Acellular Biomaterials Associated with Autologous Bone Marrow-Derived Mononuclear Stem Cells Improve Wound Healing through Paracrine Effects

Abstract

Wound healing is a complex process of repair that involves the interaction between different cell types and involves coordinated interactions between intracellular and extracellular signaling. Bone Marrow Mesenchymal Stem Cells (BMSCs) based and acellular amniotic membrane (AM) therapeutic strategies with the potential for treatment and regeneration of tissue. We aimed to evaluate the involvement of paracrine effects in tissue repair after the flap skin lesion rat model. In the full-thickness flap skin experiment of forty Wistar rats: A total of 40 male Wistar rats were randomized into four groups: group I: control (C; n = 10), with full-thickness lesions on the back, without (BMSCs) or AM (n = 10); group II: injected (BMSCs; n = 10); group III: covered by AM; group IV-injected (AM + BMSCs; n = 10). Cytokine levels, IL-1, and IL-10 assay kits, superoxide dismutase (SOD), glutathione reductase (GRs) and carbonyl activity levels were measured by ELISA 28th day, and TGF-β was evaluated by immunohistochemical, the expression collagen expression was evaluated by Picrosirius staining. Our results showed that the IL-1 interleukin was higher in the control group, and the IL-10 presented a higher mean when compared to the control group. The groups with BMSCs and AM showed the lowest expression levels of TGF-β. SOD, GRs, and carbonyl activity analysis showed a predominance in groups that received treatment from 80%. The collagen fiber type I was predominant in all groups; however, the AM + BMSCs group obtained a higher average when compared to the control group. Our findings suggest that the AM+ BMSCs promote skin wound healing, probably owing to their paracrine effect attributed to the promotion of new collagen for tissue repair.

Keywords: antioxidant system; experimental animal models; human acellular amniotic membrane; paracrine; stem cells; wound healing.

Figure 1

Experimental design: amniotic membrane collection, stem cell isolation, process membrane decellularization, flap skin surgery, implant, euthanasia, and histopathological analysis.

Figure 2

Flap skin exposition with a 30 × 50 mm (150 mm²) defect made with a scalpel.

Figure 3

Characteristics of BMSCs. Flow cytometry Histograms of Bone marrow mononuclear cells. The histograms show that the population is positive for anti-CD45 and CD34.

Figure 4

Depicts the signs of re-epithelialization, angiogenesis, and fibrosis with remodeling in four different groups: control (I), stem cell (II), amniotic membrane (III), and stem cell + amniotic membrane (IV). The samples were taken from the center of the lesion and analyzed using HE staining (hematoxylin-eosin) at 200× magnification. All groups, from I to IV, exhibit indications of complete re-epithelialization, as evidenced by a well-formed epidermis (indicated by a black arrow) over mature fibrous tissue (indicated by an asterisk). Angiogenesis is represented by numerous newly formed vessels (indicated by a black arrow) amidst fibrous stroma (indicated by an asterisk). Additionally, fibrosis with remodeling is represented by dense connective tissue containing thick, pink fibers that penetrate newly formed vessels (indicated by an asterisk).

Figure 5

Effect of AM and BMSCs on Interleukin marker IL-1. Results are expressed as mean [interquartile range] ± SEM (N = 10). p <  0.05 denoted statistical significance in comparison to the control group. BMSCs: Bone Marrow Mesenchymal Stem Cells; AM: acellular human amniotic membrane.

Figure 6

Effect of AM and BMSCs on Interleukin marker IL-10. Results are expressed as mean [interquartile range] ± SEM (N = 10). p < 0.05 denoted statistical significance in comparison to the control group. BMSCs: bone marrow mononuclear stem cells; AM: acellular human amniotic membrane.

Figure 7

Representative immunohistochemical staining images of the expression of TGF-β showed the lowest percentages in the control (A), BMSCs (B), AM (C) and BMSCs + AM (D) groups on the 28th day of the experiments.

Figure 8

Effect of AM and BMSCs on carbonyl activity. Results are expressed as mean [interquartile range] ± SEM (N = 10). p < 0.05 denoted statistical significance in comparison to the control group. BMSCs: bone marrow mononuclear stem cells; AM: acellular human amniotic membrane.

Figure 9

Effect of AM and BMSCs on SOD superoxide dismutase activity. Results are expressed as mean [interquartile range] ± SEM (N = 10). p < 0.05 denoted statistical significance in comparison to the control group. BMSCs: bone marrow mononuclear stem cells; AM: acellular human amniotic membrane.

Figure 10

Collagen content analysis and flap skin area after 28 days. Collagen deposition quantified as Picrossirius Red positive areas, control group; Collagen type III content BMSCs group. Collagen type III content, acellular amniotic membrane. Collagen type III content, bone marrow mononuclear stem cells + acellular amniotic membrane group. Arrows indicate collagen type III. Results are shown as mean ± standard deviation. p < 0.0001. Images 20×, scale bar = 20 μm.