Tissue graft composition and method

PURDUE RESEARCH FOUNDATION

METHOD FOR PREPARING A FABRIC GRAFT COMPOSITION

DESCRIPTIVE MEMORY

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This invention relates to a method for preparing a tissue graft composition from a segment of the small intestine. Said tissue graft composition comprises the submucosal tunic of a segment of the small intestine of a warm-blooded vertebrate wherein the submucosal tunic is separated from the muscularis tunic and which has at least the luminal portion of the mucosal tunic. Tissue graft composition has shown excellent mechanical characteristics as well as non-allergenicity and non-thrombogenicity characteristics in applications such as vascular autografts, vascular allografts, and vascular heterografts.

BACKGROUND OF THE INVENTION

Tissue grafting materials have now achieved considerable clinical and economic significance. It is estimated that in 19 alone $ 130 million was spent on vascular grafts alone, not including coronary artery bypass grafts. However, success rates for vascular graft processes are low compared to most other surgical techniques. For example, a cumulative patency of 5,5X is considered excellent for diameter vascular grafts. These low success rates result largely from small materials from one or more shortcomings. ^:physical or functional grafts commonly used in the clinic.

Identifying suitable tissue graft materials is particularly difficult because these materials must have a number of disparate properties. For example, vascular graft materials should not only have mechanical stability in continuous stress situations, but should also have one. adequate porosity for capillaryization, pressure resistance similar to that of host tissue, and non-thrombogenic Zeta potentials). In addition, they are negative (so they must be non-ergogenic, non-carcinogenic, and inexpensive to manufacture).

Few, if any, tissue graft materials have all the desirable properties. The bibliographic references of research and development in the field of grafts <ascu 1 ares ref 1 ec have a significant constant effort to overcome the commonly known principles of the recent grafts._owJ vKieiNM.

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Both autogenous synthetic materials were used for vascular grafts. Among synthetics, expanded polytetrafluoroethylene (PTFE) is a commonly used vascular graft material, particularly for small vessel bypass surgery. However, expanded PTFE grafts are susceptible to neointimal hyperplasia and late graft thrombosis (for example, 6-year patency rates of approximately 5-7 ° C for the bypass grafts). PTFE grafts are reported to have even lower success rates when used in venous circulation.

Another synthetic material - Dacron ^R - is often used for large diameter vascular graft processes (eg infrarenal aortic grafts>. Knitted Dacron however has a re-activatively high porosity and should be pre-coagulated prior to implantation to avoid heavy bleeding. This process pre-coagulation is not always practical or successful R. Woven Dacron, although less porous, has a pressure resistance of only 207 than found in the normal aorta. Finally, Dacron® grafts act poorly on small diameter arteries or veins where blood flow is relatively slow.

One of the most significant problems associated with the use of synthetic products as tissue graft materials is that the synthetic materials have low resistance to infection. Synthetic graft rates are those of infection following immolation at a rate of 667 '. Synthetic materials have a tendency to accommodate microorganisms similar to their interstices and, as docaments, are extremely refractory to antibacterial therapy. Explantation of infected synthetic grafts is virtually inevitable.

More recently, synthetic skin connections using human cells live 4,604,346, 4,546,500, 4,539,716 researchers have reported equivalent blood vessel preparation as. See US Patents Nos. , 4,485,097, and 4,485,096.

Among the autogenous materials, the saphenous vein, the human umbilical vein, the inverted small intestine, and the radial artery were all used, but each of these materials also had significant deficiencies. The saphenous vein may be inappropriate in size for certain processes or may not be available due to injury due to disease. In addition, the saphenous vein may present unacceptable varicosities and suffer from accelerated atherogenesis after arteriolization. Both umbilical grafts and inverted small bowel grafts are rapidly affected by thrombosis and subsequent aneurysm formation. Finally, the radial artery has limited utility because it is difficult to harvest and may deteriorate after graft implantation.

It is therefore an object of this invention to provide a tissue graft material which does not exhibit many of the shortcomings associated with many clinically used graft materials.

Another object of this invention is to provide a method for preparing a new tissue graft material from a small intestine section.

Still another object of this invention is to provide a method for using a novel multipurpose tissue graft immaterial for autograft, allograft and heterograft applications.

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Still another object of this invention is to provide a method for using one. new composition and tissue grafting to replace blood vessels.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 is a cross-section of the small intestine.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a tissue graft composition comprising mainly the submucosal tunic of a small intestine segment of a warm-blooded vertebrate. The submucosal tunic is separated from the muscularis tunic and at least the luminal portion of the mucosal tunic of the small intestine cut. Because the present tissue graft composition has been found to have excellent functional characteristics in applications such as grafts; vascular grafts and as vascular allografts, it is anticipated that the tissue graft compositions of this invention will find wide use even as heterografts in both vascular and other tissue graft applications. The applicants found that a. The present tissue graft composition has multiple physical and biological characteristics that make it particularly suited for tissue graft applications.

In a preferred embodiment of this invention, the tissue graft material comprises submucosal tissue and basilar mucosal tissue separated from a small intestine section, most preferably from the jejunum, a portion of the small intestine extending between the duodenum and the ileum. The small intestine, prior to its manipulation (lamina separation) to

ORIGINAL BAD mucosa, particularly a. mucosal lamina muscularis Ε and stratum compactum F. Those layers collectively are hereinafter referred to as the Submucosa dei Intestino Delgado (SIS).

An SIS autograft according to this invention can be prepared, for example, by first resecting an autogenous proximal jejunum segment following a midline laparotomy incision. The fasted resected segment is then wrapped in surgical sponges that have been soaked in physiological saline (physiological fluid). After the intestinal anastomosis is completed, the resected intestinal segment is prepared according to the method of this invention hereinafter described for use as tissue graft material. Similarly, allografts are prepared from intestinal tissue removed from organ / tissue donors of the same species. Heterografts may be prepared, for example, from feline, porcine or bovine intestinal tissue recovered from animals sacrificed in slaughterhouse operations. So far, minimal morphological differences have been found in the intestinal tissues of different species. Indeed, the histological aspect of human graft tissue according to this invention was found to be nearly identical to that of dog.The only recognizable morphological difference was a slightly less dense stratum compactum in human tissue.

The tissue graft material of this invention is prepared by scraping the intestinal tissue to remove the outer layers including both the serous and muscular tunics (layers B and C in Fig. 1) and the inner layers including at least the lumen portion. .inalis (layer G) of the mucous membrane (layers E to G in Fig 1). Under mild scraping conditions the mucous tunic is delaminated between the stratum compactum (layer F) and the basal bad blade of layer G. More particularly, following the removal of any mesenteric tissues from the intestinal segment using, for example, Adson-Brown forceps and Metzenbaum scissors, the serous technique and muscular tunic (outer tissue layers) are scraped off the intestinal segment using a longitudinal rubbing motion with a scalpel handle and moistened gauze. Following eversion of the intestinal segment, the lurninal portion of the mucous membrane is separated from the underlying tissue using the same rubbing motion. Care must be taken to avoid perforation of the submucosa. Any tissue appendages of the separate layers remaining on the graft surface are also removed. Optionally, the intestinal segment may be first everted, then released from the luminal layers, and then reinserted in its primitive orientation for removal of the serous and muscularis tunics. The graft material is comprised of a translucent, whitish tissue tube approximately 0.1 mm thick, typically consisting of the submucosal tunic together with the mucosal lamina muscularis and stratum compactum. For the preparation of the vascular graft, the prepared graft is everted to its primitive orientation so that the stratum compactum serves as the graft's lurninal surface.

Prepared graft material is typically washed with saline and placed in a neomycin sulphate solution at 107 ° C for approximately 20 minutes, and after that time the graft material is ready for use. Grafts are applied using routine surgical procedures commonly used for tissue graft applications. For use in non-vascular tissue graft applications, the tubular graft material may be cut longitudinally and wound to form a tissue patch. In fact, the total tissue separation process previously described r 3

ORIGINAL BAD may be performed on prepared intestinal tissue patches by cutting the intestinal segment longitudinally and unrolling it to form a pre-graft patch. Prepared tissue graft patches may be used, for example, as a skin graft material or for the repair of other body tissue defects by adapting them for surgical application of a tissue graft patch having physical and functional characteristics. of the present graft composition.

For use in vascular grafts, the graft should have a diameter roughly equal to the diameter of the recipient blood vessel. This is accomplished by manipulating the tissue graft to. defining a cylinder having a diameter approximately equal to that of the recipient blood vessel and otherwise suturing or tightly closing the tissue graft longitudinally a. for forming said vascular graft. Thus, for example, a vascular graft may be prepared by selecting a sterile glass rod having an outside diameter equal to that of the recipient blood vessel and introducing the glass rod into the graft lumen. 0 Redundant tissue is then assembled and the desired lumen diameter is achieved by suturing along the length of the graft (for example, using two continuous suture lines or a single interrupted suture line) or using other well-known tissue closure techniques recognized in the art. .

Consistent with the objectives of this invention, the SIS composition has highly desirable mechanical properties for tissue graft materials, including low porosity index, high malleability, and a high burst pressure point. As for porosity, anyone skilled in the art will appreciate that the tissue graft material should have a sufficiently low porosity to prevent intraoperative bleeding while having a porosity.

ORIGINAL BAD high enough to allow the extension of a newly developed vasa vasorum through the graft material to feed the. neointima and the luminal surface. The porosity of a graft material is typically measured in terms of

-1 ml of water passed per cm min with a top pressure of 120 mm Hg. The porosity index of SIS graft material is 10, much lower than that of other graft materials currently known in the art. (Woven Dacrori ^, for example, has a porosity index of 5Õ). However despite this low porosity index, the SIS is still sufficiently porous to allow neocapilarization to occur within the SIS graft. In vascular graft applications, SIS compositions allow the formation of blood-filled capillaries within the graft extending to the luminal surface four days shortly after surgery.

As regards graft pressure resistance, a direct relationship between pressure resistance and patency has been described in this art. Ideally a graft material should have at least a pressure resistance similar to that of the tissue it replaces. The longitudinal pressure resistance of SIS graft material was measured using a simple tensile test. The initial gauge length was formed with two ink marks 5.5 cm apart. Elongation and applied force were measured when the samples were loaded at a strain rate of 32 cm / cm / min., Providing the following results:

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SIS graft pressure resistance: 0.045 cm / N per cm

Resistance to normal dog aorta pressure: 00.07 cm / N per cm length

Thus, SIS graft materials actually have greater pressure resistance than that of the normal aorta. This constitutes significant progress over prior art in the field of vascular grafts. All currently available synthetic grafts are 3 to 10 times less resistant to pressure than the natural artery and proportionally more prone to thrombosis than the natural artery. The prior art method for compensating for this pressure resistance mismatch is to use a graft material with a diameter larger than the adjacent natural artery. This technique, however, caused additional problems. The velocity of the buck is slower through the graft segment with a larger diameter. Therefore, there is less shear force on the graft wall. Under these conditions, platelet and fibrin deposition with subsequent thrombosis is more likely. On the contrary, because SIS material demonstrates such high pressure resistance, isodiametric SIS grafts can be used without these problems.

The point at which tubular pressure could be present in the present SIS graft material was found to have burst pressure far beyond what it found physiologically. A burst test was performed by connecting a graft segment to 25 mm diameter cylinders and pressurizing with nitrogen gas at a constant flow rate. Fo one of

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ORIGINAL BAD used two flow rates. At the lower flow rate, the pressure initially increased and then dropped and stabilized as the gas outlet through the graft wall balanced with the gas inlet. With the highest flow rate, the pressure immediately rose to burst conditions at approximately 400 mm Hg, indicating that the graft material can easily withstand the continuous pulsatile pressures encountered in the use of physiologically normal vascular grafts.

EXAMPLES

Example 1. Small Intestine Submucosa as a Large Diameter Arterial Graft

A series of experiments were performed that tested the ability of three different small bowel configurations to serve as vascular grafts to the dog's infrarenal aorta. The first experiment used a full-thickness, non-inverted jejunum segment with either an intact mesenteric neurovascular delivery or a free, isolated segment as the graft material. The intestinal mucosa constituted the blood-graft interface. The 4 dogs in this experiment all died within 13 hours of surgery by graft segment thrombosis and bleeding from the suture lines.

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The second experiment used an inverted jejunum segment as a graft with the. serous tunic, serving as san queerer. Use 2 c esnestae pe ri nce. „The first dog was thrombosed 4 hours after surgery, and the second dog died of acute bleeding at the proximal anastomosis site 4 days- after surgery.

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The third experiment tested a. use of only a portion of the intestinal wall as a graft material. It was collected from each. A free segment of the upper portion of the jejunum was then removed and most of the mucosa was removed by roughly scraping the luminal surface with a scalpel handle. By the same process, the muscular serosa and tunic were then removed. The tissue remaining after this apparently brutal manipulation of the intestinal segment consisted of a section of submucosa and basilar mucosa with ΙΟΘμ thickness. This graft was then placed in the infrarenal aorta of 15 dogs and had remarkable success. The following results from this third experiment are summarized below.

Thirteen of the 15 dogs kept patent grafts until they were sacrificed. Eleven dogs were sacrificed at various times after surgery ranging from 4 days to 1 year. animals showed no signs of infection, aneurysm formation or graft thrombosis. The insu

Graft debris observed in two dogs was caused by technical error, including misapplication of the metal binding staples and poor anastomotic technique. Two animals remain alive at the time of writing and are being monitored for long-term graft patency information.

The patency of the grafts was verified by positive contrast radiography within four to seven days after. surgery, and then every 6 a. 8 weeks In addition, graft patency was clinically monitored for the presence of a strong femoral pulse and a. no hind paw edema.

Eleven of the dogs that maintained patent grafts were sacrificed at various time intervals after surgery (4 ,, 7,

ORIGINAL BAD and 14 days, and 9, 11, 13, 17, 26, 44, and 52 weeks). Immediately before sacrificing, the animals underwent an additional angiography to confirm graft patency and to provide a comparative radiograph for evaluation of graft aneurysm dilatation, stenosis, and aneurysm formation. The orc dogs all revealed complete patency with no evidence of harmful luminal changes.

Macroscopic observation of these graft segments revealed a bright luminal surface with randomly combined red and white areas with no evidence of thrombus formation. »There was accumulation of surrounding hard connective tissue which confluent with the graft wall. . All specimens examined before b months after surgery revealed no evidence of endothelial cell growth on the graft surface. The surface of these grafts was covered by a flat, moderately dense and organized layer of collagen,

Histopathological examination of specimens at 26, 44 and 52 weeks revealed endothelial cells that partially covered a thin layer (approximately 500p) of densely organized fibrin. All tissue was infiltrated with blood-filled capillaries, and the outer edge of the original graft material could not be distinguished from the surrounding connective tissue. Scanning electron microscopy of the luminal surface revealed a layer of flattened cells, which cannot be distinguished from endothelial cells with elongated pseudopodia. Electron microscopic evaluation of the transmission of these graft segments also suggested the presence of endothelial cells covering the luminal surface. In addition, the presence of Factor VIII: Related Antigen, detected by immunofluorescent staining, remained. also the

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endothelial origin of these. luroinal graft surface. Graft material was also tested for the presence of endothelial cells by testing for the presence of endothelium-derived relaxing factor. Acetylcholine was applied to the surface of the graft specimens and the effluent was collected. The effluent was found by observing smooth muscle relaxation in a rat aorta preparation to contain endothelium-derived relaxing factor.

The blood pressure in the cephalic portion, the distal portion and within the SIS graft was determined in each of the 10 sacrificed dogs. The pressures were identical at the 3 locations in each dog, reflecting the absence of detrimental hemodynamic effects from the SIS graft material.

The following laboratory parameters were measured before surgery, one day after surgery, and then at various additional times for subsequent months in all dogs: hematocrit, prothrombin time, partially activated thromboplastin time, platelet count, formula and complete count, and an abbreviated chemical profile of the serum. The results obtained with these laboratory tests at all times revealed that the animals were normal. These animals were treated with low doses of heparin (or IV units) during the surgical procedure, but were not given anticoagulant during the postoperative period. The absence of any changes in coagulation tests and platelet counts was particularly encouraging given the relatively hyperactive coagulation system of the dog compared to that of man.

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Example 2. Small Intestine Submucosa as a Graft

Small Diameter Arterial

This experiment involved the implantation in eighteen dogs of a total of 33 grafts in both the femoral and carotid arteries. Thirty-three of the thirty-six grafts remained patent. Laboratory measurements were made on these animals identical to those of the first study and no abnormalities were observed. In addition, conventional 2-dimensional ultrasound imaging was used to measure patency. and the diameter of the vessel in cross section.

Pathological examination of the graft tissue from a dog sacrificed four days after surgery revealed a non-thrombotic luminal surface and a moderately stenotic proximal anastomosis. Histological examination revealed the early presence of blood - filled capillaries in the graft wall, which represents a natural defense of the organism against the. infection. Five of these patients remained alive at the time of writing for further evaluation. The longest surviving dog in this study is now 7 months old after surgery.

Example 3, Small Intestine Submucosa as a Venous Graft

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In this experiment, the SIS graft was placed in the posterior vein (analogous to the inferior vena cava in man) of two into the anterior vena cava (analogous to the superior vena cava in that of five dogs. the posterior vena cava grafts remained patent for only 11 and 14 days respectively, anatomopathological examination revealed that the failure of the s was due to technical errors in which the site of the bad ORSGÍNAL inferior anastoroosis was stenosed (S ω in diameter at compared with the AI ​​mm of adjacent diameter in the natural vena cava and the proximal graft). In addition, the luminal surfaces of both grafts were covered by a non-thrombotic pseudoepithelium composed of tightly bound fibrin and immature collagen connective tissue.

Anterior vena cava grafts remained patent until three dogs were sacrificed on days 7, 14, and 21, respectively, after surgery. Two of the dogs remain alive at the time of writing with patent grafts at 7 weeks postoperatively. The proximal suture line in the three dogs revealed evidence of early thrombosis where a graft border had been reversed, which was causing a turbulent blood flow, but the remaining portion of the graft was non-thrombotic. In addition, pathological and histological examination revealed that the graft was coated with a smooth, shiny red surface that looked similar to the previous graft studied in previous experiments.

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Example 4 Small Intestine Submucosa as an Arterial Graft

SIS has been used as a large diameter allograft in the dog's aorta. Allografts were performed in the same manner as previously described for our study of aortic grafts. At the time of writing, the test animals are only 8 weeks old after surgery, but show no signs of thrombosis, infection or graft aneurysm formation (as documented by angiograms).

Em xem pio 5. Small Intestine Submucosa as a

Arterial heterograft □ SIS has been used as a heterograft in dogs. The feline origin SIS graft was prepared according to the procedures described hereinabove and was placed on a dog. At the time of writing, the test animal was two weeks old after surgery and showed no harmful signs.

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