Porcine small intestine submucosa (SIS) is not an acellular collagenous matrix and contains porcine DNA: possible implications in human implantation

Porcine small intestinal submucosa (SIS) has been recommended as a cell-free, biocompatible biomaterial for the repair of rotator cuff tendon tear. However, we have observed noninfectious edema and severe pain in patients who have undergone SIS implantation for tendon repair. The aim of this study was to conduct an independent assessment of the safety and efficacy of Restore SIS membrane. The Restore orthobiologic implant was examined by histology and the nested PCR technique using porcine immunoreceptor DAP12 gene to examine if SIS membrane contained porcine cells or DNA, respectively. The material was also implanted into mice and rabbits for the evaluation of biological reaction and inflammatory response. Restore SIS was found to contain multiple layers of porcine cells. Chloroacetate esterase staining showed that some of these cells were mast cells. Nested PCR of the DAP12 gene demonstrated that Restore SIS contained porcine DNA material. Subcutaneous implantation of Restore SIS membrane in mice, and in rabbits for rotator cuff tendon repair, showed that the membrane caused an inflammatory reaction characterized by massive lymphocyte infiltration. In conclusion, Restore SIS is not an acellular collagenous matrix, and contains porcine DNA. Our results contradict the current view that Restore SIS is a cell-free biomaterial, and that no inflammatory response is elicited by its implantation. We suggest that further studies should be conducted to evaluate the clinical safety and efficacy of SIS implant biomaterials.     

脱细胞猪小肠黏膜下层的组织相容性

目的:研究脱细胞猪小肠黏膜下层(small intestinal submucosa,SIS)的组织相容性。方法:酶消化联合去垢剂制备脱细胞猪SIS。将其植入SD大鼠脊柱旁肌肉内,组织学检测植入后不同时间(1、2和4周)样品周围组织反应。另取乳胶植入,做为阳性对照。结果:经脱细胞处理后,猪SIS仅存纤维结构,未见细胞残留。植入后1周,脱细胞SIS形态完整,可见宿主细胞(成纤维细胞和炎性细胞)长入。2周时,可见脱细胞SIS断裂。至4周时,脱细胞SIS降解,新生细胞外基质形成,并可见毛细血管样结构。随植入时间的延长,炎性细胞逐渐减少。在不同时间点,乳胶均可完整取出,未见宿主细胞长入,其周围形成纤维囊。结论:提示脱细胞猪SIS具有良好的组织相容性。     

Bupivacaine-enhanced small intestinal submucosa biomaterial as a hernia repair device

Management of post-surgical pain following herniorrhaphy remains a clinical challenge and novel methods to deliver analgesic compounds could be of great benefit. Because there is great interest in the use of natural biomaterials for hernia repair, we investigated the biocompatibility of a natural biomaterial, porcine small intestinal submucosa (SIS), which was impregnated with bupivacaine (SIS-B) via immersion in a solution of poly(lactic-co-glycolic acid) (PLGA). Groups of Sprague Dawley rats underwent surgical creation of a ventral abdominal wall defect with subsequent repair using either SIS or SIS-B. Analysis of serial blood samples showed peak bupivacaine levels (83?ng/mL) were achieved 16?h after implantation of SIS-B. One month after surgery, the rats were euthanized and implant sites harvested for mechanical strength testing and histological analysis. At the time of necropsy, adhesion extent and tenacity was greater in SIS-B rats, with 90% of SIS-B rats have adhesion to the implant site compared to only 75% of SIS rats. Microscopically, SIS implant sites were characterized by small amounts of residual SIS surrounded by mild-to-moderate chronic inflammation. In contrast, rats treated with SIS-B, residual SIS-B was surrounded by a ring of acute inflammatory cells and an outer ring of chronic inflammatory cells, possibly due to bupivacaine or residual PLGA. Mechanical strength testing of the harvested implant sites showed no significant (p?≤?0.05) difference between SIS and SIS-B implants. In summary, bupivacaine is readily elaborated from SIS-B; and impregnation of SIS with bupivacaine does not substantially alter the biocompatibility of the biomaterial or its mechanical strength following implantation.     

Macroporous biodegradable natural/synthetic hybrid scaffolds as small intestine submucosa impregnated poly(D,L-lactide-co-glycolide) for tissue-engineered bone

Poly(D,L-lactide-co-glycolide) (PLGA), a biodegradable synthetic polymer, is widely used in a variety of tissue-engineered applications, including drug-delivery systems. However, the PLGA scaffolds, macroporous and three-dimensional structure, are difficult to cell attachment and in-growth due to surface hydrophobicity. In order to introduce in new bioactive functionality from porcine small intestine submucosa (SIS) as natural source for PLGA, we fabricated SIS-powder-impregnated PLGA (SIS/PLGA) hybrid scaffolds. Fabrication parameters, including ratios of SIS, PLGA and salt, were optimized to produce the desired macroporous foam. The scaffolds had a relatively homogeneous pore structure, good interconnected pores from the surface to core region and showed an average pore size in the range 69.23-105.82 microm and over 90% porosity. The SIS/PLGA scaffolds degraded with a rate depending on the contents of the SIS. After the fabrication of the SIS/PLGA hybrid scaffolds the wettability of the scaffold was greatly enhanced, resulting in uniform cell seeding and distribution. So, it was observed that BMSC attachment to the SIS/PLGA scaffolds increased gradually with increasing SIS contents. Scaffolds of PLGA alone and SIS/PLGA were implanted subcutaneously under dorsal skin of athymic nude mouse to observe the osteoconductivity. It was found from the result that the effects of the SIS/PLGA scaffolds on bone formation are stronger than control PLGA scaffolds. In summary, the SIS/PLGA scaffolds have osteoconductive effects to allow remodeling and replacement by osseous tissue.     

Low-molecular-weight peptides derived from extracellular matrix as chemoattractants for primary endothelial cells.

The development of synthetic and naturally occurring scaffolds for tissue engineering applications has included strategies to promote attachment of specific cell types, control the rate of scaffold degradation, encourage angiogenesis, or otherwise modulate the host response. We have reported that bioscaffolds developed from porcine small intestinal submucosa (SIS) facilitate the constructive remodeling of tissues and recruit marrow-derived cells that persist long after the acute inflammatory stages have resolved. We have not yet determined which cells are recruited, the eventual fate of these cells, or via what mechanisms the events occur. We now have analyzed various molecular weight fractions of acid-hydrolyzed SIS by both functional and morphologic methods and have determined that fraction 4 (5 to 16 kDa) possesses chemoattractant activity for primary murine adult liver, heart, and kidney endothelial cells in vitro. Addition of fraction 4 to Matrigel plugs promoted in vivo vascularization when the plugs were implanted subcutaneously in mice. These results indicate that small-molecular-weight peptides derived from the degradation of porcine SIS are biologically active in the recruitment of murine endothelial cells in vitro and in vivo.     

The role of MMP-I up-regulation in the increased compliance in muscle-derived stem cell-seeded small intestinal submucosa

We have previously observed that muscle-derived stem cells (MDSC) seeded onto porcine small intestinal submucosa (SIS) increase the mechanical compliance of the engineered tissue construct [Lu SH, Sacks MS, Chung SY, Gloeckner DC, Pruchnic R, Huard J, et al. Biaxial mechanical properties of muscle-derived cell seeded small intestinal submucosa for bladder wall reconstitution. Biomaterials 2005;26(4):443-9]. To date, however, the initial remodeling events which occur when MDSC are seeded onto SIS have yet to be elucidated. One potential mechanism responsible for the observed increase in mechanical compliance is the release of matrix metalloproteinase-I (MMP-I). To investigate this finding, MDSC ( approximately 1x10(6)) were cultured on single-layer SIS cell culture inserts (4.7 cm2) for 1-10 days. MDSC MMP-I activity on SIS in the supernatant at 1, 3, 5, 7, and 10 days was determined using a collagenase assay kit. MMP-I activity of the MDSC/SIS was significantly higher (p<0.0025) after one day in culture compared to specimens collected from subsequent time points and the unseeded control. To further study the initial remodeling events, the impact of MMP-I on mechanical compliance was examined. SIS was incubated with 0.16 U/mL collagenase-I for 3, 4.5, 5, and 24h, then biaxial mechanical testing was performed. After 5h of digestion with collagenase-I, mechanical compliance under 1 MPa peak stress was increased by 7% in the circumferential direction, compared to control SIS. These findings suggest that the release of MMP-I in response to initial seeding on SIS and subsequent breakdown of collagen fibers is the mechanism responsible for an increase in mechanical compliance.     

Massive inflammatory syndrome and lymphocytic immunodeficiency in KARAP/DAP12-transgenic mice

KARAP/DAP12 is a broadly distributed transmembrane signaling polypeptide with an immunoreceptor tyrosine-based activation motif, and is non-covalently associated with a variety of activating surface receptors. We report here the characterization of transgenic mice that overexpress KARAP/DAP12 polypeptides in both myeloid and lymphoid compartments. KARAP/DAP12-transgenic mice present, in a transgene dose-dependent manner, a complex phenotype characterized by two independent and spontaneous hematological abnormalities: (i) a severe lymphopenia and (ii) a massive inflammatory syndrome associated with neutrophilia and lung infiltration by multinucleated macrophages. These myeloid abnormalities observed in KARAP/DAP12-transgenic mice indicate that KARAP/DAP12-driven signals are critically involved in inflammation, and constitute an essential target to control the resolution of inflammatory disorders based on monocytes/macrophages and neutrophils.     

Macroporous biodegradable natural/synthetic hybrid scaffolds as small intestine submucosa impregnated poly(D,L-lactide-co-glycolide) for tissue-engineered bone

—Poly( D , L-lactide-co-glycolide) (PLGA), a biodegradable synthetic polymer, is widely used in a variety of tissue-engineered applications, including drug-delivery systems. However, the PLGA scaffolds, macroporous and three-dimensional structure, are difficult to cell attachment and in-growth due to surface hydrophobicity. In order to introduce in new bioactive functionality from porcine small intestine submucosa (SIS) as natural source for PLGA, we fabricated SIS-powder-impregnated PLGA (SIS/PLGA) hybrid scaffolds. Fabrication parameters, including ratios of SIS, PLGA and salt, were optimized to produce the desired macroporous foam. The scaffolds had a relatively homogeneous pore structure, good interconnected pores from the surface to core region and showed an average pore size in the range 69.23–105.82 μm and over 90% porosity. The SIS/PLGA scaffolds degraded with a rate depending on the contents of the SIS. After the fabrication of the SIS/PLGA hybrid scaffolds the wettability of the scaffold was greatly enhanced, resulting in uniform cell seeding and distribution. So, it was observed that BMSC attachment to the SIS/PLGA scaffolds increased gradually with increasing SIS contents. Scaffolds of PLGA alone and SIS/PLGA were implanted subcutaneously under dorsal skin of athymic nude mouse to observe the osteoconductivity. It was found from the result that the effects of the SIS/PLGA scaffolds on bone formation are stronger than control PLGA scaffolds. In summary, the SIS/PLGA scaffolds have osteoconductive effects to allow remodeling and replacement by osseous tissue.     

Enhanced Toll-like receptor responses in the absence of signaling adaptor DAP12

DAP12 is a signaling adaptor containing an immunoreceptor tyrosine-based activation motif (ITAM) that pairs with receptors on myeloid cells and natural killer cells. We examine here the responses of mice lacking DAP12 to stimulation through Toll-like receptors (TLRs). Unexpectedly, DAP12-deficient macrophages produced higher concentrations of inflammatory cytokines in response to a variety of pathogenic stimuli. Additionally, macrophages deficient in spleen tyrosine kinase (Syk), which signals downstream of DAP12, showed a phenotype identical to that of DAP12-deficient macrophages. DAP12-deficient mice were more susceptible to endotoxic shock and had enhanced resistance to infection by the intracellular bacterium Listeria monocytogenes. These data suggest that one or more DAP12-pairing receptors negatively regulate signaling through TLRs.     

Histologic findings after in vivo placement of small intestine submucosal vascular grafts and saphenous vein grafts in the carotid artery in dogs.

A small caliber vascular graft from porcine small intestine submucosa (SIS) was implanted in a canine carotid artery (n = 24) and compared with an autogenous saphenous vein graft that was implanted in the contralateral carotid artery. In this study, four grafts were evaluated at the following times after surgery: 2, 7, 14, 28, 90, and 180 days. One SIS graft thrombosed at 2 days, two SIS and two saphenous vein grafts were thrombosed at 90 days, and one SIS and one saphenous vein graft were thrombosed at 180 days. At 2 days after implant, the luminal surface of the SIS graft was covered by a thin (30 mu) fibrin meshwork. By 14 days after surgery, endothelial cells on the fibrin meshwork were staining for FVIII-related antigen. Smooth muscle cells were observed in the new intima (fibrin meshwork) by 28 days. At 90 days, both types of graft had arterialized with an intima covered by endothelium, a smooth muscle media, and marked adventitial fibrosis. Similar histology was observed at 180 days. These results indicate that this SIS graft was similar to saphenous vein graft in the dog.     

Biocompatibility tests of components of an implantable cardiac assist device.

A permanently implantable in-series left ventricular assist device, the dynamic aortic patch (DAP), has been tested in chronic animal experiments. The DAP replaces a section of the intrathoracic aortic wall. Hemothorax and hematocele at the implantation site have been complications in recent experiments. Primary postoperative hemorrhage was ruled out, and the biocompatibility of all components was therefore examined. Dacron velour, Teflon felt, conductive polyurethane, segmented polyether polyurethane, and Teflon-coated polyester fiber sutures were implanted in the pleural cavities of dogs and tested in vitro by culturing canine saphenous vein explants on them. In vivo experiments demonstrated that all components elicited mild to moderate inflammatory reactions, but hematocele occurred only when the components were implanted in the aorta with direct blood contact and exposed to arterial blood pressures. In vitro, cells were cultured on all components with no signs of toxic reactions. These results indicated that the host tolerated all implant components without major inflammatory responses. However, histological data indicated that chronic slow bleeding into or through the Dacron velour in contact with the arterial blood serum could account for hemothorax or hematocele formation. Therefore, a configuration of the assist device using materials impermeable to blood may obviate these difficulties.     

NKG2D-mediated signaling requires a DAP10-bound Grb2-Vav1 intermediate and phosphatidylinositol-3-kinase in human natural killer cells

NKG2D is an important immunosurveillance receptor that responds to stress-induced ligand expression on tumors and virus-infected cells. Human natural killer cells express NKG2D and require the transmembrane adaptor DAP10 to initiate their full cytotoxic activation. However, DAP10 has no immunoreceptor tyrosine-based activation motif and thus the mechanism of recruiting 'downstream' effector proteins is unclear. We show here that binding of the p85 subunit of phosphatidylinositol-3- kinase to DAP10 could not by itself trigger cell-mediated cytotoxicity and that binding of an intermediate consisting of the DAP10 binding partner Grb2 and the effector molecule Vav1 (Grb2-Vav1) to DAP10 was sufficient to initiate tyrosine-phosphorylation events. For full calcium release and cytotoxicity to occur, both Grb2-Vav1 and p85 had to bind to DAP10. These findings identify a previously unknown mechanism by which NKG2D-DAP10 mediates cytotoxicity and provides a framework for evaluating activation by other receptor complexes that lack immunoreceptor tyrosine-based activation motifs.     

Improved method for the isolation and purification of human islets of langerhans using Liberase enzyme blend

To determine the effects of procedural modifications, 23 human islet isolations were analyzed. Isolations were divided into two groups based on the enzyme used. The influence of Liberase™, with an improved method of mechanical disassociation of pancreas, was compared to an automated method using Sevac collagenase. Pancreases were processed within 10 h of cross clamping. Following ductal injection of the enzyme, tissue was placed in the digestion chamber for disassociation. Purification was accomplished using a COBE 2991 cell processor and continuous gradients of Hypaque EuroFicoll.

Isolations in Group I (Sevac) had an average yield of 138,602 ± 128,364 islet equivalents (IE) (2083 ± 1679 IE/g) with a purity of 85 ± 11%. Group II (Liberase) showed an average yield of 389,586 ± 191,161 IE (5,958 ± 3,083 IE/g) with a purity of 90 ± 6.8%. Viability was confirmed by fluorescein diacetate and propidium iodide staining, static incubations, and perifusions. In conclusion, the combination of the enzyme blend, Liberase, and a more gentle system of disassociation has proven to be a more productive method of islet isolation with higher purity than the previously published methods.     

Galalpha(1,3)Gal epitope in porcine small intestinal submucosa

Small intestinal submucosa (SIS) is a naturally occurring, acellular biomaterial derived from porcine jejunum, which promotes constructive tissue remodeling when applied as a xenogeneic graft material. Galactosyl-alpha(1,3)galactose (Gal) is a cell-associated epitope responsible for hyperacute rejection of porcine whole-organ xenografts in primates. Because SIS is harvested from porcine tissue, it may contain the Gal epitope. The goals of this study were to determine if Gal is present in SIS and, if it is present, to determine if human serum complement can be activated in vitro following exposure to porcine-derived SIS. SIS was probed for Gal by immunohistochemical methods and by lectin-peroxidase staining. SIS stained strongly positive with human serum, which contains naturally occurring antibodies to Gal, followed by anti-immunoglobulin G (IgG) or anti-IgM peroxidase conjugate. Blocking with the lectin I-B(4), which is specific for the Gal epitope, decreased the intensity of staining. Exposure of SIS to alpha-galactosidase reduced staining to negligible amounts. The Gal epitope is distributed transmurally throughout the SIS material. Subtyping of the immunoglobulins that bind to SIS showed that IgG(2) is the major immunoglobulin of human plasma that binds to SIS. SIS did not activate complement in vitro as measured by radioimmunoassay for C3a.     

异种脱细胞真皮替代睑板材料重建眼睑的可行性

背景：眼睑后层重建是眼睑重建的重点和难点,其中睑板替代物更是研究的焦点。异种脱细胞真皮作为一种新型的组织工程材料,在国内外烧伤整形领域,正得到广泛的研究和应用。 目的：观察异种(猪)脱细胞真皮植入兔眼睑后的组织相容性极其组织病理学变化。 方法：剥取健康小白猪全层皮肤20 cm×20 cm,制备异种(猪)脱细胞真皮基质。同时制备兔睑板全层缺损模型并植入脱细胞真皮基质,观察大体情况,并分别于第1,2,3周取移植交界处眼睑组织光镜下观察组织学的改变。 结果与结论：大体观察未见明显排斥反应及眼睑的变形;光镜下1周时可见局部炎症细胞浸润,2周时炎症细胞减少,3周时正常纤维组织长入,逐渐分割代替植入的胶原纤维,炎症反应消失。提示异种脱细胞真皮免疫原性低,并可引导新生胶原的生长,是一种良好的睑板替代物。     

Increased TLR responses in dendritic cells lacking the ITAM-containing adapters DAP12 and FcRgamma

The inhibitory effect of DAP12 on macrophages has been revealed by examining myeloid cells from DAP12-deficient mice. In this report, we demonstrate that both DAP12 and the FcepsilonRIgamma-chain (FcRgamma) are required for negative regulation of TLR responses in bone marrow-derived dendritic cells (DC). Loss of both DAP12 and FcRgamma enhanced the pro-inflammatory cytokine production and maturation of DC after TLR stimulation, resulting in a greater percentage of DC that produced IL-12 p40, TNF, and IL-6, and expressed high levels of MHC class II, CD80, and CD86. Whereas DC lacking only DAP12 showed some increased TLR responses, those lacking only FcRgamma had a greater enhancement of maturation and cytokine production, though to a lesser extent than DC lacking both DAP12 and FcRgamma. Additionally, antigen-specific T cell proliferation was enhanced by DAP12(-/-)FcRgamma(-/-) DC relative to wild-type DC after maturation. Similar to DAP12(-/-)FcRgamma(-/-) DC, Syk-deficient DC also had increased inflammatory cytokine production, maturation, and antigen presentation. These results confirm the inhibitory effect of immunoreceptor tyrosine-based activation motif (ITAM) signaling in myeloid cells and show that DC and macrophages differ in their dependence on the ITAM-containing adapters DAP12 and FcRgamma for negative regulation of TLR signaling.     

Evaluation of decellularization protocols for production of tubular small intestine submucosa scaffolds for use in oesophageal tissue engineering

Small intestine submucosa (SIS) has emerged as one of a number of naturally derived extracellular matrix (ECM) biomaterials currently in clinical use. In addition to clinical applications, ECM materials form the basis for a variety of approaches within tissue engineering research. In our preliminary work it was found that SIS can be consistently and reliably made into tubular scaffolds which confer certain potential advantages. Given that decellularization protocols for SIS are applied to sheet-form SIS, it was hypothesized that a tubular-form SIS would behave differently to pre-existing protocols. In this work, tubular SIS was produced and decellularized by the conventional peracetic acid–agitation method, peracetic acid under perfusion along with two commonly used detergent–perfusion protocols. The aim of this was to produce a tubular SIS that was both adequately decellularized and possessing the mechanical properties which would make it a suitable scaffold for oesophageal tissue engineering, which was one of the goals of this work. Analysis was carried out via mechanical tensile testing, DNA quantification, scanning electron and light microscopy, and a metabolic assay, which was used to give an indication of the biocompatibility of each decellularization method. Both peracetic acid protocols were shown to be unsuitable methods with the agitation-protocol-produced SIS, which was poorly decellularized, and the perfusion protocol resulted in poor mechanical properties. Both detergent-based protocols produced well-decellularized SIS, with no adverse mechanical effects; however, one protocol emerged, SDS/Triton X-100, which proved superior in both respects. However, this SIS showed reduced metabolic activity, and this cytotoxic effect was attributed to residual reagents. Consequently, the use of SIS produced using the detergent SD as the decellularization agent was deemed to be the most suitable, although the elimination of the DNase enzyme would give further improvement.     

Human helper T cell activation and differentiation is suppressed by porcine small intestinal submucosa

A cell-free biomaterial derived from porcine small intestinal submucosa (SIS) has been used successfully in many models as a xenogeneic scaffolding material without generating immune-mediated inflammatory reactions. We investigated whether this absence of inflammation is due to the presence of porcine transforming growth factor beta (TGF-beta) activity found in SIS that may have immunosuppressive properties on helper T (Th) cell subset activation and differentiation. We used in vitro models for the generation of human Th1 and Th2 cells to investigate the influence of SIS. We found that SIS partially suppressed Th1 cell expansion and secretion of interleukin 12 (IL-12) and interferon gamma (IFN-gamma) in a TGF-beta-dependent manner, but Th1 cell expansion and IFN-gamma secretion could be fully overcome by addition of recombinant IL-12. The suppression by SIS of Th cell activation also involved the induction of Th cell apoptosis. In addition, SIS completely abolished the generation of Th2 cells in vitro, but this effect of SIS was not reversed by neutralizing TGF-beta antibodies. Our results indicate the presence in SIS of factors that can suppress Th cell activation through both the inhibition of IL-12 secretion and the induction of Th cell apoptosis. We established further that these factors include TGF-beta and at least one other factor.     

Brain and bone damage in KARAP/DAP12 loss-of-function mice correlate with alterations in microglia and osteoclast lineages

Human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy, also known as Nasu-Hakola disease, has been described to be associated with mutations affecting the immunoreceptor tyrosine-based activation motif-bearing KARAP/DAP12 immunoreceptor gene. Patients present bone fragilities and severe neurological alterations leading to presenile dementia. Here we investigated whether the absence of KARAP/DAP12-mediated signals in loss-of-function (KDelta75) mice also leads to bone and central nervous system pathological features. Histological analysis of adult KDelta75 mice brains revealed a diffuse hypomyelination predominating in anterior brain regions. As this was not accompanied by oligodendrocyte degeneration or microglial cell activation it suggests a developmental defect of myelin formation. Interestingly, in postnatal KDelta75 mice, we observed a dramatic reduction in microglial cell numbers similar to in vitro microglial cell differentiation impairment. Our results raise the intriguing possibility that defective microglial cell differentiation might be responsible for abnormal myelin development. Histomorphometry revealed that bone remodeling is also altered, because of a resorption defect, associated with a severe block of in vitro osteoclast differentiation. In addition, we show that, among monocytic lineages, KARAP/DAP12 specifically controls microglial and osteoclast differentiation. Our results confirm that KARAP/DAP12-mediated signals play an important role in the regulation of both brain and bone homeostasis. Yet, important differences exist between the symptoms observed in Nasu-Hakola patients and KDelta75 mice.     

Minimally immunogenic decellularized porcine valve provides in situ recellularization as a stentless bioprosthetic valve

Currently used bioprosthetic valves have several limitations such as calcification and functional deterioration, and revitalization through cellular ingrowth is impossible. To overcome these obstacles, we have developed a minimally immunogenic tissue-engineered valve that consists of an unfixed, decellularized porcine valve scaffold capable of being spontaneously revitalized in vivo after implantation. Porcine aortic root tissue was decellularized using detergents such as sodium lauryl sulfate and Triton X-100. The porcine valve was treated very gently and plenty of time was allowed for constituents to diffuse in and out of the matrix. In a preliminary study, a piece of decellularized porcine valve tissue was implanted into the rat subdermal space for 14 and 60 days and the structural integrity and calcification were evaluated. As an in vivo valve replacement model, the decellularized porcine valve was implanted in the pulmonary valve position in dogs and functional and histological evaluation was performed after 1, 2, and 6 months. Histological examination showed that the newly developed detergent treatment effectively removed cellular debris from the porcine aortic tissue. Decellularized porcine valve tissue implanted subdermally in rats showed minimal inflammatory cell infiltration and calcification. In the valve replacement model, spontaneous reendothelialization and repopulation of the medial cells were observed within 2 months, and good valve function without regurgitation was observed by echocardiography up to 6 months. The minimally immunogenic decellularized porcine valve proved effective in mitigating postimplant calcification and provided a suitable matrix for revitalizing prostheses through in situ recellularization, cellular ingrowth, and tissue remodeling.