American Association of Plastic Surgeons

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Nawal Khan, MD1, Leonardo Alaniz, BBA2, Arya Sherafat, BS3, Tania Nasrollahi, BS2, Mary A. Ziegler, PhD2, Gregory Evans, MD, FACS2, Alan D. Widgerow, MBBCh, MMed, FACS2.
1Wyckoff Heights Medical Center, Brooklyn, NY, USA, 2University of California Irvine, Orange, CA, USA, 3University of California Riverside, Riverside, CA, USA.

Purpose: Advancements in tissue engineering have allowed scientists to explore the possibility of generating human cartilage to minimize the need for complicated reconstructive surgeries. Human auricular cartilage can be decellularized to serve as a scaffold while maintaining its biochemical and mechanical properties, thus providing a framework structurally and functionally mimics the native ear. Adipose stem cells (ASCs) co-cultured with chondrocytes induce chondrogenesis. Platelet-rich plasma (PRP) contains a variety of growth factors that also promote chondrocyte proliferation and the secretion of cartilaginous matrix. Here, we decellularized human auricular cartilage and seeded it with auricular chondrocytes in the presence of ASCs with and without PRP and assessed chondrogenesis.
Methods: Auricular chondrocytes (AuCs) were isolated from discarded microtia cartilage. Adipose stem cells were isolated from healthy adult lipoaspirate. Human cadaver ears were dissected down to the cartilage, and a decellularization protocol was used that preserves the structure of the native matrix. 1 The tissue was then prepared for recellularization by placing it in peracetic acid followed by a rinsing solution. Four 6 mm biopsy punches were made to test each seeding condition as follows: no cells; AuCs alone, AuCs + ASCs (at a ratio of 1:9), and AuCs + ASCs + 0.1% PRP. The tissue was presoaked in chondrocyte medium after which it was seeded with a total of 1 million cells or just medium (negative control). The medium was replaced twice a week for 4 weeks. The tissues were fixed and prepared for staining to assess tissue structures before and after decellularization and following recellularization. Hematoxylin and eosin (HE) staining
was done to assess cellular content, Masson’s trichrome, Alcian blue and Verhoff’s staining were done to assess collagen, glycosaminoglycans (GAGs), and elastin, respectively. Finally, collagen II was assessed via immunohistochemistry.
Results: HE staining of decellularized cadaver cartilage showed successful decellularization as evidenced by reduction in nuclei compared to native cartilage. The essential ECM components were preserved, namely collagen and elastin. However, GAGs were reduced after decellularization. Upon recellularization, there was more cell attachment when the AuCs were seeded with ASCs. The addition of PRP yielded an attachment that permitted the cells to infiltrate the tissue compared to just residing on the periphery. Recellularization also induced cartilage deposition and GAG accumulation. Qualitatively, ECM assessments by Masson’s Trichrome and Alcian blue were similar when ASCs and chondrocytes were seeded together with and without PRP. However, type II collagen deposition increased after recellularization, but it was only statistically significant when AuCs were seeded with ASCs and PRP.
Conclusion: Decellularized human cadaver ears are a suitable scaffold for recellularization. Neo-chondrogenesis shows greater success when AuCs are seeded with ASCs and PRP. Further in vivo experiments are warranted to assess function, structure, and stability of the recellularized tissue after implantation.
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