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. 2013 Nov;9(11):8802-14.
doi: 10.1016/j.actbio.2013.06.021. Epub 2013 Jun 27.

Thiol-ene Michael-type formation of gelatin/poly(ethylene glycol) biomatrices for three-dimensional mesenchymal stromal/stem cell administration to cutaneous wounds

Kedi Xu  1 , David Antonio Cantu, Yao Fu, Jaehyup Kim, Xiaoxiang Zheng, Peiman Hematti, W John Kao
Affiliations

Thiol-ene Michael-type formation of gelatin/poly(ethylene glycol) biomatrices for three-dimensional mesenchymal stromal/stem cell administration to cutaneous wounds

Kedi Xu et al. Acta Biomater. 2013 Nov.

Erratum in

Abstract

Mesenchymal stromal/stem cells (MSCs) are considered promising cellular therapeutics in the fields of tissue engineering and regenerative medicine. MSCs secrete high concentrations of immunomodulatory cytokines and growth factors, which exert paracrine effects on infiltrating immune and resident cells in the wound microenvironment that could favorably promote healing after acute injury. However, better spatial delivery and improved retention at the site of injury are two factors that could improve the clinical application of MSCs. In this study, we utilized thiol-ene Michael-type addition for rapid encapsulation of MSCs within a gelatin/poly(ethylene glycol) biomatrix. This biomatrix was also applied as a provisional dressing to full thickness wounds in Sprague-Dawley rats. The three-way interaction of MSCs, gelatin/poly(ethylene glycol) biomatrices, and host immune cells and adjacent resident cells in the wound microenvironment favorably modulated wound progression and host response. In this model we observed attenuated immune cell infiltration, lack of foreign giant cell (FBGC) formation, accelerated wound closure and re-epithelialization, as well as enhanced neovascularization and granulation tissue formation by 7 days. The MSC entrapped in the gelatin/poly(ethylene glycol) biomatrix localized cell presentation adjacent to the wound microenvironment and thus mediated the early resolution of inflammatory events and facilitated the proliferative phases in wound healing.

Keywords: Cell-based therapy; Foreign body response; Inflammation; Macrophages; Mesenchymal stem cells.

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Figures

Figure 1
Figure 1
Synthesis and characterization of thiolated gelatin. A: Synthesis procedure of thiolated gelatin macromolecule with a two-step reaction between lysyl residue of gelatin, cysteamine and PEG-bis-NHS linker. (1) Synthesis of PEG-bis-NHS via a carbonate linker. (2) Synthesis of thiol modified PEG (NHS-PEG-SH). (3) Synthesis of thiol modified gelatin (type B gelatin, bloom 225) (gel-PEG-SH). (4) Crosslinking of thiol modified gelatin (gel-PEG-SH) with PEGda via Michael-type addition. B: 1H NMR of the thiolated gelatin (type B, bloom 225, Gel-PEG-SH).
Figure 2
Figure 2
Gelatin/poly(ethylene glycol) hydrogel bulk characterization. A: Swelling characterization of 10% gelatin/poly(ethylene glycol) biomatrices (n = 3) immersed in PBS at 37°C. B: Enzymatic degradation of 10% gelatin/poly(ethylene glycol) biomatrices (n = 3) by type I collagenase (collagenase concentrations: ▪: 0.1mg/mL, ▲: 0.4 mg/mL, ●: 1 mg/mL). C: Rheological characterization of gelatin/poly(ethylene glycol) hydrogels with various polymer concentrations (G′ ■ storage modulus and G″ □ shear modulus at pH 8.5, 15%w/w gel-PEG-SH; at pH 8.5, 12% w/w gel-PEG-SH; at pH 8.5, 10% w/w gel-PEG-SH; at pH 8.5, 8% w/w gel-PEG-SH.) G′ ■ at pH 8.5, 15% w/w gel-PEG-SH was statistically greater than all other gel-PEG-SH concentrations); G′ ■ at pH 8.5, 12% w/w gel-PEG-SH was statistically greater than 10% w/w gel-PEG-SH and 8% w/w gel-PEG-SH; G′ ■ at pH 8.5, 10% w/w gel-PEG-SH was statistically greater than 8% w/w gel-PEG-SH. G″ was not statistically significant amongst different polymer concentrations. *p <0.001.
Figure 3
Figure 3
3D cell encapsulation viability evaluation and the retention of hBM-MSC differentiation capability. A: LIVE/DEAD® staining of hBM-MSCs encapsulation within a gelatin/poly(ethylene glycol) biomatrix, Matrigel®, or type I collagen for 4 days (20x magnification). B: hBM-MSC were encapsulated in gelatin/poly(ethylene glycol) biomatrices for 3 days and released by collagenase digestion. After re-plating and growing to confluency, hBM-MSC were switched to separate differentiation medias and were subsequently tested for adipocyte-, chondrocyte-, osteoblast-lineage differentiation after 16 days (left), 16 days (middle), 14 days (right) respectively (20x magnification). Oil red O–staining shows lipid droplets (left: red color), Safranin-O staining shows cartilage-specific glycosaminoglycans (middle: reddish-pink ECM), von Kossa staining shows deposits of calcium crystals (right: black dots).
Figure 4
Figure 4
Representative histological sections of intramuscular injections with gelatin/poly(ethylene glycol) hydrogels for 1, 4, and 7 days (both 10x and 40x magnification). M = muscle; H = remnants of the gelatin/poly(ethylene glycol) hydrogel; F = fibroblast; NV = neovascularization; black arrows = PMNs; white arrows = monocyte/macrophages.
Figure 5
Figure 5
Wound closure and extent of re-epithelialization A: The wound closure (%) means ± SD (N = 3) observed for three different treatment groups (sham wound ●, gelatin/poly(ethylene glycol) hydrogel ■, and MSC-gelatin/poly(ethylene glycol) biomatrix ▲) after 4 or 7 days post-surgery. Wound closure (%) was calculated as: [(original wound area - wound area post-surgery)/original wound area] x100%. A probability p < 0.05 was considered statistically significant using student’s t-test. The MSC-gelatin/poly(ethylene glycol) biomatrix treatment demonstrated greater wound closure than the sham control (§p = 0.019) at 7 days. B: 7 day full-thickness wounds for sham = sham control (no observed re-epithelialization), Gel-PEG = gelatin/poly(ethylene glycol) hydrogel, Gel-PEG-MSC = MSC-gelatin/poly(ethylene glycol) biomatrix. A total of 5 widths were taken at random locations within the wound and averaged, the means ± SD were taken into account amongst the groups (n = 3). Both the gelatin/poly(ethylene glycol) hydrogel (§p = 0.011) and the MSC-gelatin/poly(ethylene glycol) biomatrix (#p = 0.0041) demonstrated statistically greater epidermal thickness than the sham control at 7 days.
Figure 6
Figure 6
H&E representative full-thickness wound images in the central area of the wound at 4 and 7 days (20x magnification). Sham = sham wound, Gel-PEG = Gelatin/poly(ethyleneglycol) hydrogel, Gel-PEG-MSC = Gelatin/poly(ethylene glycol) biomatrix, E = Epidermis (immature), D = dermis (remodeling).
Figure 7
Figure 7
Number of PMNs (A), mononuclear cells (B), fibroblasts (C), and keratinocytes (D) in each observed wound image area harvested from the central area of the wound (20x magnification) for each treatment group (n = 3) at 4 and 7 days. Sham = Sham wound, Gel-PEG = Gelatin/poly(ethylene glycol) hydrogel, MSC-gelatin/poly(ethylene glycol) biomatrix. Number of mononuclear cells per observed wound image area with the gelatin/poly(ethylene glycol) hydrogel treatment was statistically greater than the MSC-gelatin/poly(ethylene glycol) biomatrix treatment (#p = 0.0036) and the sham control (§p = 0.037) at 4 days. Number of mononuclear cells per observed wound image area with the gelatin/poly(ethylene glycol) hydrogel treatment at 4 days was statistically greater than (*p = 0.011) 7 days for the same treatment. Number of fibroblasts per observed wound image area with the MSC-gelatin/poly(ethylene glycol) hydrogel treatment at 7 days was statistically greater than (§p = 0.038) at 4 days for the same treatment. Number of keratinocytes per observed wound image area for the gelatin/poly(ethylene glycol) hydrogel treatment (§p = 0.00095) and the MSC-gelatin/poly(ethylene glycol) biomatrix (#p = 0.00043) was statistically greater than the sham control at 7 days.
Figure 8
Figure 8
Representative full-thickness wound images in the central area of the wound at 4 and 7 days (20x magnification). Macrophages were immunostained for CD163 (an anti-inflammatory macrophage marker) and the wound area was counterstained with hematoxylin. Sham = sham wound, Gel-PEG = Gelatin/poly(ethylene glycol) hydrogel, Gel-PEG-MSC = Gelatin/poly(ethylene glycol) biomatrix, E = Epidermis (immature), D = dermis (remodeling), H = Gelatin/poly(ethylene glycol) hydrogel.
Figure 9
Figure 9
Representative full-thickness wound images in the central area of the wound at 4 and 7 days (20x magnification). Macrophages were immunostained for CD68 (a pan-macrophage marker) and the wound area was counterstained with hematoxylin. Sham = sham wound, Gel-PEG = Gelatin/poly(ethylene glycol) hydrogel, Gel-PEG-MSC = Gelatin/poly(ethylene glycol) biomatrix, E = Epidermis (immature), D = dermis (remodeling), H = Gelatin/poly(ethylene glycol) hydrogel.
Figure 10
Figure 10
Number of positive macrophages for specific cell surface markers in each observed wound image area harvested from an area adjacent to the central area of the wound (20x magnification) for each treatment group (n = 3) at 4 and 7 days and the macrophage M2 percentage (CD163+/CD68+). A: Number of CD163+ cells (an anti-inflammatory macrophage marker) per observed wound area from image taken from an area adjacent to the central area of the wound (20x magnification) for each treatment group (n = 3) at 4 and 7 days. Sham = Sham wound, Gel-PEG = Gelatin/poly(ethylene glycol) hydrogel, Gel-PEG-MSC = MSC-gelatin/poly(ethylene glycol) biomatrix. Number of CD163+ cells per observed wound image area was statistically greater (p = 0.0064) for the sham control at 4 days as compared to 7 days for the same treatment group and number of CD163+ cells per observed wound image area was also statistically greater for the gelatin/poly(ethylene glycol) biomatrix at 4 days as compared to 7 days for the same treatment group (#p = 0.038). B: Number of CD68+ cells (an pan-macrophage marker) per observed wound image area taken from an area adjacent to the central area of the wound (20x magnification) for each treatment group (n = 3) at 4 and 7 days. Number of CD68+ cells per observed wound image area was statistically greater for the gelatin/poly(ethylene glycol) hydrogel treatment (§p = 0.0028) and gelatin/poly(ethylene glycol) biomatrix (#p = 0.037) than the sham control at 4 days. C: The M2 percentage (CD163+/CD68+) from the number of CD163+ and CD68+ cells from respective observed wound image areas (Figure 11 and Figure 12) at 4 and 7 days for each treatment group (n = 3). The M2 percentage (CD163+/CD68+) was statistically greater for the sham wound when compared to the gelatin/poly(ethylene glycol) treatment (§p = 0.014) at 4 days and was also statistically greater than the sham wound at 7 days (#p = 0.0011).
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