Introduction: Peripheral arterial disease can lead to critical limb ischemia with high risks of amputation and mortality. A promising treatment involves delivering adipose-derived stem cells (ASCs) to stimulate revascularization and functional recovery. However, when delivered via intramuscular injection in saline, substantial ASC loss occurs. To improve ASC retention and survival, a modular delivery gel was developed composed of: i) methacrylated glycol chitosan (MGC) functionalized with GGGGRGDS, and ii) a diacrylate triblock copolymer of poly(ethylene glycol) (PEG) and poly(trimethylene carbonate) (PTMC) to improve mechanical robustness. A thermal initiator system of ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) was optimized for in situ gelation following injectable delivery.
Methods: MGC was prepared as previously reported[1]. The N terminus of GGGGRGDS peptide was acrylated and conjugated to MGC via Michael addition. Acrylated PEG-PTMC of varying block lengths was prepared by ring-opening polymerization of TMC initiated by PEG (4, 10, or 20 kg·mol-1) (TMC:PEG MW = 1:10 – 1:2)) using HCl as a monomer activator, followed by reaction with acryloyl chloride. Polymer structure and functionalization were confirmed by 1H NMR and GPC. To form gels, APS and TEMED (2.5 to 10 mM) were added to solutions of MGC and PEG-PTMC. Initiation sensitivity and crosslinking time were assessed by rheometry at 20 °C and 37°C. Gels were equilibrated in PBS, after which the swelling ratio and mechanical properties were measured. Isolated bovine ASCs[2], expanded to passage 2, were suspended in the pre-polymer solutions. APS/TEMED was added, and the suspension injected via 25G needle into molds and crosslinked by heating (37°C). ASC viability (LIVE/DEAD staining) and metabolic activity (MTT assay) were monitored over 7 days.
Results: The MGC had a 5% degree of methacrylation and 5% degree of peptide functionalization, while the PEG-PTMC had > 99% acrylation. Pre-polymer solutions showed no gelation over 10 min at 20 °C (Fig. 1a). Upon heating, gelation time was directly related to initiator concentration (Fig. 1b).
The swelling, equilibrium compressive modulus, and maximum strain of PEG-PTMC hydrogels was dependent on the overall molecular weight and PEG/PTMC ratio (Fig. 2). By blending MGC with PEG-PTMC, gel stiffness was significantly reduced to the range of native tissue (~10-15 kPa), while improving elastic resiliency under dynamic loading. Further, the metabolic activity of encapsulated ASCs was maintained over 7 d of culture.
Discussion: Blending of MGC with PEG-PTMC facilitated adjustment of gel mechanical properties while improving resiliency under dynamic loading. The APS/TEMED crosslinking approach provided an injectable delivery method with high stimuli sensitivity. Sustained ASC metabolic activity in culture suggests the gel could promote ASC survival and retention, thereby promoting their regenerative effects in ischemic regions.
References:
[1] B. G. Amsden, A. Sukarto, D. K. Knight, and S. N. Shapka, “Methacrylated glycol chitosan as a photopolymerizable biomaterial.,” Biomacromolecules, vol. 8, no. 12, pp. 3758–66, Dec. 2007.
[2] Y. Zhao, S. D. Waldman, and L. E. Flynn, “The effect of serial passaging on the proliferation and differentiation of bovine adipose-derived stem cells,” Cells Tissues Organs, vol. 195, no. 5, pp. 414–427, 2012.