Neurorestorative effect of urinary bladder matrix-mediated neural stem cell transplantation following traumatic brain injury in rats.

Traumatic brain injury (TBI) is a leading cause of cell death and disability among young adults and lacks a successful therapeutic strategy. The multiphasic injuries of TBI severely limit the success of conventional pharmacological approaches. Recent successes with transplantation of stem cells in bioactive scaffolds in other injury paradigms provide new hope for the treatment of TBI. In this study, we transplanted neural stem cells (0.5x10(5) cells/µl) cultured in a bioactive scaffold derived from porcine urinary bladder matrix (UBM; 4 injection sites, 2.5µl each) into the rat brain following controlled cortical impact (CCI, velocity, 4.0 m/sec; duration, 0.5 sec; depth, 3.2mm). We evaluated the effectiveness of this strategy to combat the loss of motor, memory and cognitive faculties. Before transplantation, compatibility experiments showed that UBM was able to support extended proliferation and differentiation of neural stem cells. Together with its reported anti-inflammatory properties and rapid degradation characteristics in vivo, UBM emerged to be an ideal scaffold. The transplants reduced neuron/tissue loss and white matter injury, and also significantly ameliorated motor, memory, and cognitive impairments. Furthermore, exposure to UBM alone was sufficient to decrease the loss of sensorimotor skills from TBI (examined 3-28 days post-CCI). However, only UBMs that contained proliferating neural stem cells helped attenuate memory and cognitive impairments (examined 26-28 days post-CCI). In summary, these results demonstrate the therapeutic efficacy of stem cells in bioactive scaffolds against TBI and show promise for translation into future clinical use.

Damage associated molecular patterns within xenogeneic biologic scaffolds and their effects on host remodeling.

The immune response is an important determinant of the downstream remodeling of xenogeneic biologic scaffolds in vivo. Pro-inflammatory responses have been correlated with encapsulation and a foreign body reaction, while anti-inflammatory reactions are associated with constructive remodeling. However, the bioactive and bioinductive molecules within the extracellular matrix (ECM) that induce this polarization are unclear, although it is likely that cellular remnants such as damage associated molecular patterns (DAMPs) retained within the scaffold may play a role. The present study investigated the immunomodulatory effects of common ECM scaffolds. Results showed that tissue source, decellularization method and chemical crosslinking modifications affect the presence of the well characterized DAMP - HMGB1. In addition, these factors were correlated with differences in cell proliferation, death, secretion of the chemokines CCL2 and CCL4, and up regulation of the pro-inflammatory signaling receptor toll-like receptor 4 (TLR4). Inhibition of HMGB1 with glycyrrhizin increased the pro-inflammatory response, increasing cell death and up regulating chemokine and TLR4 mRNA expression. The present study suggests the importance of HMGB1 and other DAMPS as bioinductive molecules within the ECM scaffold. Identification and evaluation of other ECM bioactive molecules will be an area of future interest for new biomaterial development.

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.

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.

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.

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.

Enhanced bone regeneration using porcine small intestinal submucosa.

Small intestinal submucosa (SIS) is an easily produced material that has been used experimentally for tissue engineering. To evaluate the ability of SIS to facilitate bone growth within a long-bone defect, a segment of the radius was surgically removed in adult, female Sprague-Dawley rats. The defect was either left unfilled or implanted with SIS, demineralized cortical bone (DMCB), or ovalbumin. The defect was evaluated radiographically and histologically after 3, 6, 12, and 24 weeks. Tissue remodeling within the defect was evident by week 3 in SIS- and DMCB-treated rats. Filling was characterized initially by infiltration of mononuclear cells and extracellular material in SIS-implanted rats and multifocal remodeling bone particles and cartilage formation in DMCB-implanted rats. Cartilage was observed as early as 3 weeks and bone as early as 6 weeks in SIS-implanted rats. Filling of the defect arose from multiple foci in DMCB-implanted rats, but was contiguous with and parallel to the ulnar shaft in SIS-implanted rats, suggesting that defect repair by SIS may be conductive rather than inductive. Rats in which the defect was left unfilled demonstrated slow but progressive filling of the defect, characterized by mononuclear cell infiltrates and fibrous extracellular material. In summary, SIS facilitated rapid filling of a long-bone defect. These results suggest that SIS may be useful as a bone repair material.

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.

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.

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.

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.

Clusters of lymphoma in ferrets.

Cluster outbreaks of lymphoma and leukemia have been associated with viral infections in many species including humans, cattle, and cats. This study describes epidemiological, clinical, and pathological features of cluster outbreaks of lymphoma in multiferret households and examines and compares the Aleutian disease virus (ADV) and feline leukemia virus (FeLV) status of cases, ferrets at risk, and controls. Three ferret groups with 21 cases of histologically diagnosed lymphoma (12.6% cumulative incidence) and their cohabitants (n = 35) were examined and compared with three control groups (n = 52) of cohabitating ferrets without lymphoma. A familial distribution was observed in one group but most cases were not consanguinous. Ferrets greater than 3 years of age developed chronic disease in two of the groups and 2-year-old adults had acute disease in the remaining group. Lymphocytosis, splenomegaly, and lymphadenopathy were prominent features. Histologically, predominantly small noncleaved cell and polymorphous lymphoid lesions were observed. All of the ferrets with lymphoma that were tested for ADV and FeLV using serology or PCR were negative. The rate of ADV antibody among cases or ferrets at risk was not significantly different from controls. None of the cluster ferrets were seropositive for FeLV p27 antigen using a monoclonal ELISA. Infection with a novel ferret virus is suspected, but an etiological agent has not yet been identified.

Small intestinal submucosa: utilization for repair of rodent abdominal wall defects.

Prosthetic graft material is often used for the repair of abdominal wall defects that result from trauma, infection, neoplastic, or congenital deformities. A new material, porcine small intestinal submucosa, has been successfully used as an arterial and venous graft material in both canine and primate animal models with graft patency and infection rates equal to autologous vein. On the basis of these studies, small intestinal submucosa was used as a graft material for the repair of a 2 x 2-cm full-thickness defect of the muscle and fascia in the rodent abdominal wall (N = 11). Two animals were euthanized at 1 week, 2 weeks, 4 weeks, 2 months, and 3 months. At the time of euthanization, no abdominal hernias were noted and only minimal intra-abdominal adhesions were observed. One animal died on postoperative day 5 as a result of a wound dehiscence. Histological analysis of the excised abdominal wall hernia repairs revealed moderate initial inflammation but with incorporation of small intestinal submucosa with minimal inflammation at 2 months. No evidence of graft-versus-host rejection was noted with hematoxylin and eosin stains and light microscopy. Porcine small intestinal submucosa merits further study as a graft material for abdominal wall replacement.

Small intestinal submucosa: utilization as a wound dressing in full-thickness rodent wounds.

Wound dressings are used as a temporary wound covering to promote wound healing, control wound exudate, and decrease wound contamination as well as evaporative water loss. A new material, porcine small intestinal submucosa, has been used successfully as an arterial and venous graft in both canine and primate animal models with graft patency and infection rates equal to autologous vein. Based on these studies, small intestinal submucosa was used as a biological wound dressing in 20 x 20 mm full-thickness wounds made on Sprague-Dawley rats. In the controls (group I, n = 12), an acrylic frame (20 x 20 mm) was sutured to the wound edges, followed by placement of a thin polyurethane film. In the small intestinal submucosa-treated animals (group II, n = 12), the wound was covered with small intestinal submucosa and then with the acrylic frame and polyurethane film. The wounds were examined both visually and histologically at postapplication days 3, 7, 14, 28, 42 and 56. In addition, the wound contraction rate of 6 animals in both groups were recorded at postapplication day 0 and then at 1 week, 1 month, 2 months, and 3 months. Histological analysis (hematoxylin-eosin and periodic acid-Schiff stains) of the small intestinal submucosa-treated wounds revealed no host-versus-graft rejection and a rate of epithelialization equal to that of the control group. The wound contraction rate was statistically significant (higher; p < .05) in the control group compared to the small intestinal submucosa-treated group. Porcine small intestinal submucosa merits further study as both a biological wound dressing and as a substrate for cultured cells.

Experimental assessment of small intestinal submucosa as a bladder wall substitute.

OBJECTIVES: This study determined the feasibility of promoting urinary bladder regeneration with porcine-derived small intestinal submucosa (SIS). METHODS: Twenty-two Sprague-Dawley rats underwent partial cystectomy with immediate bladder augmentation with SIS. Bladders were harvested for histologic evaluation at 2, 4, 8, 12, 24, and 48 weeks. RESULTS: Histologically at 2 weeks, there was infiltration of the graft material with viable host cells consisting of fibroblasts, macrophages, and blood vessels covered by complete mucosal urothelium comprised of transitional cells. During the next 10 weeks, collagen formation and maturation were noted, and by the end of 12 weeks, the SIS graft was comprised of a mature collagen matrix admixed with thinly scattered disorganized smooth muscle bundles and covered by normal urothelium. At 48 weeks, all three layers of the normal bladder (urothelium, smooth muscle, and serosa) were present and were grossly and microscopically indistinguishable from the normal rat urinary bladder. CONCLUSIONS: This study further supports the concept of bladder regeneration and suggests that SIS may be a viable material for bladder augmentations.

The use of xenogeneic small intestinal submucosa as a biomaterial for Achilles tendon repair in a dog model.

A study was conducted to evaluate the tissue response to a xenogeneic biomaterial when this material was used to repair an experimentally induced Achilles tendon defect in the dog. Twenty dogs had a 1.5 cm segmental defect of the Achilles tendon created surgically which was then repaired with acellular connective tissue derived from porcine small intestinal submucosa (SIS). The animals were sacrificed at 1, 2, 4, 8, 12, 16, 24, and 48 weeks and the neotendons examined for uniaxial longitudinal tensile strength, morphologic appearance, hydroxyproline (collagen) content, and disappearance of the originally implanted SIS material over time. The contralateral normal Achilles tendons served as controls as did four additional dogs that had a 1.5 cm segmental Achilles tendon defect created surgically without subsequent surgical repair with SIS. Results showed the SIS remodeled neotendons to be stronger than the musculotendinous origin or the boney insertion (> 1000 N) by 12 weeks after surgery and to consist of organized collagen-rich connective tissue similar to the contralateral normal tendons. The four dogs in which no SIS was implanted showed inferior strength at the comparable time points of 4, 8, 12, and 16 weeks. Immunohistochemical studies suggest that the SIS biomaterial becomes degraded within the first eight weeks and serves as a temporary scaffold around which the body deposits appropriate and organized connective tissue. SIS is a promising biomaterial worthy of further investigation for orthopedic soft tissue applications.

Healing comparison of small intestine submucosa and ePTFE grafts in the canine carotid artery.

Continuing investigation of small-diameter vascular graft materials suggests that unacceptable graft complications continue and that the ideal material has not yet been found. We compared healing of xenogeneic small diameter grafts (3.5 to 5.0 mm diameter) made from porcine small intestine submucosa (SIS) implanted in the carotid artery to expanded polytetrafluorethylene (ePTFE) in the contralateral carotid in 8 dogs. Two dogs were sacrificed for graft evaluation at 7, 28, 90, and 180 days after surgery. Only one SIS graft was occluded at 28 days and the other 7 were patent. Six of 8 ePTFE grafts were occluded with thrombi. One was patent at 7 and one at 90 days. At 7 days post-implant, the luminal surface of the SIS graft was covered by a thick (30 microns), compact fibrin meshwork. By 28 days endothelial cells were seen completely covering the fibrin meshwork which stained for FVIII-related antigen. Smooth muscle cells were observed in the neo-intima. Most ePTFE grafts had fibrin on the luminal surface which formed fibrin thrombi with platelets and numerous red blood cells. Complete endothelial coverage of the ePTFE grafts was not observed by 180 days. There was not a pronounced neointima seen on the luminal surface of the graft. The vasa vasorum was present in the fibrous capsule surrounding the ePTFE graft, but it did not penetrate into the graft as seen in the SIS graft. At 90 days the SIS vascular graft had the histological appearance similar to a normal artery. The SIS graft potency and healing characteristics were superior to the synthetic ePTFE graft and warrant further investigation.