Supramolecular hydrogels encapsulating bioengineered mesenchymal stem cells for ischemic therapy

Abstract

We developed supramolecular hyaluronate (HA) hydrogels to encapsulate genetically engineered mesenchymal stem cells (MSCs) for the treatment of limb ischemia. In vivo angiogenic factors could be produced stably by the bioengineered MSCs (BMSCs) within the supramolecular hydrogels showing effective vascular repair and enhanced blood perfusion.

Fig. 1. Schematic illustration for (A) sequential synthesis of mCB[6]-HA using mono-amine CB[6] and the preparation of mCB[6]/DAH hydrogels, and (B) in vivo tests using BMSCs with the analysis by LDI to investigate ischemic limb reperfusion. Black arrows indicate the ligation point for hind limb surgery.

Fig. 2. ¹H NMR analysis for (A) mCB[6], the formation of its inclusion with spermine, and (B) mCB[6]-HA.

Fig. 3. (A) Genetic modification of MSCs using recombinant adenoviral (rAd) vectors in the presence of Fe³⁺ ions to express VEGF-A and HGF. (B) VEGF-A expression levels and (C) HGF expression levels of BMSCs (1 × 10⁵ cells per mL) without and with the mCB[6]/DAH–HA hydrogels on PET membrane transwell inserts (mesh size = 8 μm) for specified periods over 2 weeks (mean ± SD, n = 3, *P < 0.0001).

Fig. 4. Laser Doppler imaging (LDI) analysis for the blood reperfusion of BALB/c ischemic limb model mice after injection of BMSCs/HGF in various carriers. (A) Representative LDI images for peripheral blood flow in groups of mice that received media only (G1), BMSC/HGF in media (G2), in matrixen (G3), and in CB[6]/DAH–HA hydrogel (G4) at day 0, 2, 10, 18 and 24. The color-coded image shows the blood flow distribution. The low or no perfusion is shown in blue and the highest perfusion is shown in red. (B) The quantitative analysis of blood flow in hind limbs expressed as the perfusion ratio of ischemic hind limb to the untreated opposite limb (n = 5, **P < 0.01 vs. the control of G1). (C) The number of animals categorized in 3 groups as fully recovered, necrotized and amputated limbs at day 24 post-injection.

Fig. 5. Laser Doppler imaging (LDI) analysis for the blood reperfusion of BALB/c ischemic limb model mice after injection of various BMSCs in mCB[6]/DAH–HA hydrogels. (A) Representative LDI images for peripheral blood flow in groups of mice that received the intact MSC (G1), BMSC/VEGF-A (G2), BMSC/HGF (G3), and BMSC/HGF + VEGF-A (G4) within mCB[6]/DAH–HA hydrogels at day 0, 3, 7, 14 and 28 post-injection. The color-coded image shows blood flow distribution as in Fig. 4(B). (B) The quantitative analysis of blood flow in hind limbs expressed as the perfusion ratio of ischemic hind limb to the untreated opposite limb (n = 6–7, ***P < 0.001 vs. the control of G1). (C) The number of animals categorized in 3 groups as fully recovered, necrotized and amputated limbs at day 28 post-injection.