ORIGINAL ARTICLE Effects of different biomaterials: Comparing the bladder smooth muscle cells on waterborne polyurethane or poly-lactic-co-glycolic acid membranes Feng Xu a , Yan Wang a , Xia Jiang b , Hong Tan b , Hong Li a , Kun-Jie Wang a, * a Department of Urology, West China Hospital, Huaxi Clinical College, SiChuan University, ChengDu, SiChuan, China b College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, SiChuan University, ChengDu, SiChuan, China Received 24 February 2011; accepted 28 March 2011 Available online 11 December 2011 KEYWORDS Biodegradation; Bladder smooth muscle cells; Poly-e-caprolactone; Waterborne polyurethane Abstract Tissue engineering materials have often been used to repair bladder damage caused by conditions, such as infection, resection, inflammation, and trauma. However, the concept of generating a functional urinary bladder using autologous cells obtained from a biopsy specimen combined with a biomaterial scaffold remains a challenge. Previously, we presented a new method for synthesizing a biocompatible, mechanically sound, nontoxic, and cross-linked waterborne polyurethane (WBPU) as a potential material for bladder regener- ation. Here, we further evaluated the response of bladder smooth muscle cells (BSMCs) seeded on WBPU membranes in comparison with the gold standard biomaterial, poly-lactic-co-glycolic acid. Specifically, we observed the BSMC attachment, proliferation, and a-actin distribution at 1 day, 3 days, and 5 days after membrane seeding. We found that significantly more cells attached and proliferated on the WBPU membranes after 3 days and 5 days of culture, and the cells exhibited greater organization and a wider distribution of a-actin compared with BSMCs cultured on poly-lactic-co-glycolic acid membranes. These preliminary data offer promise for the use of WBPU biomaterials in bladder tissue engineering. Copyright ª 2011, Elsevier Taiwan LLC. All rights reserved. * Corresponding author. Department of Urology, West China Hospital, Huaxi Clinical College, Sichuan University, ChengDu, Sichuan 610040, China. E-mail address: [email protected](K.-J. Wang). Available online at www.sciencedirect.com journal homepage: http://www.kjms-online.com Kaohsiung Journal of Medical Sciences (2012) 28, 10e15 1607-551X/$36 Copyright ª 2011, Elsevier Taiwan LLC. All rights reserved. doi:10.1016/j.kjms.2011.06.031
Transcript
Kaohsiung Journal of Medical Sciences (2012) 28, 10e15
Available online at www.sciencedirect.com
journal homepage: http: / /www.kjms-onl ine.com
ORIGINAL ARTICLE
Effects of different biomaterials: Comparing the bladdersmooth muscle cells on waterborne polyurethane orpoly-lactic-co-glycolic acid membranes
Feng Xu a, Yan Wang a, Xia Jiang b, Hong Tan b, Hong Li a, Kun-Jie Wang a,*
aDepartment of Urology, West China Hospital, Huaxi Clinical College, SiChuan University,ChengDu, SiChuan, ChinabCollege of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering,SiChuan University, ChengDu, SiChuan, China
Received 24 February 2011; accepted 28 March 2011Available online 11 December 2011
Abstract Tissue engineering materials have often been used to repair bladder damagecaused by conditions, such as infection, resection, inflammation, and trauma. However, theconcept of generating a functional urinary bladder using autologous cells obtained froma biopsy specimen combined with a biomaterial scaffold remains a challenge. Previously, wepresented a new method for synthesizing a biocompatible, mechanically sound, nontoxic,and cross-linked waterborne polyurethane (WBPU) as a potential material for bladder regener-ation. Here, we further evaluated the response of bladder smooth muscle cells (BSMCs) seededon WBPU membranes in comparison with the gold standard biomaterial, poly-lactic-co-glycolicacid. Specifically, we observed the BSMC attachment, proliferation, and a-actin distribution at1 day, 3 days, and 5 days after membrane seeding. We found that significantly more cellsattached and proliferated on the WBPU membranes after 3 days and 5 days of culture, andthe cells exhibited greater organization and a wider distribution of a-actin compared withBSMCs cultured on poly-lactic-co-glycolic acid membranes. These preliminary data offerpromise for the use of WBPU biomaterials in bladder tissue engineering.Copyright ª 2011, Elsevier Taiwan LLC. All rights reserved.
t of Urology, West China Hospital, Huaxi Clinical College, Sichuan University, ChengDu, Sichuan
Approximately 12,710 people die from urinary bladdercancer diseases in the United States per annum [1,2]. Aradical cystectomy is often the treatment of choice andclearly indicates a need for transplantation of replacementmaterials to improve the subsequent quality of life.Through tissue engineering, the concept of generatinga functional urinary bladder using autologous cells obtainedfrom a biopsy specimen is attractive. As such, there havebeen promising reports with regard to urinary bladderregeneration [3e5].
Tissue engineering materials have often been used torepair bladder damage caused by conditions, such asinfection, resection, inflammation, and trauma. Some ofthese materials include bladder allografts, placenta,extracellular matrix, and pericardium [6e10]. In thesecases, the results have been unsatisfactory mainly becauseof the poor biocompatibilities and mechanical properties ofthe materials [10]. Additional disadvantages for the use ofsuch biological materials include their limited availabilitystemming from ethical issues and the transfer of diseasesfrom donor tissues. For these reasons, the use of syntheticmaterials for bladder reconstruction has been the focus ofrecent studies.
Poly-lactic-co-glycolic acid (PLGA) is a commonly usedbiomaterial in the field of tissue engineering [11]. Morerecently, however, waterborne polyurethanes (WBPUs) havebeen gaining popularity [12]. The latter are biodegradablenontoxic polyurethanes that are typically synthesized usingaliphatic diisocyanate, hydrolytically degradable polyesterdiol, and various chain extenders by solution polymerizationor bulk polymerization [13e20]. The preparation proceduresfor WBPUs has received increasing attention owing to thecontinuous reduction in costs and its control of volatileorganic compound emissions, thus, being more environ-mentally friendly [21e24]. To obtain WBPUs with goodbiocompatibility and satisfactory mechanical properties,a new method for the preparation of nontoxic cross-linkedWBPUs was designed using isophorone diisocyanate (IPDI),poly-e-caprolactone (PCL), polyethylene glycol (PEG), 1,4-butandiol, and L-lysine, without any other organic agentsinvolved in the synthetic process [25]. Currently, weprepare WBPU materials in our collaborative laboratory thathave good physical, biocompatible, and biodegradableproperties [25].
In the present study, we compared the biocompatibilityof WBPU with that of the commonly used PLGA. Specifi-cally, we cultured bladder smooth muscle cells (BSMCs) onboth materials and then observed the BSMC attachment,proliferation, and a-actin distribution during culture for 5days.
Materials and methods
A series of biodegradable WBPUs based on IPDI, PCL, PEG,and a chain extender were synthesized using a two-steppolymerization procedure. In the first step, IPDI and 1%stannous octoate were added to a solution of stirred PCLand PEG at 70�C under a dry nitrogen atmosphere. Afterstirring for 60 minutes at 70�C, 1,4-butandiol was added to
the melting reaction mixture for 2 hours at 62�C. Theprepolymer was then poured into an L-lysine solution foremulsification with high-speed stirring (800 revolutions perminute). Simultaneously, a diluted sodium hydroxide solu-tion was added dropwise into the polymer-water solution toneutralize the carboxyl groups of the chain extenderL-lysine at room temperature for 3 hours.
Briefly, a Vicryl knitted mesh made of polyglactin 910(90:10 copolymer of glycolic acid and lactic acid, PLGA;Ethicon, Somerville, NJ, USA) was immersed in an acidicbovine collagen solution (Type I, pH 3.2, 0.5 wt%) andfrozen at 80�C for 12 hours. The frozen solution was thenfreeze-dried under a vacuum of 0.2 Torr for 24 hours toallow the formation of a collagen sponge. The collagensponge was further cross-linked by treatment with glutar-aldehyde vapor saturated with 25% glutaraldehyde aqueoussolution at 37�C for 4 hours. Subsequently, the sponge wastreated with 0.1 mol/L glycine aqueous solution to blockunreacted aldehyde groups. Following a wash with deion-ized water and subsequent freeze-drying, the polymer-collagen hybrid mesh was prepared.
BMSCs were obtained from patients who underwenta radical cystectomy for bladder cancer. The ethicscommittee of our medical institution approved the project.The harvested smooth muscle layers were cut into smallpieces with scissors and placed in a separate dish for 1 hourat 37�C. After the explants were allowed to adhere to thebottom of the dish, Dulbecco’s modified Eagle’s mediumcontaining 10% fetal bovine serum was carefully pipettedinto the dish taking care to avoid detachment of theexplants. After 3e5 days, cells were observed to grow fromthe explants and were trypsinized and passaged to a newdish. The BSMCs in the primary cultures became confluentafter 2e3 weeks. The cultured BSMCs were seeded onto thePLGA and WBPU materials at a density of 5� 106 cells/cm2
and cultured for 1 week.Regarding theanalysis of cell proliferation, 2-(2-methoxy-
4-nitropheny)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) is thought to provide a more accurateevaluation than 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT) because WST-8 can bereduced to soluble formazan by dehydrogenases in mito-chondria and has minimal toxicity toward cells [25]. There-fore, cell proliferation was assayed using the WST-8 dye(Beyotime Institute of Biotechnology, Beijing, China)according to themanufacturer’s instructions. Briefly, 5� 103
cells/well were seeded into the wells of a 96-well flat-bottomed plate containing WBPU or PLGA membranes andgrown at 37�C for 2 hours. The absorbanceswere determinedat 450 nm using a spectrometer.
Light phase-contrast microscopy was used to assessthe morphological characteristics of the cultured BSMCs.The distribution of BSMC-a-actin confirms the smoothmuscle origin of the cells [26]. BSMCs were incubatedwith a mouse anti-human a-smooth muscle actin (a-SMA)monoclonal antibody (1:100; clone 1A4; Dako (Denmarkdako Company)) for 30 minutes. After rinsing, the cellswere incubated with alkaline phosphatase-conjugatedrabbit anti-mouse IgG (1:30; Dako) for 30 minutes,rinsed, and incubated for 15 minutes with BCIP/NBT (5-bromo-4-chloro-3- indolyl phosphate/nitroblue tetrazo-lium) chromogen for visualization of positive staining.
Figure 2. Proliferation of human bladder smooth musclecells seeded on poly-lactic-co-glycolic acid (PLGA) and water-borne polyurethane (WBPU) membranes after 1 day, 3 days,and 5 days of culture. Each bar represents the meanpercentage of cell proliferation� standard deviation at eachtime point (nZ 8 in each group). *Indicates p< 0.05, signifi-cant differences between the cell proliferation on the WBPUand PLGA membranes after 3 days and 5 days of culture.
12 F. Xu et al.
Subsequently, the cells were rinsed with distilled waterand cover-slipped using Faramount aqueous mountingmedium (Dako). Using this technique, the population ofcells expressing BSMC-a-actin could be visualized andevaluated.
Statistical analyses were performed using SPSS 11 soft-ware (SPSS, Inc., Chicago, IL, USA). Descriptive statisticswere used to describe the means and standard deviations.The significance of differences between values was deter-mined using an independent t test, and the level ofsignificance was set at a p value less than 0.05.
Results
Smooth muscle cells in the primary cultures containingpieces of bladder grew in organized arrangements (Fig. 1A).Immunofluorescence labeling with an antibody againsta-SMA identified areas of BSMCs throughout the culturesthat demonstrated high homogeneity (Fig. 1B).
After culture for 3 days and 5 days, the numbers of cellsattached to the biomaterial surfaces were significantlygreater for the WBPU membranes than for the PLGAmembranes (p< 0.05) (Figs. 2 and 3).
The morphologies and cell densities visualized by a-SMAstaining at 1 day, 3 days, and 5 days after cell seeding areshown in Fig. 4. The apparent density of a-actin staining inthe BSMCs on the WBPU membranes was greater than thatin the cells on the PLGA membranes. In addition, the BSMCswere well organized on the WBPU membranes, particularlyafter culture for 5 days. The distribution trends of BSMC-a-actin corresponded with the trends in cell attachment onthe two biomaterials at the same time points, and theWBPU membranes were more favorable than the PLGAmembranes.
Discussion
In this study, we aimed to evaluate BSMC growth andmorphology on WBPU membranes in comparison with PLGA,a commonly used biomaterial for bladder repair. We basedthe synthesis and fabrication methods for the WBPU and
Figure 1. Bladder smooth muscle cells (BSMCs) in primary cultprimary culture with small pieces of bladder exhibit good organizaa-smooth muscle actin identifies areas of BSMCs in an equivalent fiMagnification: A and B, �200.
PLGA membranes on a previous study [27], in which thefilms were created using chemical methods. In addition, wefabricated both types of membranes to have the samesurface topography. Cell culture experiments on thesepolymeric membranes provided evidence that BSMC adhe-sion, growth, and a-actin distribution were greater in cellsseeded on WBPU membranes than in cells seeded on PLGAmembranes.
BSMC-a-actin is the most commonly used marker forBSMC phenotypic characterization. Some studies haveshown that the expression of BSMC-a-actin is regulated bypolypeptide growth factors and the extracellular matrix[28,29]. The expression levels of this contractile proteinhave been used as an index of smooth muscle cell pheno-typic shifts, as well as a qualitative index for smooth musclecell density. Notably, BSMC-a-actin staining was morepronounced in cells seeded on WBPU membranes than incells seeded on PLGA membranes after 3 days and 5 daysof culture and corresponded to the trends in the cell
ure and immunofluorescence labeling (arrows). (A) BSMCs intion. (B) Immunofluorescence staining with an antibody againsteld of view and demonstrates high homogeneity of the culture.
Figure 3. Inverted phase-contrast microscopic images of bladder smooth muscle cells (BSMCs) seeded on the poly-lactic-co-glycolic acid (PLGA) and waterborne polyurethane (WBPU) membranes (arrows). Human BSMCs were cultured on PLGA (A, C,and E) and WBPU (B, D, and F) membranes and cultured for 1 day, 3 days, and 5 days. The cells appear to exhibit a greater densityon the WBPU membranes after 3 days and 5 days of culture compared with the cells on the PLGA membranes. Magnification: AeF,�200.
Biomaterials and bladder smooth muscle cells 13
numbers quantified at these time points. Interestingly,we observed that the cells were better organized on theWBPU membranes after culture for 5 days. The initial cellattachment was greater on the WBPU membranes than onthe PLGA membranes, and the cells proliferated morequickly on the WBPU membranes than on the PLGAmembranes.
The greater distribution of BSMC-a-actin within cellsseeded on the WBPU membranes and the faster BSMCproliferation rate both suggest that WBPUs support BSMCgrowth better than the more commonly used material PLGA.
It is very difficult to repair and regenerate damagedtissues and organs using the corresponding normal humantissues owing to the limitations of donation. WBPUs withfeatured properties can be designed to meet the require-ments of a specific extracellular matrix with particular
biochemical and mechanical characteristics. WBPUmembranes represent good candidates for the regenerationof tissues and organs because of their versatility and widerange of structures and properties. Such WBPU mem-branes can represent the “ideal” scaffolds for bladderregeneration if they can show a proper degradation rateand the degradation products are nontoxic. In addition,WBPUs can promote the integration of the scaffold into thenewly formed tissue.
Our purpose in this study was to develop superiorbiomaterials for bladder repair and reconstruction. Inshort- to medium-term cell cultures, WBPU materials willprovide essential mechanical support for cell adhesion andbetter proliferation. In long-term cell cultures, syntheticWBPU materials will be slowly degraded and replaced bythe BSMCs.
Figure 4. Distributions of bladder smooth muscle cell (BSMC)-a-actin visualized by immunofluorescence staining (arrows). Themorphology, a-actin distribution, and organization of human BSMCs cultured on the poly-lactic-co-glycolic acid (A, C, and E) andwaterborne polyurethane (WBPU) membranes (B, D, and E) after 1 day, 3 days, and 5 days, respectively, are shown. The cell densityappears to be greater on the WBPU membranes after 3 days and 5 days of culture. Magnification: AeE, �200.
14 F. Xu et al.
Above all, we know that BSMC adhesion, proliferation,and a-actin expression were superior on WBPU membranescompared with PLGA membranes. The present findingsclearly showed that the BSMCs had better compatibilitywith the WBPU membranes. WBPUs have excellentbiodegradation properties and the degradation rate can bewell controlled [25]. Therefore, these biomaterials witha controllable degradation rate can be used in the corre-sponding part of soft tissue engineering. We consider thatthere is a great potential for the use of WBPUs in thebiomaterials field in the near future.
In summary, we successfully seeded human BSMCs onWBPU membranes and observed that their attachment andsubsequent proliferation were greater on WBPU membranesthan on PLGA membranes. These preliminary data showpromise for the use of WBPU materials in bladder tissueengineering.
Acknowledgment
The authors thank the National Natural Science Foundationof China (30872593) for supporting this work.
References
[1] Bouhout S, Perron E, Gauvin R, Bernard G, Ouellet G,Cattan V, et al. In vitro reconstruction of an autologous,watertight, and resistant vesical equivalent. Tissue Eng Part A2010;16:1539e48.
[2] American Cancer Society. Cancer facts & figures. Atlanta, GA:American Cancer Society, Inc., http://www.cancer.org/docroot/STT/stto.aspS; 2004.
[3] Zani D, Simeone C, Arrighi N, Antonelli A, Moroni A, Ferrari M,et al. Tissue engineering in urinary bladder: morphological
and functional characterization. Arch Ital Urol Androl 2009;81:17e20.
[4] Atala A. Future trends in bladder reconstructive surgery.Semin Pediatr Surg 2002;11:134e42.
[5] Falke G, Caffaratti J, Atala A. Tissue engineering of thebladder. World J Urol 2000;18:36e43.
[6] Frederiksen H, Davidsson T, Gabella G, Uvelius B. Nervedistribution in rat urinary bladder after incorporation ofacellular matrix graft or subtotal cystectomy. Scand J UrolNephrol 2008;42:205e12.
[7] Akbal C, Lee SD, Packer SC, Davis MM, Rink RC, Kaefer M.Bladder augmentation with acellular dermal biomatrix ina diseased animal model. J Urol 2006;176(4 Pt 2):1706e11.
[8] Domingos AL, Tucci Jr S, Garcia SB, de Bessa Jr J, Cologna AJ,Martins AC. Use of a latex biomembrane for bladderaugmentation in a rabbit model: biocompatibility, clinical andhistological outcomes. Int Braz J Urol 2009;35:217e24.
[9] Walker BR, Gardner MP, Gatti JM, Lowichik A, Snow BW,Cartwright PC. Bladder augmentation in dogs using the tissuecapsule formed around perivesical tissue expander. J Urol2002;168(4 Pt 1):1534e6.
[10] Miller DC, Thapa A, Haberstroh KM, Webster TJ. Enhancedfunctions of vascular and bladder cells on Poly-Lactic-Co-Glycolic Acid Polymers with nanostructured surfaces. IEEETrans Nanobioscience 2002;1:61e6.
[11] Hsu SH, Tseng HJ, Lin YC. The biocompatibility and antibac-terial properties of waterborne polyurethane-silver nano-composites. Biomaterials 2010;31:6796e808.
[13] Ganta SR, Piesco NP, Long P, Gassner R, Motta LF, Papworth GD.Vascularization and tissue infiltration of a biodegradable poly-urethane matrix. J Biomed Mater Res A 2003;64:242e8.
[15] Yeganeh H, Lakouraj MM, Jamshidi S. Synthesis and character-ization of novel biodegradable epoxy-modified polyurethaneelastomers. J Polym Sci A Polym Chem 2005;43:2985e96.
[16] Marcos-Fernandez A, Abraham GA, Valentın JL, San RJ.Synthesis and characterization of biodegradable non-toxicpoly(ester-urethane-urea)s based on poly(e-caprolactone)and amino acid derivatives. Polymer 2006;47:785e98.
[17] Skarja GA, Woodhouse KA. In vitro degradation and erosion ofdegradable, segmented polyurethanes containing an amino
[18] Guan JJ, Sacks MS, Beckman EJ, Wagner WR. Synthesis,characterization, and cytocompatibility of elastomeric,biodegradable poly(ester-urethane) ureas based on poly(-caprolactone) and putrescine. J Biomed Mater Res 2002;61:493e503.
[19] Stankus JJ, Guan JJ, Fujimoto K, Wagner WR. Microintegratingsmooth muscle cells into a biodegradable, elastomeric fibermatrix. Biomaterials 2006;27:735e44.
[20] Kim BS, Kim BK. Enhancement of hydrolytic stability andadhesion of waterborne polyurethanes. J Appl Polym Sci 2005;97:1961e9.
[21] Yang JE, Kong JS, Park SW, Lee DJ, Kim HD. Preparation andproperties of waterborne polyurethane urea anionomers. I.The influence of the degree of neutralization and counterion.J Appl Polym Sci 2002;86:2375e83.
[22] Zhang SB, Lu HT, Zhang H, Wang B, Xu YM. Waterborne poly-urethanes: spectroscopy and stability of emulsions. J ApplPolym Sci 2006;101:597e602.
[23] Lee SK, Kim BK. High solid and high stability waterbornepolyurethanes via ionic groups in soft segments and chaintermini. J Colloid Interface Sci 2009;336:208e14.
[24] Jiang X, Li JH, Ding MM, Tan H, Ling QY, Zhong YP, et al.Synthesis and degradation of nontoxic biodegradable water-borne polyurethanes elastomer with poly(e-caprolactone) andpoly(ethylene glycol) as soft segment. Eur Polym J 2007;43:1838e46.
[25] Li XT, Zhang Y, Chen GQ. Nanofibrous polyhydroxyalkanoatematrices as cell growth supporting materials. Biomaterials2008;29:3720e8.
[26] Ram-Liebig G, Ravens U, Balana B, Haase M, Baretton G. Newapproaches in the modulation of bladder smooth muscle cellson viable detrusor constructs. World J Urol 2006;24:429e37.
[27] Garat C, Van Putten V, Refaat ZA, Dessev C, Han SY,Nemenoff RA. Induction of smooth muscle a-actin in vascularsmooth muscle cells by arginine vasopressin is mediated by c-Jun amino-terminal kinases and p38 mitogen-activatedprotein kinase. J Biol Chem 2000;275:22537e43.
[28] Drumm MR, York BD, Nagatomi J. Effect of sustained hydro-static pressure on rat bladder smooth muscle cell function.Urology 2010;75:879e85.
[29] Tock J, Van Putten V, Stenmark KR, Nemenoff RA. Induction ofSM-alpha-actin expression by mechanical strain in adultvascular smooth muscle cells is mediated through activationof JNK and p38 MAP kinase. Biochem Biophys Res Commun2003;301:1116e21.
