Preclinical Assessment of Tissue Effects by Gastrointestinal Endoscope Tip Temperature.

Background: Endoscope tips are heated by their electrical and illuminating components. During the procedure, they might get in close or even direct contact with intestinal tissues. Objective: To assess endoscope tip and tissue temperature as well as histopathologic changes of gastrointestinal (GI) tissues when exposed to the heated tip of GI endoscopes. Methods: The endoscope tip temperatures of four GI endoscopes were measured for 30 minutes in a temperature-controlled chamber. The temperature of ex vivo porcine GI tissues was measured for 5-, 15-, and 120-minute exposure to endoscope tips within a climate chamber to control environmental factors (simulation of fever as worst-case). Exposed tissues were histopathologically examined afterward. Control samples included untreated mucosa, tissue samples exposed to endoscope tips for 120 minutes, as well as tissue samples thermally coagulated with a bipolar high-frequency probe. Results: Actual endoscope tip temperatures of 59 to 86°C, dependent on the endoscope type, were measured. After 10 to 15 minutes, the maximum temperatures were reached. Maximum tissue temperatures of 44 to 46°C for 5 and 15 minutes, as well as up to 50°C for 120 minutes, were recorded dependent on tissue and endoscope type. No direct heat-induced histopathologic tissue alterations were observed in the 5- and 15-minute samples. Conclusions: Both clinically relevant and a worst-case control were tested. Even though elevated temperatures were recorded, no heat-related tissue alterations were detected. This overall supports the safety profile of GI endoscopy; however, the study findings are limited by the ex vivo setting (no metabolic tissue alterations accessible, no blood flow) and small sample number.

18F-labeling and initial in vivo evaluation of a Hitomi peptide for imaging tissue transglutaminase 2.

INTRODUCTION: Tissue transglutaminase 2 (TG2) is a calcium-dependent enzyme which cross-links proteins. It is overexpressed in many diseases and plays a key role in tissue remodeling, including cell adhesion and migration. Overexpression of TG2 in breast cancer is a marker for patients at risk of recurrence. Non-invasive imaging of TG2 can therefore play an important role in patient management. TG2 probes labeled with the positron emitters 11C and 18F have thus far not found widespread application due to purity and metabolism issues. Our approach was to radiolabel a TG2 selective, 13-mer amino acid peptide, which was modified with a 5-azidopentanoic acid group at the N-terminus via a copper free click chemistry approach. METHODS: Radiochemistry was performed and fully automated using an iPhase FlexLab module. We produced the radiolabeling synthon [18F]FBz-DBCO from [18F]SFB and DBCO-amine. After HPLC purification, [18F]FBz-DBCO was reacted with the modified peptide and the putative radiotracer purified by HPLC. In vivo imaging using the radiolabeled amine was performed in mice bearing either TG2 expressing MDA-MB-231 or non-TG2 expressing MCF-7 xenografts as negative control. Expression of the target was confirmed using immunohistochemistry and western blot techniques. RESULTS: We obtained 9 ± 2 GBq of the radiolabeled peptide from 55 ± 5 GBq of fluorine-18 in an overall synthesis time of 160 min from end of bombardment (EOB), including HPLC purification and reformulation. Small animal PET/MR imaging showed that visualization of MDA-MB-231 tumors using the radiolabeled peptide could only be achieved due to differences in clearance between tumor and surrounding tissue. In the MCF-7 xenograft model, radiotracer clearance from tumor and surrounding tissue occurred at a similar rate, thus making it impossible to visualize MCF-7 tumors. The presence of TG2 in MDA-MB-231 tumors and absence in MCF-7 tumors was confirmed by immunohistochemistry staining and western blot analysis. CONCLUSION: A fully automated synthesis of a TG2 selective, 13-amino-acid peptide modified with 5-azido pentynoic acid at the N-terminal was established using [18F]FBzDBCO as a prosthetic group. Although our results show that radiolabeled peptides have potential as imaging agents for TG2, more research needs to be performed to improve radiotracer kinetics.

Rapid and precise delivery of cells in the urethral sphincter complex by a novel needle-free waterjet technology.

OBJECTIVES: To investigate the therapy of stress urinary incontinence in a preclinical setting cells were injected into the urethrae of minipigs; however, cells injected by William's needle were frequently misplaced or lost; thus, we investigated if needle-free cell injections using a novel waterjet technology facilitates precise injections in the urethral sphincter complex. MATERIALS AND METHODS: Porcine adipose tissue-derived stromal cells (pADSCs) were isolated from boars, expanded, labelled, and injected in the sphincter of female pigs by waterjet employing two different protocols. After incubation for 15 min or 3 days, the urethrae of the pigs were examined. Injected cells were visualised by imaging and fluorescence microscopy of tissue sections. DNA of injected male cells was verified by polymerase chain reaction (PCR) of the sex-determining region (SRY) gene. Cell injections by William's needle served as controls. RESULTS: The new waterjet technology delivered pADSCs faster and with better on-site precision than the needle injections. Bleeding during or after waterjet injection or other adverse effects, such as swelling or urinary retention, were not observed. Morphologically intact pADSCs were detected in the urethrae of all pigs treated by waterjet. SRY-PCR of chromosomal DNA and detection of recombinant green fluorescent protein verified the injection of viable cells. In contrast, three of four pigs injected by William's needle displayed no or misplaced cells. CONCLUSION: Transurethral injection of viable pADSCs by waterjet is a simple, fast, precise, and yet gentle new technology. This is the first proof-of-principle concept study providing evidence that a waterjet injects intact cells exactly in the tissue targeted in a preclinical in vivo situation. To further explore the clinical potential of the waterjet technology longer follow-up, as well as incontinence models have to be studied.

Hydrojet-based delivery of footprint-free iPSC-derived cardiomyocytes into porcine myocardium.

The reprogramming of patient´s somatic cells into induced pluripotent stem cells (iPSCs) and the consecutive differentiation into cardiomyocytes enables new options for the treatment of infarcted myocardium. In this study, the applicability of a hydrojet-based method to deliver footprint-free iPSC-derived cardiomyocytes into the myocardium was analyzed. A new hydrojet system enabling a rapid and accurate change between high tissue penetration pressures and low cell injection pressures was developed. Iron oxide-coated microparticles were ex vivo injected into porcine hearts to establish the application parameters and the distribution was analyzed using magnetic resonance imaging. The influence of different hydrojet pressure settings on the viability of cardiomyocytes was analyzed. Subsequently, cardiomyocytes were delivered into the porcine myocardium and analyzed by an in vivo imaging system. The delivery of microparticles or cardiomyocytes into porcine myocardium resulted in a widespread three-dimensional distribution. In vitro, 7 days post-injection, only cardiomyocytes applied with a hydrojet pressure setting of E20 (79.57 ± 1.44%) showed a significantly reduced cell viability in comparison to the cells applied with 27G needle (98.35 ± 5.15%). Furthermore, significantly less undesired distribution of the cells via blood vessels was detected compared to 27G needle injection. This study demonstrated the applicability of the hydrojet-based method for the intramyocardial delivery of iPSC-derived cardiomyocytes. The efficient delivery of cardiomyocytes into infarcted myocardium could significantly improve the regeneration.

A novel waterjet technology for transurethral cystoscopic injection of viable cells in the urethral sphincter complex.

AIMS: In a recent preclinical study, we noticed that injection of cells in the urethral sphincter by needle through a cystoscope under visual control frequently yielded in misplacement or loss of cells. We, therefore, investigated if a needle-free waterjet device delivers viable cells under defined settings, including injection volume and pressure, fluid velocity and transportation media, precisely through the urothelium and connective tissue close to the sphincter muscle without full penetration of the sphincter apparatus. METHODS: Mesenchymal stromal cells (MSCs) were prepared for needle-free waterjet injections. Upon injections into liquids cell viability and yield were investigated by trypan blue dye exclusion. Upon injection into cadaveric urethral tissue samples, cells were isolated from the urethrae and expanded to prove that this novel method delivered viable cells into the tissue. MSC injections by William's needle served as controls. RESULTS: Waterjet injections of MSCs into isotonic cell culture medium resulted in equal or better yields of viable cells when compared with needle injections. Upon injection in urethral tissue samples, the waterjet technology facilitated fast and precise injections of viable cells through urothelial, mucosal and submucosal layers to reach the sphincter muscle. By controlling the injection pressure, loss of cells due to insufficient thrust or unintended full penetration was avoided. CONCLUSIONS: Needle-free waterjet injections deliver cells in the urethra faster and more precisely when compared with needle injections without compromising their viability. This is the first proof-of-concept study providing evidence that a waterjet transports viable cells precisely into the targeted tissue.

Cryobiopsy in the Upper Urinary Tract: Preclinical Evaluation of a Novel Device.

OBJECTIVE: To develop a novel device for cryobiopsy of the upper urinary tract (UUT) and to evaluate its feasibility in a standardized preclinical setting. MATERIALS AND METHODS: Flexible cryoprobes (diameter 0.9 mm; cooling agent CO2) were developed and used to extract biopsies in porcine UUTs. Cryosamples obtained by ureterorenoscopy were systematically compared with biopsy specimens obtained with standard of care devices in terms of physical characteristics (deflection angle and irrigation flow rates) and histologic criteria (assessability). RESULTS: Irrigation flow rates were significantly higher with introduced BIGopsy (2.8 ± 0.1) compared with standard forceps (0.94 ± 0.06; P < .001) and cryoprobe (1.1 ± 0.1; P < .001). Angular deflection was significantly reduced by the inserted cryoprobe (130.7° ± 1.2° vs 166.9° ± 1.1° [BIGopsy] or 161.4° ± 1.9° [standard forceps]; both P < .001). Significantly larger UUT tissue samples were obtained by the cryoprobe (mean specimen area 7.5 ± 2.5 vs 4.6 ± 2.5 mm² [BIGopsy] or 1.4 ± 1.4 mm² [standard forceps]; both P < .001). No crush artifacts were observed in cryosamples. Superior histologic assessability scores were achieved in samples obtained by the cryoprobe (mean 2.8 ± 0.8) and BIGopsy (2.3 ± 1.9) when compared with standard forceps (0.4 ± 0.9; P < .001). CONCLUSION: Cryobiopsy in the UUT is feasible and represents a viable new option to improve the diagnostic accuracy of histopathologic evaluation. Larger and more representative tissue samples can be obtained using a cryoprobe and artifacts may be avoided. Further optimization of the probe will reduce possible restrictions of ureterorenoscopy handling when the device is inserted.