Ring and vessel formation.
The Alloderm decellularized dermis was incorporated into our tissue engineered vessel protocol28–30 as depicted in Fig. 1. The Alloderm material exhibited some additional stiffness compared to fresh dermis tissue determined by observation, likely due to the proprietary treatment protocol for commercialization31. The human dermal fibroblasts were able to infiltrate the Alloderm material once seeded. Alloderm did not hinder ring formation and integrated into the lumen side of the ring structures and vessels.
Ring mechanics.
Circumferential ring tensile mechanics significantly improved with inclusion of Alloderm in the rings and vessels. Average stress-strain curves for rings without Alloderm, rings with Alloderm and Alloderm donuts are shown in Fig. 2 and material properties are summarized in Table 1. Average elastic modulus, ultimate tensile strength and failure strength for rings without Alloderm (n = 5) were 89.1 ± 27.5 kPa, 177 ± 21.4 kPa and 101 ± 34.8 kPa, respectively. Rings with Alloderm (n = 5) had an elastic modulus of 6630 ± 1510 kPa and an ultimate tensile strength of 1770 ± 221 kPa. Alloderm rings exhibited two main rupture points, consisting of the Alloderm completely tearing first at 1500 ± 372 kPa, followed by the remaining cells and hydrogel structure tearing to failure at 6.75 ± 3.25 kPa (Supplemental Video 1). Average elastic modulus, ultimate tensile strength and failure strength for Alloderm donuts alone (n = 4) were 8250 ± 3360 kPa, 4730 ± 628 kPa and 4390 ± 848 kPa, respectively. The percent elongation of rings, Alloderm rings and Alloderm donuts was 310 ± 29.8%, 162 ± 48.3% and 89.9 ± 16.3%, respectively.
Table 1
Average Circumferential Ring Mechanical Properties
Group
|
E (kPa)
|
UTS (kPa)
|
FS Primary
(kPa)
|
FS Secondary (kPa)
|
Percent Elongation (%)
|
Standard Rings (n = 5)
|
89.1 ± 27.5a,c
|
177 ± 21.4a,c
|
101 ± 34.8a,c
|
N/A
|
310. ± 29.8a,c
|
Alloderm Rings (n = 5)
|
6628 ± 1506a
|
1765 ± 221b,a
|
1498 ± 372b,a
|
6.75 ± 3.25
|
162 ± 48.3b,a
|
Alloderm Alone (n = 4)
|
8254 ± 3358c
|
4731 ± 848b,c
|
4388 ± 848b,c
|
N/A
|
89.9 ± 16.3b,c
|
a Statistically significant difference between standard rings and Alloderm rings (E: p ≤ 0.0010; UTS: p < 0.0001; FS Primary: p < 0.01; Percent Elongation: p < 0.001) |
b Statistically significant difference between Alloderm rings and Alloderm alone (E: not significant; UTS: p < 0.0001; FS Primary: p < 0.0001; Percent Elongation: p < 0.05) |
c Statistically significant difference between standard rings and Alloderm alone (E: p < 0.0001; UTS: p < 0.0001; FS Primary: p < 0.0001; Percent Elongation: p < 0.001) |
Vessel mechanics.
Engineered vessel tensile mechanics significantly improved with inclusion of Alloderm. Average stress-strain curves for rings without Alloderm, rings with Alloderm and Alloderm donuts are shown in Fig. 3a-e and summarized in Table 2. Interestingly, initial (FS1) and complete (FS2) failure points were noted for the vessel groups, indicating the point of failure of the first and last ring, respectively. These two failure points are of importance to note because the initial point of failure is vital information for clinical application, as is the catastrophic point of complete vessel failure. Average elastic modulus and ultimate tensile strength for vessels without Alloderm (n = 5) were 79.4 ± 11.6 kPa and 67.9 ± 9.78 kPa, respectively. The primary failure point and secondary failure point of standard rings were 49.8 ± 27.1 kPa and 3.22 ± 1.47 kPa, respectively. Average elastic modulus and ultimate tensile strength for Alloderm vessels (n = 5) were 3720 ± 686 kPa and 1500 ± 334 kPa, respectively. The primary failure point and secondary failure point for Alloderm vessels were 1397 ± 301 kPa and 4.77 ± 1.73 kPa, respectively (Supplemental Video 2). To compare the contribution of the Alloderm material alone in the vessels, 6 Alloderm donuts were adhered with Vetbond and tensile tested. Average elastic modulus and ultimate tensile strength of the 6 Alloderm donuts (n = 4) were 11.3 ± 1.96 MPa and 5049 ± 333 kPa, respectively. The primary failure point and secondary failure point were 4790 ± 348 kPa and 119 ± 39.2 kPa, respectively. Percent elongation for vessels without Alloderm, Alloderm vessels and 6 Alloderm donuts alone were 511 ± 65.0%, 286 ± 56.1% and 105 ± 12.3%, respectively. To evaluate the Alloderm vessels compared to native vessels, human saphenous vein was also mechanically tested. Interestingly, the Alloderm vessels surpassed circumferential tensile mechanics of native human saphenous vein (n = 5), which exhibited an elastic modulus of 2980 ± 410 kPa, ultimate tensile strength of 1060 ± 155 kPa and failure strength of 416 ± 157 kPa. The native human vessel had an average percent elongation of 120 ± 19.6%.
Table 2
Average Circumferential Vessel Material Properties
Group
|
E (kPa)
|
UTS (kPa)
|
FS1 (kPa)
|
FS2 (kPa)
|
Percent Elongation (%)
|
Standard Vessels (n = 5)
|
79.4 ± 11.6a,b,c
|
67.9 ± 9.78a,b,c
|
49.8 ± 27.1a,b
|
3.22 ± 1.47b
|
511 ± 64.9a,b,c
|
Alloderm Vessels (n = 5)
|
3721 ± 687a,d
|
1504 ± 262a,d,e
|
1397 ± 301a, d,e
|
4.77 ± 1.73d
|
286 ± 56.1a,d,e
|
Alloderm Alone (n = 4)
|
11396 ± 1962b,d,f
|
5049 ± 333b,d,f
|
4795 ± 384b, d,f
|
119 ± 39.2b,d
|
3726 ± 733b,d
|
Human Saphenous Vein (n = 6)
|
2979 ± 409c,f
|
1059 ± 155c,e,f
|
416 ± 157e,f
|
N/A
|
821 ± 141c,e
|
a Statistically significant difference between Standard Vessels and Alloderm Vessels (E: p < 0.0001; UTS force: p < 0.0001; FS Primary force: p < 0.0001; FS secondary: not significant; Percent Elongation: p < 0.0001) |
b Statistically significant difference between Standard Vessels and Alloderm Alone (E: p < 0.0001; UTS force: p < 0.0001; FS Primary force: p < 0.0001; FS secondary: p < 0.0001; Percent Elongation: p < 0.0001) |
c Statistically significant difference between Standard Vessels and Diabetic Human Saphenous Vein (E: p < 0.001; UTS force: p < 0.0001; FS Primary force: not significant; Percent Elongation: p < 0.0001) |
d Statistically significant difference between Alloderm Vessels and Alloderm Alone (E: p < 0.0001; UTS force: p < 0.0001; FS Primary force: p < 0.0001; FS secondary: p < 0.0001; Percent Elongation: p < 0.0001) |
e Statistically significant difference between Alloderm Vessels and Diabetic Human Saphenous Vein (E: not significant; UTS force: p < 0.05; FS Primary force: p < 0.0001; Percent Elongation: p < 0.0001)
f Statistically significant difference between Alloderm Alone and Diabetic Human Saphenous Vein (E: p < 0.0001; UTS force: p < 0.0001; FS Primary force: p < 0.0001; Percent Elongation: not significant)
|
Longitudinal tensile mechanics representing strength along the length of the vessels was not significantly different in vessels without Alloderm compared to vessels with Alloderm (Fig. 3f-g, Table 3). The longitudinal elastic modulus, ultimate tensile strength and failure strength for vessels without Alloderm (n = 4) were 26.1 ± 13.5 kPa, 11.2 ± 6.05 kPa and 1.54 ± 0.304 kPa, respectively. The longitudinal elastic modulus, ultimate tensile strength and failure strength for vessels with Alloderm (n = 3) were 10.7 ± 8.09 kPa, 3.47 ± 1.61 kPa and 1.41 ± 1.08 kPa, respectively.
Table 3
Longitudinal Vessel Material Properties
Group
|
E (kPa)
|
UTS (kPa)
|
FS (kPa)
|
Standard Vessels (n = 5)
|
26.1 ± 13.5
|
11.2 ± 6.05a
|
1.54 ± 0.304
|
Alloderm Vessels (n = 5)
|
10.7 ± 8.09
|
3.47 ± 1.61a
|
1.41 ± 1.08
|
aStatistically significant difference between Standard Vessels and Alloderm Vessels (E: no significance; UTS: p < 0.05; FS: not significant) |
The force required to strain the engineered rings, engineered vessels and Alloderm alone to failure (Supplemental Fig. 1; Supplemental Tables 1 and 2) provide insight into the resultant mechanics and the effects of the difference in cross-sectional area on strength calculations. Significant differences in forces to obtain circumferential elasticity, tensile strength and failure strength were found between standard rings and Alloderm alone (p < 0.001). Standard rings had an average elastic force, ultimate tensile force and failure force of 0.127 ± 0.134 N, 0.273 ± 0.134 N and 0.162 ± 0.104 N, respectively. Alloderm alone had an average elastic force, ultimate tensile force and failure force of 21.9 ± 9.31 N, 12.7 ± 2.94 N and 11.8 ± 3.27 N, respectively. Although significant differences were seen in comparing material properties of Alloderm rings to Alloderm alone, when comparing associated forces no significant differences were found between ultimate tensile strengths (p = 0.102), and between Alloderm ring primary failure force and Alloderm alone failure force (p = 0.793). This indicates that the larger thickness of the Alloderm rings compared to the Alloderm donuts is responsible for the different in calculation of strength due to the difference in cross-sectional area. Alloderm rings had an average elastic force, ultimate tensile force, primary failure force, and secondary failure force of 56.3 ± 6.58 N, 15.1 ± 0.677 N, 12.7 ± 1.94 N, and 0.0558 ± 0.0207 N, respectively. This indicates structural integrity of Alloderm was not comprised when in the rings, but rather the reduced mechanical properties can be attributed to increased cross-sectional area. Between standard rings and Alloderm rings a significant difference was found between elastic force, ultimate tensile force and primary failure strength force (p < 0.001). These force outputs indicate the superior tensile mechanics from inclusion of Alloderm into the rings. Using an independent t-test with equal variances not assumed, a significant difference was found between standard rings failure strength and Alloderm ring secondary failure strength (p < 0.01), however, this can also be attributed to increased cross-sectional area. Using an independent t-test with equal variances not assumed, no significant difference was found between the standard ring’s failure force and Alloderm ring secondary failure force (p = 0.084). This indicates that the cells in the fibrin gel maintain their mechanical properties regardless of the inclusion of Alloderm. All together, these results indicate that Alloderm rings are a composite material comprised with material properties of strength from the Alloderm and ductility from the ring of cells organized in the fibrin gel.
Rings and vessels histological analysis.
Histological analysis of rings provided pertinent information on cellular and ECM protein content and organization in the engineered tissue. Vessel histology showed cellular and ECM organization across the multi-ring structure. Cross-sectional ring samples were stained with multiple stains. Hematoxylin and eosin was used to visualize overall cellular structure by staining nuclei deep purple and cytoplasm and extracellular matrix pink. Masson’s Trichrome and Picrosirius red stains were used to visualize ECM content and organization, by staining collagen blue and red, respectively. DAPI stains were used to clearly demarcate cell density and location.
In the standard rings (Fig. 4a-c), fibroblasts self-organized into a band of cells surrounded by a layer of the fibrin gel. In rings with Alloderm (Fig. 4d-f), the organization of the ring from the lumen outward was first the Alloderm, followed by cells, then fibrin gel, and lastly more cells lining the outer diameter. Average ring thickness without and with Alloderm was 0.964 ± 0.170 mm and 2.35 ± 0.198 mm, respectively. Cell nuclei were seen located in the fibrin gel and Alloderm indicating cell migration. In rings with Alloderm, Trichrome stain showed a large blue band at the Alloderm, indicating its significant collagen content. In addition, a thin blue band was seen on the outer diameter suggesting collagen deposition by the cells in the Alloderm rings which was not evident in standard rings. This collagen deposition pattern was confirmed in Picrosirius red stained sections where the red collagen-stained areas were seen co-located with the cells and in parts of the hydrogel. In contrast, little positive collagen stain was seen in the standard rings without Alloderm. The same dense blue and red collagen network is seen in the Trichrome and Picrosirius red stains of Alloderm alone (Fig. 4g-i). Both trichrome and picrosirius red stains of the groups showed Alloderm alone had the highest percentage of area stained positive for collagen followed by Alloderm rings and then standard rings. Trichome stains of standard rings and Alloderm rings showed a significant difference (p < 0.001) in quantified collagen, 18.8 ± 4.77% and 65.6 ± 14.9%, respectively. Picrosirius red stains of rings without Alloderm and with Alloderm also showed significant differences (p < 0.001) in quantified collagen as 21.7 ± 8.27% and 66.7 ± 11.4%, respectively. Both ring groups additionally showed significant differences (p < 0.001) in percent area stained collagen from trichrome, 100. ± 0.00%, and picrosirius red, 100. ± 0.00%, in Alloderm alone.
Longitudinal cross sections of vessels with and without Alloderm stained for H&E (Fig. 4j-k) showed successive rings composed of cells, fibrin gel, and Alloderm for the Alloderm rings. Areas of cellular density were evident by the purple nuclei stains. Standard vessels without Alloderm showed cellular organization as dense pockets of cells, whereas Alloderm vessels show more evenly distributed layers of cells.
DAPI fluorescent stains of cell nuclei in Alloderm rings showed the organization of the cells along the outer diameter of the rings (Fig. 5). In comparison, DAPI stains for Alloderm alone were negative indicating the absence of cells. Lack of positive stained DAPI in Alloderm alone indicates cell infiltration in Alloderm rings is from the ring making process rather than possible nuclear remnants from the original material of the decellularized Alloderm.
Polarized light images of a Picrosirius red stained standard ring, Alloderm ring and Alloderm alone (Fig. 6) allowed for further assessment of collagen organization in the rings. Collagen fiber thickness can be visualized using polarized light microscopy of Picrosirius red stained tissues. More mature, thicker fibers appear orange to red whereas less mature, thinner fibers appear green to yellow. Rings without Alloderm primarily exhibited mature red collagen fibers in the region around the cells surrounded by areas of less mature yellow and orange collagen in the fibrin hydrogel. In the Alloderm alone, polarized light showed a dense red network of mature collagen. In the Alloderm ring, a dense red-orange network of collagen is seen in the Alloderm area, along with a lighter region of red fibers and orange fibers deposited by the cells surrounding the Alloderm. These results clearly show the enhanced collagen content provided by the inclusion of the Alloderm into the engineered tissue. Polarized light quantifications for percent area of fibers shows significant differences (p < 0.01) in red fibers for all three groups, in yellow fibers for all three groups, and in green fibers (p < 0.01) between standard rings and Alloderm alone and between standard rings and Alloderm rings. Standard rings contained 15.8 ± 7.85, 58.2 ± 13.9 and 19.0 ± 8.77 percent area of red, yellow and green collagen fibers, respectively, which was the highest yellow and green fiber content. Alloderm alone contained 91.3 ± 5.55, 7.49 ± 4.91 and 0.757 ± 0.791 percent area of red, yellow and green collagen fibers, respectively, which contained the highest red fiber composition. Alloderm rings contained 73.7 ± 5.49, 22.9 ± 5.41 and 3.47 ± 1.72 percent area of red, yellow and green collagen fibers, respectively, which contained higher red fiber content than similar green fiber content as Alloderm alone.
Suture retention and burst pressure.
No significant differences were found between vessels with and without Alloderm for suture retention (Fig. 7). Average suture retention for vessels without Alloderm (n = 3) was 7.73 ± 2.01 gram-force. Average suture retention for vessels with Alloderm (n = 3) was 9.83 ± 2.25 gram-force. In both vessel groups, suture retention failure points occurred in the area between the rings, at about 1 to 2 rings above the suture. However, there was a significant difference between burst pressure between vessels with and without Alloderm, with values of 51.3 ± 2.19 mmHg and 47.0 ± 1.14 mmHg, respectively (Fig. 8).