Implant for autologous soft tissue reconstruction using an adipose-derived stem cell- colonized alginate scaffold  Tobias Hirsch, Christine Laemmle, Bjoern Behr, Marcus Lehnhardt, Frank Jacobsen, Dirk Hoefer, Maximilian Kueckelhaus  Journal of Plastic, Reconstructive & Aesthetic Surgery  Volume 71, Issue 1, Pages 101-111 (January 2018) DOI: 10.1016/j.bjps.2017.08.009 Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 1 Protoscaffold structure. a–c: Prior to lyophilization; d–f: after lyophilization. a: Freshly gelled hydrogel; b: light microscopy of hydrogel; c: hydrogel surface captured by scanning electron microscopy; d: cut ready-to-use protoscaffold after lyophilization; e: variation of structures after lyophilization; f: macroscopic image of porous formation. Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 2 Cytotoxicity of alginate scaffolds. The ddH2O-washed scaffolds demonstrated significantly less cytotoxic effects compared to untreated scaffolds throughout all media dilutions (33%, 22%, 14.8%, and 9.9%) (*). Dilutions were prepared by the addition of regular DMEM. There was no significant difference between ≤2 h and ≤4 h washing cycle (#) (p < 0.001). Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 3 Viability of hASCs in alginate scaffolds. a + b: Grouped and round-shaped hASCs within the scaffold structure after 72 h of incubation; c + d: within the scaffold structure after 7 days of incubation; a: DAPI-fluorescence staining of cell nuclei; b: light microscopy of MTT-stained viable cells; c: calcein–fluorescence stain of vital cells; d: live (calcein)/dead (PI)–fluorescence stain. Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 4 Adipogenic differentiation of human, murine, and porcine MSCs. Cells were differentiated in cell culture for 21 days. a: hASCs; b: M2; c: pASCs. Intracellular lipid vacuoles stained with Oil Red O at 21 days. Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 5 VEGF secretion by pASCs in cell culture and in alginate scaffolds during adipogenic differentiation. In cell culture, undifferentiated pASCs released significantly more VEGF than differentiated pASCs from day 7 onward (#). Although there was no significant difference in VEGF release between pASCs on day 1 of differentiation and undifferentiated pASCs, VEGF content was significantly higher on day 1 of differentiation (*). pASCs in alginate scaffolds did not generally release more VEGF than pASCs in cell culture. During differentiation in the scaffold, there also was no significant difference compared to the undifferentiated control, but the VEGF content of native pASCs on day 1 was significantly higher than that on day 21 (+) (p < 0.05). Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 6 Angiogenic potential in ovo: after CAM removal from the egg and storage in formalin, images were produced via stereomicroscopy. The number of vessels were then counted using Image J software. Group sizes were as follows: pASC-colonized scaffolds without collagen, n = 6; pASC-colonized scaffolds with collagen, n = 21; cell-free alginate scaffolds, n = 5; cell-free alginate scaffolds with collagen, n = 16. Cell-free alginate scaffolds with and without collagen served as negative controls. a: pASC-colonized alginate scaffold in ovo immediately prior to explantation of the CAM; b: explanted CAM with collagen-containing pASC-colonized alginate scaffold; c: the negative control of a cell-free collagen-containing alginate scaffold demonstrates a lower density of embryonic vascular density than stem cell-colonized alginate scaffolds on the macroscopic level; d: pASC-colonized scaffolds with and without collagen demonstrated significantly more afferent vessels on the CAM than their respective negative controls (NC, #, §). The comparison between pASC-colonized scaffolds with and without collagen (*), and direct comparison with the respective negative controls (+) did not demonstrate significant differences. # = p < 0.05; § = p < 0.001; * = p > 0.05; + = p > 0.05. Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 7 HUVEC tube sizes after conditioning with media from pASC-colonized scaffolds. Incubation of HUVECs with media from pASC-colonized alginate scaffolds and collagen-containing pASC-colonized alginate scaffolds led to significantly longer tubes than with their respective negative controls. There was no significant difference between pASC-colonized alginate scaffolds and collagen-containing pASC-colonized alginate scaffolds, as well as no significant difference with the negative controls. Tubes in positive control in combination with growth factor-enhanced media were significantly larger and tubes in negative controls in combination with growth factor-enhanced media were significantly smaller than those in pASC-colonized scaffolds (p1 = 1. quartile; p3 = 3. quartile; p < 0.001). Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 8 HUVEC tube sizes after conditioning with media from adipogenic pASCs differentiated in cell culture or in alginate scaffolds. Media from pASCs differentiated in alginate scaffolds (blue stripes) lead to significantly longer tube formation than media from pASCs differentiated in cell culture (yellow stripes) on days 1 and 14 of differentiation (#). In the scaffold group, the 21-day differentiated pASCs resulted in the significantly longest tubes (*). The shortest tubes were observed in the 21-day undifferentiated negative control (§). In the cell culture group, 21-day differentiated pASCs led to the longest tubes, which were significantly longer than tubes from negative control from day 1 (+) (PC = positive control; NC = negative control; d = day; p1 = 1. quartile; p3 = 3. quartile) (* = p < 0.05) (# = p < 0.001) (+ = p < 0.001) (§ = p < 0.05). Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Supplemental Figure 1 Chemical reaction of internal alginate gelation. Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Supplemental Figure 2 Protoscaffold weight loss due to alternating hydration and lyophilization. a: Weight development of the wet scaffold after five cycles of hydration and lyophilization demonstrating significant weight loss compared to its state immediately after gelling; b: weight development of the dry scaffold showing a similar development (p < 0.001). Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Supplemental Figure 3 Adipogenic differentiation of various MSCs in alginate scaffolds. Calcein staining on day 7 displayed a high density of vital mesenchymal stem cells. hASCs, M2, and pASCs were differentiated into adipocytes, which showed intracellular triacylglycerides on day 21 (bright yellow with Nile red staining). The negative controls (HF ASTs and MSCs cultured in reduced media) did not differentiate into adipocytes. After 21 days, only the phospholipids of the cell membranes were stained red-orange with Nile red staining. Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Supplemental Figure 4 VEGF secretion from pASC-colonized alginate scaffolds. Alginate scaffolds were colonized with a pASC or collagen-enriched cell suspension of 9 × 104 cells and cultured for 3 days. The media was then screened for VEGF secretion over 24 h. There was no significant difference in VEGF content between the two groups. Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Supplemental Figure 5 Histology of pASC-colonized alginate scaffolds. a: Overview; b: pASC-colonized scaffold; c: collagen-containing pASC-colonized scaffold; d: uncolonized scaffold; e: collagen-containing uncolonized scaffold; f: fluorescence staining showing agglutinin (red) and nuclei (blue). The overview (a) shows the CAM (C) with blood vessel (B) and the alginate scaffold. The arrows in b, c, d, and e indicate cells. The presence of cells in the uncolonized scaffolds indicate the migration of cells from the CAM into the scaffold. Journal of Plastic, Reconstructive & Aesthetic Surgery 2018 71, 101-111DOI: (10.1016/j.bjps.2017.08.009) Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions