Figure 1.
Biomaterial construct prior to implantation.
A: Polyurethane based polycaprolactone construct seeded with chondrocytes. B: SEM picture of porous PCL construct, magnification 48×. C: Live/dead staining was performed with fluoresceindiacetate and propidium jodid using confocal laser scanning microscopy. D: Cultivated cartilage cell biomaterial construct (black arrow) implanted subcutaneously above the panniculus carnosus; day 0 postoperative. Scale bars = 1 cm.
Figure 2.
A: The implantation of the cultured auricle (white arrow) took place on the right side of the abdomen above the panniculus carnosus in a skin flap measuring 150×80 mm. A silicon foil was sewn onto the abdominal fascia to prevent neovascularization from the abdominal wall. Saphenous artery and greater saphenous vein were distally anastomosed turned in the cranial direction and attached below the abdominal skin flap. *indicates the original position of A. saphena and V. saphena magna. B: Four-sided flap excision. To proof the ability of the neovascularized cultivated 3D cartilage cell construct (black arrow) to survive solely perfused by the newly implanted axial vascular pedicle the abdominal flap was excised four-sided, leaving the vascular pedicle (white arrow) intact. C: Macroscopic view after free microsurgical transplantation. Healed skin flap three weeks after free microsurgical transplantation. Black arrow indicates the integrated neovascularized cultured auricle. Scale bars = 5 cm.
Table 1.
Harvesting of cartilage, cell culture, and seeding of biomaterials.
Figure 3.
Microangiographic images of prefabricated 3D skinflaps after neovascularisation.
A–J: Images show vasculature of abdominal skin flaps, with integrated 3D cartilage constructs, perfused by Micropaque ® through the implanted vascular pedicle. K: The micro-angiographic control was performed using a Radifluor 120– TORR (Philips Electronic Instruments, Los Angeles, USA). Scale bars = 1 cm.
Figure 4.
Angiographically quantified neovascularization in prefabricated 3D flaps. This boxplot shows the quantitative neovascularization of the 10 test flaps in comparison to 6 native abdominal skin flaps (control). To quantify the neovascularization in prefabricated 3D flaps 25 evenly spaced standard integral lines were placed corresponding to the 15-cm flap length. Vessel quantity was determined by counting the total number of times that vessels perfused by Micropaque ® intersected the integral lines over the entire surface of the flap under 2× magnification as described previously [14]. There was a significant difference between experimental and control group, ***p = 0.0002, Mann-Whitney Test (two-tailed).
Figure 5.
A (study group): Stitched full cross section of the cultured auricle (black+white line). B: Implanted vascular pedicle (saphenous artery and greater saphenous vein), original magnification, x5. C+E: Low power view of the center of the cultivated cartilage cell biomaterial construct shows GAG, black arrows indicate ingrown vessels (C: Alcian blue counterstained with Kernechtred, original magnification, x50; E: Alcian blue staining, original magnification, x50). D+F: Periphery of cultivated cartilage cell biomaterial construct shows vital cartilage and good integration in the skin flap. White arrow indicates an ingrown vessel (Alcian blue counterstained with Kernechtred, original magnification, D×10; C×200). G (control group): Stitched full cross section of the cultured auricle (black+white line). H+J: Low power view of the center of the cultivated cartilage cell biomaterial construct shows necrosis with no GAG (H: Alcian blue counterstained with Kernechtred, original magnification, ×50; J: Alcian blue staining, original magnification, ×50). I+K: Periphery of cultivated cartilage cell biomaterial construct shows small areas of vital cartilage and good integration in the skin flap. (Alcian blue counterstained with Kernechtred, original magnification, I×10, K×200). L: Image indicates the performed sectional planes within the biomaterial construct. All showed histological images are from section plane E. Scale bars = 400 µm.