1. Introduction

1. Small Intestine
1. Composed of several sections, the jejunum is used in experimental studies.
2. from inside to outside: tunica mucosa, muscularis mucosa of the tunica mucosa, stratum compactum, tunica muscularis, serosa muscularis (Matsumoto, Holmes et al. 1966)
1. mucosa: absorbs nutrients/secretes digestive enzymes
2. submucosa: collagen, structure of SI
3. muscularis: circumferencial and longitudinal layers, provide for peristalsis movement
2. submucosa structure, purpose, use
1. acellular, composed of the stratum compactum and muscularis mucosa of the tunica mucosa
3. Submucosa used for experiments other than vascular
1. Abdominal Wall Repair: did ½ thickness & full thickness grafts in 12 dogs. Used porcine SIS and polypropylene mesh contralaterally. Two from each group were sacrificed at 1,2,4 months. Polypropylene adhered to tissue better, but SIS worked in all cases. No SIS remained at 4 months. Polypropylene always showed signs of inflammatory response, SIS didn’t most of the time. (Clarke, Lantz et al. 1996)
2. Cranial Dural Grafting: Used 20 rats, took cranial dura from both sides, replaced one with SIS. 3 died, 8 were killed at 7 days, 9 at 28 days. There was "increased thickness, increased vascularity, and greater cellular infiltration of the SIS-treated defects…" (Cobb, Badylak et al. 1996)
3. Achille’s tendon repair:
1. Used 20 dogs, sacrificed at 1,2,4,8,12, 16, 24, 48 weeks. Put porcine SIS in a 1.5-cm gap made in Achille’s tendon. Contralateral was control- normal tendons. They were braced for 5 weeks, then the brace was removed. By 8 weeks, all could put their weight on the leg. By 12 weeks, the SIS neotendon was as strong as contralateral by uniaxial testing. Staining: by 2 weeks, SIS <10% of neotendon. No SIS by 12 weeks. (Badylak, Tullius et al. 1995)
2. Used 36 rabbits. Put braids of SIS in a 1.5 cm gap. Two weeks of immobilization, then varying mobilization. Maximum mobilization showed most cross sectional area. (Hodde, Badylak et al. 1997)
4. Canine Bladder Wall: Used 8 beagles, replaced 5 x 4 cm patches for 15 months. They did electrical stimulation experiments, histology, and uniaxial tensile tests (preconditioning 5 cycles) on the explants. They found that SIS-regenerated bladder was weaker than native SIS. All dogs voided normally throughout and didn’t develop stones. (Kropp, Sawyer et al. 1996)

2. Animals used and Autogenous/Xenogeneic tissue
1. porcine into dogs (Sandusky, Badylak et al. 1992; Hiles, Badylak et al. 1995; Sandusky, Lantz et al. 1995)
2. autografts, dogs (Krippaehne, Hunt et al. 1962; Matsumoto, Holmes et al. 1966; Matsumoto, Holmes et al. 1966; Lawler, Foster et al. 1971; Badylak, Lantz et al. 1989; Lantz, Badylak et al. 1990; Lantz, Badylak et al. 1992; Badylak, Coffey et al. 1994)
3. dogs, no implantation (Herbert, Badylak et al. 1993)
4. pigs, no implantation (Badylak 1993; Ferrand, Kokini et al. 1993; Hiles, Badylak et al. 1993; Prevel, Eppley et al. 1994)
5. porcine into rats (Prevel, Eppley et al. 1994)

 

3. Vessel replaced
1. infrarenal aorta in dogs (Krippaehne, Hunt et al. 1962; Badylak, Lantz et al. 1989; Badylak, Coffey et al. 1994; Hiles, Badylak et al. 1995)
2. carotid artery in dogs (Sandusky, Badylak et al. 1992; Sandusky, Lantz et al. 1995):
3. inferior and superior vena cava in dogs (Matsumoto, Holmes et al. 1966; Matsumoto, Holmes et al. 1966)
4. thoracic aorta and thoracic inferior vena cava in dogs (Lawler, Foster et al. 1971)
5. carotid and femoral artery in dogs (Lantz, Badylak et al. 1990)
6. superior vena cava into dogs (Lantz, Badylak et al. 1992)

 

4. Graft Preparation
1. (Sandusky, Badylak et al. 1992; Ferrand, Kokini et al. 1993; Herbert, Badylak et al. 1993; Hiles, Badylak et al. 1993; Badylak, Coffey et al. 1994; Prevel, Eppley et al. 1994; Sandusky, Lantz et al. 1995)(these all refer back to (Badylak 1993)):
1. Mesenteric tissues are removed
2. inverted, tunica mucosa is removed
3. inverted, serosa and tunica muscularis is removed. ("pre-existing lymphatic and vascular channels are evident" listed in (Prevel, Eppley et al. 1994))
4. rinsed in saline and placed in 10% neomycin sulfate solution (15-20 min) (Badylak, Lantz et al. 1989; Lantz, Badylak et al. 1990; Lantz, Badylak et al. 1992; Hiles, Badylak et al. 1993; Badylak, Coffey et al. 1994; Prevel, Eppley et al. 1994)
5. rinsed in saline and placed in 5% neomycin sulfate solution (Ferrand, Kokini et al. 1993):
2. Same as above 1-3 (Badylak), then sterilized by 0.1% peracetic acid. Stored in gentamycin-phosphate-buffered saline until surgery (Hiles, Badylak et al. 1995)
3. (Matsumoto, Holmes et al. 1966):
1. group 1 of 10 dogs, unaltered SIS: lumen occluded within 72 hours
2. group 2 of 9 dogs, mucosa removed: lumen occluded within 72 hours
3. group 3 of 40 dogs: mesentery removed: all doing fine. No narrowing noted, no thrombosis, inflammation, or adhesion
1. 8 dogs, inversion alone
2. 18 dogs, mucosa removed and graft inverted
3. 14 dogs, mucosa, serosa, and muscularis were stripped (unable to see clear division between the wall of the vena cava and the graft without microscopy)
4. (Matsumoto, Holmes et al. 1966): all grafts rinsed with 2% neomycin solution
1. group 1: 10 dogs: inferior or superior vena cava, unaltered, all occluded within 72 hours
2. group 2: 9 dogs: mucosa scraped off, not inverted, all occluded within 72 hours
3. group 3: 40 dogs: all inverted
1. 8 dogs: simple inversion only
2. 18 dogs: mucosa was removed by abrasion
3. 14 dogs: only submucosa remained
5. (Lawler, Foster et al. 1971): submucosa only
1. mucosa and muscularis removed with dull instrument
2. grafts preserved in 70% alcohol
3. soaked in 10% neomycin solution prior to storage in alcohol
4. group 1: 8 dogs: preserved in thoracic aorta: aneurysm present in all animals but 6-12 months ones
5. group 2: 5 dogs: fresh in thoracic aorta: one died of suture ine disruption at 7 days, and two others of aneurysm rupture at 1 and 6 months. Aneurysm developed within a year in the remaining two grafts.
6. group 3: 19 dogs: 7(fresh) and 12 (preserved) in thoracic inferior vena cava: all were doing fine 6 to 18 months
7. group 4: 12 dogs: preserved in abdominal inferior vena cava: all thrombosed within three weeks.
6. Microvasculature (Prevel, Eppley et al. 1994)
1. less than 0.95 mm diameter, femoral artery used, 42 rats, all with SIS died within one hour
2. source and treatment of SIS
1. xenogeneic, porcine SIS
2. 6 groups: untreated SIS, SIS soaked in heparin, systemic heparin of rats, SIS crosslinked to heparin, SIS crosslinked to urokinase, untreated autologous epigastric vein
7. submucosa only (Krippaehne, Hunt et al. 1962)
1. submucosa taken off the muscle layer, mucosa scraped off, then treated with neomycin
2. had 2 series (autografts and homografts), 6 groups each with 3 animals sacrificed at 5, 11, 18, 26,40, 60 days (18 animals died and were excluded from the study)
5. Drugs given to animals
1. (Hiles, Badylak et al. 1995):
1. aspirin to each dog every morning for 3 weeks, beginning with surgery
2. heparin prior to aortic cross-clamping
3. cephazolin (500 mg) intravenously during surgery and twice a day for 14 days
4. coumadin as needed.
2. (Sandusky, Badylak et al. 1992):
1. heparin during surgery
2. dicumarol daily beginning on day 2 after surgery until day 60
3. one aspirin daily until day 60
3. (Matsumoto, Holmes et al. 1966): None mentioned. (?)
4. (Matsumoto, Holmes et al. 1966): heparain postoperatively for 14 days
5. (Badylak, Coffey et al. 1994; Prevel, Eppley et al. 1994): heparin given 2 min before surgery
6. (Sandusky, Lantz et al. 1995):
1. heparain sodium allowed to circulate 2 min before surgery
2. cephalothin sodium on day of surgery and for 7 days thereafter
3. warfarin sodium(adjusted as needed) and aspirin given orally for 8 weeks
7. (Lantz, Badylak et al. 1990; Lantz, Badylak et al. 1992):
1. heparain sodium allowed to circulate 2 min before surgery and for 24 hours after
2. cephalothin sodium on day of surgery and for 7 days thereafter
3. warfarin sodium(adjusted as needed) and aspirin given orally for 8 weeks
8. no antibiotics or anticoagulants used. Temporary carotid-femoral artery bypass with Silastic tubing was employed during aortic grafting. (Lawler, Foster et al. 1971)
9. no fibrinolysins or anicoagulants used (Krippaehne, Hunt et al. 1962)
6. Immunochemistry
1. stained with hematoxyline and eosin, monoclonal antibody for PSIS (Hiles, Badylak et al. 1995):
2. hematoxylin and eosin, Verhoeff’s elastic-Martius-scarlet-blue-trichrome stain (elastic membrane and connective tissue components), periodic acid-Schiff-alcian blue (endothelial and smooth muscle cells), FVIII-related antigen for smooth muscle actin for canine cells (Sandusky, Badylak et al. 1992)
3. hematoxylin and eosin and modified Verhoeff’s elastic-Martius-scarlet-blue-trichrome stain, antibodies against both FVIII-related antigen and smooth muscle actin (Lantz, Badylak et al. 1992; Sandusky, Lantz et al. 1995)
4. by hydroxyproline assay, porcine submucosa is 40% collagen dry weight (Badylak 1993)
7. Mechanical testing
1. (Hiles, Badylak et al. 1995):
1. pressure-volume testing: porcine, room temp saline bath, pressurized with saline. 5 cycles of preconditioning, then ramp to 500 mmHg. Measured the outer diameter during test.
2. circumferential properties: after pressure-volume, rings of 3-5 mm width were cut. They were strained to rupture at 1 inch/min by putting two tubes in the ring, and pulling outward.
1. results:

1. dogs a uniaxial stress-strain test was done with a whole tube of SIS. Stress versus strain is pretty uniform, and is only slightly concave up. Also given is a list of Young’s Modulus for many dog and human aortas as gathered by other people, for matching purposes. (Herbert, Badylak et al. 1993)

9. Porosity
1. (Hiles, Badylak et al. 1993): tests on porcine submucosa, with pressure on mucosal wall to see how much water goes through.
1. 15 cut pieces at 120 mm Hg for 6 min
2. 6 cut pieces at 80, 120, and 160 mm Hg (randomized) for 6 min
3. unidirectional tests where the specimens were put in tension (if flat) and tubular specimens were pressurized at 60,80, 100, and 120 mm Hg
4. Results: SIS porosity is 1000 times less than knitted polyester, and 100 times less than woven polyester. The uniaxial tension had no effect.

Applied Pressure (mm Hg)	Porosity(mL/(min cm2))
80.0	0.4
120.0	0.58
160.0	0.79

Applied Uniaxial Tension (N)	Porosity (mL/(min cm2))
0.0	0.82
5.4	0.81

Applied Pressure (mm Hg)	Tubular Porosity (mL/(min cm2)
60	0.15
80	0.23
100	0.27
120	0.33

Graft Material	Porosity Index (mL/(min cm2)
PTFE	0
MKII	1.3*10-3
H.U.V.	5*10-3
SIS	0.52
Woven Polyester	300
Knitted Polyester	1500

1. (Ferrand, Kokini et al. 1993): directional and preconditioning tests on porcine submucosa, using weight of pig, test order, and test location as possible parameters
1. Directionality: 35 sections from 13 pigs with 3 min each, in both directions of same section, recording which side after which side.
2. Preconditioned: 13 samples cut in sheets and tested as in 1.
3. Test duration: 5 samples were tested for 53 min straight, recording a 3-min flow every ten minutes. It was found that the flow decreased with time.
4. Results: The test order, test location, and pig weight did not matter. Serosal porosity is significantly greater than mucosal porosity. Preconditioning increases the serosal, but not mucosal.

9. Compared to ePTFE, Dacron, saphenous vein
1. (Sandusky, Badylak et al. 1992):

Time at Death (Days)	SIS survivors		Saphenous Vein survivors
2	3/4		4/4
7		4/4	4/4
14	4/4		4/4
28	4/4		4/4
90	2/4		2/4
180	3/4		3/4
Total	20/24		21/24

1. (Sandusky, Lantz et al. 1995):

Time at Death	SIS survivors	ePTFE survivors
7	2/2	1/2
28	1/2	0/2
90	2/2	1/2
180	2/2	0/2
Total	7/8	2/8

10. Infection Studies

1. (Matsumoto, Holmes et al. 1966): The SI sections they took out of the dogs were found to be infected with three bacteria, among them E. coli. All the grafts-to-be were rinsed in 2% neomycin for at least 5 minutes, but swabs of those grafts after the rinse showed that 18 of 59 grafts still contained E. coli. In the 10 dogs operated upon one week after grafting, there was no evidence of infection and cultures taken from the outward facing mucosa were negative. Serial blood cultures taken on the first three postoperative days were negative in all 59 dogs.
2. (Badylak, Coffey et al. 1994): Each graft was intentionally inoculated with 100 million Staphylococcus aureus organisms. All SIS dogs were negative. Five of 9 dogs with ePTFE grafts were positive for the S. aureus. The ePTFE dogs had a higher temperature (103.2 compared with 101.6).