Efficient in vivo vascularization of tissue-engineering scaffolds.

2.1. Cells Human Dermal Microvascular Endothelial Cells (HMVEC, single donor lot; Lonza) were grown in EGM-2 MV medium (Lonza).

Human Pulmonary artery Smooth Muscle

Cells (hPASMC, single donor lot; Lonza) were grown in SmGm medium (Lonza). Primary cells were used between passage 3–7.

Phoenix A retroviral packaging cells

(ATCC) were grown in Dulbecco’s Modified Eagle Medium (DMEM) (4500 mg/ml glucose) (Sigma Aldrich) supplemented with 10% FBS (Euro Clone/PAA), 5% Penicillin/Streptomycin (Sigma Aldrich) and 5% L-Glutamine (Sigma Aldrich).

Retroviral transduction Phoenix

A retroviral packaging cells were transfected with GFP or RFP retroviral vectors ( Evensen et al., 2009 ) as per Swift et al ( Swift et al., 1999 ). Briefly, subconfluent Phoenix A were transfected by CaCl 2 -precipitation in the presence of chloroquine (Sigma Aldrich). Virus was harvested in EGM-2 MV or SmGm medium 48 hours post transfection and added to subconfluent HMVEC or hPASMC (passage 3–5) with 5 μg/ml protamine sulfate (Sigma Aldrich) for 16 hours. Transduced GFP-expressing HMVEC and RFP-expressing hPASMC were purified by flow cytometric sorting on a FACSAria Cell Sorter (BD Biosciences). In vitro organotypic blood vessel system A microtiter plate format in vitro organotypic blood vessel system assay was conducted as described ( Evensen et al., 2009 ). Briefly, 6 000 GFP-expressing HMVEC and 50 000 hPASMC cells were co-seeded in EGM-2MV medium in a 96-well plate. Co-cultures were imaged after 72 hours using a fully automated high throughput fluorescence microscope (BD Pathway 855). 2.4.

Experimental animals

For all experiments, non-obese mice with severe combined immunodeficiency disease (NOD/SCID) (Gade Institute/Taconic Farms) were used. The animals were approximately 6–8 weeks of age at the time of scaffold implantation. All experiments were approved by the Norwegian Animal Research Authority, and conducted according to the European Convention for the Protection of Vertebrates Used for Scientific Purposes. 2.5. Scaffold preparation Poly-L Lactic Acid (PLLA) scaffolds were produced by a solvent-casting particulate-leaching technique, previously described by Nor et al ( Nor et al., 2001 ) One gram of PLLA (Resomer L 206 S, Boehringer Ingelheim) was dissolved in 20 ml chloroform (Sigma) to yield a 5% solution. NaCl (Fisher scientific) was sieved trough a test sieve (Retsch) with a pore size of 450μm, and 3,45 g of the sieved NaCl was distributed in silanised glass beakers. The NaCl was mixed with the 5% PLLA solution, and the solvent was left to evaporate. Thereafter, the scaffolds were leached for 48 h with ddH 2 O to wash out the NaCl, and then dried and cut into 6×6×1 mm pieces. Scaffolds were sterilized in a descending alcohol row from 100-70% EtOH, and kept in sterile PBS until implantation. 2.6.

Tissue engineering model

Prior to implantation, scaffolds were dried briefly on sterile paper and filled with a total of 1×10 6 cells in 36 μl of 50:50 EGM-2 MV and growth factor reduced phenol red-free Matrigel (BD). The scaffolds were left at 37 °C for 30 min, for the Matrigel to solidify. Scaffolds were seeded with 1×10 6 HMVEC alone, 1:1 (500 000 HMVEC: 500 000 hPASMC) or 1:4 (200 000 HMVEC: 800 000 hPASMC) ratios. For each experimental group, 5 mice were implanted with two scaffolds each. Acellular scaffolds containing Matrigel only were implanted into 4 mice; and scaffolds seeded with 1×10 6 hPASMC only were implanted into 5 mice, to provide negative controls for histology and imaging. NOD/SCID mice were anesthetized with an intramuscular injection of 20μl 1:2 concentration of Rompun (Xylazin) (20mg/ml) (Bayer Health Care) and Narketan (Ketamin) (100mg/ml) (Vétoquinol) in the thigh muscle. A 2.5 cm incision was made on the back of the mouse, and scaffolds were placed in skin flaps at the flanks. After 7, 14 or 21 days, the mice were sacrificed by cervical dislocation after deep Isoflurane (Schering-Plough) anesthesia, and scaffolds were recovered for fixation. 2.7.

Histological staining

Scaffolds were fixed in 10% paraformaldehyde (PFA), and subsequently embedded for paraffin sectioning. Sections from the middle part of the scaffold were deparaffinized and stained with monoclonal mouse-anti human CD31 (M0823, DAKO), and monoclonal mouse anti-human smooth muscle actin ((M0851, DAKO), visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB) (EnVision ™ detection system, K5007, DAKO) and counterstained with Hematoxylin (DakoREAL ™ Hematoxylin, S2020, DAKO). Antibody specificity was validated using sections from acellular and hPASMC only implants. 2.8.

Functional multiphoton fluorescence microscopy analysis

In order to specifically label perfused human endothelial cells in the scaffolds, 200 μl of UEA-1 Lectin-FITC (Sigma-Aldrich) (1 mg/ml in sterile 0.9% NaCl) was injected into the lateral tail vein 30 min before sacrifice ( Holland et al., 2005 ) Scaffolds were excised and immediately imaged using a Multiphoton Microscope (Leica SP5). Acquired serial images were analyzed using 3D image analysis software (IMARIS 6.3). 2.9.

Flow cytometry analysis

Scaffolds were removed 30 min after injection with UEA-1 Lectin-FITC and homogenized using a syringe in a collagenase A solution (Sigma-Aldrich) (22U/ml). Cells were centrifuged at 1000 rpm for 5 minutes, washed with PBS and filtered with a 40 μm pore size filter. Cells were then washed one more time, resuspended in PBS and analysed by flow cytometry for FITC fluorescence. Propidium iodide staining (1 μg/ml, Molecular Probes) was performed in order to exclude dead cells from the analysis. 2.10.

Image analysis

To quantify the number of vessels per mm 2 and the vessel diameter, histological sections of scaffolds were examined with a light microscope (Leica). Using the image processing program AnalySIS, the outlines of anti-hCD31-staining vessels in five fields of view of a section were encircled manually at 200 × magnification. The number of vessels from five fields of view was used to calculate the vessel diameter and number of vessels per mm 2 . Vessels were subdivided into 5 categories based on diameter: