Collagenase Clostridium histolyticum for Peyronie's disease: a contemporary atlas of complications and their management.

INTRODUCTION: Collagenase Clostridium histolyticum (CCH) remains the only Food and Drug Administration-approved medical treatment for Peyronie's disease (PD). The initial IMPRESS I and II trials (Investigation for Maximal Peyronie's Reduction Efficacy and Safety), which led to Food and Drug Administration approval, revealed a rate of treatment-related adverse events as high as 84%. Studies fail to provide clear definitions of complications. OBJECTIVES: To review complications, provide a CCH complication atlas, and propose management strategies for commonly encountered complications. METHODS: We performed a literature review using PubMed. A photographic atlas was provided regarding complications in patients in a high-volume CCH center for PD. RESULTS: Complications were identified and classified by nature and severity. We followed a standardized previously published grading system for hematomas. Complications include bruising, swelling, hematoma formation, back pain, and, rarely, corporal rupture. Complications were discussed, and hematomas were graded by penile surface area. Complication photographs were graded and displayed. Treatment-related adverse effects do not affect overall results. CONCLUSION: Recognizing and grading complications associated with CCH therapy for PD is crucial for effective patient management and informed decision making. A standardized grading system allows for consistency in reporting and comparing hematoma complication rates across studies and patient populations. Herein we provide images that will help clinicians identify and confidently manage common complications that may occur in any CCH program.

Combination clomiphene citrate and anastrozole duotherapy improves semen parameters in a multi-institutional, retrospective cohort of infertile men.

In men with impaired semen parameters, empiric medical therapies such as clomiphene citrate, a selective estrogen receptor modulator (SERM), and anastrozole, a selective aromatase inhibitor, are often employed. The effects of jointly administering these agents on semen parameters are not well understood. Here, we describe the findings of our multi-center, retrospective cohort study of men with idiopathic primary or secondary infertility. Twenty-one men were treated with combination therapy (anastrozole and clomiphene) and 69 men were treated with monotherapy (anastrozole). Patients with pre-treatment normozoospermia and recent or current exogenous testosterone therapy were excluded. Baseline and post-treatment semen and sex hormone parameters were compared among groups. The median follow-up duration was 91 days [interquartile range (IQR), 64-117 days]. Following treatment, 43% of men in the combination therapy group demonstrated normozoospermia, compared to 25% in the monotherapy group. Furthermore, men in the combined group demonstrated marked improvements in total motile sperm count (TMSC) [11.3 vs. 2.1 million (M), P=0.03]. There were no significant differences in hormone levels among the two groups following treatment. Combination therapy with clomiphene citrate and anastrozole was associated with modest benefits in post-treatment semen parameters, when compared to anastrozole monotherapy. These benefits may contribute to improvements in pregnancy outcomes with less invasive assisted reproductive technologies, such as intrauterine insemination (IUI). Future investigations with larger sample sizes and prospective study designs are necessary.

Potential primary prevention of Peyronie's disease post prostatectomy?-retrospective analysis of peri-operative multi-modal penile rehabilitation.

The surgical management of prostate cancer through radical prostatectomy has the potential to impact patients' sexual function, including erectile dysfunction and Peyronie's disease (PD). Historical data suggests the incidence of PD in post-prostatectomy patients is higher than in the general population at 15.9%. Our study objective was to measure the rate of the development of PD among patients that receive penile rehabilitation (PR) regimen prior to and immediately after radical prostatectomy. In this study, we retrospectively reviewed the charts of 581 patients who were diagnosed with prostate cancer, treated with radical prostatectomy, and engaged in a PR program. Patients with the diagnosis of PD prior to prostatectomy were excluded from this study. The PR program consists of daily tadalafil, L-citrulline, and weekly vacuum erectile device with the option of intracavernosal injections if patients fail to respond to the regular regimen. We found the incidence of PD to be 2.9%, suggesting that PR regimens programs may be associated with a reduced incidence of PD in post-prostatectomy patients. Ten (out of 17) patients were diagnosed with PD after 2 years of follow up. The return of erections was not statistically different among patients who developed PD and the rest of the patients. Prospective, multi-institutional trials will be required to elucidate whether PR can prevent the development of PD in this patient population.

Tissue anisotropy and collagenomics in porcine penile tunica albuginea: Implications for penile structure-function relationships and tissue engineering.

The tunica albuginea (TA) of the penis is an elastic layer that serves a structural role in penile erection. Disorders affecting the TA cause pain, deformity, and erectile dysfunction. There is a substantial clinical need for engineered replacements of TA, but data are scarce on the material properties and biochemical composition of healthy TA. The objective of this study was to assess tissue organization, protein content, and mechanical properties of porcine TA to establish structure-function relationships and design criteria for tissue engineering efforts. TA was isolated from six pigs and subjected to histomorphometry, quantification of collagen content and pyridinoline crosslinks, bottom-up proteomics, and tensile mechanical testing. Collagen was 20 ± 2%/wet weight (WW) and 53 ± 4%/dry weight (DW). Pyridinoline content was 426 ±131 ng/mg WW, 1011 ± 190 ng/mg DW, and 45 ± 8 mmol/mol hydroxyproline. Bottom-up proteomics identified 14 proteins with an abundance of >0.1% of total protein. The most abundant collagen subtype was type I, representing 95.5 ± 1.5% of the total protein in the samples. Collagen types III, XII, and VI were quantified at 1.7 ± 1.0%, 0.8 ± 0.2%, and 0.4 ± 0.2%, respectively. Tensile testing revealed anisotropy: Young's modulus was significantly higher longitudinally than circumferentially (60 ± 18 MPa vs. 8 ± 5 MPa, p < 0.01), as was ultimate tensile strength (16 ± 4 MPa vs. 3 ± 3 MPa, p < 0.01). Taken together, the tissue mechanical and compositional data obtained in this study provide important benchmarks for the development of TA biomaterials. STATEMENT OF SIGNIFICANCE: The tunica albuginea of the penis serves an important structural role in physiologic penile erection. This tissue can become damaged by disease or trauma, leading to pain and deformity. Treatment options are limited. Little is known about the precise biochemical composition and biomechanical properties of healthy tunica albuginea. In this study, we characterize the tissue using proteomic analysis and tensile testing to establish design parameters for future tissue engineering efforts. To our knowledge, this is the first study to quantify tissue anisotropy and to use bottom-up proteomics to characterize the composition of penile tunica albuginea.

A middle-aged man with hyperandrogenic state.

Hyperandrogenism secondary to testicular cancer typically arises in patients in whom Leydig cell hyperplasia or neoplasia can be identified. Additionally, benign and malignant adrenocortical tumors can also present with signs and symptoms of hyperandrogenism. We report a case of a 40-year-old gentleman who experienced several months of weight gain, worsening gynecomastia, and mood changes secondary to high testosterone and estradiol levels. Workup initially was negative for testicular malignancy and positive for a benign-appearing lesion in the adrenal gland. Despite adrenalectomy, symptoms continued to persist and ultimately a testicular cancer without Leydig cell involvement was identified.

Multiple courses of intralesional collagenase injections for Peyronie disease: a retrospective analysis.

BACKGROUND: In the original clinical trials evaluating intralesional collagenase Clostridium histolyticum for Peyronie disease (PD), treatment protocols were limited to 8 injections. AIM: We sought to describe our single-center experience with the use of multiple rounds (>8 injections) of intralesional collagenase in patients with PD. METHODS: We conducted a retrospective analysis of all patients with PD receiving intralesional collagenase injections at our institution from October 2015 through December 2020. Some patients who completed 1 round of treatment elected to undergo additional rounds (16 or 24 injections) based on persistent curvature and presence of penile plaque. Clinical improvement was defined as a 20% reduction in penile curvature from the start of a given round of treatment to the end of that round of treatment. We measured erect penile curvature before and after each round and collected demographics, medical and surgical history, curvature outcomes, and treatment-related adverse events. OUTCOME: The primary outcome was the reduction in penile curvature after multiple rounds of treatment with intralesional collagenase injections in patients with PD. RESULTS: A total of 330 patients underwent intralesional collagenase injections for PD, of whom 229 completed at least 8 injections and underwent pre- and posttreatment erect penile goniometry. An overall 42.8% (98/229), 38.6% (22/57), and 12.5% (1/8) of patients achieved clinical improvement after 1 round of therapy (8 injections), 2 rounds (16 injections), and 3 rounds (24 injections), respectively. Mean degree and mean percentage improvement of penile curvature for the start and end of each round of treatment were 8.3° and 16.4% (after 1 round), 7.2° and 16.8% (after 2 rounds), and 3.3° and 8.1% (after 3 rounds). Bruising was the most common complication, with an incidence of at least 50% in each round. CLINICAL IMPLICATIONS: Knowledge of patient responses to multiple rounds of intralesional collagenase injections may help guide physicians in management and counseling of patients regarding PD treatment options. STRENGTHS AND LIMITATIONS: This is the first study to evaluate multiple rounds (>8 injections) of intralesional collagenase for PD. Limitations include retrospective analysis and smaller sample size among patients undergoing 3 rounds (24 injections). CONCLUSION: For patients who did not achieve clinical improvement after 1 round of treatment, an additional round may be beneficial. However, no real improvement was observed for patients undergoing a third round.

Temporal Changes of Clomiphene on Testosterone Levels and Semen Parameters in Subfertile Men.

PURPOSE: Clomiphene citrate (CC) is prescribed off-label in men to improve testosterone and sperm parameters, but the duration of treatment needed to reach maximal benefit remains unclear. Our objective was to examine temporal effects of CC on total testosterone (TT) and semen analysis (SA) using longitudinal follow-up data in treated men. MATERIALS AND METHODS: We analyzed an IRB-approved database of men treated with CC (25 mg q.d. or 50 mg q.o.d.) from January 2016 through May 2021. We identified patients with 3, 6, 9, and 12 month follow-up data for TT and 3, 6, and 9 month follow-up SA. Mean absolute changes in TT and sperm concentration compared to baseline were calculated, along with 95% confidence intervals. Men with prior genitourinary procedures or hormone therapy were excluded. Paired t-tests were used to compare TT and sperm concentration at each time point to baseline (alpha=0.05). RESULTS: One hundered thirty-four men received CC, mean age 37.7 years (SD 6.7, range 24-52). TT at all follow-ups (3, 6, 9, and 12 months) were available for 25 men, and SA at 3, 6, and 9 months for 26 men. Baseline TT was 358±145 ng/dL and sperm concentration was 13±17.2 M/mL. Significant improvement in TT was identified at 3 months (62.7 ng/dL, 95% CI: 0.49-125.0, p=0.048), additional benefit at 6 months (181.8 ng/dL, 95% CI: 114.1-249.5, p<0.01), and plateau at 9 and 12 months. Improvement in sperm concentration was first observed at 9 months (20.7 M/mL, 95% CI: 10.2-31.2, p<0.01). Semen volume and sperm motility did not change. CONCLUSIONS: Duration of treatment with clomiphene may impact testosterone and sperm concentration, and the historical 3 month milestone may be insufficient for clinical and research evaluation. Men taking CC may experience plateau in TT at 6 months and first benefit in sperm concentration at 9 months.

Acute Lower Back Pain after Intralesional Injection of Collagenase Clostridium histolyticum for Peyronie's Disease.

PURPOSE: The safety label for collagenase Clostridium histolyticum was updated to include postinjection acute lower back pain as an adverse event observed with intralesional therapy for Peyronie's disease. Incidence and causality are unknown. We assessed frequencies and temporal associations for this adverse event in a large cohort. MATERIALS AND METHODS: Data on all men undergoing collagenase injections for Peyronie's disease at our institution from October 2015 through December 2020 were retrospectively assessed. The study included 330 patients, 300 completing at least 1 full course (8 injections). Measured outcomes included incidence and timing of back pain, and associations with demographics and comorbidities. RESULTS: Of 330 patients, 19 (5.8%) experienced at least 1 episode of postinjection acute lower back pain. Of 300 who completed at least 1 full course of 8 injections, 4 (1.3%) reported back pain within the 8-injection course. A subset underwent additional rounds (16 or 24 injections). Back pain increased to 8.7% (13/149) during a second round, 6.9% (3/43) during a third. No association was found with age, diabetes or back pain history. Most cases occurred shortly after injection; all were self-limited or resolved with a single dose of ketorolac. CONCLUSIONS: This single-center, retrospective analysis suggests that intralesional collagenase injections for Peyronie's disease may cause acute lower back pain in up to 6% of patients. Patients may benefit from counseling regarding this risk. Incidence rises with additional rounds of treatment. Prospective safety data regarding >8 injections do not exist. No patient had long-term sequelae of back pain.

Initial gonadotropin levels and sperm parameters differentiate the response to clomiphene citrate in subfertile men.

Background: Efficacy of clomiphene citrate (CC) in the treatment of male subfertility remains unclear, with inconsistent results in the literature and limited guidance from professional organizations. We sought to stratify the response to clomiphene in men based on their initial gonadotropins and semen parameters. Methods: We conducted a retrospective analysis of 234 patients from an academic center who took CC for subfertility. Patients with pre-treatment and 3 months follow-up total testosterone (TT) and semen analyses were included. Patients with previous hormone therapy, genitourinary surgery, prior success in conceiving pregnancy, or only one semen analysis were excluded. Primary outcomes were magnitudes of improvement in TT and semen parameters at 3 months. Student's t-test (alpha =0.05) was used for univariate analyses; multivariable linear regression was used for multivariate analysis. Results: One hundred and thirty-seven patients met inclusion criteria. Thirty-four percent of patients experienced improvement in sperm concentration after 3 months of CC treatment, 13% decreased, and 53% showed no change. Using a pre-treatment TT cutoff of 300 ng/dL and gonadotropin thresholds of 7 miU/mL, initial TT did not affect magnitude of improvement in semen parameters, while lower initial gonadotropins showed statistical improvement across all outcomes. Multivariate analysis showed pre-treatment follicle stimulating hormone (FSH) was inversely correlated with improvement in TT [odds ratio (OR): 2.64e-05, 95% confidence interval (CI): 1.32e-09 to 5.28e-01, P=0.04] and sperm concentration (OR: 0.22, 95% CI: 5.70e-02 to 8.48e-01, P=0.03). We also provide initial gonadotropin cutoffs that suggest statistical benefit from CC use. Conclusions: Men with lower gonadotropin levels may expect greater degree of improvement in both hormone and semen parameters with use of CC. Men with azoospermia do not benefit based on semen analyses alone. Degree of non-azoospermia does not affect magnitude of improvement. CC had decreasing efficacy at higher initial gonadotropin levels. These data may provide guidance in stratifying and counseling men for CC treatment.

3D-printed phantoms to quantify accuracy and variability of goniometric and volumetric assessment of Peyronie's disease deformities.

Characterization of Peyronie's disease (PD) involves manual goniometry and penile length measurement. These techniques neglect volume loss or hourglass deformities. Inter-provider variability complicates accuracy. Using 3D-printed models, we aimed to evaluate measurement accuracy and variability and establish computational assessment workflows. Five digital phantoms were created: 13.0 cm cylinder, 13.0 cm hourglass cylinder, 15.0 cm cylinder with 40° angulation, 12.0 cm straight penis, and 12.9 cm PD penis with 68° angulation and hourglass. Lengths, volumes, and angles were determined computationally. Each phantom was 3D-printed. Ten urology providers determined lengths, angles, and volumes with measuring tape, goniometer, and volume calculator. Provider versus computational measurements were compared to determine accuracy using t-tests or Wilcoxon rank-sum tests. No significant differences were observed between manual assessment of length of penile models and designed length in penile models. Average curvature angles from providers for bent cylinder and PD phantoms were 38.3° ± 3.9° (p = 0.25) and 57.5°â€†±â€†7.2° (p = 0.006), respectively. When assessing for volume, hourglass cylinder and bent cylinder showed significant differences between designed volume and provider averages. All assessments of length, angle, and volume showed significant provider variability. Our results suggest manual measurements suffer from inaccuracy and variability. Computational workflows are useful for improved accuracy and volume assessment.

Risk factors for penile fracture compared with a surgical control cohort in the United States: the role of substance abuse.

Penile fracture (PF) is a surgical emergency. Given its rarity, we queried a national cohort over an 11-year period to study the temporal and demographic variations in presentation, evaluation, and management of patients with PF compared with a cohort of control patients. The National Inpatient Sample was queried between the years 2005 and 2016 for patients with a diagnosis of PF. Appendectomy patients were selected as a control cohort, given the non-discriminatory nature of this disease. Clinical and demographic data of the patients were compared with that of controls. Presenting symptoms, rates of surgical repair, and rates of associated surgical procedures were evaluated in the PF cohort. During the study period, 5802 patients were hospitalized for PF. The annual incidence of PF remained unchanged at 1.0-1.8 cases per 100 000 hospitalizations over the study period. Compared with the control cohort, PF patients were more likely to be younger (38.7 years vs 41.2 years, P ≤ 0.001), have lower rates of comorbidities except erectile dysfunction (1.4% vs 0.1%, P ≤ 0.001), and were more likely of Black race (25.4% vs 6.2%, P ≤ 0.001). Notably, PF patients had significantly higher rates of substance abuse (26.4% vs 18.1%, P ≤ 0.001), despite no difference in the diagnosed psychiatric disorders. PF rarely presented with hematuria (3.5%); however, urethral evaluation was performed in 23.1%, most commonly with cystoscopy (19.2%). PF occurs more commonly in a younger, healthier male population, and among minorities. Importantly, rates of substance abuse appear to be higher in the PF cohort compared with those of controls.

Efficacy of a preprostatectomy multi-modal penile rehabilitation regimen on recovery of postoperative erectile function.

To evaluate the efficacy of a novel, multi-modal, preoperative approach to postprostatectomy penile rehabilitation (PR), we performed a retrospective review of patients who underwent nerve-sparing robotic-assisted laparoscopic prostatectomy (NS-RALP). All patients were evaluated at a comprehensive, academic sexual medicine clinic between 2016 and 2017. The "prehabilitation" PR group (n = 106) consisted of men who were seen in the pre-op period and began tadalafil and L-citrulline 2 weeks prior to surgery. Vacuum erectile device (VED) therapy was started at 1-month post-op. These interventions were continued throughout the 12-month follow-up period. Individuals refractory to these therapies could start treatment with intracavernosal injections. The postprostatectomy PR group (n = 25) consisted of men who were not seen in the pre-op period and started the above therapies immediately following their first visit. A higher percentage of men in the prehabilitation group reported return of erectile function within 12 months (56% vs. 24%, P = 0.007). The prehabilitation group also showed better compliance with PR (PDE5i [96% vs. 64%, P < 0.001], L-citrulline [93% vs. 49%, P < 0.001], and VED [55% vs. 20%, P < 0.001]). Seventy-eight percent of men who attended 4-5 follow-up visits reported return of erectile function. Our results suggest that men undergoing a preoperative protocol show superior recovery of erectile function following NS-RALP. Further studies with prospective designs are warranted.

Defining a "High Volume" Radical Cystectomy Hospital: Where Do We Draw the Line?

BACKGROUND: Centralization of radical cystectomy (RC) to "high volume" centers can lead to decreased morbidity but also limits access to care. In the context of centralization, there is a need to systematically define the hospital volume cutoffs for this procedure. OBJECTIVE: To systematically examine the effect of hospital volume on inpatient complications of RC for bladder cancer and to define a threshold to minimize RC morbidity. DESIGN, SETTING, AND PARTICIPANTS: This was a retrospective analysis of data for 6790 adults undergoing RC for nonmetastatic bladder cancer during 2008-2011 from the National Inpatient Sample (weighted population estimate of 33 249 RCs in the USA during this period). INTERVENTION: RC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Overall and major complications were defined according to International Classification of Diseases (9th revision) diagnosis and procedure codes. To define the relationship between hospital volume and morbidity, logistic regression analyses within a generalized estimating equation framework with restricted cubic splines were used. RESULTS AND LIMITATIONS: The inpatient complication rate was 4769/6790 (70.2%), of which 1572/6790 (23.2%) were major complications. Restricted cubic spline analysis revealed a significant inverse nonlinear association between hospital volume and complications. The odds of complications decreased with increasing volume, with a plateau at 50-55 cases/yr for any complications (p=0.024) and 45-50 cases/yr for major complications (p=0.007). CONCLUSIONS: The relationship between hospital volume and RC morbidity is nonlinear, with a plateau for the complication rate at 50-55 cases/yr. Restricting RC to centers with such high thresholds will restrict access to care. There is a need to identify and publish best practices from high-volume centers in quality improvement initiatives to improve morbidity at low-volume centers. PATIENT SUMMARY: There is a nonlinear relationship between the annual number of radical cystectomy procedures performed at a hospital and the inpatient complication rate. Complications decrease with increasing hospital volume and reach a plateau at 50-55 cases per year, beyond which the incremental benefit of increasing volume is minimal.

A modified technique for intralesional injection of collagenase Clostridium histolyticum for Peyronie's disease results in reduced procedural morbidity using a standardized hematoma classification rubric.

PURPOSE: Early clinical trials of injectable collagenase Clostridium histolyticum (CCh) for Peyronie's disease (PD) demonstrated safety and efficacy. Since then, modified injection protocols have been proposed. Adverse events-such as bruising, swelling, hematoma, and corporal rupture-exceed 50% in many studies, but lack of standardization of hematoma severity limits conclusions about the relative safety of protocols. We propose a modification of the standard injection technique that aims to decrease the rates of adverse events. We further describe a hematoma classification rubric that may standardize safety assessment. METHODS: A modified injection procedure, termed the "fan" technique, was employed in the treatment of PD. All men receiving CCh from January 2016 through January 2019 at a single institution were included in an institutional review board (IRB) approved database. Treatment outcomes and adverse events were retrospectively assessed. A three-tiered hematoma classification rubric was devised to standardize reporting of hematoma, which was defined as concurrent bruising and swelling at the site of injection without loss of erection. RESULTS: Using the fan technique, 152 patients received 1323 injections. Eight hematomas (5.3% of all patients, 0.6% of all injections) were observed. The number of grade I, grade II, and grade III hematomas were 3, 2, and 3, respectively. Bruising or swelling not meeting the definition of hematoma was seen in 54.6% and 27.0% of patients, respectively. There were zero corporal ruptures. CONCLUSION: A modified injection technique results in reduced procedural morbidity. A hematoma classification system provides clarity and standardization to the assessment of safety in PD treatment. Further clinical studies with control arms are required to verify these findings.

Discontinuation from Collagenase Clostridium histolyticum Therapy for Peyronie's Disease: Review and Single-Center Cohort Analysis.

INTRODUCTION: Intralesional injection therapy with collagenase Clostridium histolyticum (CCh) is an effective treatment option for Peyronie's disease (PD), but it carries risks, costs, and the need for multiple visits, which may cause patients to discontinue therapy prematurely. AIMS: To identify and summarize the current literature on CCh discontinuation and present our experience with CCh discontinuation. METHODS: We performed a PubMed review of existing literature on discontinuation from CCh therapy and retrospectively analyzed our prospectively maintained Institutional Review Board-approved CCh database for January 2016-December 2018. Demographic information, clinical outcomes, and communication logs were collected. Reasons for discontinuation of therapy were assessed. A logistic regression to identify factors influencing dropout was performed. MAIN OUTCOME MEASURES: Documentation of discontinuation statistics in published literature, and rates of and reasons for discontinuation in a single-institution cohort. RESULTS: Our literature review identified 15 studies with specified cohort sizes. Of these, 10 specifically quantified discontinuation rates, which ranged from 13% to 56%. Combined, these studies show a 20% dropout rate. Dissatisfaction with therapy was the most common reason for dropout. In our cohort, 100 men completed a course of 8 CCh injections. Twelve men (10.7%) discontinued therapy, including 4 due to relocation, 3 due to cost, 1 due to a hematoma, 1 due to early satisfaction, 2 due to no perceived improvement, and 1 due to a demanding work schedule. Hematoma formation was a predictor of dropout in our cohort (odds ratio 8.74; P = .037). CONCLUSION: Additional focus must be placed on quantifying and evaluating CCh discontinuation. Our findings show that a majority of men complete a full course of 8 injections; most of the few men who dropped out of therapy did so due to relocation. Counseling to reduce CCh discontinuation should focus on initial sexual function, adverse events, and expectations. Amighi A, Eleswarapu SV, Mendhiratta N, et al. Discontinuation from Collagenase Clostridium histolyticum Therapy for Peyronie's Disease: Review and Single-Center Cohort Analysis. Sex Med Rev 2019;7:690-698.

TRPV4 channel activation improves the tensile properties of self-assembled articular cartilage constructs.

A persistent hurdle in the field of tissue regeneration is to produce tissues with biochemical and biomechanical properties robust enough to meet the aggressive physiological demands of the native milieu. In an effort to improve these properties tissues grown in vitro are often subjected to mechanical stimuli that aim to recapitulate the in vivo physiology. These mechanical stimuli are thought to produce downstream alterations in intracellular ion concentrations, which ultimately give rise to increased biosynthesis. There is mounting evidence that these perturbations in the cellular microenvironment are regulated by the Ca(2+)-permeable transient receptor potential vanilloid 4 (TRPV4) channel. In this study we examined the effects of targeted TRPV4 activation on self-assembled articular cartilage constructs. The objectives of this study were: (i) to determine whether TRPV4 activation would enhance self-assembled constructs; (ii) to identify an optimal treatment time window for TRPV4 activation; and (iii) to compare TRPV4 activation which Na(+)/K(+) pump inhibition, which has previously been shown to improve the construct tensile properties. This study employed a two phase approach. In Phase I self-assembled constructs were grown for 4weeks and subjected to treatment with the TRPV4 agonist 4α-phorbol-12,13-didecanoate (4α-PDD) during three treatment time windows: t=6-10, t=10-14, and t=14-18days. Treatment for t=10-14days produced an 88% increase in collagen and a 153% increase in tensile stiffness. This treatment window was carried forward to Phase II. In Phase II we performed a head to head comparison between TRPV4 activation using 4α-PDD and Na(+)/K(+) pump inhibition using ouabain. Treatment with 4α-PDD produced improvements on a par with ouabain (91-107% increases in tensile stiffness). The results of this study demonstrate the effectiveness of ion channel modulation as a strategy for improving engineered tissues. To our knowledge this is the first study to examine TRPV4 channel activation in tissue engineering.