Human Adipose-Derived Stem Cell-Conditioned Medium Promotes Vascularization of Nanostructured Scaffold Transplanted into Nude Mice

Abstract

Several studies have been conducted on the interaction between three-dimensional scaffolds and mesenchymal stem cells for the regeneration of damaged tissues. Considering that stem cells do not survive for sufficient time to directly sustain tissue regeneration, it is essential to develop cell-free systems to be applied in regenerative medicine. In this work, by in vivo experiments, we established that a collagen-nanostructured scaffold, loaded with a culture medium conditioned with mesenchymal stem cells derived from adipose tissue (hASC-CM), exerts a synergic positive effect on angiogenesis, fundamental in tissue regeneration. To this aim, we engrafted athymic BALB-C nude mice with four different combinations: scaffold alone; scaffold with hASCs; scaffold with hASC crude protein extract; scaffold with hASC-CM. After their removal, we verified the presence of blood vessels by optical microscopy and confirmed the vascularization evaluating, by real-time PCR, several vascular growth factors: CD31, CD34, CD105, ANGPT1, ANGPT2, and CDH5. Our results showed that blood vessels were absent in the scaffold grafted alone, while all the other systems appeared vascularized, a finding supported by the over-expression of CD31 and CDH5 mRNA. In conclusion, our data sustain the capability of hASC-CM to be used as a therapeutic cell-free approach for damaged tissue regeneration.

Keywords: angiogenesis; cell culture; cell-free therapy; in vivo experiment; regenerative medicine; secretome; tissue engineering.

Figure 1

Pictures of mice’s back immediately after the grafting and the retrieval. (A) INTEGRA^® Flowable Wound Matrix grafting; (B) scaffold removal after 28 days with magnification on the newly formed vessels entering the scaffold.

Figure 2

hASC-conditioned medium (A) and cell protein extract (B) characterization. VEGF-A, HIF-1α, IL-6, and TGFβ1 are evaluated. Protein amounts are given as ng/1 × 10⁶ cells. The results are expressed as mean ± S.D. n = 2.

Figure 3

Representative images of macroscopic scaffold analysis after animal sacrifice. All the samples appeared yellow, soft on palpation and of comparable size. (A) FWM alone: the vascularization is almost absent. (B) FWM combined with hASCs. (C) FWM combined with cell protein extract. (D) FWM combined with cell-conditioned medium. In formulations (B–D), the vascularization was more evident and present also inside the scaffold.

Figure 4

Representative microscopic images of scaffold specimens stained with hematoxylin–eosin. (A) FWM alone: observe the presence of collagen fibers and the absence of vessels. (B) FWM combined with hASCs. (C) FWM combined with cell protein extract. (D) FWM combined with cell-conditioned medium. In panels (B–D), in addition to collagen fibers, the presence of numerous cell nuclei (probably of fibroblasts) and capillaries full of erythrocytes (indicated by the blue arrows) are notable.

Figure 5

Evaluation and classification of newly formed vessels in the scaffolds represented as number of vessel/mm². Although some discrepancies were appreciated among the three formulations, no statistically significant differences were observed in the number of capillaries. No blood vessels were found in the FWM alone. The results are expressed as mean ± S.D. n = 5.

Figure 6

Real-time PCR of vascular growth factors in scaffold after 28 days from the grafting. mRNA expression of CD31 and CDH5 appeared statistically significantly higher in the scaffold loaded with the conditioned medium (black bar). No significant differences were observed for CD34, CD105, ANGPT1, and ANGPT2. n = 12; * p < 0.05.