Biomedical subjects

S F Badylak

Publications and source records attributed to S F Badylak.

At least 19 recordsLinked to original sources

PubMed2006

Thrombolysis vs. bleeding from hemostatic sites by a prourokinase mutant compared with tissue plasminogen activator.

BACKGROUND: A single site mutant (M5) of prourokinase (proUK) was developed to make proUK less vulnerable to spontaneous activation in plasma. This was a problem that seriously compromised proUK in clinical trials, as it precluded proUK-mediated fibrinolysis at therapeutic concentrations. METHODS AND RESULTS: After completing dose-finding studies, 12 anesthetized dogs with femoral artery thrombosis were given either M5 (2.0 mg kg(-1)) or tissue plasminogen activator (t-PA) (1.4 mg kg(-1)) by i.v. infusion over 60 min (20% administered as a bolus). Two pairs of standardized injuries were inflicted at which hemostasis was completed prior to drug administration. Blood loss was quantified by measuring the hemoglobin in blood absorbed from these sites. Thrombolysis was evaluated at 90 min and was comparably effective by both activators. Rethrombosis developed in one t-PA dog. The principal difference found was that blood loss was 10-fold higher with t-PA (mean approximately 40 mL) than with M5 (mean approximately 4 mL) (P = 0.026) and occurred at more multiple sites (mean 2.7 vs. 1.2). This effect was postulated to be related to differences in the mechanism of plasminogen activation by t-PA and M5 in which the latter is promoted by degraded rather than intact (hemostatic) fibrin. In addition, two-chain M5 was efficiently inactivated by plasma C1 inactivator, an exceptional property which helped contain its non-specific proteolytic effect. CONCLUSIONS: Intravascular thrombolysis by M5 was accompanied by significantly less bleeding from hemostatic sites than by t-PA. This was attributed to the proUK paradigm of fibrinolysis being retained at therapeutic concentrations by the mutation.

Animals↗

PubMed2001

Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response.

BACKGROUND: Porcine small intestinal submucosa (SIS) is an acellular, naturally derived extracellular matrix (ECM) that has been used for tissue remodeling and repair in numerous xenotransplantations. Although a vigorous immune response to xenogeneic extracellular matrix biomaterials is expected, to date there has been evidence for only normal tissue regeneration without any accompanying rejection. The purpose of this study was to determine the reason for a lack of rejection. METHODS: Mice were implanted s.c. with xenogeneic tissue, syngeneic tissue, or SIS, and the graft site analyzed histologically for rejection or acceptance. Additionally, graft site cytokine levels were determined by reverse transcriptase polymerase chain reaction and SIS-specific serum antibody isotype levels were determined by ELISA. RESULTS: Xenogeneically implanted mice showed an acute inflammatory response followed by chronic inflammation and ultimately graft necrosis, consistent with rejection. Syngeneically or SIS implanted mice, however, showed an acute inflammatory response that diminished such that the graft ultimately became indistinguishable from native tissue, observations that are consistent with graft acceptance. Graft site cytokine analysis showed an increase in interleukin-4 and an absence of interferon-gamma. In addition, mice implanted with SIS produced a SIS-specific antibody response that was restricted to the IgG1 isotype. Reimplantation of SIS into mice led to a secondary anti-SIS antibody response that was still restricted to IgG1. Similar results were observed with porcine submucosa derived from urinary bladder. To determine if the observed immune responses were T cell dependent, T cell KO mice were implanted with SIS. These mice expressed neither interleukin-4 at the implant site nor anti-SIS-specific serum antibodies but they did accept the SIS graft. CONCLUSIONS: Porcine extracellular matrix elicits an immune response that is predominately Th2-like, consistent with a remodeling reaction rather than rejection.

Animals↗

PubMed2001

Small bowel tissue engineering using small intestinal submucosa as a scaffold.

BACKGROUND: Small intestinal submucosa (SIS) is an extracellular matrix used in tissue engineering studies to create de novo abdominal wall, urinary bladder, tendons, blood vessels, and dura mater. The purpose of this study is to evaluate the feasibility of using SIS as a scaffold for small bowel regeneration in an in situ xenograft model. MATERIALS AND METHODS: Twenty-three dogs had a partial defect created on the small bowel wall which was repaired with a SIS patch. Four dogs underwent small bowel resection with placement of an interposed tube of SIS. The animals were followed 2 weeks to 1 year. RESULTS: Three of the 23 dogs with SIS placed as a patch died shortly after surgery due to leakage from the site. The other 20 dogs survived up to time of elective necropsy with no evidence of intestinal dysfunction. At necropsy, the bowel circumference in the patched area had no stenosis. Histological evaluation showed the presence of a mucosal epithelial layer, varying amount of smooth muscle, sheets of collagen, and a serosal covering. Architecturally, the layers were not well organized in the submucosal region. An abundance of inflammatory cells was present in the early postoperative period but receded with time. All 4 dogs with a tubular segment of SIS interposed had significant problems. One had partial obstruction at 1 month, and 3 died in the early postoperative period due to leakage. CONCLUSIONS: This preliminary study suggests that SIS patches can be used for small bowel regeneration. Tubular segmental replacement is not feasible at this time.

Animals↗

PubMed2001

In vivo degradation of 14C-labeled small intestinal submucosa (SIS) when used for urinary bladder repair.

The rate of in vivo degradation was determined for a naturally occurring biomaterial derived from the extracellular matrix of the small intestinal submucosa (SIS). The SIS was labeled by giving weekly intravenous injections of 10 microCi of 14C-proline to piglets from 3 weeks of age until the time of sacrifice at 26 weeks. The resultant SIS prepared from these pigs contained approximately 10(3) fold more 14C than unlabeled tissues. The labeled SIS was used to repair experimental defects in the urinary bladder of 10 dogs. The animals were sacrificed at post-operative times ranging from 3 days to 1 year and the remodeled urinary bladder tissue was harvested for evaluation of 14C by a combination of liquid scintillation counting and accelerator mass spectrometry. The remodeled tissue contained less than 10% of the 14C (disintegrations per minute/gram tissue wet weight) at 3 months post-surgery compared to the SIS biomaterial that was originally implanted. The SIS scaffold was replaced by host tissue that resembled normal bladder both in structure and function. After implantation, 14C was detected in highest concentrations in the blood and the urine. The SIS bioscaffold provides a temporary scaffold for tissue remodeling with rapid host tissue remodeling, degradation, and elimination via the urine when used as a urinary bladder repair device.

Animals↗

PubMed2001

Marrow-derived cells populate scaffolds composed of xenogeneic extracellular matrix.

INTRODUCTION: The source of cells that participate in wound repair directly affects outcome. The extracellular matrix (ECM) and other acellular biomaterials have been used as therapeutic scaffolds for cell attachment and proliferation and as templates for tissue repair. The ECM consists of structural and functional proteins that influence cell attachment, gene expression patterns, and the differentiation of cells. OBJECTIVE: The objective of this study was to determine if the composition of acellular matrix scaffolds affects the recruitment of bone marrow-derived cellular elements that populate the scaffolds in vivo. METHODS: Scaffolds composed of porcine tissue ECM, purified Type I collagen, poly(L)lactic coglycolic acid (PLGA), or a mixture of porcine ECM and PLGA were implanted into subcutaneous pouches on the dorsum of mice. The origin of cells that populated the matrices was determined by first performing bone marrow transplantation to convert the marrow of glucose phosphate isomerase 1b (Gpi-1(b)) mice to cells expressing glucose phosphate isomerase 1a (Gpi-1(a)). RESULTS: A significant increase in Gpi-1(a) expressing cells was present in sites implanted with the porcine ECM compared to sites implanted with either Type I collagen or PLGA. Use of recipient mice transplanted with marrow cells that expressed beta-galactosidase confirmed that the majority of cells that populated and remodeled the naturally occurring porcine ECM were marrow derived. Addition of porcine ECM to the PLGA scaffold caused a significant increase in the number of marrow-derived cells that became part of the remodeled implant site. CONCLUSION: The composition of bioscaffolds affects the cellular recruitment pattern during tissue repair. ECM scaffolds facilitate the recruitment of marrow-derived cells into sites of remodeling.

Animals↗

PubMed2001

Porcine small intestinal submucosa (SIS): a bioscaffold supporting in vitro primary human epidermal cell differentiation and synthesis of basement membrane proteins.

The growth pattern of human epidermal cells, fibroblasts or Swiss mouse 3T3/J2 fibroblasts cultured upon the extracellular matrix (ECM) derived from small intestinal submucosa (SIS) was evaluated. The cell/SIS composites were grown submerged, then maintained in air/liquid interface for 2, 7, 10 or 14 days. The presence of differentiation-related keratins 10, 14 and 16, FN, laminin, collagen type VII and collagen type IV was determined by immunohistochemical methods in SIS alone and in the SIS/cell composite. Only FN could be detected in SIS alone. SIS supported the formation of an epithelial structure with suprabasal expression of K16 and regional suprabasal expression of K10. The epidermal cells were K14 positive and tended to 'invade' the SIS to various degrees. Following the growth of epidermal cells and fibroblasts on the SIS substratum, immunolabeling of FN, laminin, collagen type VII and collagen type IV was observed in a cell-associated pattern. The fibroblasts commonly invaded the SIS, when co-cultivated with epidermal cells on the opposite side of the SIS. The ability of SIS to support epidermal cell/fibroblast attachment, migration and/or proliferation and differentiation with deposition of basement membrane (BM) components indicates that the composite model may be useful for studying cell-matrix interactions and for investigation as a dermal substitute.

3T3 Cells↗

PubMed2001

Vascular endothelial growth factor in porcine-derived extracellular matrix.

An extracellular matrix (ECM) derived from the submucosa of the porcine small intestine (SIS) has been shown to induce angiogenesis and host tissue remodeling when used as a xenogeneic bioscaffold in animal models of wound repair. In the present study, we compared the in vitro effects of SIS ECM extracts to several purified angiogenic growth factors on human dermal microvascular endothelial cell (HMEC) growth patterns. The SIS ECM was shown to induce tube formation from HMEC in a three-dimensional fibrin-based angiogenesis assay in a manner similar to that caused by the addition of vascular endothelial growth factor (VEGF). This tube formation was blocked in the presence of anti-VEGF neutralizing antibody. Western blots and ELISA procedures showed that the SIS ECM contains as much as 0.77 ng VEGF/g SIS. The closely related endothelial cell mitogen, platelet-derived growth factor (PDGF), was not detectable in the SIS extracts. We conclude that VEGF is present in the SIS extracellular matrix. The role of VEGF in SIS-induced wound repair remains unknown, but its presence in the ECM makes it a possible contributor to the angiogenic effect of SIS when this ECM is used as a tissue repair scaffold in animal models of wound repair.

Animals↗

PubMed2000

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.

Animals↗

PubMed1999

Porcine small intestinal submucosa as a dural substitute.

BACKGROUND: The continuing search for the ideal dural substitute is currently directed toward collagen preparations. Xenogeneic porcine small intestinal submucosa (SIS), a naturally occurring extracellular matrix rich in collagen, has been successfully used as a soft tissue graft in several body organ systems, including preliminary studies as a dural substitute in the rat. METHODS: Eight dogs underwent temporoparietal craniotomy and dural resection with replacement by SIS. Five dogs had contralateral procedures without SIS grafting. Three dogs had contralateral SIS grafts placed 2 months after the initial procedure. Histologic assessment was obtained at 7, 30, 60, 90, and 120 days. Cerebrospinal fluid (CSF) cytological examination and routine serum chemistry preceded sacrifice. RESULTS: Histologic evaluation showed initial graft infiltration by mononuclear round cells, spindle-shaped cells within an eosinophilic staining extracellular matrix, and neovascularity. Complete resorption of the graft was evident by 60 days. This pattern is consistent with the previously described incorporation and remodeling of the SIS graft at other sites. CSF cytology and routine serum chemistry at the time of sacrifice were normal. Response to repeat grafting was identical to that of initial exposure. There was no clinical or histologic evidence of sensitization or graft rejection. No evidence of adverse effect on the underlying cerebral cortex was observed. CONCLUSIONS: Porcine small intestinal submucosa demonstrates a favorable biologic response as a dural substitute in the canine model. It is a promising biomaterial for dural replacement.

Animals↗

PubMed1998

Multilaminate resorbable biomedical device under biaxial loading.

The design and test of a multilaminate sheet developed for a hernia repair application is presented. As biomaterial applications become more complex, characterization of uniaxial properties becomes insufficient and biaxial testing becomes necessary. A measure of the in-plane biaxial strength of the device is inferred from a ball burst test. The results of this test for different thicknesses of the device are correlated with the uniaxial strength of the material. A biaxial test such as the ball burst test is more indicative of the properties of a planar material than would be a uniaxial test. The interactions in the biaxial mode of failure are of value and can be related back to a classical uniaxial tensile test from the ball burst test. The material used in this study to fabricate the device was a resorbable biomaterial called small intestinal submucosa (SIS). The effects of rehydration on the stiffness and associated ball burst properties of the SIS device were also measured. It is shown that at a rehydration time of 5 min from a reference dry state, steady-state mechanical properties are reached.

Biocompatible Materials↗

PubMed1998

Application and evaluation of the alamarBlue assay for cell growth and survival of fibroblasts.

Cell proliferation assays are essential to developing an understanding of the molecular mechanisms that modulate cell growth and differentiation. In this paper, we describe the application of alamarBlue, a new and versatile metabolic dye, for the detection of Swiss 3T3 fibroblast proliferation and/or survival. As a redox indicator, alamarBlue is reduced by reactions innate to cellular metabolism and, therefore, provides an indirect measure of viable cell number. Various assay parameters were optimized for a 96-well format to achieve a detectable range of fibroblast cell number from 100 to 20,000 cells/well, which is similar to that obtained with traditional (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and [3H]thymidine assay techniques. Standard (reference) curves generated with a known fibroblast stimulator were used to facilitate quantitation and comparison of unknown test substances. The alamarBlue assay offers the advantages of technical simplicity, freedom from radioisotopes, versatility in detection, no extraction, and excellent reproducibility and sensitivity. We anticipate that this simple and versatile alamarBlue assay, when used alone or in conjunction with other bioassays, will be a useful tool for investigating the complex mechanisms of cellular proliferation.

3T3 Cells↗

PubMed1998

Rabbit urethral regeneration using small intestinal submucosa onlay grafts.

OBJECTIVES: To determine if small intestinal submucosa (SIS) can evoke urethral regeneration. METHODS: Twenty male white New Zealand rabbits were assigned to one of three experimental groups. Group 1 (n = 4) underwent simple urethrotomy and closure. Group 2 (n = 8), a second control group, underwent an onlay urethroplasty with a graft of full-thickness preputial skin from the host rabbit. Group 3 (n = 8) underwent an onlay urethroplasty with an SIS graft. RESULTS: All eight SIS onlay grafts promoted regeneration of the normal rabbit epithelium supported by a well-vascularized collagen and smooth muscle backing. Preputial free onlay grafts maintained a keratinizing squamous cell epithelium with a poor supportive backing, which resulted in the formation of urethral diverticulum. CONCLUSIONS: SIS onlay patch grafts for urethroplasty promote rabbit urethral regeneration.

Animals↗

PubMed1998

Small intestinal submucosa: a rapidly resorbed bioscaffold for augmentation cystoplasty in a dog model.

The extracellular matrix (ECM) of porcine small intestinal submucosa (SIS) has been shown to serve as a resorbable scaffold for tissue repair and remodeling in several body locations including the urinary bladder. The rate of resorption and extent of SIS degradation are unknown. Nine dogs were divided into three equal groups. Approximately 40% of the anterior dome of the urinary bladder was resected in each dog and replaced with porcine SIS. One group of dogs was sacrificed at each of 4, 8, and 12 weeks after surgery and the fate of the implanted SIS determined by immunohistochemical methods using a monoclonal antibody specific for porcine-derived SIS. By 4 weeks after surgery, only scattered remnants of SIS were present in the remodeled urinary bladder and these positively staining foci were surrounded by an extensive new host derived ECM and neovascularization. There was a continuous layer of transitional epithelium on the luminal surface by 4 weeks. No evidence for the originally implanted SIS could be found at either 8 or 12 weeks and bundles of organized smooth muscle cells were present at the operative site. In summary, SIS is rapidly and extensively degraded when used as a bioscaffold for augmentation cystoplasty in the dog model.

Animals↗

PubMed1998

Small intestinal submucosa: a substrate for in vitro cell growth.

The extracellular matrix (ECM) of the small intestinal submucosa (SIS) was harvested by removing the superficial layers of the mucosa and the external muscular layers. The remaining 80 microns thick sheet was disinfected and sterilized by methods which removed all cellular components. The SIS-ECM, retaining its native 3-dimensional microarchitecture and composition, was evaluated for its ability to support in vitro cell growth. Six separate cell types were seeded either alone or in coculture with other cells upon this matrix, grown in selected media, a examined daily for time periods ranging from 48 h to 2 weeks. The six cell types tested were NIH Swiss mouse 3T3 fibroblast, NIH 3T3/j2 fibroblasts, primary human fibroblasts, primary human keratinocytes, human microvascular endothelial cells (HMECs), and an established rat osteosarcoma (ROS) cell line. All cell types showed the ability to attach a proliferate. All fibroblast cell line and the keratinocytes proliferated and/or migrated into the 3-dimensional scaffold of the SIS matrix. The ROS cells and the HMECs were confined in their growth pattern to the surface of the matrix. Coculturing of NIH 3T3/J2 fibroblasts and primary human keratinocytes resulted in a distinctive spatial orientation of the two cell types. The fibroblast populated the mid-substance of the 3-dimensional matrix and the keratinocytes formed an epidermal structure with rete ridge-like formation and stratification when the composite was lifted to an air liquid interface in culture. In summary, SIS provides a substratum with a 3-dimensional scaffold that allows for cell migration and spatial organization. The substratum is suitable for in vitro studies of the interaction between epithelial or mesenchymal cells and a naturally occurring extracellular matrix.

3T3 Cells↗

PubMed1997

Identification of extractable growth factors from small intestinal submucosa.

When implanted as a biomaterial for tissue replacement, selected submucosal layers of porcine small intestine induce site-specific tissue remodeling. Small intestinal submucosa (SIS), as isolated, is primarily an acellular extracellular matrix material. In an attempt to discover the components of small intestinal submucosa which are able to induce this tissue remodeling, the material was extracted and extracts were tested for the ability to stimulate Swiss 3T3 fibroblasts to synthesize DNA and proliferate. Each of the four different extracts of small intestinal submucosa had measurable cell-stimulating activity when analyzed in both a whole cell proliferation assay (alamarBlue dye reduction) and a DNA synthesis assay ([3H]-thymidine incorporation). Proteins extracted from SIS with 2 M urea induced activity profiles in the two assays which were very similar to the activity profiles of basic fibroblast growth factor (FGF-2) in the assays. As well, the changes in cell morphology in response to the extracted proteins mimicked the changes induced by FGF-2. Neutralization experiments with specific antibodies to this growth factor confirmed the presence of FGF-2 and indicated that it was responsible for 60% of the fibroblast-stimulating activity of the urea extract of small intestinal submucosa. Western blot analysis with a monoclonal antibody specific for FGF-2 detected a reactive doublet at approximately 19 kDa and further confirmed the presence of FGF-2. Cell stimulating activity of proteins extracted from SIS with 4 M guanidine was neutralized by an antibody specific for transforming growth factor beta (TGF beta). Changes in the morphology of the fibroblasts exposed to this extract were nearly identical to changes induced by TGF beta. Although no reactive protein band was detected at 25 kDa in nonreduced western blot analysis, several bands were reactive at higher molecular weight. The identity of this TGF beta-related component of small intestinal submucosa is unknown. Identification of FGF-2 and TGF beta-related activities in SIS, two growth factors known to significantly affect critical processes of tissue development and differentiation, provides the opportunity to further elucidate the mechanisms by which this extracellular matrix biomaterial modulates wound healing and tissue remodeling.

3T3 Cells↗

PubMed1996

Intestine submucosa and polypropylene mesh for abdominal wall repair in dogs.

Continuing investigations of abdominal body wall reconstruction materials suggest that unacceptable implant complications continue and that the ideal material has not yet been found. This pilot study compared xenogeneic (porcine) small intestine submucosa (SIS) with polypropylene mesh (PPM) for repair of created partial-thickness (six dogs) and full-thickness (six dogs) abdominal wall defects. Postoperative clinical evaluation of all dogs showed no evidence of implant failure. Dogs were euthanized at 1, 2, and 4 months after surgery. The SIS implants were completely replaced by host tissue at 4 months as determined by immunohistochemistry. The resultant repair was well-organized, smooth, dense collagenous connective tissue that was well incorporated into the adjacent fascia and skeletal muscle fiber bundles. In the full-thickness defect dogs, omentum covered a significantly larger portion of PPM (P = 0.001) and was more firmly attached to PPM (P = 0.0001) compared to SIS/connective tissue repair. We conclude that xenogeneic SIS can be used as an abdominal body wall repair material in the dog and warrants further investigations.

Abdominal Muscles↗

PubMed1996

Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations.

PURPOSE: To evaluate small intestinal submucosa (SIS) as a possible bladder augmentation material. MATERIALS AND METHODS: Nineteen male dogs underwent 35 to 45% partial cystectomy with immediate augmentation with SIS grafts. All dogs were evaluated pre- and postoperatively with blood chemistries, urine cultures, intravenous urograms, cystograms and cystometrograms. Postoperatively (1 to 15 months), bladders were examined with routine histology and image analysis. RESULTS: All dogs survived their intended survival period without morbidity. All results were normal. Histologically, all 3 layers (mucosa, smooth muscle, serosa) of the normal bladder showed evidence of regeneration. CONCLUSIONS: Small intestinal submucosa acts as a scaffold for bladder augmentation through regeneration and could be a potential option for bladder reconstruction.

Animals↗

PubMed1996

Histology after dural grafting with small intestinal submucosa.

BACKGROUND: The search for the ideal dural substitute continues, inasmuch as available materials have significant limitations. Xenogeneic porcine small intestinal submucosa (SIS) has been successfully used as a soft tissue graft in several body organ systems, and it was logical to evaluate its use as a dural replacement. METHODS: Twenty rats underwent bihemispheric craniectomy with dural resection. SIS onlay grafting on one side was performed. Histologic assessment was obtained at 7 and 28 days after dural grafting and included descriptive evaluation and quantitative scoring of graft-site thickness, vascularity, and cellular density. The total scores for the respective groups were compared using the Student's t test, significance being accepted for a p value < 0.05. RESULTS: Histologic evaluation showed graft infiltration by spindle-shaped mononuclear cells, deposition of connective tissue, and neovascularity. This pattern is consistent with the previously described incorporation and remodeling of the SIS graft at other sites. A significant difference between the histologic scores of the SIS graft site and control site was found at 7 days (3.4 +/- 0.8 versus 0.1 +/- 0.1) and at 28 days (4.6 +/- 1.1 versus 2.2 +/- 0.5). No evidence of adverse effect on the underlying cortex was observed. CONCLUSIONS: The results of this preliminary study utilizing porcine SIS as a dural substitute are promising and therefore justify further chronic studies.

Animals↗