Conditioned medium-electrospun fiber biomaterials for skin regeneration

Abstract

Conditioned medium (CM) contains variety of factors secreted by cells, which directly regulate cellular processes, showing tremendous potential in regenerative medicine. Here, for the first time, we proposed a novel regenerative therapy mediated by biodegradable micro-nano electrospun fibers loaded with highly active conditioned medium of adipose-derived stem cells (ADSC-CM). ADSC-CM was successfully loaded into the nanofibers with biological protection and controllable sustained-release properties by emulsion electrospinning and protein freeze-drying technologies. In vitro, ADSC-CM released by the fibers accelerated the migration rate of fibroblasts; inhibited the over proliferation of fibroblasts by inducing apoptosis and damaging cell membrane; in addition, ADSC-CM inhibited the transformation of fibroblasts into myofibroblasts and suppressed excessive production of extracellular matrix (ECM). In vivo, the application of CM-biomaterials significantly accelerated wound closure and improved regeneration outcome, showing superior pro-regenerative performance. This study pioneered the application of CM-biomaterials in regenerative medicine, and confirmed the practicability and significant biological effects of this innovative biomaterials.

Keywords: Biomaterials; Conditioned medium; Electrospun fiber; Regenerative medicine; Skin regeneration.

Graphical abstract

Fig. 1

Structure and function diagram of MPF loaded with ADSC-CM. (a) Preparation process of ADSC-CM loaded MPF. CM of ADSCs obtained by liposuction are freeze-dried and encapsulated inside HA nanoparticles, which constitutes the core of MPF@CM. (b) The pro-scarless skin regeneration effect of MPF@CM. ADSC-CM is released from the MPF@CM and acts on the surrounding fibroblasts to accelerate wound healing by promoting cell migration, and inhibits scar formation by limiting the over proliferation, inhibiting pathological differentiation of fibroblasts, and suppressing excessive ECM deposition.

Fig. 2

ADSC-CM loaded by MPF @ CM effectively release in vitro. (a) Surface morphology of membrane composed of MPF@CM. (b) The core-shell structure of MPF@CM observed by TEM. (c) Core and shell diameters of fibers (Average ± SD, n = 100 for each group). (d) Ultraviolet spectrophotometry of releasing buffer of fibers collected on day 7 and (f) the release rate of MPF@CM for 7 days (Average ± SD, n = 6 for each group, *** denotes P < 0.001 by One-way ANOVA followed by Tukey's multiple comparison test). (e) The shades of color with the changes of concentration of ADSC-CM encapsulated in HA nanoparticles.

Fig. 3

Morphology and survival rate of fibroblasts seeded on electrospun membranes. (a) Morphology of fibroblasts with cytoskeleton staining seeded on electrospun membranes for 3 days. Fibroblasts seeded on electrospun membranes for (b) 1 day and (c) 3 days are stained with live-dead staining kits that make live cells green and dead cells red. The survival rate of fibroblasts cultured for (d) 1 day and (e) 3 days on electrospun membranes (Average ± SD, n = 6 for each group).

Fig. 4

MPF@CM significantly inhibits fibroblast proliferation. (a) Morphology of fibroblasts cultured for 1 day and 3 days. (b) The flow cytometry and (d) analysis of apoptosis rate (Average ± SD, n = 3 for each group, * denotes P < 0.05 by One-way ANOVA followed by Tukey's multiple comparison test). (c) CCK8 cell proliferation assay (Average ± SD, n = 3 for each group, * denotes P < 0.05 and ** denotes P < 0.01 by One-way ANOVA followed by Tukey's multiple comparison test). (e) LDH cytotoxicity detection. (Average ± SD, n = 3 for each group, ** denotes P < 0.01 and *** denotes P < 0.001 by One-way ANOVA followed by Tukey's multiple comparison test).

Fig. 5

MPF@CM promotes the migration of fibroblasts and inhibits the expression of scar-related protein. (a) Morphological and (c) quantitative analysis of HS fibroblasts migration area on 0-h, 6-h, 12-h, and 24-h (Average ± SD, n = 6 for each group, * denotes P < 0.05, ** denotes P < 0.01 and ***denotes P < 0.001 by One-way ANOVA followed by Tukey's multiple comparison test). (b) Western blot analysis of Col I, Col III, and α-SMA. Quantitative real-time PCR analysis of (d) Col I, (e) Col III, and (f) α-SMA. (Average ± SD, n = 3 for each group, * denotes P < 0.05, ** denotes P < 0.01 and *** denotes P < 0.001 by One-way ANOVA followed by Tukey's multiple comparison test).

Fig. 6

MPF@CM promotes wound healing and inhibits scar formation in vivo. (a) Compared with the control and MPF, MPF@CM promotes skin regeneration in vivo. Statistical analysis of the healing of (b) skin defect and (c) scar area (Average ± SD, n = 3 for each group, * denotes P < 0.05, ** denotes P < 0.01 and *** denotes P < 0.001 by One-way ANOVA followed by Tukey's multiple comparison test).

Fig. 7

Histological analysis of scar tissues. (a) HE staining of epidermal layer and (b) Masson staining of dermal collagen deposition. Immunohistochemical staining of (c) Col I, (d) Col III, (e) MMP1, (f) TIMP1, (g) VEGF, and (h) ɑ-SMA. Statistical analysis of (i) epidermal thickness, (j) dermal collagen deposition, and the expression of (k) Col I, (l) Col III, (m) MMP1, (n) TIMP1, (o) VEGF, and (p) ɑ-SMA. (Average ± SD, n = 3 for each group, * denotes P < 0.05, ** denotes P < 0.01 and *** denotes P < 0.001 by One-way ANOVA followed by Tukey's multiple comparison test).