Healing wounds with fat by leveraging 3D adipose organoid produced adipokines to activate fibroblasts using a TGF-beta1 independent mechanism.

Introduction:: The idea that adipose tissues can contribute to wound healing has gained significant attention over the past decade. Plastic surgeons initially started performing adipose micrografting as a way to add volume to skin defects, however, more recently, it has become apparent that these tissue micrografts also have biological mechanisms. Unfortunately, very little is known about which cell types and which biological factors in adipose are responsible for the observed improvements in wound healing. Adipose tissues are composed of a continuum of cell types ranging from adipocyte stem cells to mature adipocytes, each of which appears to play important roles in regulating extracellular matrix (ECM) remodeling. Recently, we developed a 3D adipose organoid system (1) that provides an innovative approach for investigating adipocyte-fibroblast interactions in the context of wound healing and tissue remodeling (2). Herein we sought to use our innovative 3D adipose organoid model to understand how the different cellular components of adipose influence fibroblast phenotype and function.


Materials and Methods:: (A) Adipose spheroids were generated by plating 20,000 adipose stromal vascular fraction derived MSC into hanging drops for 3 days and then transferred to ultra-low adherent 24-well plates with 5 spheroids per well. Spheroids were then either differentiated into pre-adipocytes or mature adipocytes by changing the media to PGM-2 or PDM-2, respectively. After 10 days of differentiation, the medium was removed, the spheroids were rinsed gently with 1X PBS, then 0.75 mL of DMEM containing 0.5% FBS, and 1% penicillin/streptomycin was added to each well. After two days, this medium was collected and termed either pre-adipocyte or adipocyte-conditioned medium (PCM or ACM). The PCM and ACM were then applied to fibroblasts to determine the effect of each on fibroblast migration, contraction, and collagen synthesis.

PCM and ACM were then screened with a multiplex adipokine assay in order to measure levels of common cytokines, hormones, and other factors commonly secreted by adipose tissues. To identify the adipokines responsible for controlling fibroblast phenotype, the ACM was heat-inactivated, molecular weight fractionated, and lipid depleted. Myofibroblast Conversion Assay was assessed by staining for α-SMA and the proportion of converted myofibroblasts was quantified by measuring fluorescence with a microplate reader.


Results, Conclusions, and Discussions:: Results

When human dermal fibroblasts were treated with ACM, but not PCM, they converted into highly contractile α-SMA expressing myofibroblasts (B) and generated large quantities of collagen. Analysis of ACM and PCM revealed adipocytes and pre-adipocytes produced remarkably different profiles of adipokines (C). Interestingly, neither ACM or PCM contained significant levels of TGF-β which is traditionally thought to be necessary for myofibroblast conversion. To investigate what other adipokine could convert fibroblasts to myofibroblasts, we heat-inactivated, lipid-depleted and protein-fractionated ACM and repeated the α-SMA induction assay. We found the factor(s) responsible for myofibroblast conversion appeared to be a protein between 30-100 kDa and lipid-associated (D). Of the adipokines profiled to be different between ACM and PCM, adiponectin fit the physiochemical description so we depleted adiponectin from the ACM and found that doing so nearly eliminated the myofibroblast-inducing potential of the ACM (E).



Discussion & Conclusions

These results shed important light on the cellular and molecular mechanisms that drive the effects of adipose micrografting on dermal fibroblasts. To our surprise, we found adipocytes themselves, rather than adipose progenitors had a more significant impact on fibroblast function. Specifically, adipocytes convert dermal fibroblasts to highly synthetic, myofibroblasts through a TGF-𝛽1 independent mechanism (2). Molecular fractionation of the ACM revealed that the bioactive factor was consistent with the most abundant protein in ACM, adiponectin (3). Adiponectin associates into trimers and other multimeric forms that are believed to have differential functions related to cell metabolism. Work is ongoing to determine which molecular form of adiponectin is responsible for directing fibroblast activity during tissue remodeling. Such information will provide a better understanding of how adipocytes and other components of fat grafts contribute to wound healing and how these different components can be optimized to provide maximum therapeutic benefit to wound healing treatment strategies.


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1.Klingelhutz, A. J. et al. Scaffold-free generation of uniform adipose spheroids for metabolism research and drug discovery. Sci Rep-uk 8, 523 (2018).

2. El-Hattab, M. Y. et al. Human Adipocyte Conditioned Medium Promotes In Vitro Fibroblast Conversion to Myofibroblasts. Sci Rep-uk 10, 10286 (2020).

3. El-Hattab, M. Y. et al. Native adiponectin plays a role in the adipocyte-mediated conversion of fibroblasts to myofibroblasts. J Roy Soc Interface 20, 20230004 (2023).