Modulating physical properties of porcine urethra with injection of novel biomimetic proteoglycans ex vivo

Abstract

Urinary incontinence is a significant challenge for women who are affected by it. We propose augmenting the tissue structure to restore normal biomechanics by molecularly engineering the tissue using a novel family of biomimetic proteoglycans (BPGs). This work examines the ability of BPGs to modulate the mechanical and physical properties of porcine urethras ex vivo to determine the feasibility of BPGs to be implemented as molecular treatment for stress urinary incontinence (SUI). We investigated compliance by performing a unique radial expansion testing method using urethras from six- to nine-month-old pigs. The urethras were injected with 0.5 ml BPG solution at three sites every approximately 120° (conc.: 25 mg ml⁻¹, 50 mg ml⁻¹ and 75 mg ml⁻¹ in 1× phosphate-buffered saline (PBS); n = 4 per group) and compared them with PBS-injected controls. Young's modulus was calculated by treating the urethra as a thin-walled pressure vessel. A water uptake study was performed by soaking 10 mm urethra biopsy samples that were injected with 0.1 ml BPG solution (conc.: 50 mg ml⁻¹, 100 mg ml⁻¹ and 200 mg ml⁻¹ in 1× PBS; n = 6 per group) in 5 ml PBS for 24 h. Although there was no significant difference in Young's modulus data, there were differences between groups as can be seen in the raw radial expansion testing data. Results showed that BPGs have the potential to increase hydration in samples, and that there was a significant difference in water uptake between BPG-injected samples and the controls (100 mg ml⁻¹ samples versus PBS samples, p < 0.05). This work shows that BPGs have the potential to be implemented as a molecular treatment for SUI, by restoring the diminished proteoglycan content and subsequently increasing hydration and improving the compliance of urethral tissue.

1. Introduction

Stress urinary incontinence (SUI) is defined as the involuntary leakage of urine and is prevalent in 56% of postmenopausal women [1]. Leakage occurs during activities that cause an increase in abdominal pressure and can be induced by coughing, exercising, lifting and in severe cases even laughing and walking. SUI is linked to a decrease in compliance and an increase in stiffness of urethral tissue [2,3]. Current treatments are limited and because they are either invasive (retropubic surgeries) or can lead to serious side effects, or even if they are only minimally invasive (bulking agents), they only remain effective for several months [4]. This introduces the need for an improved treatment option. Biomimetic proteoglycans (BPGs) are novel molecules that are composed of an enzymatically resistant polyacrylic acid (PAA) core (250 kDa) with covalently attached chondroitin sulfate (CS) bristles (22 kDa) [5,6], which may have the potential to molecularly engineer the urethra by restoring the depleted proteoglycan content in the tissue.

1.1. Mechanical properties

The female urethra is an intricate tubular structure which during the filling phase of the bladder undergoes passive and active closure mechanisms [7]. Continence is dependent on the ability of the urethra to resist dilation as urine accumulates inside the bladder [7,8]. One way of evaluating continence is to investigate the underlying mechanical properties of the urethra. However, the mechanical properties of the urethra are not well documented in the literature and need to be studied further.

The mechanical properties of the urethra are affected by both smooth and striated muscles and the surrounding connective tissue. Jankowski et al. [9] investigated the urethral pressure–diameter response of different regions of the female rat urethra, and proposed and validated a system to characterize the biomechanical function of the urethra in vivo.

A study conducted by Trowbridge et al. [10] with 82 nulliparous women (aged 21–70 years) investigated how ageing affects the lower urinary tract. Experimental procedures included urethral hypermobility, urodynamics, vaginal closure force, pelvic organ quantification and uroflow. The results revealed a correlation between age and maximum urethral closure pressure; with increasing age the maximum urethral closure pressure decreased approximately 15 cm-H₂O per decade (p < 0.001). Additionally, it was discovered that increasing age did not affect levator function as well as urethral support and organ support. A decrease in maximal urethral closure pressure with increasing age may be attributable to the organizational changes that occur with ageing in tissue. The morphological analyses of paraurethral connective tissue by Falconer et al. [2] showed that the ultrastructure of continent and incontinent individuals is different, with thicker collagen fibrils, higher cross-linking of collagen fibrils and a lower proteoglycan–collagen ratio in incontinent women. Specifically, the results revealed that the collagen content from paraurethral connective tissue biopsy samples in continent individuals was 13.5 ± 3.3 µg mg⁻¹ wet weight and 17.9 ± 6.2 µg mg⁻¹ wet weight for incontinent individuals. The proteoglycan contents were 1.96 ± 0.57 µg mg⁻¹ wet weight and 1.86 ± 0.48 µg mg⁻¹ wet weight for continent and incontinent individuals, respectively. This resulted in a significantly lower proteoglycan–collagen ratio for incontinent individuals, p = 0.03 [2]. These structural and biochemical differences may lead to a less compliant tissue, making it more difficult for the musculature to ensure complete closure of the urethra [2].

1.2. Biomimetic proteoglycans

The Marcolongo laboratory synthesizes and characterizes a novel family of BPGs, which are resistant to enzymatic degradation and which may have the ability to molecularly engineer the extracellular matrix (ECM). BPGs mimic the three-dimensional bottle brush architecture and hydrating properties of natural proteoglycans, and are composed of an enzymatically resistant PAA core with covalently attached, naturally occurring CS bristles (approx. 22 kDa) [5,6,11]. CS is a linear sulfated chain of polysaccharides that is commonly attached to proteins. BPG250 is composed of an approximately 250 kDa PAA core and with the attached CS bristles has a molecular weight of approximately 1600 kDa; it is displayed in the atomic force microscopy (AFM) image in figure 1b [6]. Briefly, the AFM images were taken on a Multimode III atomic force microscope (BrukerNano) with tapping mode implementing nanosized silicon tips.

Figure 1.

AFM images of (a) aggrecan, a natural proteoglycan, and (b) BPG250. BPGs are composed of CS chains that are covalently linked to an enzymatically resistant PAA core. BPGs highly resemble the three-dimensional bottlebrush architecture of natural proteoglycans [6]. (Online version in colour.)

The molecular characteristics of the BPGs are provided in table 1. The characteristics of the natural proteoglycan aggrecan are also listed for comparison.

Table 1.

Molecular characteristics of the natural proteoglycan aggrecan, and proteoglycan mimics BPG10 and BPG250.

	molecular weight (MW)	number of bristles	bristle-to-bristle spacing	total MW
natural aggrecan	approximately 200–250 kDa	approximately 100	3–5 nm	approximately 2000 kDa
BPG10	approximately 10 kDa	approximately 7–8	3–4 nm	approximately 160–180 kDa
BPG250	approximately 250 kDa	approximately 60	approximately 14 nm	approximately 1600 kDa

Although natural aggrecan has a higher molecular weight than both BPG10 and BPG250, the water uptake of aggrecan is approximately 38% compared with approximately 60–62% for BPGs [5,6]. This increased water uptake for BPGs could potentially be due to the higher charge density of BPG molecules. Unlike BPGs, natural aggrecan has a non-charged core and the charge depends on the covalently linked GAG chains. BPGs have an anionic PAA core in addition to the anionic GAG side chains and may have an overall higher charge density as a result of this.

1.3. Water uptake properties of polymers

Water uptake properties are an important aspect of polymers in a wide range of fields including composite materials, fire-extinguishing gels, cement, textile processing, packaging, agriculture, sensors, personal care products and biomedical engineering [12,13]. In biomedical fields, examining the water uptake properties of polymers is vital to better understanding their capabilities for tissue engineering, drug delivery, regenerative medicine and wound-healing applications [12–14]. Furthermore, the water uptake properties of polymers provide insight into other polymer features. Generally, the water uptake of polymers, as well as the properties of cross-linked polymer networks such as hydrogels, can be used as an indication of the hydrolytic degradation rate of the polymer(s) [14,15]. The rate of hydrolysis typically decreases as the water uptake properties decrease [15]. An important characteristic of a biopolymer intended to function as a proteoglycan mimic, and to molecularly engineer the urethra for SUI, is its ability to attract water to the surrounding tissue. Studies have shown that the urethral tissue of incontinent women is associated with a decrease in volume and compliance of urethral tissue, possibly due to a decreased proteoglycan–collagen ratio [2,3].

Previously, the water uptake of CS, BPG10 and BPG250 has been analysed using a thermal gravimetric analyser (TGA) at 37°C, 90% relative humidity, over a 24 h time period [5,6]. The water uptake was approximately 41% for CS, approximately 62% for BPG10 and approximately 60% for BPG250 compared with approximately 38% for natural aggrecan [5,6]. Overall, each BPG molecule contains approximately 16% of additional charged groups compared with aggrecan, which may be attributed to the higher water uptake of BPGs [5].

As of now, the water uptake properties of BPGs injected into porcine urethra ex vivo have not been studied. In this paper, water uptake experiments of urethral tissue injected with BPGs are conducted, attempting to simulate the response of BPGs implanted into tissue in vivo, and we investigate their ability to attract water to surrounding tissue.

Incontinence is linked to a decrease in volume and compliance of urethral tissue [2,3]. The purpose of this study is to evaluate the mechanical properties and water uptake properties of porcine urethras injected with BPG250 ex vivo. It is hypothesized that incorporation of BPG250 in tissue will increase the compliance and hydration of the tissue through enhanced water uptake properties of the anionic BPGs.

2. Methods

2.1. Synthesis of biomimetic proteoglycans

Chondroitin sulfate (CS-4; MW 22 kDa), PAA (MW 250 kDa), N-hydroxysulfosuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide were purchased from Sigma-Aldrich; 50 kDa molecular weight cut-off (MWCO) membrane was obtained from Spectrum Labs; and 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH) was purchased from Thermo Fisher Scientific. The synthesis of BPG250 was achieved by grafting CS chains onto PAA backbones via N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysulfosuccinimide sodium salt (NHS) coupling as previously described [6]. For retention studies and confocal imaging, BPG250 was tagged with DCCH, a fluorescent molecule [6].

2.2. Radial expansion testing of biomimetic proteoglycan-injected porcine urethras

A radial expansion test (RET) was developed to study urethral tissue mechanics. Frozen urethras from the cadavers of six- to nine-month-old pigs were allowed to thaw and were cut into approximately 50 mm long pieces using a no. 22 scalpel blade. After the urethral samples were equilibrated in 1× phosphate-buffered saline (PBS) for 1 h, they were injected transurethrally, approximately 1–2 mm into the tissue wall, with either PBS (control samples) or BPG250 solution using a 21 gauge needle. Solution (0.5 ml) was injected into the tissue at three sites at approximately every 120°. Three different BPG solutions were tested: 25 mg ml⁻¹, 50 mg ml⁻¹ and 75 mg ml⁻¹ in 1× PBS solution, n = 4, for all sample groups. Afterwards, the injected samples were allowed to soak in 1× PBS for an additional 2 h. Radial expansion testing was then conducted by placing a 16 French balloon catheter inside the lumen of the urethra, and a Monarch pressure syringe was used to measure the urethral wall pressure as the balloon was inflated in 1 ml increments.

2.3. Water uptake study and polymer retention of biomimetic proteoglycan-injected porcine urethras

Frozen urethras from the cadavers of six- to nine-month-old pigs were defrosted at room temperature prior to experimentation. The distal urethra and bladder neck were removed from the tissue specimen. The urethras were then sliced open once in the longitudinal direction to obtain approximately 20 mm (width) × 60 mm (length) × 3 mm (thickness) rectangular tissue samples. Biopsy punches were used to obtain 10 mm disc-shaped urethral samples (four biopsy samples per urethra). A pilot study determined the time the urethral biopsy samples needed to equilibrate (pre-soak) in 1× PBS. After the equilibrium pre-soak (5 h), the urethral samples were injected with fluorescently labelled BPG250. The most distal biopsy sample of each urethra served as a control sample and was injected with 0.1 ml 1× PBS. The other three biopsy samples were injected with 0.1 ml of the following BPG250-DCCH concentrations: 50 mg ml⁻¹, 100 mg ml⁻¹ and 200 mg ml⁻¹. Table 2 summarizes the experimental matrix.

Table 2.

Experimental design: n = 4 biopsy punches per porcine urethra. One biopsy sample from each group served as a control, the other three were injected with BPG250.

	10 mm biopsy punch samples (n = 4 biopsy punches per urethra); (n = 6 for each sample condition) distal → proximal
	no. 1 control	no. 2 BPG250-DCCH	no. 3 BPG250-DCCH	no. 4 BPG250-DCCH
2 urethras	0.1 ml PBS	0.1 ml conc.: 50 mg ml−1	0.1 ml conc.: 50 mg ml−1	0.1 ml conc.: 50 mg ml−1
2 urethras	0.1 ml PBS	0.1 ml conc.: 100 mg ml−1	0.1 ml conc.: 100 mg ml−1	0.1 ml conc.: 100 mg ml−1
2 urethras	0.1 ml PBS	0.1 ml conc.: 200 mg ml−1	0.1 ml conc.: 200 mg ml−1	0.1 ml conc.: 200 mg ml−1

Throughout the duration of the study, each biopsy sample was soaked in 5 ml of 1× PBS. Biopsy samples were weighed 24 h after injection. The percentage water uptake, percentage BPG250 retention and percentage swelling ratios were calculated. Water uptake refers to the amount of water the biopsy samples can hold, retention is the amount of injected BPG250 that remains in the biopsy samples (the amount of BPG250 that does not diffuse out of tissue), and the swelling ratio (very similar to water uptake) pertains to the amount of water the biopsy sample holds; however, the weight of injected BPG250 is subtracted from the overall results. One-way ANOVA analyses were performed to test for statistical significance. After the 24 h weight was obtained, the BPG250 retention inside the polymer was calculated and the biopsy samples were fixed with embedding matrix in an embedding mould, frozen and cryosectioned into 5 µm thick sections. Samples were imaged using an Olympus FV100 microscope with a 4′,6-diamidino-2-phenylindole (DAPI) filter.

A weighing error experiment was conducted, where five equilibrated biopsy punch samples were weighed every 5 min over a 40 min time period. The resulting weighing error was calculated, and any weight change of the biopsy samples in that range was not attributed to the influence of BPGs.

2.4. Analysing the amount of biomimetic proteoglycans retained in the tissue

Emission and excitation sweeps of BPG250-DCCH solutions dissolved in PBS were conducted to find the emission and excitation wavelengths of BPG250-DCCH. A standard fluorescence curve using a set of BPG250-DCCH dilutions was obtained with the following concentrations (BPG250-DCCH dissolved in 1× PBS): 4 mg ml⁻¹, 2 mg ml⁻¹, 1 mg ml⁻¹, 0.5 mg ml⁻¹, 0.25 mg ml⁻¹, 0.125 mg ml⁻¹, 0.0625 mg ml⁻¹, 0.03125 mg ml⁻¹, 0.015625 mg ml⁻¹, 0.0078125 mg ml⁻¹ and 0 mg ml⁻¹ (1× PBS control). The BPG250-DCCH concentrations in this dilution experiment are in correspondence with the experiment outlined in §3.3, where 10 mm porcine urethral biopsy punches injected with 0.1 ml BPG250-DCCH (concentrations: 200 mg ml⁻¹, 100 mg ml⁻¹, 50 mg ml⁻¹) were placed in 5 ml of 1× PBS for 24 h. For the highest injection concentration (200 mg ml⁻¹, volume: 0.1 ml), if 100% of the BPG250-DCCH molecules diffused out of the biopsy sample and into the 5 ml 1× PBS solution, the BPG250-DCCH concentration in 1× PBS would be 4 mg ml⁻¹ ((200 mg ml⁻¹ × 0.1 ml)/5 ml) = 4 mg ml⁻¹). Therefore, the highest concentration for the dilution experiment to analyse the amount of BPG250-DCCH that diffused out of the tissue is 4 mg ml⁻¹.

The linear portion of the standard curve was then used to analyse the PBS solution that the biopsy samples were soaking in to determine the amount of BPG250-DCCH that diffused out of the tissue. The equation of the standard curve is

$$y=111504\hspace{0.17em}\hspace{0.17em}\ast \hspace{0.17em}\hspace{0.17em}X+3557.$$ 2.1

2.5. Confocal imaging

After the 24 h time points, biopsy samples were submerged in embedding matrix in embedding moulds, frozen and cryosectioned into approximately 5 µm thick sections. Confocal images of BPG250-DCCH-injected porcine biopsy samples were taken to image the distribution of BPGs in the urethral tissue. Imaging of the unstained urethra sections was conducted using an Olympus confocal FV1000 microscope with a DAPI filter at a laser intensity of 0.3%.

2.6. Statistical analysis

All statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). The results in the RET graphs are displayed as the mean ± 1 s.d. A one-way analysis of variance (ANOVA) was used to test for statistical significance between the different BPG250 concentrations for the RET results, radial moduli, percentage BPG250 retention, percentage water uptake and percentage swelling ratio data.

3. Results

3.1. Radial expansion testing

Results of the RET are displayed in figure 2. The schematic in the figure represents the assumed behaviour of collagen fibrils as the balloon is inflated inside the urethra in 1 ml increments. Early on in the experiment, from approximately 0 to 2 ml inflation volume, collagen organization is random and the collagen fibrils begin to slide past each other. The random alignment of collagen results in mechanical anisotropy of tissue [16]. At around 3 ml, collagen fibrils start to detangle and align in the orientation of the radial strain. In the approximately 5–8 ml region, collagen fibrils are straight and aligned in the direction of the force. Inflation volumes greater than approximately 9 ml start to induce damage to the collagen fibrils and the continuous increase of tension eventually causes them to break.

Figure 2.

Radial expansion testing results of PBS-injected control samples (n = 4) and BPG250-injected samples at three different concentrations (n = 4 for each condition). The collagen schematic represents the assumed collagen fibril alignment based on experimental observations. (Online version in colour.)

The data points of the four sample groups lie relatively close to each other at 1 ml and begin to deviate around approximately 2 ml, with a significant difference between the 50 and 75 mg ml⁻¹ sample sets. Starting at 4 ml, the data show that when the balloon was inflated the urethral wall of the 50 mg ml⁻¹ sample group exerted the least amount of pressure on the balloon, while the PBS group exerted the highest amount of pressure. Statistical difference resulted at 4, 5 and 6 ml between the PBS and 50 mg ml⁻¹ samples.

3.2. Calculation of radial moduli

In order to evaluate the underlying mechanical properties of the PBS- and BPG250-injected samples, the radial moduli were calculated, where the urethra was treated as a thin-walled pressure vessel. Several assumptions were made for the calculations, as follows.

• (1)Tissue is linear elastic, homogeneous and isotropic.
• (2)Outer diameter of the balloon catheter = inner diameter of thin-walled pressure vessel.
• (3)A balloon catheter is cylindrical.
• (4)There is a limited contact point of balloon with tissue.
• (5)
  Vessel is axisymmetric

  $${\sigma }_1=\frac{\hspace{0.17em}pr}{t}\hspace{0.17em},$$ 3.1

  where σ₁ is the hoop stress, p is the pressure of the urethral wall exerted on the balloon catheter, r is the radius of the balloon catheter and t is the thickness of the urethra.
• (6)There is no longitudinal stress (σ₂) since the urethra is an open vessel.
• (7)
  The strain, ɛ, is radial

  $$\epsilon =\frac{\mathit{\Delta }r}{r_i},$$ 3.2

  where Δr is the change in radius and r_i is the initial radius of the balloon (inflation volume = 0 ml).

The radial moduli, E, of the samples were then calculated using the following equation:

$$E=\hspace{0.17em}\frac{{\sigma }_1}{\epsilon }.$$ 3.3

The diameter of the balloon catheter was measured as the balloon was inflated in 1 ml increments. The diameters were then converted to radii, and the values are displayed in figure 3.

Figure 3.

Radius of a 16 French balloon catheter as it is inflated in 1 ml increments.

The calculated radial moduli are displayed in figure 4, and are 177 ± 18, 177 ± 35, 184 ± 40 and 146 ± 23 kPa for PBS, 25 mg ml⁻¹, 50 mg ml⁻¹ and 75 mg ml⁻¹, respectively. There was no statistical significance between the groups.

Figure 4.

Radial moduli that were calculated from the RET experiment. There is no statistical significance between groups. (Online version in colour.)

3.3. Evaluating fluorescence of BPG250-DCCH dissolved in 1× PBS to quantify BPG250 retention in tissue

The fluorescence of a set of dilutions of BPG250-DCCH solutions was tested to obtain a standard curve. The equation of the linear regression of the standard curve (equation (2.1); figure 5) was used to evaluate the concentration of BPG250 that diffused from the biopsy samples into the PBS solution,

$$X=\frac{Y-3557}{111504},$$ 3.4

where X is the concentration of BPG250 that diffused out of the tissue and Y is the intensity of the PBS solution the biopsy sample was soaking in. The concentration of BPG250 was then converted to a weight to quantify the amount of BPG250 retained in the biopsy samples based on the fluorescence intensity of the PBS solution and the amount of injected BPG250 (table 3).

Figure 5.

Standard curve of BPG250-DCCH (R² = 0.9819) to correlate fluorescent intensity with concentration of BPG250-DCCH.

Table 3.

Summary table (values used for calculations for BPG250 retention in tissue).

sample group	soaking solution volume (PBS)	injection volume	amount of injected BPG250	concentration of BPG250 in PBS if all injected molecules diffused out of tissue
50 mg ml−1	5 ml	0.1 ml	5 mg	1 mg ml−1
100 mg ml−1	5 ml	0.1 ml	10 mg	2 mg ml−1
200 mg ml−1	5 ml	0.1 ml	20 mg	4 mg ml−1

Figure 6 displays the amount of retained BPG250 as raw numbers calculated from the fluorescence intensity (in milligrams), and as a percentage relative to the amount injected: 5 mg, 10 mg and 20 mg, for 50 mg ml⁻¹, 100 mg ml⁻¹ and 200 mg ml⁻¹, respectively. BPG250 retention was highest for the 200 mg ml⁻¹ samples (approx. 84% retention) with statistical significance compared with the 50 mg ml⁻¹ sample group (approx. 59% retention), p < 0.01. Samples (100 mg ml⁻¹) had an average BPG250 retention of approximately 76%.

Figure 6.

Quantification of BPG250 retained in porcine tissue in (a) mg and (b) as a percentage, with statistical significance between 50 mg ml⁻¹ and 200 mg ml⁻¹ samples, p < 0.01. (Online version in colour.)

3.4. Evaluating water uptake properties of BPG250-injected porcine tissue

The weighing error experiment resulted in a 5% weighing error of samples, and any water uptake below 5% is not considered to be caused by BPGs. Figure 7 displays the percentage water uptake and percentage swelling ratios for the PBS control group and the different BPG250 groups after 24 h. Water uptake and swelling ratios were calculated using the following equations:

$$\text{\%}\hspace{0.17em}\mathrm{water}\hspace{0.17em}\hspace{0.17em}\mathrm{uptake}=\left(\frac{\mathrm{weight}\hspace{0.17em}\hspace{0.17em}\mathrm{of}\hspace{0.17em}\hspace{0.17em}\mathrm{tissue}\hspace{0.17em}\hspace{0.17em}\mathrm{after}\hspace{0.17em}\hspace{0.17em}24\hspace{0.17em}\hspace{0.17em}\mathrm{h}-\hspace{0.17em}\hspace{0.17em}\mathrm{weight}\hspace{0.17em}\hspace{0.17em}\mathrm{of}\hspace{0.17em}\hspace{0.17em}\mathrm{equilibrated}\hspace{0.17em}\hspace{0.17em}\mathrm{tissue}}{\mathrm{weight}\hspace{0.17em}\hspace{0.17em}\mathrm{of}\hspace{0.17em}\hspace{0.17em}\mathrm{equilibrated}\hspace{0.17em}\hspace{0.17em}\mathrm{tissue}}\right)\times 100,$$ 3.5

$$\text{\%}\hspace{0.17em}\mathrm{swelling}\hspace{0.17em}\mathrm{ratio}=\left(\frac{\mathrm{weight}\hspace{0.17em}\mathrm{of}\hspace{0.17em}\mathrm{tissue}\hspace{0.17em}\mathrm{after}\hspace{0.17em}24\hspace{0.17em}\mathrm{h}-\mathrm{amount}\hspace{0.17em}\mathrm{of}\hspace{0.17em}\mathrm{injected}\hspace{0.17em}\mathrm{BPG}250-\mathrm{weight}\hspace{0.17em}\hspace{0.17em}\mathrm{of}\hspace{0.17em}\hspace{0.17em}\mathrm{equilibrated}\hspace{0.17em}\hspace{0.17em}\mathrm{tissue}}{\mathrm{weight}\hspace{0.17em}\mathrm{of}\hspace{0.17em}\mathrm{equilibrated}\hspace{0.17em}\mathrm{tissue}}\right)\times 100.$$ 3.6

Figure 7.

(a) Percentage water uptake, (b) percentage swelling ratio and (c) percentage swelling ratio versus BPG250 retention. There is a significant difference for percentage water uptake between PBS samples (22%) and 100 mg ml⁻¹ samples (37%), p < 0.05. (Online version in colour.)

Out of all the sample groups, the samples injected with a BPG250 concentration of 100 mg ml⁻¹ had the highest percentage water uptake. There is statistical significance for percentage water uptake between PBS samples (22%) and 100 mg ml⁻¹ samples (37%), p < 0.05; 50 mg ml⁻¹ and 200 mg ml⁻¹ had an increase in water uptake of 27% and 30%, respectively. There is no statistical significance between groups for percentage swelling ratio.

3.5. Confocal images of biopsy samples (water uptake study)

Confocal images of control and BPG250-DCCH-injected biopsy samples were taken in the mid-substance of the urethra to qualitatively show the presence of BPG250-DCCH in the urethral tissue (figure 8). BPG250-DCCH seems to be present in the interstitial tissue.

Figure 8.

Confocal images of PBS and BPG250-DCCH-injected biopsy samples at 50 mg ml⁻¹, 100 mg ml⁻¹ and 200 mg ml⁻¹ (scale bars = 200 µm). (Online version in colour.)

4. Discussion

4.1. Radial expansion

Mechanical and biochemical properties of the urethra are still far from being fully understood with only a select number of studies and publications examining these properties; some of the major topics are the mean static measurements of resting urethra to evaluate elastance in continent women, a biochemical study examining the different morphologies of urethral tissue in continent and incontinent women, examination of the urethral support and closure mechanisms, the effect of ageing on the maximum urethral closure pressure, and the pressure–diameter response of different urethral regions [2,7,9,10,17]. The radial expansion technique in this study was developed in an attempt to better understand the underlying properties of the urethra and subsequently molecularly engineer the tissue to improve its compliance. Radial expansion testing is unique and has never been implemented before to test urethral tissue.

One way of analysing the mechanical properties of biological tubular structures, such as blood vessels and urethra, is in terms of compliance and elastance [7,18]. Compliance of soft tissue is the measure of how easily a tubular tissue expands when filled with volume and elastance is the reciprocal of compliance [7,19]. Lose [7] investigated the mechanical properties of the resting urethra under standardized static conditions and examined the elastance of the urethra to evaluate the urethral closure function during the bladder fill phase. Lose reported the elastance of the mid-urethra and distal urethra to be 1.27 and 1.05 cmH₂O mm⁻², respectively.

RET is a way to analyse the compliance of the porcine urethra. Overall, out of the three tested BPG concentrations, 50 mg ml⁻¹ showed the greatest improvement in tissue compliance, since the tissue exerted the lowest amount of pressure on the balloon as the volume was increased. It is hypothesized that the 50 mg ml⁻¹ BPG concentration, injected at three sites in each urethra, allowed for the maximum water uptake and therefore resulted in the greatest improvement in tissue compliance, while at the highest tested BPG concentration (75 mg ml⁻¹) the compliance of the porcine tissue was reduced. This result could be due to a maximum water uptake limit that was reached with the injection of 75 mg ml⁻¹, where the maximum threshold was surpassed, forcing some of the water to diffuse back out of the tissue. For the 25 mg ml⁻¹ sample group, the results indicate that the injected BPG concentration was too low to improve the tissue compliance because the number of BPG molecules present in the tissue was not high enough to achieve the maximum water uptake.

There are several limitations when evaluating the radial expansion data and attempting to quantify the radial moduli and compliance of the urethra, which may explain why there is no significance difference between the groups examined here. In hindsight, the syringe used to inflate the balloon catheter did not have the required sensitivity to increase the volume in smaller increments and measure the corresponding urethral wall pressure. As a result, there was a lack of sufficient data points to adequately analyse the moduli and the effect of BPGs on the mechanical properties. As the balloon inflates, it causes the urethral wall to expand and induces significant organizational changes inside the urethral wall. The mechanical properties of the tissue are controlled by the organization of ECM proteins.

Pressure–volume measurements indicate there is a difference in compliance properties between control and BPG-injected groups that are not statistically different when data are resolved to a modulus value. There may be a limitation in the assumption of a thin-walled pressure vessel. However, it should be noted that there were differences in the mechanical behaviour between groups, which can be observed in the raw data of the curves in figure 2. The moduli of control and BPG-injected tissue are comparable to the moduli of soft tissue cited in the literature: rat aorta approximately 600 kPa, and human aorta approximately 10 kPa [20]. Another study reported the modulus of sow urethra to be approximately 10–20 kPa [21].

4.2. Water uptake study

Versican is a large ECM proteoglycan (greater than 1000 kDa) with 12–15 anionic CS side chains and is associated with paraurethral connective tissue [2,22–24]. Versican is involved in a variety of functions including cell proliferation, adhesion and migration, spacing out collagen fibrils and contributing to matrix hydration [2,24–26]. The biochemical analysis by Falconer et al. [2] revealed that the proteoglycan–collagen ratio was significantly reduced for incontinent individuals (p = 0.03), which is hypothesized to lead to an overall stiffer tissue. The major goal of this study was to examine if BPG250 can be introduced and retained in tissue samples and enhance the water uptake properties. Increased water uptake and hydration of tissue may be indicative of increased compliance.

The percentage retention of BPG250 was highest for the 200 mg ml⁻¹ samples with statistical significance compared with 50 mg ml⁻¹ injected samples (p < 0.01). Increased percentage retention of BPG250 in tissue with increasing concentration may be explained by the anionic charge properties of the molecule. In order to inject BPG250 in the porcine biopsy samples BPG250 was dissolved in 1× PBS and then injected into the samples. The presence of salt ions from the PBS solution may decrease the electrostatic repulsion between BPG250 molecules and enable them to agglomerate [27]. The mobility of large BPG250 aggregates in the ECM of the biopsy sample is likely to be reduced in comparison with a single BPG250 molecule. The 200 mg ml⁻¹ samples contained a higher number of BPG250 molecules than the 50 mg ml⁻¹ samples, and therefore probably had a larger number of BPG250 aggregates which decreased their ability to redistribute inside the collagen matrix. An important point to address is that, although the injections were aimed towards the centre of the biopsy sample, the BPG250 solution spread randomly throughout the tissue. It is likely that single BPG250 molecules close to the surface of the biopsy samples were able to diffuse out. This likelihood is reduced for the larger BPG250 aggregates that may become entangled in the collagen fibrils of the porcine sample. The presence of the fluorescent tag is hypothesized to not have an influence on BPG250 transport because the DCCH molecules are relatively small in comparison with the BPG250 molecules.

Versican and the cartilage proteoglycan aggrecan (approx. 2000 kDa) are two of the main large sulfated proteoglycans [28,29]. BPG250 with a molecular weight approximately 1600 kDa is similar in size to aggrecan, a proteoglycan known for its hydrating properties in the ECM of cartilage which generates large osmotic swelling pressures [30,31]. These swelling pressures are attributed to the highly anionic nature of the molecule and its large size, which does not make it possible for aggrecan to redistribute itself in a collagen network [31].

Like aggrecan, versican is a highly anionic molecule due to its negatively charged CS side chains [32,33]. Structural changes to versican affect its anionic charge density, which may influence the viscoelastic characteristics and volume of the tissue [32]. Osmotic disruptions due to decreased sulfation and loss of fixed charge of large proteoglycans are linked to detrimental effects on the normal functioning of tissue. Versican is also found in the ECM of normal blood vessels. Decreased sulfation of versican leads to osmotic disruptions in the matrix, which may make atherosclerotic lesions more susceptible to thrombosis [34]. In cartilage, the high osmotic pressure generated by the anionic charge density of CS bristles of aggrecan is essential for the mechanical properties, resistance to compression and lubrication of the normal joint, whereas cartilage in an osteoarthritic joint lacks high aggrecan concentrations and a high degree of sulfation of aggrecan [30,35].

Osmosis is the net movement of water from a region of lower solute (particle) concentration to a region of higher solute (particle) concentration [36,37]. More than 90% of ions from the interstitial fluid and plasma in the body that are involved in osmotic activities are sodium ions [36]. Therefore, sodium ions play a key role in managing bodily fluids through osmotic processes. Sodium ions are also the most plentiful ions present in the extracellular fluid and govern the volume of the ECM [36]. In the body, osmotic gradients are established via the presence of sodium cations and the ion concentration in a region is balanced out with water [36].

Per cent water uptake was highest for the 100 mg ml⁻¹ samples with a significant difference compared with the PBS sample group (p < 0.05). Like the natural hydrating proteoglycan of urethral tissue versican, BPG250 composed of a PAA core with natural CS bristles has a highly anionic charge density. The highly negatively charged BPG molecules attract positive ions from the PBS soaking solution into the biopsy sample, which caused osmotic pressure imbalances. Since BPGs are too large to relocate themselves water molecules account for these imbalances and cause the porcine biopsy samples to increase their water uptake and subsequently swell. AT a molecular level, integration of BPG250 into the matrix and the subsequent swelling of the matrix is hypothesized to decrease the collagen density because the BPGs and water molecules space out the collagen fibrils. An in vivo study where BPG250 was injected into rabbit urethra (0.5 ml injection of 200 mg ml⁻¹ BPG250 solution) showed noticeable bulking spots at the injection site [38]. The study also revealed there was no adverse histological response induced by injection of BPG250 into the urethra, and no morphological differences between the control and BPG250-injected groups [38].

Biopsy punches with a 10 mm diameter were used to prepare the porcine samples for this experiment. The reason the 200 mg ml⁻¹ sample group had a lower percentage water uptake may be because of the sample size and the limited amount of water it can hold, causing a portion of the water to diffuse back out of the sample. The swelling ratio is very similar to the percentage water uptake; however, the weight of the injected BPGs was not incorporated in the calculations. The percentage swelling ratio versus BPG250 retention graph shows that 100 mg ml⁻¹ samples had the highest water uptake for BPGs that were retained in the tissue. The values were lowest for the 200 mg ml⁻¹ sample group, which again might be an indication there was a swelling limit for samples of this size.

Advantages of the water uptake experiment were that this technique allowed for the direct contact of BPG250-injected porcine biopsy samples with water. The results provided an increased understanding of how incorporation of BPGs in the ECM can be used to molecularly engineer the properties of tissue. Possible mistakes for weighing errors of the biopsy samples were accounted for with the weighing error experiment, which excluded any weight change of samples up to 5%. Limitations for this technique are that the experiment cannot be conducted over an extended period of time because the tissue begins to break down. A further limitation is that while the use of biopsy punches resulted in similarly sized samples and allowed for experimental repetition and direct comparison between samples, the effect of volume and concentration used in the experiment cannot be directly translated to the influence BPG250 may have on tissue properties in vivo. When incorporated into the ECM in vivo there is a larger, extended matrix for BPGs to integrate into which may require an increased amount of BPG molecules to achieve modified tissue properties.

The results strongly suggest that BPG250 has the ability to be retained in porcine urethra and incorporation of the molecules affects the water uptake properties of tissue. Implantation of BPG250 and the anionic charge density increases the hydration of the surrounding tissue. A molecular treatment for SUI needs to have the ability to reverse the degenerate properties of urethral tissue that are linked to SUI—a decrease in compliance and volume of the tissue. BPG250 has the potential to improve compliance of the tissue by restoring depleted proteoglycan contents and increasing the hydration and volume of the surrounding tissue.

5. Conclusion

This work explored the physical and mechanical properties of BPGs injected into porcine urethra ex vivo. The radial expansion technique that was developed is unique and has not been used to test the mechanical properties of urethral tissue before. Although there were no significant differences in the Young's modulus, there were differences between groups as can be seen in the raw RET data. RET has the ability to measure localized mechanical properties of the urethra where BPGs were injected, as opposed to tensile testing, which tests the entire tissue under longitudinal extension. The results suggest that, by introducing BPGs into urethral tissue and subsequently adding charge density, there is increased hydration and thus improvements to compliance of the tissue. Like the natural proteoglycan versican, BPGs have the ability to be retained in the ECM of the samples, probably because of their relatively large size. In summary, this work demonstrated that BPGs have the potential to be implemented as a molecular treatment for SUI by restoring the diminished proteoglycan content and increasing the hydration and improving the compliance of the tissue.

Data accessibility

This article has no additional data.

Authors' contributions

A.S.K. carried out the study presented. K.P. and M.S.M. gave guidance for the study.

Competing interests

We declare we have no competing interests.

Funding

The authors thank the Coulter Foundation for funding.