Advanced Review
Nanostructured bladder tissue replacements Young Wook Chun,1 Hojean Lim,2 Thomas J. Webster1 and Karen M. Haberstroh1∗ The interaction between cells or tissues and natural or synthetic materials which mimic the natural biological environment has been a matter of great interest in tissue engineering. In particular, surface properties of biomaterials (regardless of whether they are natural or synthetic) have been optimized using nanotechnology to improve interactions with cells for regenerative medicine applications. Specifically, in vivo and in vitro studies have demonstrated greater bladder tissue growth on polymeric surfaces with nanoscale to submicron surface features. Improved bladder cell responses on nanostructured polymers have been correlated to unique nanomaterial surface features leading to greater surface energy which influences initial protein interactions. Moreover, coupled with the observed greater in vitro and in vivo bladder cell adhesion as well as proliferation on nanostructured compared to conventional synthetic polymers, decreased calcium stone formation has also been measured. In this article, the importance of nanostructured biomaterial surface features for bladder tissue replacements are reviewed with thoughts on future directions for this emerging field. 2010 John Wiley & Sons, Inc. WIREs Nanomed Nanobiotechnol 2011 3 134–145 DOI: 10.1002/wnan.89
INTRODUCTION
N
anotechnology has largely emerged in the last decade of the 20th century based on the development of new enabling technologies for medicine. Nanotechnology provides the opportunity to directly control initial protein interactions on implants which dictate cell functions, through the reliable, repeatable creation of biologically inspired nanoscale surface features.1 Along this line, nanotechnology has been used to develop better implantable devices for treating diseased cells or tissues to improve human health.2,3 In particular, it is noted that cells elicit specific, distinct reactions depending on implant surface features since proteins preferentially adsorb to biomaterials based on their associated surface energy which can be easily controlled through nanotechnology. For example, it was recently reported4 that nanoscale surface structures compared to submicron surface features on biomaterials led to the increased ∗ Correspondence 1
to: Karen
[email protected]
Division of Engineering, Brown University, Providence, RI, USA
2 Department
of Neuroscience, College of William and Mary, Williamsburg, VA, USA DOI: 10.1002/wnan.89
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adsorption of fibronectin, a major component in the bladder extracellular matrix that accelerates bladder cellular responses. Therefore, a key design parameter for achieving maximal cellular responses on tissue engineering materials is the degree of nanometer surface features, easily controlled by a number of nanotechnology-based processes. Along this line, this review demonstrates the importance of specific nanostructured biomaterial surface features that mimic that of native bladder tissue in bladder tissue replacements and how such surface features enhance cellular responses by increasing implant surface energy, thereby leading to better initial protein interactions.
BLADDER INJURY AND HEALING Injury to the bladder is uncommon (e.g., resulting from only about 8–10% of pelvic fractures).5 Because the bladder is located within the bony structures of the pelvis, it is protected from most external forces. However, it has been reported that injury may occur if there is a strike to the pelvis that is severe enough to break bones and cause bone fragments to penetrate the bladder wall.6 Also, congenital deformities or abnormalities (such as
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exstrophy and myelomeningocele) can lead to a variety of bladder injuries causing partial or complete lack of sensation, leg paralysis, and urinary tract infections.7,8 In addition, high-grade bladder cancer (carcinoma) has been difficult to remove through conventional methods.9 If left untreated or when recurring even after removal, this form of cancer will seemingly progress to an invasive stage. These problematic bladders have been conventionally treated by cystoplasty using natural tissue replacements and bladder reconstruction procedures such as omental cystoplasty, peritoneal cystoplasty, colocystoplasty, gastrocystoplasty, and ureter cystoplasty for bladder augmentation or the partial replacement of bladder tissue10,11 when conventional treatments (such as immunotherapy) have failed. However, such cystoplasties have generally involved the incorporation of intestinal mucosa into the urinary tract and have sometimes generated potential complications that arose from such incorporation. For example, cystoplasties have demonstrated an approximate 600fold increased risk of malignancy in the anastomosed gastrointestinal tissue compared to the risk of bladder cancer.12 As well as increasing malignancy, it has been reported that recurrent urinary tract infections, calculus formation, metabolic disorders, transplant perforation, excessive mucous production, and poor healing with urinary fistula formation can ensue.13,14 Other naturally occurring biopolymer matrices such as the skin, placenta, dura, small intestinal submucosa (SIS), and acellular tissue matrix have been developed. In particular, SIS is a collagen based biomaterial obtained from the porcine small intestine and has been successfully utilized to promote the regeneration of the bladder.15–17 As well as SIS, acellular tissue matrices derived from full-thickness porcine bladders have shown promise. The scaffold is produced by a detergent and enzymatic extraction process which removes all cellular constituents from the bladder extracellular matrix. The resulting material consists primarily of collagen and elastin.18 However, although still under investigation, some have suggested that these materials have not served as the panacea for all bladder tissue replacements or augmentations because of poor mechanical and structural properties during the remodeling process. To overcome such potential mechanical deficiencies, recent studies have reported that cells seeded in a collagen gel contract the gel, thus, improving scaffold strength.19 Alternatively, it has been shown that through plastic compression, excess fluids were removed from collagen gels resulting in a more dense and, therefore, mechanically stronger structure whose tensile properties approach that of native tissue.20,21 Vo lu me 3, March/April 2011
For example, Engelhardt et al. determined greater bladder urothelial cell proliferation and effective distribution in vitro using plastic compressed collagen gels.22 Although showing promise, the development of these natural polymers has been at the initial stage and needs to be pursued more for clinical trials to improve bladder regeneration. Of course, high mechanical strength can be easily created and controlled by utilizing synthetic polymeric scaffolds. Such synthetic polymeric materials offer major potential advantages over natural materials in that they can be fabricated on a large scale, to degrade following implantation at predetermined rates (or not at all), can be designed to mimic the material properties of the native tissue they are designed to replace, and can avoid potential xenogeneic or pathogenic contaminants. In terms of these advantages, synthetic polymers [such as poly glycolic acid (PGA), poly l-lactic acid (PLLA), poly lactic–glycolic acid (PLGA), poly-ε-caprolactone (PCL), and polyurethane (PU)] have been employed in composites with various collagens to maximize mechanical and physical properties important for bladder tissue engineering applications.23–26 On the synthetic side of material design, nanotechnology has been utilized to improve many properties of bladder tissue engineering materials. It has been demonstrated that an optimal bladder tissue response to synthetic materials could be achieved by enhancing the interactions between bladder cells (especially, urothelial cells which comprise the urothelium) and the biomaterial through nanotechnology. Specifically, recent research efforts27–29 have shown the importance of biomaterial surface roughness for bladder regeneration when such roughness mimics the natural nanometer roughness of the bladder itself. Specifically, the urothelium and smooth muscle layers contain two major cells of the bladder which are surrounded by numerous nanostructured proteins (such as collagen and elastin). Researchers have found that nanosized features on the surface of synthetic materials are related to the degree of interaction between proteins and cells and, eventually, can be used to increase cell functions (such as their adhesion).4,29–33
BLADDER MATERIAL NANOMETER SURFACE FEATURES AND ASSOCIATED SURFACE ENERGY It has been widely noted that protein adsorption on surfaces is affected by implant chemistry, topography, roughness, etc.33,34 Accordingly, cellular responses to an implant are dictated by the types of proteins
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that initially adsorb to the implant surface and the bioactivity of those proteins once adsorbed, which, in turn, are directly influenced by the protein adsorption process. One of the important characteristics of such protein adsorption on materials is the ability to freely wet the implant surface with biological solutions. At the liquid–material surface interface, if the liquid molecules have a stronger attraction to the solid surface molecules than to each other (i.e., the adhesive forces are stronger than the cohesive forces), wetting of the surface occurs. Alternately, if the liquid molecules are more strongly attracted to each other than the solid surface molecules (i.e., the cohesive forces are stronger than the adhesive forces), the liquid containing the proteins beads up and does not completely wet the surface.35,36 One way to quantify the surface wetting characteristics of a material is to measure the contact angle of a liquid drop placed on its surface.36 The contact angle is the angle formed at the material and liquid interface. The wetting ability of the liquid is a function of the surface energy of the implanted material–liquid interface. The surface energy across a liquid–solid interface is a measure of the energy required to form the new liquid surface at the solid interface. Therefore, increasing surface area by creating various topographical features or changing surface chemistry by functionalization of the implant with charged species increases (especially at the nanometer level) surface energy to change the type of adsorbed proteins (Figure 1). Recent published work has demonstrated that such increased surface roughness (at the nanometer and submicron levels) improved the adsorption of select proteins important for bladder cell functions. According to such studies, increasing the roughness on surfaces (such as titanium used as bladder stents) through the use of e-beam evaporation, anodization, and other nanotechnology-based processes increased vitronectin adsorption (by 20%) compared to nanosmooth features. Moreover, when compared to micron scale titanium roughness, titanium nanoroughness improved the growth of various tissues (such as vascular, skin, cartilage, etc.). As with titanium surfaces, polymers (e.g., polycarbonate-urethane, PCU) created with increased roughness, for example, by adding carbon nanotubes (CNTs) with dimensions less than 100 nm, increased the adsorption of fibronectin without changing implant surface chemistry (Figure 2). Because increased adsorption of collagen I or IV and laminin as well as fibronectin has enhanced bladder cell adhesion to actively synthesize deoxyribonucleic acid synthesis for metabolism (Table 1, Figure 3, 37), such data represent a critical biological event that 136
can be easily controlled by using nanomaterials to create nanostructured roughness beneficial for bladder applications.30 In particular, the use of polymers and of nanostructured surfaces encourages the growth of bladder tissue for numerous bladder applications.38 Accordingly, it has been very meaningful to compare bladder cellular responses (specifically, smooth muscle and urothelial cells) on currently used bladder patches with micron, submicron and nanostructured surfaces, as will be described in the next section.
BLADDER SMOOTH MUSCLE CELLS The bladder wall has various types of tissue layers (such as the mucosa, muscle submucosa, and perivesical fat). Each layer has several types of cells (e.g., more than four kinds of cells reside in the adventitia and serosas while the perivesical fat has urothelial and smooth muscle cells). Specifically, smooth muscle cells in the bladder play an important role in the stretching and relaxing of the bladder as needed. To regenerate the bladder muscle layer, biodegradable polymers have become popular. PLGA, PCL and PU have successfully promoted the growth of bladder smooth muscle and urothelial cells in vitro.39–42 Moreover, cell-engineered matrices created by placing the urothelial cells on one side of PGA coated with PLGA (50:50) and the bladder muscle cells on the opposite side in vitro were recently successfully tested in vivo.43 Biodegradable three-dimensional matrices have also been created by isolating and expanding autologous bladder urothelial and muscle cells in vitro and then implanting such cells with polymer matrices in patients with end-stage bladder disease. Data demonstrated that the margin between the tissue engineered matrices and the native bladder tissue grossly disappeared and a lumen consisting of urothelial cells surrounded by submucosa and muscle were observed. Such research showed the promise of cell based constructs and their application to regenerate bladders in patients with bladder disease.44 However, notwithstanding the aforementioned outstanding results, many conventional synthetic biodegradable polymers and natural scaffolds investigated to date have shown limited success in bladder tissue regeneration applications due to their poor mechanical stability, adverse tissue and immune responses, poor urothelial regeneration, excessive stone formation, etc. as mentioned in the previous chapter. Thus, it is necessary to develop bladder organ replacements with alternative strategies to combat these problems, but staying with the strategy to utilize biodegradable polymers in which such materials
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(a) Flat
50 nm
500 nm Flat
300 100
(b)
Nano
20 nm
500 nm Nano
300 100
(c)
Submicron
300 nm
FIGURE 1 | Unique surface roughness, area, and 5 µm
Submicron
3
8
** 6
12 10
4
8 6
2
4 2
**
0 Flat
Nano Submicron Substrate structures
are quickly and efficiently replaced by healthy host tissue. As mentioned, it is known that bladder cells respond differently to nanodimensional, micron, and flat surface features; this could be because as mentioned the surfaces of these nanomaterials mimic their natural environment (i.e., extracellular matrix proteins in bladder tissue are nanodimensional). To date, the investigation of smooth muscle cell functions on biodegradable polymers has provided evidence that the topography of such surfaces affect Vo lu me 3, March/April 2011
0
Surface area diff. (%)
14
(e) 72 Surface energy (mJ/m2)
16
(d)
70
**
68 66
*
64
*
62 60 58 56
**
54
44 42 40 38 36 34 32 30 28 26 24 22 20 18 16
Contact angle (θ)
1
Roughness (RMS, nm)
energy for nanostructured titanium (a material commonly used in bladder stent applications) compared nanosmooth titanium. Contact angles demonstrated increased hydrophilicity from (a) flat to (b) nanometer and (c) submicron (increasing from left to right) surface featured titanium. (d) Titanium surface roughness [root mean square (RMS) as measured by atomic force microscopy] and surface area (%) measurements showed small differences between flat and nanometer rough (0.7%) titanium samples while submicron rough titanium showed greater increases in RMS values (more than 10 nm) and surface area (7%) compared to flat titanium surfaces. (e) Compared to flat titanium (54 mJ/m2 ), nanometer rough titanium (62 mJ/m2 ) and submicron rough (69 mJ/m2 ) titanium showed greater increments in surface energy. All error bars are mean ± SEM; n = 3; *p < 0.01 (compared to flat titanium) and **p < 0.01 (compared to nanometer titanium).30
Flat
Nano Submicron Substrate structures
cellular adhesion and proliferation compared to conventional polymers. For example, Thapa et al. designed biodegradable polymers [such as Food and Drug Administration (FDA) approved PLGA, PCL, and PU] with micron, submicron and nanometer surface features by a novel chemically etching technique breaking ester and ether bonds via NaOH and HNO3 treatments, respectively.45 According to their report, human smooth muscle cell functions on PLGA, PCL, and PU with nanodimensional surface
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(a)
(e)
µm 2
(b) 500 nm
(f)
µm 2 1
(c) 500 nm
22 20 18 16 14 12 10 8 6 4 2 0
45 40 35 30 25 20 15 10 5 0
***
** * 0
5 10 15 20 25 30 35 40 CNTs in PCU (weight %)
**
***
*
0
5 10 15 20 25 30 35 40 CNTs in PCU (weight %)
µm 2 1
(d) 500 nm
µm 2 1
features in a range of 50–100 nm were greater than on respective conventional polymers consisting of nanosmooth surface features. In comparison with micron or submicron surfaces, nanosurfaces promoted bladder smooth muscle cell functions (e.g., elastin synthesis and collagen production) compared to micron and submicron respective polymers. Importantly, the micron, submicron and nanostructured polymers compared in such studies had similar chemistry, thus, providing strong evidence that it was the polymeric nanostructured surface features alone that promoted bladder smooth muscle cell functions. It was hypothesized in such studies that any material could promote bladder smooth muscle cell function by simply figuring out a way to create nanometer features on such polymers. 138
Surface energy (mJ/m2)
surface energy, and fibronectin (FN) adsorption on greater compositions of carbon nanotubes (CNTs) in polycarbonate-urethane (PCU)/CNTs composites. (a) Smooth surfaces had root mean square (RMS) values of 1.9 (70.14) nm for pure PCU. (b) Surfaces with RMS values of 2.5 (70.09) nm were obtained by adding 13% (weight) CNTs in PCU. (c) Surfaces with RMS values of 7.3 (71.96) nm were obtained by adding 20% CNTs in PCU. (d) Surfaces with RMS values of 17.7 (72.89) nm were obtained by adding 33% (weight) CNTs in PCU. (e) Increased effective RMS values by RMSeffective = SN × RMSfiltered . (f) Surface energy versus composition: the Owens–Wendt equation (circle) showed that greater surface energies were obtained by increasing the weight percentages of CNTs in PCU. Inset images (contact angles) show increased hydrophilicity with higher percentages of CNT in composites (i.e., high effective nanosurface roughness). (g) Increased effective FN adsorption: higher percentage (weight) of CNTs in PCU shows greater FN adsorption (by FNeffective = SN × FNmeasured ) after surface area normalization. All X:Y composites represent weight ratios of CNTs in PCU (on x-axis). All error bars are mean ± SEM; n = 5; *p < 0.1 (compared to control PCU), **p < 0.05 (compared to PCU), and ***p < 0.01 (compared to PCU). (Reprinted with permission from Ref 4. Copyright 2007 Elsevier).
1
(g) FNeff adsorption (normalized)
FIGURE 2 | Increased effective roughness,
reff (RMSeff, nm)
500 nm
1.0
*
0.8
**
***
0.6 0.4 0.2 0.0
0
5 10 15 20 25 30 35 40 CNTs in PCU (weight %)
To confirm the importance of nanometer topography on bladder tissue growth, they also created nanostructured polymer casts consisting of PLGA and PU; these casts possessed the same topography as the original nanostructured polymers, but in the absence of a chemical change caused by the original chemical etching technique used to create the materials. It was found that the nanometer topography alone (not chemistry change due to chemical etching) enhanced the function of bladder smooth muscle cells (Figure 4). Also, Pattison et al. created three-dimensional nanostructured PLGA and PU scaffolds using similar chemical etching techniques via NaOH and HNO3 soaking as previously stated and demonstrated greater elastin and collagen production by bladder smooth muscle cells (under a mechanical environment similar
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TABLE 1 Summary of Deoxyribonucleic Acid Synthesis, Metabolic Activity, and Initial Cell Attachment of Normal Human Urothelial Cells Grown on Protein Coatings37
DNA Synthesis1
mean4
Metabolic Activity2
p-value (n)
Type IV collagen
0.17 ± 0.06
0.004 (44)
Type I collagen
0.08 ± 0.03
0.02 (40)
Fibronectin
0.21 ± 0.06
Laminin
0.27 ± 0.06
Initial Attachment3
mean4
p-value (n)
mean4
p-value (n)
0.00 ± 0.04
0.92 (25)
0.05 ± 0.01
