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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.
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).
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| Name | Type | Description | Arm Group Labels | Other Names |
|---|---|---|---|---|
| Transurethral Resection of Prostate | Procedure | 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. |
|
| 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 |
| 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. |
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Inclusion Criteria:
Exclusion Criteria:
Only male patients have prostate
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Male patient requiring TURP for Benign Prostatic Hyperplasia with prostate weight evaluated preoperatively greater than or equal to 30 grams
| Name | Role | Phone | Extension | |
|---|---|---|---|---|
| Nicolas Berte, Dr | Contact | 00333673004195 | n.berte@chru-nancy.fr | |
| Jean-Louis Lemelle, PHD | Contact | 0033383154729 | jl.lemelle@chru-nancy.fr |
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| PubMed Identifier | Type | Citation | Retractions |
|---|---|---|---|
| 24579029 | Background | 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. | |
| 25446136 | Background | 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. |
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Individual data are not necessary for this study
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| ID | Term |
|---|---|
| D016135 | Spinal Dysraphism |
| D001750 | Urinary Bladder, Neurogenic |
| D001746 | Bladder Exstrophy |
| D007021 | Hypospadias |
| ID | Term |
|---|---|
| D009436 | Neural Tube Defects |
| D009421 | Nervous System Malformations |
| D009422 | Nervous System Diseases |
| D000013 | Congenital Abnormalities |
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| ID | Term |
|---|---|
| D020728 | Transurethral Resection of Prostate |
| ID | Term |
|---|---|
| D011468 | Prostatectomy |
| D013521 | Urologic Surgical Procedures, Male |
| D013520 | Urologic Surgical Procedures |
| D013519 | Urogenital Surgical Procedures |
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Bladder biopsy obtained from the bladder neck during Transurethral Resection of Prostate.
Maximum 3 biopsies measuring about 0.5*0.3*0.2 cm.
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.
| 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:
| 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 |
| 24053725 | Background | 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. |
| 24697150 | Background | 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. |
| D009358 | Congenital, Hereditary, and Neonatal Diseases and Abnormalities |
| D009461 | Neurologic Manifestations |
| D001745 | Urinary Bladder Diseases |
| D014570 | Urologic Diseases |
| D052776 | Female Urogenital Diseases |
| D005261 | Female Urogenital Diseases and Pregnancy Complications |
| D000091642 | Urogenital Diseases |
| D052801 | Male Urogenital Diseases |
| D012816 | Signs and Symptoms |
| D013568 | Pathological Conditions, Signs and Symptoms |
| D014564 | Urogenital Abnormalities |
| D010409 | Penile Diseases |
| D005832 | Genital Diseases, Male |
| D000091662 | Genital Diseases |
| D013514 | Surgical Procedures, Operative |