脱细胞血管基质制备和异体移植的实验研究

目的研究建立无细胞血管基质制备的新方法,并探讨脱细胞血管异体移植的可行性。方法采用不同去垢剂[1％Triton X-100、1％十二烷基磺酸钠(SDS)]多步骤对血管进行脱细胞处理,并通过组织学和力学观测,建立兔动脉血管脱细胞的方法;并进行脱细胞血管的异体移植。结果经低渗溶液和去垢剂1％TritonX-100、1％SDS等多步骤处理,兔胸主动脉血管的细胞基本脱除,细胞外基质保持完好,血管的弹性、韧性和力学强度仅轻度降低;用该法制备的兔颈动脉(直径约2．0mm)进行异体移植,经2个月观察,全部通畅。结论经去垢剂TritonX-100、SDS加低渗溶液和蛋白酶抑制剂处理的多步法,可以脱除血管的细胞成分,细胞外基质和力学特性保持完好,是一种较好的方法;用该法制备的兔颈动脉(直径约2．0mm)进行异体移植,初步取得成功。     

Decellularization reduces the immune response to aortic valve allografts in the rat

OBJECTIVES: Cryopreserved valve allografts used in congenital cardiac surgery are associated with a significant cellular and humoral immune response. This might be reduced by removal of antigenic cellular elements (decellularization). The aim of this study was to determine the immunologic effect of decellularization in a rat allograft valve model. METHODS: Brown Norway and Lewis rat aortic valves were decellularized with a series of hypotonic and hypertonic buffers, protease inhibitors, gentle detergents (Triton X-100), and phosphate-buffered saline. Valves were implanted into Lewis rats in syngeneic and allogeneic combinations. Cellular (CD3 and CD8) infiltrates were assessed with morphometric analysis, and the humoral response was assessed with flow cytometry. RESULTS: Morphometric analysis identified a significant reduction in CD3 + cell infiltrates (cells per square millimeter of leaflet tissue) in decellularized allografts compared with that seen in nondecellularized allografts at 1 (79 +/- 29 vs 3310 +/- 223, P < .001), 2 (26 +/- 11 vs 109 +/- 20, P = .004), and 4 weeks (283 +/- 122 vs 984 +/- 145, P < .001). Anti-CD8 staining confirmed the majority of infiltrates were cytotoxic T cells. Flow cytometric mean channel fluorescence intensity identified a negative shift (abrogated antibody formation) for decellularized allografts compared with nondecellularized allografts at 2 (19 +/- 1 vs 27 +/- 3, P = .033), 4 (35 +/- 2 vs 133 +/- 29, P = .001), and 16 weeks (28 +/- 2 vs 166 +/- 54, P = .017). CONCLUSIONS: Decellularization significantly reduces the cellular and humoral immune response to allograft tissue. This could prolong the durability of valve allografts and might prevent immunologic sensitization of allograft recipients.     

A size-matching heterotopic aortic valve implantation model in the rat

BACKGROUND: Structural failure of cardiac valve allografts may be related to technical factors such as size mismatch, resulting in early intimal proliferation and fibrosis or immunological reactions against the transplanted valves, featuring lymphocytic infiltration. OBJECTIVE: To develop a heterotopic aortic valve implantation model in the rat to study the immunological factors leading to graft failure in the setting of a technical adaptation for size mismatch. METHODS: Syngeneic (WAG-WAG or DA-DA) and allogeneic (WAG-BN or WAG-DA) rat strain combinations were used to study the effect of the allogeneic response on valve properties. An end-to-side anastomosis was made between the U-shaped aortic root graft and the recipient's abdominal aorta to resolve the problems of size matching. RESULTS: No animals suffered from ischemic or neurological complications during the study period. One hundred percent survival and patency of the aortic grafts were achieved at the end of a 21-day observation period. In the syngeneic group 9 of 10 valves were still competent when assessed during retrograde injection. In contrast, 2 of 10 allogeneic valve grafts were competent on postoperative Day 21. Microscopic evaluation revealed no fibrosis or intimal thickening in the syngeneic valve grafts while the allogeneic valve grafts demonstrated rejection-like morphology. CONCLUSION: The absence of fibrosis and intimal thickening in the syngeneic transplanted valve grafts indicates that this implantation model is not influenced by nonimmunological-based structural changes. Therefore, this new model enables us to study the association between donor-directed immune responses and allograft degeneration in a technically unbiased manner.     

Histopathology of `fresh'' human aortic valve allografts

Gavin, J. B., Barratt-Boyes, B. G., Hitchcock, G. C., and Herdson, P. B. (1973).Thorax, 28, 482-487. Histopathology of `fresh' human aortic valve allografts. Six aortic valve allografts were studied histologically after having functioned in patients as aortic valve replacements for 14 to 442 weeks. The grafts initially had been collected under sterile conditions from cadavers and stored in Hanks's balanced salt solution for 2 to 24 days before use. All grafts showed a cellular reaction along the host graft interface characterized by macrophages, lymphocytes, and organizing granulation tissue, and there was a progressive replacement of the donor aortic sleeve by host collagenous tissue. Sheaths of cellular, avascular host tissue extended from the margins of all grafts over their intimal surfaces and, in those which had been in place more than 36 weeks, this tissue had resulted in thickening of the proximal parts of one or more cusps. In one graft this thickening extended almost to the free margins of the cusps. While the leaflets of the graft at 14 weeks were virtually acellular, older grafts contained cellular areas with active fibroblasts in the proximal regions of the cusps as well as acellular regions which generally were more distally placed. Macrophages were always present along the interface between cellular and acellular areas. These observations indicate that there is a gradual replacement fibrosis of the graft by the host which proceeds in different grafts, and even different cusps in the same graft, at different rates.     

Heart valve dysfunction resulting from cellular rejection in a novel heterotopic transplantation rat model

Abstract Structural failure of heart valve allografts may be related to technical factors or immunological reactions. To circumvent nonimmunological factors a new rat implantation model was developed to study whether alloreactivity results in histopathological changes and valve dysfunction. Syngeneic (WAG‐WAG, DA‐DA) and allogeneic (WAG‐BN, WAG‐DA) transplantation was carried out using this new technique, and the function of explanted valves was assessed 21 days later by retrograde comptence testing. Additionally, grafts were examined using standard histological and immunohistochemical techniques. There was no leakage during retrograde injection in nine of tem syngeneic and two of ten allogeneic grafts. Microscopically, syngeneic valves appeared normal without fibrosis or intimal thickening, although CD8⁺ lymphocytes and macrophages were found in necrotic myocardial rim and adventitia. In contrast, allogeneic valves were deformed and noncellular, with extensive infiltration of CD4⁺, CD8⁺ and CD68⁺ cells in adventitia and media. Absence of fibrosis and intimal thickening in syngeneic transplanted valves indicated circumvention of nonimmunological factors. Allogeneic valve transplantation induces cellular infiltration in the graft with subsequent graft failure.     

Experimental aortic valve heterotransplantation

Pig aortic valves were transplanted into the descending aorta of 41 dogs. Ficin-digested valve grafts and fresh untreated grafts were associated with poor results and extremely short survival of the recipient animals. When freeze-dried valves were transplanted the mortality was reduced and the survival time increased, but there was a marked tendency for the grafts to rupture through the heterologous aortic wall cuff. If a homologous aortic cuff was used the survival time was increased, but rupture through the aortic wall cuff still occurred. The freeze-dried valve grafts eventually became almost acellular. Thrombus, which was commonly present on the valves, rarely showed organization. The valve cusps tended to remain thin and delicate, in one dog for as long as eight months after operation.     

去细胞处理对化学去细胞异体神经免疫原性的影响

目的研究化学去细胞异体神经的免疫学和制备方法,以期进一步降低同种异体神经移植的免疫反应。方法取SD大鼠的坐骨神经,经4．0％Triton X-100和3．0％脱氧胆酸钠消化进行去细胞处理。将化学去细胞和新鲜的SD大鼠坐骨神经植入Wistar大鼠皮下,通过免疫组化方法观察移植物及周围组织中的CD3、CIM和CD8阳性T细胞浸润程度,评价免疫反应强度。将犬的尺神经按上述方法进行去细胞处理,按去细胞处理次数分为3个组,分别从去细胞程度、髓鞘染色程度、GAG免疫组化染色和神经基底板层结构完整性方面进行组织学观察。结果与新鲜异体神经移植比较,化学去细胞异体神经植入后可明显降低大鼠的免疫原性,但仍有轻度细胞介导的免疫反应存在。经化学去细胞处理的神经,其残余神经轴突髓鞘的染色强度不随去细胞处理次数增加而明显减弱,GAG染色强度也无改变,但神经基底板层结构的破坏程度随去细胞处理次数的增加而增强。结论移植经化学去细胞处理的异体神经后,可有轻微的免疫反应,诱发免疫反应的物质可能与糖蛋白成分有关,残余的髓鞘组分不随去细胞处理次数增加而明显减少。     

Prognostic Importance of Viability and A Study of A Second Set Allograft Valve: An Experimental Study

An experimental study of transplantation of first and second set aortic valve allografts in heterotopic infrarenal aortic position in inbred rats with varying histocompatibility is described. On the 20th and 50th postoperative days, there was deposition of donor-specific antibodies on the allograft endothelia of the weakly allogeneic, non-MHC group, as evidenced by immunofluorescence studies, whereas the nonviable grafts showed no identifiable antibodies on their endothelia. The endothelia were mostly lost at day 100; previous focal mononuclear cellular infiltrates disappeared. The ground substances, however, were maintained until the 250th day. In the nonviable allografts, the media was completely acellular on the 50th day with collagen disintegration and changes in ground substance on the 100th day. Loss of endothelia and replacement by fibrin deposits with transient focal cellular infiltrations were the most significant early microscopic findings. Acellularity, fibrous neointima, and leaflet thickening due to varying degrees of fibrosis with changes in ground substance and focal infiltrations of macrophages around degenerated collagenous matrix of aortic valve allografts are the significant late changes. The second set allograft valves showed no difference in the rate or type of healing, but early degeneration while the second set skin grafts underwent accelerated rejection, thus confirming prior sensitization. This finding confirms the weak antigenicity of cardiac valve allografts. Therefore, the use of cardiac valve allografts for secondary valve replacement might be favorable if properly used. Limitation of antigen incompatibility by considering at least the ABO matching and reduction of a recipient's immunological reactivity might be a proper step to achieve a longer survival of viable allografts.     

Antigenicity of fresh and cryopreserved rat valve allografts

Aortic valve allografts have demonstrated excellent clinical performance, but the importance of antigenic differences between donor and recipient is largely unknown. To determine the antigenicity of aortic valve grafts, rat aortic valves with a short portion of thoracic aorta were transplanted into the abdominal aorta of recipient rats. Valves were used immediately after harvest (fresh) or following cryopreservation. Three weeks after this procedure, the recipient rats received a skin graft from a rat of a strain syngeneic to that of the aortic valve donor. Additional groups of rats were subjected to sham operation (sham) followed three weeks later by skin grafting. Recipient rats were of the Lewis strain. Donor rats were of the Lewis, F344 (weakly allogeneic, RT1-compatible, non-RT1-incompatible), LBN F1 (moderately allogeneic, one-haplotype-identical and one-haplotype-incompatible at both the RT1 and non-RT1 loci), or BN (strongly allogeneic, RT1 and non-RT1-incompatible) strain. Time to skin graft rejection was measured. Among rats receiving the F344 grafts, the time to skin graft rejection (mean +/- SD) was sham: 9.1 +/- 1.0 days, fresh: 7.1 +/- 1.2 days, cryopreserved: 6.9 +/- 0.7 days. Among rats receiving the LBN F1 grafts, the corresponding times were sham: 7.8 +/- 0.8 days, fresh: 5.6 +/- 0.5 days, cryopreserved: 5.4 +/- 0.5 days. Among rats receiving the BN grafts, the corresponding times were sham: 7.1 +/- 0.3, fresh: 4.5 +/- 1.0 days, and cryopreserved: 4.3 +/- 0.7 days. Significant differences (P less than 0.05) existed between sham and fresh and between sham and cryopreserved, but not between fresh and cryopreserved. Significant differences (P less than 0.05) also existed between each histocompatibility grouping. It is concluded that aortic valve allografts in rats are antigenic and produce recipient sensitization. Cryopreservation does not diminish this sensitization. The degree of antigenicity is related to the degree of histoincompatibility between donor and recipient. Both RT1 and non-RT1 antigens appear to play a role in this process.     

Heart valve transplantation: the urgent need for non‐immunogenic porcine heart valve matrices

Heart valve replacement is a life‐saving procedure conducted worldwide approximately 300 000 times per year. Used mechanical devices and bioprosthetic valves, however, exhibit severe drawbacks. They either request for lifelong anticoagulation therapy, or display an early failure due to calcification and stenosis. Furthermore, their use in pediatric surgery requests reoperations since these prosthesis lack the ability to grow. An optimal heart valve substitute that exhibits unlimited durability, perfect hemodynamics (no need for anticoagulation) and the capacity to grow has been identified by implantations of decellularized allogenic heart valve matrices. Such grafts are revitalized in vivo by cells of the recipient, which form a functional endothelium and a living interstitium. However, the remaining obstacle is the limited availability of human donor heart valves. Until now, xenogenic substitutes (porcine origin) failed due to acute or chronic rejection when implanted into humans as well as in the sheep model. Thus, we aim to generate a porcine derived decellularized matrix, free of immunogenic epitopes, for endogenous tissue regeneration and tissue engineering. In a first step, we developed analytical tests to quantify residual α(1,3)Gal epitopes, known to be responsible for hyper‐acute rejection reactions, and other xenoantigens detected by natural human anti‐pig antibodies, which are present on matrices treated by our standard decellularization protocol. Since extracellular matrix is insoluble with employed the inhibitory ELISA technique using the commercial available anti‐αGal antibody M86 and natural anti‐pig antibodies isolated from the serum of healthy volunteers. In brief, porcine kidneys were perfused with human plasma. Unbound antibodies were washed away and bound xenoantibodies were eluted using 3.5 M NaSCN. After dialysis against PBS antibodies were used to perform the inhibitory ELISA as follows. Native as well as decellularized heart valve tissue was crushed in liquid nitrogen and incubated with a defined amount of antibodies. Unbound antibodies were separated from matrix bound antibodies by centrifugation and quantified. M86 ELISA was performed using αGal‐BSA (Dextra) as solid antigen and HRP conjugated goat anti‐mouse IgM antibody as detection antibody. In the case of natural human anti‐pig antibodies, the total amount of human IgG was quantified using a human IgG ELISA (Mabtech). In our experiments comparing native and decellularized porcine heart valves, we found that standard decellularization removes already 70% of the αGal epitopes. In heart valves from α1,3‐galactosyltransferase KO (GalT‐KO) pigs the binding of M86 antibody is comparable to the extent as binding of M86 is found in human pulmonary artery tissue. In respect to general xenoantigens reduced binding of xenoantibodies after decellularization was detected. However, human as well as GalT‐KO pig derived tissue bind less xenoantibodies as tissue derived from normal pigs. Comparing only GalT‐KO and human tissue, the human tissue binds less xenoantibodies indicating the existence of non‐Gal antigens on GalT‐KO tissue. In conclusion, we found strong evidence that xenoantigens including αGal and non‐Gal xenoantigens are not only present on cells and cell debris but also on decellularized heart valve matrixes that are in fact insoluble extra cellular matrix proteins. Thus, decellularization causes a reduction of the antigenicity, however, the degree of this removal might be dependent on the decellularization protocol. Modification of the current decellularization protocol as well as enzymatic treatment against glycocalyx structures, which are believed to be hard candidates as xenoantigens may result in an additional reduction.     

Early failure of the tissue engineered porcine heart valve SYNERGRAFT™ in pediatric patients

Objectives: The first tissue engineered decellularized porcine heart valve, Synergraft™ (Cryolife Inc., USA) was introduced in Europe as an alternative to conventional biological valves. This is the first report of the rapid failure of these new grafts in a small series. Materials and methods: In 2001, 2 model 500 and 2 model 700 Synergraft™ valves were implanted in four male children (age 2.5–11 years) in the right ventricular outflow tract as a root. Two patients had a Ross operation and two had a homograft replacement. Results: The cryopreserved Synergraft™ valves appeared macroscopically unremarkable at implantation. Recovery from surgery was uneventful and good valve function was demonstrated postoperatively. Three children died, two suddenly with severely degenerated Synergraft™ valves 6 weeks and 1 year after implantation. The third child died on the 7th day due to Synergraft™ rupture. Subsequently the fourth graft was explanted prophylactically 2 days after implantation. Macroscopically all four grafts showed severe inflammation starting on the outside (day 2 explant) leading to structural failure (day 7 explant) and severe degeneration of the leaflets and wall (6 weeks and 1 year explant). Histology demonstrated severe foreign body type reaction dominated by neutrophil granulocytes and macrophages in the early explants and a lymphocytic reaction at 1 year. In addition significant calcific deposits were demonstrated at all stages. Surprisingly pre-implant samples of the Synergraft™ revealed incomplete decellularization and calcific deposits. No cell repopulation of the porcine matrix occurred. Conclusion: The xenogenic collagen matrix of the Synergraft™ valve elicits a strong inflammatory response in humans which is non-specific early on and is followed by a lymphocyte response. Structural failure or rapid degeneration of the graft occurred within 1 year. Calcific deposits before implantation and incomplete decellularization may indicate manufacturing problems. The porcine Synergraft™ treated heart valves should not be implanted at this stage and has been stopped.     

Immunogenicity, Antigenicity, and Endothelial Viability of Aortic Valves Preserved at 4°C in a Nutrient Medium

In patients undergoing aortic valve replacement, allograft valves stored at 4 degrees C in a nutrient medium have been associated with excellent immediate and long-term results. The effects of this method of prolonged storage on the antigenic, immunological and cellular characteristics of these grafts are incompletely understood. This study was designed to study these phenomena in rat aortic valves subjected to antibiotic sterilization and stored for up to 3 weeks in RPMI containing 10% fetal calf serum. Selected valves from Brown Norway rats were implanted heterotopically into the abdominal aorta of Lewis rats. Other valves were studied prior to transplantation. Antigenicity was determined by immunocytochemical staining using monoclonal mouse antibodies directed at Class I and Class II rat antigens. Immunogenicity was determined by duration of second-set skin graft survival following heterotopic aortic valve implant. Endothelial cell viability was determined by flow cytometric analysis of endothelial cells harvested from aortic valve allografts by collagenase digestion. Only fresh valves and valves stored for 1 day were positive for Class I antigens; no valves were positive for Class II antigens. Duration of skin graft survival was prolonged with greater duration of storage, but grafts remained immunogenic after 21 days of storage. Endothelial cell viability declined from 95% in the fresh valves to 64% after 21 days of storage. With prolonged duration of allograft valve storage at 4 degrees C, there is an attenuation of antigenicity, immunogenicity, and endothelial cell viability. Loss of endothelial cells may contribute to the changes in immunological responses to the valve allografts. The expression of antigens on the endothelial surface is not a reliable predictor of immunological response.     

Development and characterization of a full-thickness acellular porcine cornea matrix for tissue engineering

Our aim was to produce a natural, acellular matrix from porcine cornea for use as a scaffold in developing a tissue-engineered cornea replacement. Full-thickness, intact porcine corneas were decellularized by immersion in 0.5% (wt/vol) sodium dodecyl sulfate. The resulting acellular matrices were then characterized and examined specifically for completeness of the decellularization process. Histological analyses of decellularized corneal stromas showed that complete cell and α-Gal removal was achieved, while the major structural proteins including collagen type I and IV, laminin, and fibronectin were retained. DAPI staining did not detect any residual DNA within the matrix, and the DNA contents, which reflect the presence of cellular materials, were significantly diminished in the decellularized cornea. The collagen content of the decellularized cornea was well maintained compared with native tissues. Uniaxial tensile testing indicated that decellularization did not significantly compromise the ultimate tensile strength of the tissue (P > 0.05). In vitro cytotoxicity assays using rabbit corneal fibroblast cultures excluded the presence of soluble toxins in the biomaterial. In vivo implantation to rabbit interlamellar stromal pockets showed good biocompability. In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the cornea has been successfully developed. The matrix is biocompatible with cornea-derived cells and has potential for use in corneal transplantation and tissue-engineering applications.     

Effect of Immunological Differences on Rat Aortic Valve Allograft Calcification

Calcification may be a cause of allograft valve degeneration. To determine whether immunological differences between donor and recipient affect the degree of calcification that occurs, adult Lewis rats received aortic valve allografts transplanted heterotopically into the abdominal aorta. All valves were transplanted immediately after harvest. The valves were not exposed to antibiotics or albumin before insertion. Valve donors were of the Lewis (syngeneic), F344 (weakly allogeneic, RT1 compatible, non-RT1 incompatible), LBN F1 (moderately allogeneic, one haplotype identical, one haplotype incompatible at the RT1 and non-RT1 loci), and Brown Norway (strongly allogeneic, RT1 and non-RT1 incompatible) strains. Valves were harvested 3-12 weeks following transplantation. Scanning electron microscopy and energy dispersion x-ray microanalysis were performed on one leaflet of each valve to evaluate calcium content. Calcium content expressed in counts (mean +/- standard error) according to donor strain were: Lewis, 1642 +/- 233; F344, 4853 +/- 1412; LBN F1, 4714 +/- 823; and Brown Norway, 4358 +/- 835. Significant differences (p less than 0.05) existed between valves from Lewis donors and those from each other strain. No differences among the other strains were statistically significant. It is concluded that syngeneic valve allografts calcify less than allogeneic grafts. However, the degree of allogenicity did not influence the magnitude of calcification.     

Decellularized native and engineered arterial scaffolds for transplantation

More than 570,000 coronary artery bypass grafts are implanted each year, creating an important demand for small-diameter vascular grafts. For patients who lack adequate internal mammary artery or saphenous vein, tissue-engineered arteries may prove useful. However, the time needed to tissue engineer arteries (7 weeks or more) is too long for many patients. Decellularized cadaveric human arteries are another possible source of vascular conduit, but limited availability and the potential for disease transmission limit their widespread use. In contrast, decellularized tissue-engineered arteries could serve as grafts for immediate implantation, as scaffolds onto which patients' cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. The goal of this study was to quantify the effects of decellularization on vascular matrix and mechanical properties. Specifically, we compared cellular elimination, extracellular matrix retention, and mechanical characteristics of porcine carotid arteries before and after treatment with three decellularization methods. In addition, for the first time, tissue-engineered arteries were decellularized. Decellularized native arteries were also used as a scaffold onto which vascular cells were seeded. These studies identified a decellularization method for native and engineered arteries that maximized cellular elimination, without greatly compromising mechanical integrity. We showed that engineered tissues could be decellularized, and demonstrated the feasibility of reseeding decellularized vessels with vascular cells.     

Durability of the viable aortic allograft

Of 581 aortic allografts implanted since 1967, 421 were analyzed for structural deterioration. This series is unique in that it includes patients from the early allograft experience. All allografts were cleanly procured, antibiotic sterilized, and either stored at 4 degrees C for up to 8 weeks or frozen to liquid nitrogen temperatures with cryopreservation to preserve the viable cusp fibroblasts. There were 25 frozen mounted aortic valves with a median time to valve failure of 12.1 years, which was not significantly different from the 12.5-year period for 114 fresh free-sewn aortic valves. The median time to valve failure was 6.6 years for 90 fresh-mounted aortic valves and 8.6 years for 192 fresh-mounted mitral valves (p = 0.05). The difference between all mounted and unmounted grafts was significant (p = 0.0001). In all groups, viable fibroblasts were present in specimens explanted up to 5 years after the operation. All specimens returned after more than 10 years were almost totally acellular. Evidence of increased collagen, suggesting that the fibroblasts survive implantation and then gradually die, was present in all specimens. This series suggests that durability of the unmounted viable allograft for aortic valve replacement is greater than for other types of tissue valves. Pre-mounted allografts for aortic or mitral valve replacement have a median survival of 8 years and are not more durable than other tissue valves.     

Procollagen synthesis by fresh and cryopreserved rat pulmonary valve grafts

OBJECTIVE: Allograft valves are frequently used in the repair of congenital cardiac anomalies. The failure rate may differ depending on the type of allograft used. Previous studies have shown that rat aortic valve grafts exhibit synthesis of procollagen, suggesting a capacity for repair and regeneration after implantation. No studies of pulmonary valve grafts in the heterotopic rat implant model have thus far been reported. This study was designed to investigate whether pulmonary valve grafts maintain in vivo viability, as demonstrated by procollagen synthesis, and whether cryopreservation, histocompatibility, or both affect this property. METHODS: Cryopreserved and fresh rat pulmonary valves were implanted into the abdominal aorta of syngeneic and allogeneic recipients. The grafts and native valves were excised 3 to 21 days after implantation. Valves were sectioned and immunohistochemically stained for procollagen. Computerized morphometry was used to calculate changes in intima, media, and adventitia as a percentage of cross-sectional area of the graft. Procollagen content was graded by semiquantitative methods. RESULTS: Pulmonary valve grafts had significantly greater collagen density in the intima and adventitia compared with native aortic and pulmonary tissues, but collagen density in the media was similar in all groups. The grafts demonstrated appreciably greater procollagen than the corresponding native valves. These findings were consistent in all grafts (ie, both fresh and cryopreserved, both syngeneic and allogeneic), irrespective of duration of implantation. CONCLUSIONS: Procollagen synthesis occurs in pulmonary valve grafts early after implantation, indicating viability of these tissues. This model of pulmonary valve implantation may have wide applicability to questions of allograft biology.     

Decellularized vein as a potential scaffold for vascular tissue engineering

PURPOSE: Current strategies to create small-diameter vascular grafts involve seeding biocompatible, compliant scaffolds with autologous vascular cells. Our purpose was to study the composition and strength of decellularized vein to determine its potential as a vascular tissue-engineering scaffold. METHODS: Intact human greater saphenous vein specimens were decellularized by using sodium dodecyl sulfate (SDS). Residual cellular and extracellular matrix composition was studied with light and electron microscopy as well as immunohistochemistry. Burst and suture-holding strength was measured in vitro by insufflation and pull-through techniques. To assess initial handling and durability of decellularized vein in vivo, a canine model was developed wherein decellularized canine jugular veins were implanted as carotid interposition grafts in recipient animals. After two weeks of arterial perfusion, these grafts were studied with duplex imaging and histologic methods. RESULTS: Human saphenous vein decellularized by using SDS was devoid of endothelial cells and >94% of the cells resident within the vein wall. Collagen morphology appeared unchanged, and elastin staining decreased only slightly. Basement membrane collagen type IV remained intact. Compared with fresh vein, decellularized vein had similar in vitro burst (2480 +/- 460 mm Hg vs 2380 +/- 620 mm Hg; P >.05) and suture-holding (185 +/- 30 gm vs 178 +/- 66 gm; P >.05) strength. Decellularized canine vein functioned well in vivo without dilation, anastomotic complication, or rupture over 2 weeks of arterial perfusion. CONCLUSIONS: Vein rendered acellular with SDS has well-preserved extracellular matrix, basement membrane structure, and strength sufficient for vascular grafting. These properties suggest proof of concept for its use as a scaffold for further vascular tissue engineering. CLINICAL RELEVANCE: The following research examines the creation of a new small-diameter bypass graft. It is clinically relevant to patients who need distal arterial bypass, coronary artery bypass, or hemodialysis access, but who do not have adequate autologous vein for their surgeries. Future investigations will involve further tissue engineering of this vascular scaffold (eg, autologous endothelial seeding of its lumen) and testing the clinical usefulness of the completed graft.     

In Vitro Generation of Atrioventricular Heart Valve Neoscaffolds

Tissue engineering of cardiovascular structures represents a novel approach to improve clinical strategies in heart valve disease treatment. The aim of this study was to engineer decellularized atrioventricular heart valve neoscaffolds with an intact ultrastructure and to reseed them with umbilical cord‐derived endothelial cells under physiological conditions in a bioreactor environment. Mitral (n = 38) and tricuspid (n = 36) valves were harvested from 40 hearts of German Landrace swine from a selected abattoir. Decellularization of atrioventricular heart valves was achieved by a detergent‐based cell extraction protocol. Evaluation of the decellularization method was conducted with light microscopy and quantitative analysis of collagen and elastin content. The presence of residual DNA within the decellularized atrioventricular heart valves was determined with spectrophotometric quantification. The described decellularization regime produced full removal of native cells while maintaining the mechanical stability and the quantitative composition of the atrioventricular heart valve neoscaffolds. The surface of the xenogeneic matrix could be successfully reseeded with in vitro‐expanded human umbilical cord‐derived endothelial cells under physiological flow conditions. After complete decellularization with the detergent‐based protocol described here, physiological reseeding of the xenogeneic neoscaffolds resulted in the formation of a confluent layer of human umbilical cord‐derived endothelial cells. These results warrant further research toward the generation of atrioventricular heart valve neoscaffolds on the basis of decellularized xenogeneic tissue.     

An innovative method to obtain porous porcine aorta scaffolds for tissue engineering

Decellularized porcine aorta (PA) is a promising biomaterial for vascular substitutes. However, decellularized PAs suffer from mechanical weakness and have less pores, which limit cellular ingrowth into the grafts and hinder the remodeling. In this study, PAs were decellularized by vacuum‐freeze‐thawing cycles and 0.3% of sodium dodecyl sulfate (SDS) buffer (VLS). Results showed that the application of vacuum‐freeze‐thawing significantly improved the decellularization efficiency of SDS while effectively preserved the mechanical function of PA tissues, decreased residual SDS, and minimized cytotoxicity. Furthermore, scanning electron microscopy (SEM) examination demonstrated that VLS generated interconnected pores with uniform distribution. In vivo subcutaneous implantation assay further demonstrated that VLS implants had less calcification and adverse inflammatory response. Moreover, VLS treatment markedly enhanced ingrowth of myofibroblasts and endothelial cells, and thereby promoted synthesis of extracellular matrix and vascularization. These results suggest that the application of vacuum‐freeze‐thawing into the decellularization process may produce a promising vascular graft candidate for tissue engineering application.