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    Tissue Engineering

    Adipose and Osteogenic MSC Line Differentiation Responses using Hair Protein Biomolecules

    (K-425) Adipose and Osteogenic MSC Line Differentiation Responses using Hair Protein Biomolecules

    Thursday, October 12, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row K - Poster # 425
    Introduction::

    Introduction:  Human hair extracts, majority as keratin and keratin associated proteins (KAPs), offer biocompatibility with potential applications in tissue engineering and in vitro cell culture.  Mesenchymal stem cells (MSCs) hold the capacity for tissue repair by differentiating into connective tissue cells such as adipocytes and osteoblasts.  The standard cultured MSCs differentiation on tissue culture plastic (TCP) employs biochemical cocktails (dexamethasone, isobutyl-1-methylxanthine, indomethacin, and insulin (DIII) for fat, and dexamethasone, ascorbic acid, and beta-glycerol phosphate (DAB) for bone), but still inefficient, unnatural, and produces different cell types; thus, the process can further be improved for differentiation control by exposing the cells to a variety of stimuli.  This study investigates the impact of incorporating hair proteins as soluble and substrate materials for MSC adipo- and osteogenesis.



    Materials and Methods:: Material and Methods:  MSC line (ASC52telo, ATCC) cells were cultured using DMEM with 5% serum (CM).  Cells were seeded on hair strands immobilized on agarose and adhesion quantified after 24 h.  Scanning electron microscopy (SEM) utilized to visualize cells.  For differentiation, soluble hair extracts: KTN (rich in keratins and KAPs) and KAP (mostly KAPs) at ~ 0.3 ng/cell were added separately without or with DIII or DAB in CM of confluent cells in 24-well TCP, replaced bi-weekly over 2 and 3 w for Oil Red O (lipid in vacuole, fat) and Alizarin Red S (calcium deposition, bone) staining, respectively.  Areas covered by the dyes were quantified and normalized.  In another experiment, MSCs were grown on KTN and KAP thin gel films separately without or with DIII or DAB and staining results quantified.

    Results, Conclusions, and Discussions:: Results and Discussion:  MSCs grown on intact reduced and delipidized hair strands (Fig. 1) showed relatively low adhesion and proliferation, but still higher compared to agarose-only control.  When cultured on hair protein extract films: KTN and KAP, MSCs demonstrated greater proliferation until complete confluency, likely due to exposure of cell-binding domains.  Cells without or with DIII or DAB exhibited similar responses compared to those on TCP, indicating that hair proteins as a substrate can support MSCs with the same capacity as the TCP.  Notably, when MSCs on TCP were treated with DIII + KAPs, they differentiated into adipocytes with significantly more fat droplets (Fig. 2, middle right).  More importantly, even without DIII but with KTN (which contains keratins and KAPs), cells were still differentiated into fat-like cells (Fig. 2, bottom left), but with different morphology!  We are currently characterizing these cells via SEM and lipid analysis.  Cells exposed to DAB, without or with KAP or KTN differentiated similarly for the calcium bone marker.

    Conclusion:  Different responses upon treatment of MSCs with hair proteins indicate bioactivity for influencing MSCs differentiation fate.  Particularly, adding KTN without the DIII adipogenic standard still led to the production of high amounts of intracellular lipid-filled vacuoles.  This MSCs behavior can be utilized for in vitro fat tissue synthesis and for fat tissue engineering, but require prior experimentation and analysis.



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