Accelerate Healing of Severe Burn Wounds by Mouse Bone Marrow Mesenchymal Stem Cell-Seeded Biodegradable Hydrogel Scaffold Synthesized from Arginine-Based Poly(ester amide) and Chitosan

Abstract

Severe burns are some of the most challenging problems in clinics and still lack ideal modalities. Mesenchymal stem cells (MSCs) incorporated with biomaterial coverage of burn wounds may offer a viable solution. In this report, we seeded MSCs to a biodegradable hybrid hydrogel, namely ACgel, that was synthesized from unsaturated arginine-based poly(ester amide) (UArg-PEA) and chitosan derivative. MSC adhered to ACgels. ACgels maintained a high viability of MSCs in culture for 6 days. MSC seeded to ACgels presented well in third-degree burn wounds of mice at 8 days postburn (dpb) after the necrotic full-thickness skin of burn wounds was debrided and filled and covered by MSC-carrying ACgels. MSC-seeded ACgels promoted the closure, reepithelialization, granulation tissue formation, and vascularization of the burn wounds. ACgels alone can also promote vascularization but less effectively compared with MSC-seeded ACgels. The actions of MSC-seeded ACgels or ACgels alone involve the induction of reparative, anti-inflammatory interleukin-10, and M2-like macrophages, as well as the reduction of inflammatory cytokine TNFα and M1-like macrophages at the late inflammatory phase of burn wound healing, which provided the mechanistic insights associated with inflammation and macrophages in burn wounds. For the studied regimens of these treatments, no toxicity was identified to MSCs or mice. Our results indicate that MSC-seeded ACgels have potential use as a novel adjuvant therapy for severe burns to complement commonly used skin grafting and, thus, minimize the downsides of grafting.

Keywords: M1- or M2-like macrophages; MSC-seeded ACgels; chronic inflammation; hydrogels synthesized from unsaturated arginine-based Poly(ester amide) and chitosan; mesenchymal stem cells; severe burn.

FIG. 1.

The chemical structure and specific physical and mechanical properties of hydrogels prepared from UArg-PEA and GMA-chitosan. (A) Chemical structure of the polymer repeat units. UArg-PEA were crosslinked with GMA-chitosan to create UArg-PEA:GMA-chitosan hybrid hydrogels (ACgels). (B) Specific physical and mechanical properties of swelled ACgels [Notes: ^a, ^b, and ^c were analyzed using a DMA Q800 Dynamic Mechanical Analyzer (TA Instrument, Inc., New Castle, DE), scanning electronic microscope (Leica Microsystems GmbH, Wetzlar, Germany), and Malvern Zetasizer Nano-ZS (Malvern Panalytical Ltd. Malvern, United Kingdom), respectively]. UArg-PEA, unsaturated arginine-based poly(ester amide); GMA-chitosan, glycidyl methacrylate chitosan.

FIG. 2.

MSCs maintained a high viability after seeded to ACgel scaffolds and cultured. (A) Illustration for seeding MSCs to ACgel scaffolds. Each swelled and blotted ACgel took up a drop of MSC medium with or without MSCs (1 × 10⁵) on a hydrophobic Parafilm by capillary force. (B) The typical microimage shown that the MSCs scaffolded by ACgels were ∼95% viable (green) at day 6 of culture based on the green-staining of living cells and red-staining of dead and dying cells. (C) MSC viabilities at 1, 3, or 6 days in vitro for MSCs seeded to ACgels or directly cultured in the plate-wells (2D-culture). (D) MSCs (%) adhered to ACgels at 15 min, 3 days, or 6 days in culture. MSCs in ACgels, plate-well surfaces, culture media, or washing media were analyzed using the live/dead cell assay kit consisted of calcein-AM and ethidium-homodimer. The MSCs were cultured (37°C, 5% CO₂) in MSC medium and stained at the end. n = 3 independent experiments. MSC, mesenchymal stem cell. Color images available online at www.liebertpub.com/scd

FIG. 3.

ACgels sustained the presence of MSCs in the 3° burn wounds of C57BL/6 J mice. (A) The photos of 3° burn wounds at (I) 0 dpb and (II) 2 dpb without excision; (III) at 2 dpb immediately after the excision of the full-thickness necrotic skin created by burn injury, and (IV) at 2 dpb immediately after covering the burn and excision generated skinless area using an ACgel with or without MSC loading. (B) On the left, a representative microimage of Dil-red tagged C57BL/6 J mouse MSCs implanted with ACgel scaffold to burn wounds at 8 dpb. Nuclei were stained blue with DAPI. On the right, Numbers of Dil-red tagged MSCs/microscopic field in the burn wounds at 8 dpb. (C) A typical microimage of burn wounds showed the ACgel residues (marked by arrows) at 8 dpb, indicating the biodegradation of ACgels. e, epidermis; d, dermis; and g, granulation tissue. The white arrow marks a red-tagged MSC. The blue arrow indicates a hydrogel residue. The full-thickness 3° burn wounds were generated on the dorsal skin of C57BL/6 J mice. MSCs carried by ACgels or medium were applied to necrotic skin excised burn wounds at 2 dpb. The ACgels were used to cover the burned and excised wounds. The whole wound areas were then covered by transparent film dressing. Data are means ± SD; n = 4; **P < 0.01 versus Dil-red tagged MSCs. DAPI, 4′-6-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/scd

FIG. 4.

MSC-seeded ACgels accelerated the closure of 3° burn wounds of mice. (A) Representative photos of burn wounds at 2 dpb (immediately after excision of necrotic skin) and at 8 dpb. (B) Wound closure as the percentage of original burn wound area. The 3° burn wounds were created on the dorsal skin of C57BL/6 J mice, covered with MSC-seeded ACgels, ACgels alone, or treated with medium control after the necrotic skin was excised at 2 dpb. Data are means ± SD; n = 4; **P < 0.01 versus medium control. Color images available online at www.liebertpub.com/scd

FIG. 5.

ACgels seeded with mouse MSCs promoted reepithelialization and granulation tissue formation in the 3° burn wounds of mice. (A) Representative microimages of H&E stained sections of burn wounds of 8 dpb. (B) Reepithelialization % (upper) and (C) granulation tissue area % (lower) compared to the sections of original burn wounds. g, granulation tissue. e, epidermis. Green arrows, reepithelization tongues; red dotted lines, the wound granulation tissue areas; black arrows, original wound edges; and scale bar, 500 μm. The wounds and treatments were the same as those in Figure 4. Data are means ± SD; n = 4; *P < 0.05 versus medium control. H&E, hematoxylin and eosin. Color images available online at www.liebertpub.com/scd

FIG. 6.

The blood vessel regeneration in the 3° burn wounds at the late inflammatory phase and early proliferative phase was enhanced by ACgels seeded with mouse MSCs or ACgels alone. The burn wound model and treatment were conducted on C57BL/6 J mice as in Figure 4. (A) Representative microimages show the CD31⁺ vessels in wounds treated with medium control, ACgels, or ACgels seeded with C57BL/6 J MSCs at 8 dpb. Negative staining showed no unspecific staining. (B) Increased CD31⁺ blood vessel area (CD31⁺ pixels/microscopic field) compared with the control. Scale bar, 50 μm. Nuclei were counter-stained blue with DAPI. e, epidermis; d, dermis; Red, CD31⁺ cells or vascular structures. Data are means ± SD; n = 4; *P < 0.05 and **P < 0.01 versus medium control. Color images available online at www.liebertpub.com/scd

FIG. 7.

Augmented levels of reparative, anti-inflammatory IL-10 and M2-like Mφs at the late inflammatory phase of burn wounds by MSC-seeded ACgels or ACgels alone. (A) Fluorescent microimages representing the presence of IL-10 (Magenta), CD206 (green, marker of M2-like Mφ), and F4/80 (red, Mφ marker) in burn wounds. (B) IL-10 levels in wounds were presented as intensity sum of IL-10⁺ pixels per microscopic field. F4/80⁺Mφs that were (C) IL10⁺, (D) CD206⁺, or (E) CD206⁺IL10⁺ were counted under confocal microscope and were presented as their percentages among total cells per microscopic field. The treatments using MSC-seeded ACgels or ACgels alone increased wound levels of IL-10, IL-10 expressing Mφs, M2-like Mφs, and IL-10 expressing M2-like Mφs compared with medium control, and MSC-seeded ACgels are more effective than ACgels alone in these actions. The burn wound model and treatment were conducted on C57BL/6 J mice. The cryosections of wounds at 8 dpb were analyzed by immunofluorescence histology. e, epidermis; d, dermis; and scale bar, 50 μm. Negative staining in (A) indicated no unspecific staining. Data are means ± SD; n = 4; *P < 0.05 and **P <0.01. Mφ, macrophage. Color images available online at www.liebertpub.com/scd

FIG. 8.

Attenuated levels of inflammatory TNFα and M1-like Mφs at the late inflammatory phase of burn wounds by MSC-seeded ACgels or ACgels alone. (A) Fluorescent microimages representing the presence of TNFα (Magenta), iNOS (green, marker of M1-like Mφ), and F4/80 (red, Mφ marker) in burn wounds. (B) TNFα levels were presented as the intensity sum of TNFα⁺ pixels per microscopic field. F4/80⁺Mφs that were (C) TNFα ⁺, (D) iNOS⁺, or (E) iNOS⁺TNFα⁺ were counted under confocal microscope and presented as their percentages among total cells per microscopic field were presented. The treatments using MSC-seeded ACgels or ACgels alone attenuated wound levels of TNFα, M1-like Mφs, TNFα expressing Mφs, and TNFα expressing M1-like Mφs compared with medium control. The 3° burn wound model and treatments were conducted on C57BL/6 J mice. The cryosections of wounds at 8 dpb were analyzed by immunofluorescence histology. e, epidermis; d, dermis; and scale bar, 50 μm. Negative staining in (A) indicated no unspecific staining. Data are means ± SD; n = 4; *P < 0.05 and **P < 0.01. iNOS, inducible nitric oxide synthase. TNFα, tumor necrosis factor α. Color images available online at www.liebertpub.com/scd