A novel needle-free WaterJet technique for injecting porcine muscle-derived cells into the urethra

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URI: http://hdl.handle.net/10900/135566
Dokumentart: Dissertation
Date: 2023-01-23
Language: English
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Aicher, Wilhelm K. (Prof. Dr.)
Day of Oral Examination: 2022-12-22
DDC Classifikation: 610 - Medicine and health
Keywords: Myoblast
License: Publishing license including print on demand
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Stress urinary incontinence is the most common type of urinary incontinence. It reduces the quality of life of patients. Actual standard surgical therapeutic modalities are offering a symptomatic relief without treating the underlying disorder. Therefore, we developed a novel cell injection technology to deliver viable cells for recovery of the function of the urethral sphincter by WaterJet (WJ, ERBE). Here we investigated if a) porcine muscle−derived cells (pMDC) could be injected by WJ in both, cadaveric samples and living porcine urethra with high viability, b) the WJ inherits the risk of full tissue penetration of the porcine urethra and thus loss of cells, and c) WJ grants improved precision of cell injection and distribution in tissues targeted. The pMDC were produced from male boars and characterized by qRT−PCR and immunofluorescence. Visualized by Calcein AM vs EthD−1, cells were injected into cadaveric porcine urethral by WaterJet vs William’s Needle. In another in vivo study, cells labelled with PKH 26 or/and BacMam, were injected in living female pigs by WaterJet using either a moderate (E60−10; n = 18) or elevated pressure (E80−10; n = 6) protocol, and follow-up (f/u) of up to 7 days. Cell injections targeted the site of the maximum urethral closure pressure (Aquarius TT, Laborie Medical). After harvesting the whole bladder and urethra, cells were traced by an In Vivo Imaging System (IVIS, PerkinElmer) and visualized by fluorescence microscopy of cryosections. Nuclei were stained by DAPI, muscular tissue by phalloidin-iFluor 488. The pSRY gene was detected by PCR. The distribution of injected pMDC was measured as X−depth, Y−width, Z−height and calculated areas in the XY-, YZ-, XZ- planes were analysed. The distance between sphincter muscle and injected cells (DISIC) was measured simultaneously. The analyses provided experimental evidence that pMDCs injected by WaterJet in vitro were viable. Our in vivo study supported that cells appeared defined cellular somata with distinct nuclei and contained intact chromosomal DNA. The success rates of WJ cell application in living animals were significantly higher (≥ 95%, n = 24) when compared to needle injections. Only one out of six samples with full penetration was observed in the WJ E80−10 group. The analyses of the 3D distribution of cells after WJ injection documented that the Y−width of the WJ E80-10 group was statistically significant wider (P = 0.0479) than that of WJ E60-10 group. The same was recorded for the cell distribution in Z-height (P < 0.0001). The YZ-plane of the WJ E80−10 group was statistically significant larger (P = 0.0005) than that of WJ E60-10 group, as well as XZ-plane (P = 0.0204). The injection depth of WJ E80-10 compared to WJ E60-10 showed a statistically significant decrease in length (P < 0.0001). This indicated that WJ E80-10 injections transported cells closer to the targeted rhabdosphincter, but at a higher risk for full penetration. We conclude that the novel WJ is a fast, precise, and easy-to-use innovative method to inject living cells in tissues with a significantly wider and diffuse distribution, with less disintegration of the tissue targeted, and at higher success rates.

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