Urothelium Tissue Engineering Using Biopsies From Transurethral Resection of Prostate (IMOPU)

Urothelium Tissue Engineering Using Bladder Mucosa From Transurethral Resection of Prostate

Different clinical conditions can require urinary bladder augmentation or replacement. Tissue engineered bladder has been clinically evaluated but is not recommended due to diverse side effects. Thus, there is a real interest for the development of regenerative approach with innovative scaffolds and cell transplantation.

The investigators propose the use of urothelial cells obtained by Trans-Urethral Resection of Prostate or bladder (TURP) to obtain a tissue engineered urothelium in association with different scaffolds.

Study Overview

• Status: Not yet recruiting
• Conditions: Hypospadias; Bladder, Neurogenic; Spina Bifida; Bladder Exstrophy; Urothelial Neoplasm
• Intervention / Treatment: Procedure: Transurethral Resection of Prostate

Detailed Description

Bladder biopsies will be obtained during cystoscopy, conserved in culture medium (DMEM®), digested by dispase and sowed on collagen-coated culture support. Keratinocyte Serum Free Medium (KSFM) will be used for proliferation. Microscopy, immunohistochemistry, RNA extraction, Reverse Transcription and quantitative Polymerase Chain Reaction (RT-qPCR) will be performed during passages. Cell culture conditions will be optimized to improve proliferation and avoid loss of differentiation. The investigators will develop scaffolds based on sodium alginate hydrogels, followed by freeze-drying to generate porous sponges (at -20°C and -80°C). Cultured cells will be associated to these original scaffolds and to other scaffolds, for example alginate hydrogels or Collagen Cell Carrier (CCC), cultivated for 28 days and analyzed. Histological and immunohistological appearance of cellularized scaffolds will be compared to assess the effectiveness of each scaffold for tissue engineering in urothelium.

Cellularized scaffolds will be studied in vitro (Transepithelial Electrical Resistance, impermeability, ability to be stitched, resistance to urine) and in vivo in ectopic location (subcutaneous location in Nude mice) or in orthotopic location (bladder augmentation in small animal).

• Study Type: Observational
• Enrollment (Anticipated): 365

Contacts and Locations

• Study Contact: Name: Nicolas Berte, Dr; Phone Number: 00333673004195; Email: n.berte@chru-nancy.fr
• Study Contact Backup: Name: Jean-Louis Lemelle, PHD; Phone Number: 0033383154729; Email: jl.lemelle@chru-nancy.fr

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years and older (Adult, Older Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: Male

• Sampling Method: Non-Probability Sample

Study Population

Male patient requiring TURP for Benign Prostatic Hyperplasia with prostate weight evaluated preoperatively greater than or equal to 30 grams

Description

Inclusion Criteria:

• Patient needing a Transurethral Resection of Prostate (TURP)
• Weight of prostate (evaluated by ultrasonography)greater than or equal to 30 grams
• Affiliation to a social security system
• Patient over the age of majority
• Patient receiving complete information on research organization without opposition to the use of biological specimen

Exclusion Criteria:

• Patient for whom no TURP is realized during endoscopic procedure
• Patient for whom no resection is realized on the bladder neck
• Patient with prostate weight estimated under 30 grams
• Patient who opposed to the realization of the study

Study Plan

How is the study designed?

Design Details

• Observational Models: Other
• Time Perspectives: Prospective

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Histological analysis of biopsy.	Histological analysis with standard coloration will assess the viability of urothelium. Signs of necrosis (ulcerations, destructions of urothelium structure) will be noted.	6 month

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Immunohistological analysis of biopsy.	Immunohistological analysis using specific antibodies will locate the urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen).	6 month
RT-qPCR analysis of biopsy.	RT-qPCR analysis will measure the level of expression (compared to housekeeping gene RPLP0) of RNA specific to urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen). This level will be compared to cultured urothelial cells.	6 month
Digestion of the biopsy and culture with adapted medium. Optimization of culture conditions.	The biopsy will be digested using dispase. Cells will be sowed on collagen coated supports and cultured using Keratinocyte Serum Free Medium (KSFM). Trypsination will be done at confluence. RT-qPCR (level of expression of each specific marker) will be realized at each passage to assess evolution during culture.	12 months
Histological analysis of the cultured cells.	Cultured cells (Outcome 4) will be analyzed in contrast phase microscopy and with standard microscopy coloration to assess their morphology, the number of cellular types and their evolution throughout passages.	12 months
Immunocytological analysis of the cultured cells.	Cultured cells (Outcome 4) will be analyzed in immunocytology to assess the type of cells (urothelial / smooth muscle / prostatic), the expression and the localization of specific markers (Outcome 2)	12 months
RT-qPCR analysis of the cultured cells.	Cultured cells (Outcome 4) will be analyzed in RT-qPCR at each passage to assess evolution during culture. The level of expression (compared to housekeeping gene RPLP0) of RNA specific to urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen) will be measured.	12 months
Development of original alginate freezed-dried scaffold	Development of an original scaffold based on sodium alginate hydrogels, followed by freeze-drying to generate porous sponges (at -20°C and -80°C). Analysis of structure, impermeability, ability to be stitched..	12 months
Association of the cultured cells with different scaffolds. Culture of cellularized scaffolds.	Previously cultured and analyzed cells will be associated to different scaffolds.; Collagen scaffolds; Alginate scaffoldsAlginate hydrogels obtained by sprayOriginal alginate freezed-dried scaffold (Outcome 4) Cellularized scaffolds will be cultured at least 28 days using previously described conditions.	36 months
Histological analysis of cellularized scaffolds	After association, cellularized scaffolds (Outcome 9) will be cultured at least 28 days. Survival rate using MTT assay will be performed. Histological analysis using standard colorations will be performed to evaluate the appearance of the cellularized scaffold, the location, the appearance and the organization of the cells.	36 months
Immunohistological analysis of cellularized scaffolds	After association, cellularized scaffolds (Outcome 9) will be cultured at least 28 days. Immunohistological analysis will be performed to evaluate the expression and location of specific markers (Outcome 2)	36 months
RT-qPCR analysis of cellularized scaffolds	After association, cellularized scaffolds (Outcome 9) will be cultured at least 28 days. RT-qPCR will be performed to assess evolution during culture in 3 dimensional conditions. The level of expression (compared to housekeeping gene RPLP0) of RNA specific to urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen) will be measured.	36 months
Biophysical analysis of cellularized scaffolds	After association, cellularized scaffolds (Outcome 9) will be cultured at least 28 days. Biophysical analysis on cellularized scaffolds will be done: Transepithelial Electrical Resistance (cellular ability to form an impermeable barrier); Impermeability (ability of the cellularized scaffold to form a water resistant barrier); Ability to be stitched (ability of the cellularized scaffold to be manipulated and stitched); Resistance to urine (ability of the cellularized scaffold to resist to chemical aggression of urine)	36 months
Implantation of the cellularized scaffold in Nude mice	Cellularized scaffolds will be implanted in subcutaneous location in Nude mice. Incubation in vivo will be done during at least 28 days. Analysis (histology, immunohistology, RT-qPCR) will be performed after 28 days of incubation to assess the survival of cells and the behavior of the cellularized scaffold in vivo. Signs of necrosis, neoangiogenesis and inflammation will be noted.	12 months
Histological analysis of implanted scaffolds	Histological analysis will be performed after 28 days of incubation on implanted scaffolds (Outcome 14) to evaluate the appearance of the cellularized scaffold, the location, the appearance and the organization of the cells. Survival rate using MTT assay will be performed. Signs of fibrosis will be noted.	12 months
Immunohistological analysis of implanted scaffolds	Histological analysis will be performed after 28 days of incubation on implanted scaffolds (Outcome 14) to evaluate Immunohistological analysis will be performed to evaluate the expression and location of specific markers (Outcome 2)	12 months
RT-qPCR analysis of implanted scaffolds	RT-qPCR analysis will be performed after 28 days of incubation on implanted scaffolds (Outcome 14) to assess evolution during in vivo conditions. The level of expression (compared to housekeeping gene RPLP0) of RNA specific to urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen) will be measured.	12 months

Collaborators and Investigators

• Sponsor: Central Hospital, Nancy, France

Publications and helpful links

General Publications

• Adamowicz J, Kowalczyk T, Drewa T. Tissue engineering of urinary bladder - current state of art and future perspectives. Cent European J Urol. 2013;66(2):202-6. doi: 10.5173/ceju.2013.02.art23. Epub 2013 Aug 13.
• Lam Van Ba O, Aharony S, Loutochin O, Corcos J. Bladder tissue engineering: a literature review. Adv Drug Deliv Rev. 2015 Mar;82-83:31-7. doi: 10.1016/j.addr.2014.11.013. Epub 2014 Nov 14.
• Garthwaite M, Hinley J, Cross W, Warwick RM, Ambrose A, Hardaker H, Eardley I, Southgate J. Use of donor bladder tissues for in vitro research. BJU Int. 2014 Jan;113(1):160-6. doi: 10.1111/bju.12285.
• Baker SC, Shabir S, Southgate J. Biomimetic urothelial tissue models for the in vitro evaluation of barrier physiology and bladder drug efficacy. Mol Pharm. 2014 Jul 7;11(7):1964-70. doi: 10.1021/mp500065m. Epub 2014 Apr 17.

Study record dates

Study Major Dates

• Study Start (Anticipated): October 1, 2018
• Primary Completion (Anticipated): October 1, 2024
• Study Completion (Anticipated): October 1, 2026

Study Registration Dates

• First Submitted: September 17, 2018
• First Submitted That Met QC Criteria: October 4, 2018
• First Posted (Actual): October 9, 2018

Study Record Updates

• Last Update Posted (Actual): October 9, 2018
• Last Update Submitted That Met QC Criteria: October 4, 2018
• Last Verified: September 1, 2018

More Information

Terms related to this study

• Keywords: Tissue engineering; Collagen scaffold; Alginate scaffold; Urothelial cells
• Additional Relevant MeSH Terms: Nervous System Diseases; Urologic Diseases; Urinary Bladder Diseases; Neurologic Manifestations; Urogenital Abnormalities; Congenital Abnormalities; Nervous System Malformations; Penile Diseases; Neural Tube Defects; Urinary Bladder, Neurogenic; Hypospadias; Spinal Dysraphism; Bladder Exstrophy
• Other Study ID Numbers: PSS2017/IMOPU-BERTE/MS

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: No
• IPD Plan Description: Individual data are not necessary for this study

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No