Ined at 300 mOsm by mixing it with the appropriate volumes of 1N NaOH, 106 phosphate buffered saline (PBS), and 16 PBS as previously described [17,19]. This collagen solution was immediately mixed with the cells and media and injected into ear molds using a syringe stop-cock system to obtain a final collagen concentration of 10 mg/mL and a final cell concentration of 256106 cells/mL. Separate acellular constructs were made through an identical process that did not involve suspension of cells in collagen. The molds were allowed to gel for 50 minutes at 37uC. After 50 minutes, the ear constructs were removed from the molds and cultured in media composed of DMEM, 12926553 10 fetal bovine serum, 100 mg/mL penicillin, 100 mg/mL streptomycin, 0.1 mM non-essential amino acids, 50 mg/mL ascorbate, and 0.4 mM L-proline. Samples were cultured in this media for 3? days until implantation. A total of 16 cell-seeded and 9 acellular samples were generated for this study. Two cell-seeded constructs were excluded from ex vivo analysis due to seroma formation.implant (,465 cm) was dissected in the loose subcutaneous areolar tissue. An acellular or cellular implant was then inserted and appropriately oriented. Incisions were closed with metallic wound clips and a sterile occlusive dressing was placed prior to recovery from anesthesia. Animals were sacrificed via CO2 asphyxiation and bilateral thoracotomy after 1 or 3 months. Constructs were harvested and their weights recorded. Construct length was measured along the lobule-helix axis. Construct width was defined as the largest dimension measured along an axis perpendicular to the lobulehelix axis (Figure 3). Half of each specimen was snap-LED-209 web frozen in liquid nitrogen for biomechanical analysis, while the remainder was fixed in 10 neutral buffered formalin for 48 hours prior to histologic analyses.Histologic analysesThe fixed portions of samples were Calyculin A web dehydrated by sequential washes in ethanol, embedded in paraffin, cut into 5 mm sections, and stained with Safranin O/Fast green to assess proteoglycan 
distribution and Verhoeff’s/Van Gieson to assess the presence of elastin fibers.Biomechanical analysisSix mm61 mm disks were cut from the central portion of frozen implants using dermal biopsy punches and thawed in PBSIn vivo implantationTen-week old male athymic nude rats (RNU; Charles River, Wilmington MA) were used for in vivo studies. Animals were anesthetized via intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg). After induction of anesthesia, the animal’s dorsum was shaved, depilated, prepped with povidone iodine, and appropriately draped. All animals received a subcutaneous injection of buprenorphine (0.1 mg/kg) and an intraperitoneal injection of cefazolin (11 mg/kg) prior to 15755315 any surgical manipulation. An incision was then made overlying the dorsum and the smallest subcutaneous pocket that would accommodate theFigure 2. Mold design based on ear anatomy. The digital images of ears (A) were used to design 7-part molds (B ) by embedding the solid images of the ear into virtual blocks. doi:10.1371/journal.pone.0056506.gFigure 3. Schematic representation of length and width measurements. Construct length was measured along the lobulehelix axis. Construct width was defined as the largest dimension measured along an axis perpendicular to the lobule-helix axis. doi:10.1371/journal.pone.0056506.gTissue Engineering of Patient-Specific Auriclescontaining protease inhibitors. Disks were placed in a cy.Ined at 300 mOsm by mixing it with the appropriate volumes of 1N NaOH, 106 phosphate buffered saline (PBS), and 16 PBS as previously described [17,19]. This collagen solution was immediately mixed with the cells and media and injected into ear molds using a syringe stop-cock system to obtain a final collagen concentration of 10 mg/mL and a final cell concentration of 256106 cells/mL. Separate acellular constructs were made through an identical process that did not involve suspension of cells in collagen. The molds were allowed to gel for 50 minutes at 37uC. After 50 minutes, the ear constructs were removed from the molds and cultured in media composed of DMEM, 12926553 10 fetal bovine serum, 100 mg/mL penicillin, 100 mg/mL streptomycin, 0.1 mM non-essential amino acids, 50 mg/mL ascorbate, and 0.4 mM L-proline. Samples were cultured in this media for 3? days until implantation. A total of 16 cell-seeded and 9 acellular samples were generated for this study. Two cell-seeded constructs were excluded from ex vivo analysis due to seroma formation.implant (,465 cm) was dissected in the loose subcutaneous areolar tissue. An acellular or cellular implant was then inserted and appropriately oriented. Incisions were 
closed with metallic wound clips and a sterile occlusive dressing was placed prior to recovery from anesthesia. Animals were sacrificed via CO2 asphyxiation and bilateral thoracotomy after 1 or 3 months. Constructs were harvested and their weights recorded. Construct length was measured along the lobule-helix axis. Construct width was defined as the largest dimension measured along an axis perpendicular to the lobulehelix axis (Figure 3). Half of each specimen was snap-frozen in liquid nitrogen for biomechanical analysis, while the remainder was fixed in 10 neutral buffered formalin for 48 hours prior to histologic analyses.Histologic analysesThe fixed portions of samples were dehydrated by sequential washes in ethanol, embedded in paraffin, cut into 5 mm sections, and stained with Safranin O/Fast green to assess proteoglycan distribution and Verhoeff’s/Van Gieson to assess the presence of elastin fibers.Biomechanical analysisSix mm61 mm disks were cut from the central portion of frozen implants using dermal biopsy punches and thawed in PBSIn vivo implantationTen-week old male athymic nude rats (RNU; Charles River, Wilmington MA) were used for in vivo studies. Animals were anesthetized via intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg). After induction of anesthesia, the animal’s dorsum was shaved, depilated, prepped with povidone iodine, and appropriately draped. All animals received a subcutaneous injection of buprenorphine (0.1 mg/kg) and an intraperitoneal injection of cefazolin (11 mg/kg) prior to 15755315 any surgical manipulation. An incision was then made overlying the dorsum and the smallest subcutaneous pocket that would accommodate theFigure 2. Mold design based on ear anatomy. The digital images of ears (A) were used to design 7-part molds (B ) by embedding the solid images of the ear into virtual blocks. doi:10.1371/journal.pone.0056506.gFigure 3. Schematic representation of length and width measurements. Construct length was measured along the lobulehelix axis. Construct width was defined as the largest dimension measured along an axis perpendicular to the lobule-helix axis. doi:10.1371/journal.pone.0056506.gTissue Engineering of Patient-Specific Auriclescontaining protease inhibitors. Disks were placed in a cy.
