Bladder Exstrophy, Bladder, Neurogenic, Hypospadias, Spina Bifida, Urothelial Neoplasm
Conditions
Keywords
Tissue engineering, Collagen scaffold, Alginate scaffold, Urothelial cells
Brief summary
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.
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).
Interventions
PROCEDURETransurethral Resection of Prostate
Transurethral resection of the prostate is a urological operation used to treat benign prostatic hyperplasia (BPH). It is performed by visualising the prostate through the urethra and removing tissue by electrocautery or sharp dissection with a resectoscope. This is considered the most effective treatment for BPH. This procedure is done with spinal or general anaesthetic. A triple lumen catheter is inserted through the urethra to irrigate and drain the bladder after the surgical procedure is complete. Outcome is considered excellent for 80-90% of BPH patients.
Sponsors
Central Hospital, Nancy, France
Study design
Observational model
OTHER
Time perspective
PROSPECTIVE
Eligibility
Sex/Gender
MALE
Age
18 Years to No maximum
Healthy volunteers
No
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
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Histological analysis of biopsy. | 6 month | Histological analysis with standard coloration will assess the viability of urothelium. Signs of necrosis (ulcerations, destructions of urothelium structure) will be noted. |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| RT-qPCR analysis of biopsy. | 6 month | 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. |
| Digestion of the biopsy and culture with adapted medium. Optimization of culture conditions. | 12 months | 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. |
| Histological analysis of the cultured cells. | 12 months | 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. |
| Immunocytological analysis of the cultured cells. | 12 months | 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) |
| RT-qPCR analysis of the cultured cells. | 12 months | 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. |
| Development of original alginate freezed-dried scaffold | 12 months | 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.. |
| Association of the cultured cells with different scaffolds. Culture of cellularized scaffolds. | 36 months | Previously cultured and analyzed cells will be associated to different scaffolds. * Collagen scaffolds * Alginate scaffolds * Alginate hydrogels obtained by spray * Original alginate freezed-dried scaffold (Outcome 4) Cellularized scaffolds will be cultured at least 28 days using previously described conditions. |
| Immunohistological analysis of biopsy. | 6 month | Immunohistological analysis using specific antibodies will locate the urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen). |
| Immunohistological analysis of cellularized scaffolds | 36 months | 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) |
| RT-qPCR analysis of cellularized scaffolds | 36 months | 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. |
| Biophysical analysis of cellularized scaffolds | 36 months | 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) |
| Implantation of the cellularized scaffold in Nude mice | 12 months | 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. |
| Histological analysis of implanted scaffolds | 12 months | 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. |
| Immunohistological analysis of implanted scaffolds | 12 months | 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) |
| RT-qPCR analysis of implanted scaffolds | 12 months | 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. |
| Histological analysis of cellularized scaffolds | 36 months | 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. |
Contacts
Primary ContactNicolas Berte, Dr
Backup ContactJean-Louis Lemelle, PHD
Outcome results
None listed