Amniotic growth factors enhanced human pre-adipocyte cell viability and differentiation under hypoxia

Abstract

One of the major drawbacks associated with autologous fat grafting is unpredictable graft retention. Various efforts to improve the survivability of these cells have been explored, but these methods are time-consuming, complex, and demand significant technical skill. In our study, we examine the use of cryopreserved amniotic membrane as a source of exogenous growth factors to improve adipocyte survivability under normal and hypoxic conditions. Human primary preadipocytes were cultured in a gelatin-ferulic acid (Gtn-FA) hydrogel with variable oxygen concentration and treated with amniotic membrane-derived condition medium (CM) for 7 days. This hydrogel provides a hypoxic environment and also creates a 3D cell culture to better mimic recipient site conditions. The O₂ concentration in the hydrogel was measured by electron paramagnetic resonance oxygen imaging (EPROI). The conjugation of FA was confirmed by FTIR and NMR spectroscopy. The cell viability and adipocyte differentiation were analyzed by alamarBlue™ assay, Oil Red O staining, and RT-qPCR. The expression of genes: Pref-1, C/EBP β, C/EBP α, PPAR-ƴ, SLC2A4, and VEGF-A were quantified. The cell viability results show that the 50% CM showed significantly higher cell pre-adipocyte cell viability. In addition, compared to normal conditions, hypoxia/CM provided higher PPAR-ƴ (p < .05), SLC2A4, and VEGF-A (p < .05) (early and terminal differentiating markers) mRNA expression. This finding demonstrates the efficacy of amniotic CM supplementation as a novel way to promote adipocyte survival and retention via the expression of key gene markers for differentiation and angiogenesis.

Keywords: adipocytes; amniotic growth factors; fat grafting; hypoxia; synergism.

FIGURE 1

(A) Differentiation of HPADs: (i) Oil Red O stain was used to qualitatively assess the state of differentiation of HPADs under commercial differentiation medium after day 1 (a), day 7(b), and day 14 (c) (ii) Gene expression of fully differentiated HPADs. HPADs were given a differentiation medium to assess the gene expression. Statistical significance was established by comparison of the experimental group (differentiation medium) and the internal control (preadipocyte medium). (B) Cell Viability assessment of HPADs cultured with CM HPADs viability was assessed via alamarBlue for 7 days while supplementing with and without amniotic CM at various concentrations. An unpaired t test was performed to establish a significant difference between experimental groups and internal control (MEM). (* indicates p < .05 p‐value)

FIGURE 2

(A) Differentiation of HPADs after supplementation with CM Oil Red O stain was used to qualitatively assess the state of differentiation of HPADs under supplementation of amniotic growth factors (B) Gene expression analysis after CM supplementation. HPADs were treated with preadipocyte media growth media, 25% CM, and 50% CM, and RNA was collected after 7 days. Statistical significance was established between the comparison of the experimental group and internal control (preadipocyte medium) (* indicates p < .05 p‐value)

FIGURE 3

(A) (i) FTIR Spectrum of Gelatin, Ferulic acid (FA), and Gelatin‐FA demonstrating the characteristic peaks of FA in Gelatin‐FA hydrogel. (ii) Raman spectrum confirming the characteristic peak of FA in Gelatin‐FA at 6.1–6.5 ppm compared with Gelatin (ii) showing the gelating of hydrogel after (a) 0, (b) 10, (c) 30, and (d) 60 min of incubation and 37°C. (B) Oxygen spectroscopy against timeline was demonstrated with corresponding oxygen images as a function of time in torr (0–200 torr)

FIGURE 4

Cytotoxicity of (A) Gelatin‐FA hydrogel and (B) Laccase after 24 incubation. (C) graph showing the normalized absorbance of HPADs under the normal and hypoxic conditions from the initial cell number in the presence of CM after 4 and 7 days of culture. (* indicates p < .05 p‐value)

FIGURE 5

Graphs showing the relative mRNA expression of (A) Pref‐1, (B) PPAR ƴ, (C) SLC2A4, and (D) VEGF‐A by adipocytes after day 1 and 7 incubation in the presence of normal and hypoxic condition supplemented with CM

FIGURE 6

Gene expression analysis of adipocyte cells under CoCl₂ hypoxic conditions HPADs was seeded in a 24 well plate and after 24 h of normal culture 100 μM of CoCl₂ was given to induce hypoxia in two experimental groups. Cells were cultured for 7 days. Statistical significance was established by comparison of the treatment group to the control, which was preadipocyte medium (* = <.05 p‐value)