthesized using a “grafting to” strategy incorporating CS bristles on PAA polymer backbones. The resultant molecules showed osmotic pressures in excess of those for the fixed charge alone. Moreover, the number and density of CS side chains influences the osmotic pressure supporting a hypothesis that the electrostatic repulsion and conformation entropy derived from the 3D macromolecular bottle-brush structure contributes to the osmotic pressure. BioAGG shows promise for mimicking physical and osmotic properties of natural aggrecan.Acknowledgment Coulter Foundation GrantI confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredOlczyk K. Zeitschrift für Rheumatologie 1994;53:19Roberts S, et.al. Spine 2000;25:3005Goupille P, et.al. Spine 1998;23:1612Le Maitre C, et.al. Biochemical Society Transactions 2007;35:652Kiani C, et al. Cell Research 2002;12:19