Kaohsiung Journal of Medical Sciences
Volume 28, Issue 1 , Pages 10-15, January 2012

Effects of different biomaterials: Comparing the bladder smooth muscle cells on waterborne polyurethane or poly-lactic-co-glycolic acid membranes

  • Feng Xu

      Affiliations

    • Department of Urology, West China Hospital, Huaxi Clinical College, SiChuan University, ChengDu, SiChuan, China
    • ,
  • Yan Wang

      Affiliations

    • Department of Urology, West China Hospital, Huaxi Clinical College, SiChuan University, ChengDu, SiChuan, China
    • ,
  • Xia Jiang

      Affiliations

    • College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, SiChuan University, ChengDu, SiChuan, China
    • ,
  • Hong Tan

      Affiliations

    • College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, SiChuan University, ChengDu, SiChuan, China
    • ,
  • Hong Li

      Affiliations

    • Department of Urology, West China Hospital, Huaxi Clinical College, SiChuan University, ChengDu, SiChuan, China
    • ,
  • Kun-Jie Wang

      Affiliations

    • Department of Urology, West China Hospital, Huaxi Clinical College, SiChuan University, ChengDu, SiChuan, China
    • Corresponding Author InformationCorresponding author. Department of Urology, West China Hospital, Huaxi Clinical College, Sichuan University, ChengDu, Sichuan 610040, China.

Received 24 February 2011; accepted 28 March 2011. published online 12 December 2011.

Article Outline

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 regeneration. 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 α-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 α-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.

Keywords: Biodegradation, Bladder smooth muscle cells, Poly-e-caprolactone, Waterborne polyurethane

 

Back to Article Outline

Introduction 

Approximately 12,710 people die from urinary bladder cancer diseases in the United States per annum [1], [2]. A radical cystectomy is often the treatment of choice and clearly indicates a need for transplantation of replacement materials to improve the subsequent quality of life. Through tissue engineering, the concept of generating a functional urinary bladder using autologous cells obtained from a biopsy specimen is attractive. As such, there have been promising reports with regard to urinary bladder regeneration [3], [4], [5].

Tissue engineering materials have often been used to repair bladder damage caused by conditions, such as infection, resection, inflammation, and trauma. Some of these materials include bladder allografts, placenta, extracellular matrix, and pericardium [6], [7], [8], [9], [10]. In these cases, the results have been unsatisfactory mainly because of the poor biocompatibilities and mechanical properties of the materials [10]. Additional disadvantages for the use of such biological materials include their limited availability stemming from ethical issues and the transfer of diseases from donor tissues. For these reasons, the use of synthetic materials for bladder reconstruction has been the focus of recent studies.

Poly-lactic-co-glycolic acid (PLGA) is a commonly used biomaterial in the field of tissue engineering [11]. More recently, however, waterborne polyurethanes (WBPUs) have been gaining popularity [12]. The latter are biodegradable nontoxic polyurethanes that are typically synthesized using aliphatic diisocyanate, hydrolytically degradable polyester diol, and various chain extenders by solution polymerization or bulk polymerization [13], [14], [15], [16], [17], [18], [19], [20]. The preparation procedures for WBPUs has received increasing attention owing to the continuous reduction in costs and its control of volatile organic compound emissions, thus, being more environmentally friendly [21], [22], [23], [24]. To obtain WBPUs with good biocompatibility and satisfactory mechanical properties, a new method for the preparation of nontoxic cross-linked WBPUs was designed using isophorone diisocyanate (IPDI), poly-e-caprolactone (PCL), polyethylene glycol (PEG), 1,4-butandiol, and l-lysine, without any other organic agents involved in the synthetic process [25]. Currently, we prepare WBPU materials in our collaborative laboratory that have good physical, biocompatible, and biodegradable properties [25].

In the present study, we compared the biocompatibility of WBPU with that of the commonly used PLGA. Specifically, we cultured bladder smooth muscle cells (BSMCs) on both materials and then observed the BSMC attachment, proliferation, and α-actin distribution during culture for 5 days.

Back to Article Outline

Materials and methods 

A series of biodegradable WBPUs based on IPDI, PCL, PEG, and a chain extender were synthesized using a two-step polymerization procedure. In the first step, IPDI and 1% stannous octoate were added to a solution of stirred PCL and PEG at 70°C under a dry nitrogen atmosphere. After stirring for 60 minutes at 70°C, 1,4-butandiol was added to the melting reaction mixture for 2 hours at 62°C. The prepolymer was then poured into an l-lysine solution for emulsification with high-speed stirring (800 revolutions per minute). Simultaneously, a diluted sodium hydroxide solution was added dropwise into the polymer-water solution to neutralize the carboxyl groups of the chain extender l-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 acidic bovine collagen solution (Type I, pH 3.2, 0.5 wt%) and frozen at 80°C for 12 hours. The frozen solution was then freeze-dried under a vacuum of 0.2Torr for 24 hours to allow the formation of a collagen sponge. The collagen sponge was further cross-linked by treatment with glutaraldehyde vapor saturated with 25% glutaraldehyde aqueous solution at 37°C for 4 hours. Subsequently, the sponge was treated with 0.1mol/L glycine aqueous solution to block unreacted aldehyde groups. Following a wash with deionized water and subsequent freeze-drying, the polymer-collagen hybrid mesh was prepared.

BMSCs were obtained from patients who underwent a radical cystectomy for bladder cancer. The ethics committee of our medical institution approved the project. The harvested smooth muscle layers were cut into small pieces with scissors and placed in a separate dish for 1 hour at 37°C. After the explants were allowed to adhere to the bottom of the dish, Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum was carefully pipetted into the dish taking care to avoid detachment of the explants. After 3–5 days, cells were observed to grow from the explants and were trypsinized and passaged to a new dish. The BSMCs in the primary cultures became confluent after 2–3 weeks. The cultured BSMCs were seeded onto the PLGA and WBPU materials at a density of 5×106 cells/cm2 and cultured for 1 week.

Regarding the analysis 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 accurate evaluation than 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) because WST-8 can be reduced to soluble formazan by dehydrogenases in mitochondria and has minimal toxicity toward cells [25]. Therefore, cell proliferation was assayed using the WST-8 dye (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer’s instructions. Briefly, 5×103 cells/well were seeded into the wells of a 96-well flat-bottomed plate containing WBPU or PLGA membranes and grown at 37°C for 2 hours. The absorbances were determined at 450nm using a spectrometer.

Light phase-contrast microscopy was used to assess the morphological characteristics of the cultured BSMCs. The distribution of BSMC-α-actin confirms the smooth muscle origin of the cells [26]. BSMCs were incubated with a mouse anti-human α-smooth muscle actin (α-SMA) monoclonal antibody (1:100; clone 1A4; Dako (Denmark dako Company)) for 30 minutes. After rinsing, the cells were incubated with alkaline phosphatase-conjugated rabbit 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 tetrazolium) chromogen for visualization of positive staining. Subsequently, the cells were rinsed with distilled water and cover-slipped using Faramount aqueous mounting medium (Dako). Using this technique, the population of cells expressing BSMC-α-actin could be visualized and evaluated.

Statistical analyses were performed using SPSS 11 software (SPSS, Inc., Chicago, IL, USA). Descriptive statistics were used to describe the means and standard deviations. The significance of differences between values was determined using an independent t test, and the level of significance was set at a p value less than 0.05.

Back to Article Outline

Results 

Smooth muscle cells in the primary cultures containing pieces of bladder grew in organized arrangements (Fig. 1A). Immunofluorescence labeling with an antibody against α-SMA identified areas of BSMCs throughout the cultures that demonstrated high homogeneity (Fig. 1B).

  • View full-size image.
  • Figure 1 

    Bladder smooth muscle cells (BSMCs) in primary culture and immunofluorescence labeling (arrows). (A) BSMCs in primary culture with small pieces of bladder exhibit good organization. (B) Immunofluorescence staining with an antibody against α-smooth muscle actin identifies areas of BSMCs in an equivalent field of view and demonstrates high homogeneity of the culture. Magnification: A and B, ×200.

After culture for 3 days and 5 days, the numbers of cells attached to the biomaterial surfaces were significantly greater for the WBPU membranes than for the PLGA membranes (p<0.05) (Figure 2, Figure 3).

  • View full-size image.
  • Figure 2 

    Proliferation of human bladder smooth muscle cells seeded on poly-lactic-co-glycolic acid (PLGA) and waterborne polyurethane (WBPU) membranes after 1 day, 3 days, and 5 days of culture. Each bar represents the mean percentage of cell proliferation±standard deviation at each time point (n=8 in each group). *Indicates p<0.05, significant differences between the cell proliferation on the WBPU and PLGA membranes after 3 days and 5 days of culture.

  • View full-size image.
  • 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 density on the WBPU membranes after 3 days and 5 days of culture compared with the cells on the PLGA membranes. Magnification: A–F, ×200.

The morphologies and cell densities visualized by α-SMA staining at 1 day, 3 days, and 5 days after cell seeding are shown in Fig. 4. The apparent density of α-actin staining in the BSMCs on the WBPU membranes was greater than that in the cells on the PLGA membranes. In addition, the BSMCs were well organized on the WBPU membranes, particularly after culture for 5 days. The distribution trends of BSMC-α-actin corresponded with the trends in cell attachment on the two biomaterials at the same time points, and the WBPU membranes were more favorable than the PLGA membranes.

  • View full-size image.
  • Figure 4 

    Distributions of bladder smooth muscle cell (BSMC)-α-actin visualized by immunofluorescence staining (arrows). The morphology, α-actin distribution, and organization of human BSMCs cultured on the poly-lactic-co-glycolic acid (A, C, and E) and waterborne polyurethane (WBPU) membranes (B, D, and E) after 1 day, 3 days, and 5 days, respectively, are shown. The cell density appears to be greater on the WBPU membranes after 3 days and 5 days of culture. Magnification: A–E, ×200.

Back to Article Outline

Discussion 

In this study, we aimed to evaluate BSMC growth and morphology on WBPU membranes in comparison with PLGA, a commonly used biomaterial for bladder repair. We based the synthesis and fabrication methods for the WBPU and PLGA membranes on a previous study [27], in which the films were created using chemical methods. In addition, we fabricated both types of membranes to have the same surface topography. Cell culture experiments on these polymeric membranes provided evidence that BSMC adhesion, growth, and α-actin distribution were greater in cells seeded on WBPU membranes than in cells seeded on PLGA membranes.

BSMC-α-actin is the most commonly used marker for BSMC phenotypic characterization. Some studies have shown that the expression of BSMC-α-actin is regulated by polypeptide growth factors and the extracellular matrix [28], [29]. The expression levels of this contractile protein have been used as an index of smooth muscle cell phenotypic shifts, as well as a qualitative index for smooth muscle cell density. Notably, BSMC-α-actin staining was more pronounced in cells seeded on WBPU membranes than in cells seeded on PLGA membranes after 3 days and 5 days of culture and corresponded to the trends in the cell numbers quantified at these time points. Interestingly, we observed that the cells were better organized on the WBPU membranes after culture for 5 days. The initial cell attachment was greater on the WBPU membranes than on the PLGA membranes, and the cells proliferated more quickly on the WBPU membranes than on the PLGA membranes.

The greater distribution of BSMC-α-actin within cells seeded on the WBPU membranes and the faster BSMC proliferation rate both suggest that WBPUs support BSMC growth better than the more commonly used material PLGA.

It is very difficult to repair and regenerate damaged tissues and organs using the corresponding normal human tissues owing to the limitations of donation. WBPUs with featured properties can be designed to meet the requirements of a specific extracellular matrix with particular biochemical and mechanical characteristics. WBPU membranes represent good candidates for the regeneration of tissues and organs because of their versatility and wide range of structures and properties. Such WBPU membranes can represent the “ideal” scaffolds for bladder regeneration if they can show a proper degradation rate and the degradation products are nontoxic. In addition, WBPUs can promote the integration of the scaffold into the newly formed tissue.

Our purpose in this study was to develop superior biomaterials for bladder repair and reconstruction. In short- to medium-term cell cultures, WBPU materials will provide essential mechanical support for cell adhesion and better proliferation. In long-term cell cultures, synthetic WBPU materials will be slowly degraded and replaced by the BSMCs.

Above all, we know that BSMC adhesion, proliferation, and α-actin expression were superior on WBPU membranes compared with PLGA membranes. The present findings clearly showed that the BSMCs had better compatibility with the WBPU membranes. WBPUs have excellent biodegradation properties and the degradation rate can be well controlled [25]. Therefore, these biomaterials with a controllable degradation rate can be used in the corresponding part of soft tissue engineering. We consider that there is a great potential for the use of WBPUs in the biomaterials field in the near future.

In summary, we successfully seeded human BSMCs on WBPU membranes and observed that their attachment and subsequent proliferation were greater on WBPU membranes than on PLGA membranes. These preliminary data show promise for the use of WBPU materials in bladder tissue engineering.

Back to Article Outline

Acknowledgment 

The authors thank the National Natural Science Foundation of China (30872593) for supporting this work.

Back to Article Outline

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 A. 2010;16:1539–1548
  2. American Cancer Society . Cancer facts & figures. Atlanta, GA: American Cancer Society, Inc.; 2004;http://www.cancer.org/docroot/STT/stto.aspS
  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;81:17–20
  4. Atala A. Future trends in bladder reconstructive surgery. Semin Pediatr Surg. 2002;11:134–142
  5. Falke G, Caffaratti J, Atala A. Tissue engineering of the bladder. World J Urol. 2000;18:36–43
  6. Frederiksen H, Davidsson T, Gabella G, Uvelius B. Nerve distribution in rat urinary bladder after incorporation of acellular matrix graft or subtotal cystectomy. Scand J Urol Nephrol. 2008;42:205–212
  7. Akbal C, Lee SD, Packer SC, Davis MM, Rink RC, Kaefer M. Bladder augmentation with acellular dermal biomatrix in a diseased animal model. J Urol. 2006;176(4 Pt 2):1706–1711
  8. Domingos AL, Tucci S, Garcia SB, de Bessa J, Cologna AJ, Martins AC. Use of a latex biomembrane for bladder augmentation in a rabbit model: biocompatibility, clinical and histological outcomes. Int Braz J Urol. 2009;35:217–224
  9. Walker BR, Gardner MP, Gatti JM, Lowichik A, Snow BW, Cartwright PC. Bladder augmentation in dogs using the tissue capsule formed around perivesical tissue expander. J Urol. 2002;168(4 Pt 1):1534–1536
  10. Miller DC, Thapa A, Haberstroh KM, Webster TJ. Enhanced functions of vascular and bladder cells on Poly-Lactic-Co-Glycolic Acid Polymers with nanostructured surfaces. IEEE Trans Nanobioscience. 2002;1:61–66
  11. Hsu SH, Tseng HJ, Lin YC. The biocompatibility and antibacterial properties of waterborne polyurethane-silver nanocomposites. Biomaterials. 2010;31:6796–6808
  12. Guan JJ, Fujimoto KL, Sacks MS, Wagner WR. Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications. Biomaterials. 2005;26:3961–3971
  13. Ganta SR, Piesco NP, Long P, Gassner R, Motta LF, Papworth GD. Vascularization and tissue infiltration of a biodegradable polyurethane matrix. J Biomed Mater Res A. 2003;64:242–248
  14. Skarja GA, Woodhouse KA. Structure property relationships of degradable polyurethane elastomers containing an amino acid-based chain extender. J Appl Polym Sci. 2000;75:1522–1534
  15. Yeganeh H, Lakouraj MM, Jamshidi S. Synthesis and characterization of novel biodegradable epoxy-modified polyurethane elastomers. J Polym Sci A Polym Chem. 2005;43:2985–2996
  16. Marcos-Fernandez A, Abraham GA, Valentın JL, San RJ. Synthesis and characterization of biodegradable non-toxic poly(ester-urethane-urea)s based on poly(e-caprolactone) and amino acid derivatives. Polymer. 2006;47:785–798
  17. Skarja GA, Woodhouse KA. In vitro degradation and erosion of degradable, segmented polyurethanes containing an amino acid-based chain extender. J Biomater Sci Polym Edn. 2001;12:851–873
  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;61:493–503
  19. Stankus JJ, Guan JJ, Fujimoto K, Wagner WR. Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix. Biomaterials. 2006;27:735–744
  20. Kim BS, Kim BK. Enhancement of hydrolytic stability and adhesion of waterborne polyurethanes. J Appl Polym Sci. 2005;97:1961–1969
  21. Yang JE, Kong JS, Park SW, Lee DJ, Kim HD. Preparation and properties of waterborne polyurethane urea anionomers. I. The influence of the degree of neutralization and counterion. J Appl Polym Sci. 2002;86:2375–2383
  22. Zhang SB, Lu HT, Zhang H, Wang B, Xu YM. Waterborne polyurethanes: spectroscopy and stability of emulsions. J Appl Polym Sci. 2006;101:597–602
  23. Lee SK, Kim BK. High solid and high stability waterborne polyurethanes via ionic groups in soft segments and chain termini. J Colloid Interface Sci. 2009;336:208–214
  24. Jiang X, Li JH, Ding MM, Tan H, Ling QY, Zhong YP, et al. Synthesis and degradation of nontoxic biodegradable waterborne polyurethanes elastomer with poly(e-caprolactone) and poly(ethylene glycol) as soft segment. Eur Polym J. 2007;43:1838–1846
  25. Li XT, Zhang Y, Chen GQ. Nanofibrous polyhydroxyalkanoate matrices as cell growth supporting materials. Biomaterials. 2008;29:3720–3728
  26. Ram-Liebig G, Ravens U, Balana B, Haase M, Baretton G. New approaches in the modulation of bladder smooth muscle cells on viable detrusor constructs. World J Urol. 2006;24:429–437
  27. Garat C, Van Putten V, Refaat ZA, Dessev C, Han SY, Nemenoff RA. Induction of smooth muscle a-actin in vascular smooth muscle cells by arginine vasopressin is mediated by c-Jun amino-terminal kinases and p38 mitogen-activated protein kinase. J Biol Chem. 2000;275:22537–22543
  28. Drumm MR, York BD, Nagatomi J. Effect of sustained hydrostatic pressure on rat bladder smooth muscle cell function. Urology. 2010;75:879–885
  29. Tock J, Van Putten V, Stenmark KR, Nemenoff RA. Induction of SM-alpha-actin expression by mechanical strain in adult vascular smooth muscle cells is mediated through activation of JNK and p38 MAP kinase. Biochem Biophys Res Commun. 2003;301:1116–1121

PII: S1607-551X(11)00152-5

doi:10.1016/j.kjms.2011.06.031

Kaohsiung Journal of Medical Sciences
Volume 28, Issue 1 , Pages 10-15, January 2012

Article Tools

* Image Usage & Resolution