Collagen production on histology demonstrated an initial increase in type III at 1 week followed by type I production at 2 weeks and type IV at 4 weeks. Gene expression of both type I and type III peaked at 2 weeks, whereas type IV continued to increase over the 4-week period. Histology demonstrated fibrillin-1 deposition at 1 week followed by elastin production at 4 weeks. Elastin gene expression significantly increased at 4 weeks, whereas fibrillin-1 decreased at 4 weeks. GAG demonstrated abundant production at each time point on histology. Gene expression of decorin significantly increased at 4 weeks, whereas versican decreased over time. Biochemical analysis showed that total collagen production was greatest at 2 weeks, and there was a significant increase in elastin and GAG production at 4 weeks. Histological characterization of MMPs showed abundant production of MMP-2 at each time point, while MMP-9 decreased over the 4-week period. Gene expression of MMP-2 significantly increased at 4 weeks, whereas MMP-9 significantly decreased at 4 weeks.

Bone marrow was harvested from gender-matched syngeneic C57BL/6 mice. BMC were isolated from bone marrow by performing density gradient centrifugation using Histopaque-1083 (Sigma). One million BMC were statically seeded onto each scaffold and incubated in RPMI-1640 culture media overnight at 37°C and 5% CO₂.

Thirty-six female C57BL/6 mice at approximately 8 weeks of age were obtained from The Jackson Laboratory (Bar Harbor, Me., USA) for this study. All experimental procedures were approved by the Yale University Institutional Animal Care and Use Committee. Seeded TEVGs were implanted as inferior vena cava (IVC) interposition grafts using microsurgical techniques as previously described [Roh et al., 2008]. Tissue-engineered neovessels were harvested 7 days (n = 12), 14 days (n = 12), and 28 days (n = 12) after surgery. Fresh specimens were used for molecular, enzymatic, and biochemical analysis (9 animals per time point). Pressure-fixed specimens were used for morphological studies (3 animals per time point).

A high-frequency ultrasound biomicroscopy system (Vevo 770; Visualsonics, Toronto, Ont., Canada) equipped with an RMV-704 transducer was used to measure the neovessel internal diameter for all mice at postoperative days 7, 14, and 28.

Explanted TEVG samples were pressure fixed in 10% formalin and embedded in paraffin. Five-micrometer sections were stained using standard techniques for Masson's trichrome, Elastica van Gieson, and Alcian blue stains. Masson's trichrome staining was used to demonstrate collagen fibers in green and cellular infiltrate in bright red. Picrosirius red staining was used to show the difference in distribution between large and thin collagen fibers.

Tissue sections were deparaffinized, rehydrated, and blocked for endogenous peroxidase activity and then for nonspecific staining. Primary antibodies used included: collagen I, III [Madri et al., 1980], and IV (Santa Cruz); Fibrillin-1 (Abcam); matrix metalloproteinase (MMP)-2 (Millipore); MMP-9 (R&D Systems); α-smooth muscle actin (Dako), and CD31 (Abcam). For immunohistochemistry (IHC), antibody binding was detected using biotinylated secondary antibodies, followed by binding of streptavidin- HRP. Color development was performed by chromogenic reaction with 3,3-diaminobenzidine (Vector). Nuclei were counterstained with hematoxylin. For immunofluorescence detection, goat-anti-rabbit IgG-Alexa Fluor 568 (Invitrogen) or a goat-anti-mouse IgG-Alexa Fluor 488 (Invitrogen) was used with subsequent 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining.

In order to determine the in vivo degradation rate of scaffold polymer, the histologic samples were observed under a polarized microscope in which remaining scaffold components were clearly demarcated by their birefringence.

The entire length of each fresh specimen TEVG (3 animals for each time point) was homogenized and lyophilized for quantitative biochemical analysis.

The collagen content was determined by measuring the hydroxyproline content. Dry weights of lyophilized samples (n = 3 for each time point) were measured and transferred to 1.8-ml cryo tubes containing 100 μl of 2 N NaOH. In separate tubes, 0 (blank), 5, 10, 15, and 20 μg of hydroxyproline standard (Sigma) dissolved in 100 μl 2 N NaOH were prepared to establish a standard curve for each experiment. Samples were hydrolyzed at 120°C for 30 min and then oxidized by adding 450 μl of chloramine-T reagent and incubating at room temperature for 25 min. After oxidation, a chromophore was developed by adding 500 μl Ehrlich's reagent to each sample and incubating at 65°C for 20 min. The absorbance of each standard and the samples was measured at 550 nm by spectrometry. The collagen content was calculated by multiplying the hydroxyproline content by 7.0 as previously described [Samuel, 2009]. Collagen production for each sample was determined by dividing the total collagen content by the total weight of each sample.

The elastin content was determined using a Fastin™ colorimetric assay (Biocolor Assays, Inc.). Dry weights of lyophilized samples were measured and transferred to 1.5-ml microcentrifuge tubes containing 100 μl 0.25 M oxalic acid. The tubes were then placed into a boiling water bath for 60 min to convert insoluble elastin to water-soluble α-elastin. A standard curve was created using controls from a kit. The elastin content in each sample was detected with spectrometry at 513 nm after precipitation and dye binding of α-elastin.

The GAG content was determined using a Blyscan™ colorimetric assay (Biocolor Assays). Dry weights of lyophilized samples were measured and digested in papain extraction reagent. A standard curve was created using controls from a kit. The GAG content in each sample was detected with spectrometry at 656 nm after precipitation and dye binding of GAG.

Tissue samples were homogenized in extraction buffer (Triton X-100). Homogenized tissue was centrifuged and supernatants recovered for analysis. Protein concentrations in lysates were determined against a standard curve (Bio-Rad, Hercules, Calif., USA). Thirty-microgram samples were mixed 1:2 with zymography sample buffer (Bio-Rad) and electrophoresed under nonreducing conditions on zymography gels (Readygel; Bio-Rad) for up to 2 h. Following electrophoresis, gels were placed in renaturation buffer (Triton X-100) for 60 min to remove SDS and then incubated at 37°C overnight in Ca²⁺ buffer. Controls were preincubated with 20 mM EDTA for 1 h, and 20 mM EDTA was also added to the substrate. Gels were stained the following day with 0.3% Coomassie blue for 1 h and then destained for up to 2 h. MMPs appear as clear bands on a blue background. Bands were compared to MMP-2 and MMP-9 standards (Chemicon).

In order to detect localization of gelatinolytic activity, in situ zymography was performed. Gelatin DQ is linked to a quenched FITC that is released upon gelatin digestion. Ten-micrometer-thick sections were cut from the same tissue which was used for substrate zymography. The cryosections were air dried for 30 min and then washed in PBS. Substrate was prepared by dissolving 1 mg DQ™ gelatin in 1.0 ml Milli-Q water, and this was further diluted 1:50 in the developing buffer containing 50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl₂, and 0.02% (w/v) Brij 35. Fifty microliters of this mixture was put on tissue sections and incubated in a dark humidity chamber for 2 h at 37°C. Sections were then rinsed in PBS. To verify the contribution of metalloproteases, control slides were preincubated with 20 mM EDTA for 1 h, and 20 mM EDTA was also added to the substrate. The level of autofluorescence in the tissue was evaluated by incubating the control section after the developing buffer was added.

RNA extraction from explanted scaffolds was performed using an RNeasy Mini Kit (QIAGEN VWR, Stockholm, Sweden) following the manufacturer's instructions. The RNA concentration of each sample was determined using a Quant-iT™ RiboGreen® RNA Reagent and Kit (Invitrogen Co., Carlsbad, Calif., USA) according to the manufacturer's instructions. Reverse transcription was carried out with a 50-μl total volume containing 1 μg RNA, 5 μl 10× TaqMan RT buffer, 11 μl MgCl₂ (25 mM), 10 μl dNTP mixture (10 mM), 2.5 μl random hexamer (50 mM), 1 μl RNase inhibitor (20 U/μl), and 1.25 μl reverse transcriptase (50 U/μl) (Applied Biosystems, Foster City, Calif., USA). RNAse-free water was added up to 50 μl. Thermal cycling parameters included incubation at 25°C for 10 min, reverse transcription at 48°C for 30 min, and inactivation at 95°C for 5 min. Predesigned and validated gene-specific TaqMan Gene Expression Assays from Applied Biosystems were used in duplicate for quantitative real-time PCR according to the manufacturer's instructions. The genes of interest for analysis included type I collagen (Col1a2, Mm00483888_m1), type III collagen (Col3a1, Mm01254458_g1), type IV collagen (Col4a3, Mm01269207_m1), elastin (Eln, Mm00514692_m1), decorin (Dcn, Mm01223999_m1), versican (Mm00490174_m1), MMP-9 (Mmp9, Mm01240563_g1), and MMP-2 (Mmp2, Mm00439498_m1) (Applied Biosystems). Real-time PCR reactions were carried out in 96-well reaction plates in the iCycler iQ Real-Time PCR Detection System (Bio-Rad) following the manufacturer's instructions. Relative expression levels were determined from collected data as threshold cycle numbers. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as an endogenous control.

Masson's trichrome staining highlighted collagen, which was associated with scaffold material over the 4-week study period. Abundant collagenous fibers formed between the polymer at early time points (fig. 3a, b), but these fibers lost their volume by 4 weeks (fig. ​(fig.3c).3c). A thin layer of collagen fibers was also noted in the medial layer of the neovessel (fig. ​(fig.3c).3c). IHC revealed an initial increase in type III collagen deposited between the scaffold fragments (fig. 3i, j) followed by type I collagen deposition (fig. 3e, f). Both type III and type I collagen deposited between scaffold fragments disappeared by 4 weeks, but a thin layer consisting of both types of collagen formed within the medial layer of the neovessel (fig. 3g, k). Type IV collagen was not detected at the luminal surface of the neovessel until the 4-week time point. When viewed using polarized light (dark-field microscopy), Picrosirius red staining revealed larger collagen fibers (possibly type I collagen) as bright yellow or orange and thinner fibers (possibly type III collagen) as green. At the 4-week time point, the predominant collagen was type I (fig. ​(fig.3o).3o). Figure ​Figure44 illustrates changes in gene expression of the collagen subtypes over the course of TEVG neovessel formation. The results indicate the trend of early induction of collagen gene expression at 1 week, which peaked for both type I and type III collagen at 2 weeks. The levels of gene expression for both collagen I and collagen III were significantly greater than those for the normal vena cava; however, there was no statistical difference at 4 weeks, which suggests that neovessl collagen gene expression will approximate that of native tissue at 4 weeks. The expression of type IV collagen was significantly lower at early time points (1 and 2 weeks) compared to that of normal vena cava and then approximated that of vena cava at 4 weeks. Total collagen quantified by a hydroxyproline assay indicated that the trend of total collagen in the TEVG peaked at 2 weeks. There was statistical significance between 2-week TEVG and normal vena cava, but the collagen content was equivalent between the 4-week TEVG and normal vena cava. This suggests that the amount collagen in the neovessel will approximate that of vena cava at 4 weeks (fig. ​(fig.4d4d).

A faint stain suggested elastic fibers near the luminal surface of the neovessels at 4 weeks. Fibrillin-1 (which presumably serves as a template for tropoelastin deposition) was upregulated during early time periods (fig. 5e–g). Figure ​Figure5i5i and j shows associated changes in the gene expression for elastin and fibrillin-1 at different times during TEVG neovessel formation. Note that the gene expression of elastin increased over time throughout the entire study period; in contrast, that of fibrillin-1 was higher than that of elastin at early time points but then decreased at 4 weeks. Overall, elastin protein production increased over time in the neovessels.

Alcian blue staining revealed the appearance of sulfated GAG in the neovessels at each time point during neovessel formation (fig. 6a–c). Figure ​Figure6e6e and f demonstrates further that the gene expression of proteoglycans differed depending on the type: there was a significant increase in decorin gene expression at the 4-week time point but no significant change in versican gene expression during the study period. Gene expression for both decorin and versican was higher in the neovessels than in vena cava, however. Finally, the Blyscan GAG assay demonstrated that there was an overall significant increase in the production of GAG in the neovessels at 4 weeks compared to earlier time points (fig. ​(fig.6g6g).

Histology showed robust production of MMP-2 at each time point (fig. 7a–c), whereas MMP-9 expression decreased over time (fig. 7e–g). MMP-9 initially appeared adjacent to scaffold fragments but shifted towards the luminal surface over time to more closely approximate expression of MMP-9 in normal vena cava. Gene expression of MMP-2 increased significantly (fig. ​(fig.7i),7i), while that for MMP-9 decreased significantly at 4 weeks (fig. ​(fig.7j).7j). Figure 8a, b, and c shows an in situ zymogram of neovessels at different time points. The enzymatic activity first appeared around scaffold fragments and then shifted to the luminal surface at 4 weeks. Figure ​Figure8d8d is a substrate zymogram, which demonstrates the gelatinolytic activity of neovessels at different time points. MMP-9 activity was detectable up to 4 weeks but showed variability between samples. MMP-2 activity was consistently detectable up to 4 weeks.