Introduction

Intact acellular extracellular matrices (ECM) have demonstrated potential in clinical applications. We hypothesized that ‘functionalizing' these scaffolds with dendrimers would improve the regenerative potential further. Dendrimers, a new class of macromolecules with a tree-like branching structure and a highly-functionalized multi-terminated surface containing functional groups can be tethered onto these scaffolds. Incorporation of dendrimers would provide additional grafting sites for the inclusion of bioactive peptides, growth factors or gene delivery. As a first step towards this goal, this study was designed to optimize the conditions for incorporation of polyamidoamine (PAMAM) “Starburst” dendrimer onto the cholecyst-derived extracellular matrix (CEM). CEM is a novel acellular scaffold currently under development in our research facility.¹ The resulting scaffolds were characterized by collagenase degradation assay, shrink temperature by differential scanning colorimetry (DSC) and Fourier Transform Infrared (FTIR) spectra.

Materials and Methods

CEM scaffolds were isolated from porcine cholecyst as previously reported.¹ Different amount of Generation 1 Starburst (PAMAM) dendrimer (0.0, 0.35, 0.7, 2.1, 4.2, 6.3, 8.4 mmoles per mg of CEM) were incorporated onto CEM scaffold using 0.0066 mM EDC/NHS cross-linking system in MES buffer (50 mM, pH5.3) by utilizing the carboxyl groups within the scaffold. For in vitro degradation, scaffolds were immersed in Tris-HCl buffer of pH 7.4 at 37 ^oC, in the presence of collagenase enzyme (10unit/mg). The percent remaining weight as a function of degradation time was recorded. To determine the shrink temperatue, a heating rate of 5^oC/min was used (25-100 ^oC) for DSC. Shrink temperatures were recorded from the DSC traces. FTIR spectra were recorded and analyzed.

Results

The incorporation of dendrimer decreased the susceptibility of these scaffolds to collagenase degradation up to an initial reactant concentration of 0.7 mM PAMAM/mg CEM. Above 0.7mM PAMAM/mg CEM, there was an increase in degradation (Fig. 1). Corresponding trends were observed with shrink temperature measurements and FTIR spectra. The shrink temperature gives an indication of scaffolds stability. There was an increase up to 0.7mM PAMAM/mg CEM and then showed a decrease thereafter (Fig 2).

Discussion

EDC/NHS crosslinking alone (without dendrimer) improved the stability of the CEM scaffold. This effect was further improved in the presence of Starburst (PAMAM) dendrimer. This indicates that the dendrimer functions as a cross-link bridge within the CEM scaffold. However, excess of PAMAM (>0.7mM PAMAM/mg CEM) resulted in deterioration of the scaffold stability. As indicated by the shrink temperature measurements, the improvement in stability can be attributed to increase in crosslinking, while the decrease could be due to excess dendrimer molecules competing for the limited carboxyl groups on the CEM scaffold. Similar trend has been reported when hexane diamine was used as crosslinking bridge for collagen.²

Conclusions

The conditions for tethering PAMAM dendrimer to CEM scaffold was optimized in this study and the resulting scaffolds was characterized using collagenase assay, DSC and FTIR.