Experimental approach to nasal septal cartilage regeneration with adipose tissue‐derived stem cells and decellularized porcine septal cartilage

Abstract

Background Cartilage shortage is a major problem in facial reconstructive surgery. Prior studies have shown that decellularized porcine nasal septal cartilage (DPNC) seeded with primary human nasal chondrocytes enabled cartilage regeneration and showed potential as a replacement material for nasal cartilage. Since adipose tissue‐derived stem cells (ASCs) are easily accessible and almost abundantly available, they appear to be a promising alternative to limited chondrocytes making the combination of DPNC and ASCs a feasible approach towards clinical translation. Thus, this study was intended to investigate the interactions between ASCs and DPNC in an in vitro model. Methods DPNCs were seeded and 3D‐cultured with primary human ASCs that were priorly characterized with trilineage differentiation and flow cytometry. Cell vitality and proliferation were evaluated by Live‐Dead, alamarBlue, and PicoGreen assays. Chondrogenic differentiation was examined by DMMB assay and cryosectioning‐based histology. Cell invasion within DPNC was visualized and quantified by fluorescent histology (DAPI, Phalloidin). Results ASCs showed good adherence to DPNC and Live‐Dead assay proved their viability over 2 weeks. AlamarBlueassay showed an increase in metabolic activity compared to 2D cultures, and PicoGreen assay demonstrated an increase of cell number within DPNC over time. Biochemical assays and histology added evidence of chondrogenic differentiation of 3D‐cultured ASCs under the influence of chondrogenic induction medium. Fluorescent image analysis showed a significant increase of cell‐occupied areas of scaffolds over time (P < .05). Conclusions DPNC scaffolds provided a suitable environment for ASCs that allowed good cell vitality, high proliferation, and chondrogenic differentiation. Thus, the use of ASCs and DPNC yields a promising alternative to the use of primary human chondrocytes. For facial cartilage tissue engineering, we regard ASCs as an attractive alternative to human nasal chondrocytes due to their better accessibility and availability. Further research will be necessary to determine long‐term effects and in vivo outcomes of ASCs and DPNC in cartilage regeneration of the face.

Differentiation Behaviour of Adipose-Derived Stromal Cells (ASCs) Seeded on Polyurethane-Fibrin Scaffolds In Vitro and In Vivo

“…By contrast, there was ossification of the constructs in vivo, as described in the literature for the same growth factor combination and other growth factors [30,63,65]. With regard to the generation of stable hyaline cartilage using ASCs, an interesting focus is certainly on the optimization of the scaffold and the additional evaluation of alternative scaffold materials [30,44,64,66] that promote neo-cartilage formation or the development of threedimensional constructs even without scaffold [67].…”

Zone-Dependent Architecture and Biochemical Composition of Decellularized Porcine Nasal Cartilage Modulate the Activity of Adipose Tissue-Derived Stem Cells in Cartilage Regeneration

“…The plastic-adherent ASCs were cultivated until 80% confluency, and were trypsinized, frozen and stored in liquid nitrogen until use. All ASCs were subjected to flow cytometry using positive mesenchymal stem cell markers and multilineage differentiation assays to prove their stem cell potential, as described previously [ 27 ]. The experiments were performed in triplicates, unless otherwise stated, with cells of passage 1 and 2.…”

“…Induction was started 7 days after seeding and scaffolds were harvested at 7, 14 and 28 days, snap-frozen in liquid nitrogen and stored at −80 °C. Both assays were performed as described previously by the authors [ 25 , 27 , 65 ].…”

“…Results were corrected for sGAG concentration of corresponding empty scaffolds, resulting in ∆ sGAG. Induction was started 7 days after seeding and scaffolds were harvested at 7, 14 and 28 days, snap-frozen in liquid nitrogen and stored at −80 • C. Both assays were performed as described previously by the authors [25,27,65].…”

“…Decellularized cartilage has the advantage of providing a high degree of biomimicry due to its composition and three-dimensional architecture derived from native cartilage [ 10 , 24 ]. Previous experiments have shown that DPNC yields a proper environment for human nasal chondrocytes and ASCs that enables cartilage regeneration [ 23 , 25 , 26 , 27 ].…”