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SPU RESEARCH GRANT UPDATE

Spring 2011
Spring 2010



SPU PROGRESS REPORT - Spring 2011

Principal Investigator: Bradley P. Kropp

Project title: SIS Modification with Hyaluronic Acid Nanoparticles

Work Accomplished

This project was designed to enhance the performance of porcine small intestinal submucosa (SIS) in promoting urinary bladder regeneration in the field of regenerative medicine. Hyaluronic acid (HA)-poly (lactide-co-glycolide) acid (PLGA) nanoparticles (NPs) will be synthesized to stabilize the porous structure of SIS, to improve surface biocompatibility and to enhance performance in tissue regeneration. Since the inception of the project on July 1, 2009, HA-PLGA NPs have been formulated, characterized, and embedded in SIS to study their effects in promoting endothelial cell growth.

Formulation and characterization of HA-PLGA NPs
PLGA NPs were synthesized using a double emulsion technique followed by modifications with polyethylene imine to produce cationic NPs. The cationic PLGA NPs were used to conjugate different amounts of anionic HA by non-covalent electrostatic attractions. The size, polydispersity index, zeta potential, and loading potential of the synthesized HA-PLGA NPs were characterized (Table 1). Surface morphology of the NPs was tested using a JOEL-JSM-880 scanning electron microscope (SEM) (data not shown).

Table 1. Characterization of HA-PLGA NPs
SampleHA (µg)PLGA (µg)Size (nm)PolydispersityZeta Potential (mV)Loading Efficiency (%)
PLGA NPs0100241.9 ± 8.40.062 ± 0.005+ 34.67 ± 3.98N/A
HA-PLGA NPs (0.2:1)20100436.8 ± 8.50.160 ± 0.048+ 24.48 ± 2.0815.5
HA-PLGA NPs (0.5:1)50100490.3 ± 6.20.202 ± 0.012+ 19.43 ± 1.5620.4
HA-PLGA NPs (1:1)100100517.3 ± 6.30.177 ± 0.078+ 7.79 ± 0.6326.2

Quantification of HA release from PLGA NPs
To determine the release of HA from the PLGA NPs, 200 µl of the HA-PLGA NPs were incubated at 37 oC in phosphate buffered saline (PBS, pH 7.4) and quantified the amounts of HA released between 6 hours and 28 days using an ELISA-based quantitation assay. The release rates ranging from 8.9% to 17.6% were observed throughout the experimental period. There were no statistical differences in HA release among and between different time points. By day 28, 81% of the loaded HA on the NPs had been released.

Endothelial cell proliferation assay
To evaluate endothelial cell response to HA, 600 µl of 1 mg/ml PLGA or HA-PLGA NPs were encased with 80 µl collagen type I, 80 µl 10 × PBS and 2 µl 1N NaOH. A 50 µl aliquot of the NPs was delivered to each well of 96-well tissue culture plates. Human microvascular endothelial cells (HMEC-1) suspended in 100 µl of growth medium were seeded at 1.0 ? 103 cells/well (day 0) and incubated at 37 şC overnight for adherence (day 1). Wells that did not receive cells were used as the baseline control. HMEC-1 responded to 1:1 ratio of HA:PLGA with significantly elevated cell proliferation, a 7.6-fold increase in cell number, as compared to a 3.7-fold increase in PLGA NPs alone or lower HA:PLGA ratios at day 5 after cell seeding. All groups reached the maximal cell growth on day 7 after cell seeding.

Endothelial cell growth on HA-PLGA-SIS
Commercial SIS and HA-PLGA NP-modified SIS (HA-PLGA-SIS) were prepared in 1.5 ml micro-centrifuge tubes. Formulated PLGA and HA-PLGA NPs were placed onto the mucosal side of SIS at a concentration of 1 mg/ml and incubated on an orbital shaker overnight. The NPs successfully fitted into the pores of SIS based on SEM images (data not shown). HMEC-1 (5.0 ? 103 cells/cm2 in 1 ml growth medium) were seeded into the inserts; and cell-SIS membranes were harvested between days 3 and 7 after seeding. Genomic DNA was isolated from these preparations to quantify endothelial growth on HA-PLGA-SIS. The HA-PLGA-SIS with 1:1 ratio of HA:PLGA supported significantly elevated cell growth (8.7 x 104 cells) as compared to PLGA-SIS (6.2 x 104 cells) on day 7 following cell seeding.

Research Plans

Quantification of endothelial cell migration to HA-PLGA NPs
Endothelial cell migration may impact angiogenesis during the process of tissue regeneration. Enhanced cell migration in vitro may be translated to accelerated wound healing and tissue regeneration in vivo. Directional cell migration will be studied in HMEC-1 by culturing the cells with HA-PLGA NPs using an assay in modified Boyden chambers.

Induction of endothelial cell capillary formation
A monolayer of endothelial cells can invade the MatrigelTM basement membrane matrix (BD Biosciences) to form capillary-like structures. The assay is attractive in the sense that one can rapidly demonstrate the presence or absence of capillary-like structures and therefore suggest the likelihood of HA-PLGA NPs in vivo vessel formation. A serial ratios of HA and PLGA used to formulate NPs as illustrated in Table 1 will be mixed with MatrigelTM; and the NP-Matrigel mixtures will be delivered to each well of 24-well tissue culture plates followed by the addition of endothelial cells using our established procedures (1). Images of capillary tube formation will be captured between 8 and 96 hours; and number of capillary tubes formed will be calculated mathematically (1). Capability of the HA-PLGA NPs in promoting capability-like structures will be compared among different ratios of HA and PLGA.

Physical characterization of HA-PLGA-SIS
Physical characteristics including matrix thickness, tensile properties, and cyclical loading of HA-PLGA-SIS will be determined using our previous described procedures (2). HA-PLGA-SIS will also be measured for their permeability to urea using a custom-built chamber (2). These parameters are important for successful bladder regeneration. We expect that HA-PLGA-stabilized SIS will not alter any of these characteristics as compared to SIS derived from distal sections small intestine.

Biocompability study of HA-PLGA-SIS
Biocompatility of HA-PLGA-SIS bioscaffold will be investigated in a rat bladder augmentation model. Sprague-Dawley rats will be subjected to hemi-cystectomy to remove 45-50% of the bladder followed by anastomosis of a bladder patch of SIS or HA-PLGA-SIS. Bladders will be excised after days 2, 7, 14, 28, and 56. Tissue regeneration will be evaluated by standard hematoxylin and eosin staining. Immunohistochemical staining will be used to quantify neutrophils, macrophages, eosinophils, and mast cells infiltration into the regenerating tissues (3). Results will be compared between SIS and HA-PLGA-SIS augmented bladders. We expect that HA-PLGA-SIS will not alter the profiles of immune cell infiltration as compared to SIS.

References

  1. Azzarello, J, Kropp, BP, Fung, KM, and Lin, HK. Age-dependent vascular endothelial growth factor expression and angiogenic capability of bladder smooth muscle cells: implications for cell-seeded technology in bladder tissue engineering. J Tissue Eng Regen Med 3: 579-589, 2009.
  2. Raghavan, D, Kropp, BP, Lin, HK, Zhang, Y, Cowan, R, and Madihally, SV. Physical characteristics of small intestinal submucosa scaffolds are location-dependent. J Biomed Mater Res A 73A: 90-96, 2005.
  3. Ashley, RA, Palmer, BW, Schultz, AD, Woodson, BW, Roth, CC, Routh, JC, Fung, KM, Frimberger, D, et al. Leukocyte inflammatory response in a rat urinary bladder regeneration model using porcine small intestinal submucosa scaffold. Tissue Eng Part A in press, 2009.

Impact Statement

Surgical urinary bladder augmentation is a complex reconstructive procedure required for various bladder dysfunctions resulting from development defects as well as stroke, spinal injury, diabetic, or multiple sclerosis. In addition, total bladder replacement is necessary for patients who are diagnosed with invasive bladder cancer undergoing total cystectomy. The goal of bladder reconstruction is to restore bladder function and to avoid potential kidney failure. The procedures involve harvest of tissue grafts (anastomosis) from a section of the small intestine (ileum), stomach, or other substitutes and attachment of the graft to the bladder to increase the size of the organ and to improve its ability to stretch. Major complications of current procedures include stone formation due to metabolite imbalance from the grafts, perforation of the gastric segment, and potential cancer development. The goal of the study is to determine whether NP-modified SIS can be used as a bioscaffold for bladder augmentation to enhance regeneration process.


Michael Hsieh, M.D.
Assistant Professor of Urology, University Tenure Line
Stanford University Medical Center
Department of Urology

Probiotics-Mediated Suppression of Vaginal Biofilm Function and Pediatric Bacteriuria

Significant progress has been made on the SPU-funded project entitled "Probiotics-Mediated Suppression of Vaginal Biofilm Function and Pediatric Bacteriuria". Specifically, the pilot clinical trial of probiotics for girls with spina bifida (Aim 1) has been completed and has been presented as an abstract at the 2009 Texas Children's Hospital Fellows' Research Day.

The clinical data will also be presented at the 2009 meeting of the European Society of Pediatric Urology. Furthermore, the in vitro experiments (Aim 2) have also been proceeding well. We have successfully established a Lactobacillus-vaginal epithelial cell co-culture model to study genitourinary probiotic biofilms.

Our data on the effects of these bioforms on vaginal epithelial cells will be submitted as an abstract for the upcoming AAP meeting. We anticipate submission of a manuscript for publication shortly after the end of the term of the SPU Grant. Finally, the support provided by the SPU has enabled me to obtain additional research funding from the Spina Bifida Association and the Thrasher Foundation.


Stacy T. Tanaka, MD
Clinical Fellow, Division of Pediatric Urology
Monroe Carell Jr. Children’s Hospital at Vanderbilt

The Role of Bone Marrow Derived Cells in Bladder Obstruction and Fibrosis

I am writing to update you on the progress our lab has made on the project “The role of bone marrow derived cells in bladder obstruction and fibrosis,” which was generously supported by the 2008-2009 Society for Pediatric Urology Research Grant.

The main goal of our proposal was to study the role of bone marrow derived cells in bladder fibrosis. Chemokines recruit bone marrow derived cells to sites of tissue injury. Blockade of chemokines in other organ systems has resulted in reduced fibrosis. In the bladder, identification of bone marrow derived cells and the chemokines involved in their recruitment may provide potential targets for antifibrotic therapies. In order to study the role of the bone marrow derived cells in bladder fibrosis, we used chimeric mice whose bone marrow cells were labeled with green fluorescent protein (GFP). Briefly, wild type C57BL/6 female mice underwent lethal irradiation. Their bone marrow was reconstituted using fetal liver cells from transgenic mice ubiquitously expressing GFP. The chimeric mice then underwent partial bladder outlet obstruction with periurethral collagen injection.

We describe below our progress on each of our specific aims:

SPECIFIC AIM 1:
To characterize the bone marrow derived cells associated with partial bladder outlet obstruction. We will establish the identity of bone marrow derived cells in the bladder after urethral obstruction and their temporal appearance in relation to histologic and physiologic changes in the bladder

  • We found that periurethral bladder outlet obstruction caused histologic changes in the bladder. At 4 weeks of obstruction, bladder smooth muscle hypertrophy was present. At 12 weeks of obstruction, increased collagen within the detrusor layer was consistently present.
     
  • We found that periurethral bladder outlet obstruction caused urodynamic changes. At 4 weeks of obstruction, mice had decreased bladder capacity but no significant difference in compliance. At 12 weeks of obstruction, bladder capacity was less than half the capacity of unobstructed mice, and mean bladder compliance was less than one-third the compliance of unobstructed mice.
     
  • Bone marrow cells are recruited to the bladder and persist long term after bladder outlet obstruction. GFP-positive bone marrow derived cells were consistently present in the urothelial and stromal layers of obstructed mice from 1 to 12 weeks after obstruction.
     
  • Although we were unable to identify any bone marrow derived myofibroblasts which we hypothesized may be responsible for fibrosis in the obstructed bladder, we have been able to use fluorescence activated cell sorting to establish the identity of some of the bone marrow derived cells. We found that less than 5% of the GFP-positive cells were also positive for the murine macrophage marker F4/80. Other possible cell identities are currently being investigated.
     
  • We also discovered that bone marrow derived cells may affect the injured bladder not only by differentiation but also by release of factors associated with hypertrophy and fibrosis. We found clusters of cells with activated EGF receptors around GFP-positive bone marrow derived cells after 12 weeks of obstruction but not in unobstructed controls. These studies are ongoing.

SPECIFIC AIM 2:
Identify mechanisms involved in recruitment of bone marrow derived cells after bladder outlet obstruction. Chemokines involved in the recruitment of bone marrow derived cells to the bladder will be identified. We will determine if blockade of chemokine / chemokine receptor interactions with neutralizing antibodies will decrease the number of recruited bone marrow derived cells, decrease fibrosis and/or preserve normal bladder function.

  • The chemokines CCL2 and CXCL12 which are associated with fibrosis in other organ systems were observed at 1 week and persisted up to 12 weeks following bladder outlet obstruction.

  •  
  • Treatment of mice with neutralizing antibodies to the chemokines CCL2 and CXCL12 remain ongoing. We will study the effect of these neutralizing antibodies on the reduction of pathologic histologic and pathologic urodynamic changes.

We were able to present our initial findings as a moderated poster at the 2008 American Academy of Pediatrics meeting in Boston. The associated manuscript has been accepted for publication in the Journal of Urology Pediatric Supplement. We hope to complete our pending experiments as indicated and submit our additional findings for publication in the near future.

Thank you again for your generous support of our research.



SPU PROGRESS REPORT - Spring 2010
Principal Investigator: Dominic Frimberger

Project title: Survival and Role Potential of Bone Marrow Stromal Cells in Bladder Regeneration

Work Accomplished

Bone marrow stromal cells (BMSCs) have been shown to be a multipotent progenitor cell with broad differentiation capabilities. In bladder regeneration, BMSCs have been shown to improve regeneration in several small animal models after being seeded onto bioscaffolds (1). However, the underlying mechanisms of this regeneration have not been fully investigated. Possibilities for the improved regeneration include in vivo differentiation of MSCs to cell types composing the bladder wall, or support of regeneration through the production of growth factors or chemokines. This project was designed to determine whether MSCs survive following transplantation or undergo in vivo differentiation.

BMSC Harvest, Culture, and Seeding on porcine intestinal submucosa (SIS)

Adult BMSCs were harvested from the femur and tibia of green fluorescence protein (GFP) transgenic Sprague-Dawley (SD) rats strain [SD-Tag(GFP)Bal]. The colleted cells were plated on four 60 mm tissue culture dishes in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The BMSC cultures were incubated at 37° C for 48 hours for adherence, the floating hematopoetic cells were removed. Cell culture media was changed every two days and cell passages were performed at approximately 90-95% confluence. All adherent cells showed GFP expression. We have found that BMSCs are relatively easy to isolate and replicate at a rapid rate. At the third passage, cells were trypsinized, harvested, and seeded on SIS at the concentration of 1x106 cells/cm2 in DMEM plus 10% FBS. The cell-composite SIS was incubated at 37° C for another 7 days, removed from the frame, and cut to the size of 1x1 cm2 for bladder augmentation in rats. Sections of the SIS were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin (H&E) as well as Masson’s trichrome. Ingrowth of BMSCs in the SIS was demonstrated with fluorescence microscopy and these stains.

Bladder Augmentation

To perform bladder augmentation, adult female SD rats were anesthetized. Following partial cystectomy to remove a 1 cm2 segment of the apical bladder, the 1 cm2 section of BMSC-composite SIS was sutured to the native bladder edges using 6-0 polyglactin 910 (Ethicon) in a water-tight fashion. The four corners of the suture line were marked with 6-0 nylon (Ethicon) suture to locate the grafts. Augmented bladders were harvested at 14, 28 and 56 days after augmentation.

At harvest, the bladders are inflated with 4% paraformaldehyde and fixed for 16 hours, and then sectioned in half vertically. One half of the bladder was embedded in optimal cutting temperature (O.C.T.) compound and frozen at -80° C. Frozen sections were cut at 5 ?m on a cryotome at -20 °C and mounted on charged microscope slides. Frozen sections were visually inspected under a fluorescent microscope to determine the presence and location of GFP expressing cells. Inspection of the area near the SIS is limited by autofluorescence of the SIS itself under fluorescence microscopy. At days 14 and 28, there were GFP-expressing cells present in the graft areas, suggesting that seeded BMSCs survived during the early stages of regeneration process. In addition, a limited number of GFP-positive cells appeared to have migrated into the surrounding submucosal tissues. At day 56, neither the smooth muscle bundles nor the urothelium show any significant GFP fluorescence.

The second half of the bladder was embedded in paraffin and sectioned at 5 ?m, mounted on glass slides, baked at 60 °C and stored at room temperature. Sections were stained with H&E as well as Masson’s trichrome per standard protocols. Histological characterization of these tissue sections did not exhibit enhancement in regeneration at days 14, 28 and 56 as compared to regenerated bladders augmented with unseeded SIS.

Research Plans

The preliminary results suggested that the BMSCs may not directly incorporate into the regenerated bladder wall. A larger number of animals will be performed to confirm our current observations. In order to further evaluate the survival of the BMSCs, earlier time points will be included and tissue sections will be stained for cell death markers including terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and activated caspase-3 as well as cell proliferation markers such as Ki-67 along with GFP staining.

It is possible that seeded BMSCs may improve regeneration by improving vascularization of the graft tissue (2). An attempt will be made to quantify number of blood vessels in regenerated bladder using by immunohistochemical staining for CD31; and results will be compared with bladders augmented with unseeded SIS. In the future, differentiation of BMSCs to bladder smooth muscle cells (SMCs) will be characterized in vitro. Several studies have suggested that BMSCs are capable of differentiation to SMC lineage (3). To accomplish this goal, molecular techniques as well as functional assays will be applied to evaluate the gene expression profiles and contractile capabilities of BMSC after in vitro induction for SMC differentiation.

References

  1. Zhang, Y, et Al. Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder. BJU Int. 2005 Nov;96 (7): 1120-5.
  2. Zhang K, et Al. Bone marrow mesenchymal stem cells induce angiogenesis and promote bladder cancer growth in a rabbit model. Urol Int. 2010;84(1):94-9. Epub 2010 Feb 17.
  3. Tian H, et Al. Differentiation of human bone marrow mesenchymal stem cells into bladder cells: potential for urological tissue engineering. Tissue Eng Part A. 2010 May;16(5):1769-79.