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From wires to waves, a novel sensor system for in vivo pressure monitoring – Scientific Reports

Design of the sensor element and sensor packaging

A piezoresistive sensor element was designed and manufactured by SINTEF17. In brief, it comprises a 2-µm thick single-crystalline silicon device layer with a complete Wheatstone bridge configuration. This device layer is anodically bonded to a glass wafer containing cavities located beneath the pressure sensing membranes46. The sensor element has been seamlessly integrated into a probe, and the materials exposed to the environment comply with biocompatibility standards, such as USP Class VI and/or ISO-10993. Further information about the sensor probe can be found in a separate publication19.

Engineering of the SDL, MICS and BLE units

The engineering of the tethered sensor data logger (SDL) was previously reported19. Briefly, it consists of the assembly of an analog digital converter, a microcontroller, and a micro-SD card. The SDL is powered by batteries or through a USB connection to a PC. The measurement data is stored on the micro-SD card, while a software interface allows for real-time streaming and local storage of the data. Unfortunately, during acute implantation experiments in pigs, the SDL data files were corrupted several times. The data presented in Fig. 1 could however be salvaged thanks to video recordings that were later digitized.

For the engineering of the implantable wireless units, MICS and BLE, an overview of their system architecture is presented in Supplementary Fig. 1a. The signal from the sensor is amplified, digitized, and stored on a low power sensor interface. Both systems were built with small commercial off-the-shelf components.

The MICS unit was obtained—as a courtesy—from the laboratory of Prof. Sawan, Department of Electrical Engineering, Polytechnique Montréal. This system is based on a MICS component powered by a battery and capable of communicating with an external base station (Sup. Fig. 1b). The assembly and communication modules were from Microsemi Corporation (Arizona, USA) conforming to the MICS standard. Four prototypes were built on a round PCB resulting in a unit of 30 mm in diameter (Sup. Fig. 1c). The MICS unit is connected to a 30-mm coin battery holder to accommodate a Panasonic CR2477 1000 mAh lithium battery. A LabVIEW (National Instruments, Texas, USA) program with a graphical user interface allows the retrieval of the data stored in the memory and the configuration of the MICS unit.

Four prototypes of the BLE unit were manually assembled on a 30 × 25 mm PCB. The Bluetooth stack ran on an MK13A module from MOKOBlue, which is based on a Nordic Semiconductor nRF5340 SOC, while analog-to-digital conversion was performed using two Texas Instruments ADS1100 ADCs. The whole system was powered by a 750 mAh lithium thionyl chloride EF651625 battery from EVE Energy Co, regulated through a Linear Technology LTC3544 quad buck regulator. Data collection was handled by a custom Python script using an nRF52840DK Development Kit as a receiver unit.

Sensor system characterization and calibration

The complete wireless sensor systems (with MICS/BLE communication means) underwent characterization and calibration. To perform the calibration, the sensor probe and MICS/BLE unit were placed inside a dedicated aluminum pressure chamber (Sup. Fig. 2a and 2b). This chamber was designed to withstand pressure and was connected to a Fluke 6270A pressure controller and calibrator via a 6 mm hose. Inside the chamber, a fixture was specifically designed and 3D-printed to support up to four sensor probes and the MICS/BLE unit. The tip of the sensor probe was immersed in water during the characterization. The chamber was then closed and partially submerged in a thermally controlled water bath set at 37 °C. Pressure was applied in increments of 25 mbar, ranging from 800 to 1200 mbar and back to 800 mbar. A representative calibration curve is presented in supplementary Fig. 2c. Using these data points, a calibration equation was derived through polynomial fitting of the pressure versus the averaged sensor output for each pressure level (Sup. Fig. 2d). This equation was subsequently used to calculate the pressure values from the sensor outputs recorded during the animal trials. Of note, for the BLE in vivo experiment, the two sensors implanted—in the bladder and the femoral artery—could not be calibrated before the implantation due to unforeseen technical issues and time constraints. Therefore, the plots presented in Fig. 3 were created using a calibration curve obtained after the implantation. The sensors were removed, cleaned with saline, dried, and underwent calibration a few days later. To compensate for value drift that may have occurred during this process, the blood pressure curve was normalized to the pressure recorded by the intraoperative monitoring system after termination of the pig. For the bladder pressure measurements, normalization was not done. Due to the production costs associated with the sensor elements and packaging, we opted to reuse sensors from a previous clinical trial.

Housings for in vivo implantation

To be fully implantable, the MICS/BLE units had to be isolated from body fluids in a manner that did not compromise data transmission. To build the housing of the MICS unit, we used titanium. Water resistance was ensured by a rubber joint at the interface of the two pieces (Fig. 2a). The top and bottom walls thinned down to 500 µm thin. The housing was assembled with four corner M2.5 T8 screws. A hole was drilled for the cable to pass through the wall of the housing. Silicon rubber (Smooth-On, Inc, Macungie, PA) and epoxy (EPO-TEK 353ND-T) were applied on the inside and the outside of the housing to maintain water resistance. For the housing of the BLE unit, the overall design was similar, although there were differences in dimensions and materials used (Fig. 3a). Instead of titanium, we utilized PEEK (polyether ether ketone), a biocompatible plastic material that supports BLE transmission. To test the water resistance, the housings were immersed in a container filled with colored water.

Animals

Animal experiments were carried out at the Section for Experimental Biomedicine at the Department of Production Animal Clinical Sciences (ProdMed), at the Norwegian University of Life Sciences (NMBU), SEARCH at NMBU, and the Centre for Clinical, Experimental Surgery, and Translational Research of the Biomedical Research Foundation of the Academy of Athens. These facilities are approved by EU standards and regulations to carry out animal experiments on large animals. In Norway, the procedure was approved by the Norwegian Animal Research Authority (Forsøksdyrutvalget) under the identification numbers 7089 and 19,435. In Greece, the research was conducted in compliance with the legal requirements for animal experimentation, in harmonization to the European Directive 2010/63, and it was approved by the Veterinary Service of the Prefecture of Athens under registration number 5522/24-10-2018. In compliance with the FELASA guidelines, all efforts were made to minimize the number of animals used and their suffering. This report has been written using the ARRIVE guidelines for animal research. All surgical procedures, post-operative procedures, and euthanasia were supervised by veterinarians. In total, we used nine female pigs weighing between 25 and 50 kg. Eight of them had a genetic background consisting of 50% Norwegian land pig, 25% Norwegian Yorkshire, and 25% Duroc, and one had a genetic background of 50% Landrace and 50% Large White female pigs. This inconsistency was a consequence of the COVID-19 pandemic; however, it is not expected to have any impact on the results.

Anesthesia and analgesia

Food was withheld from pigs 12 h prior to the induction of anesthesia. Anesthesia for acute experiments was performed as previously described47. For short-term survival experiments, the pigs were premedicated with dexmedetomidine 80 µg/kg (0.5 mg/ml Dexdomitor, Orion) and midazolam 0.6 mg/kg (5 mg/ml Dormicum, Roche) intramuscularly. An auricular vein was catheterized prior to induction of anesthesia by propofol 1.2 mg/kg intravenously. The pigs were endotracheally intubated and connected to an anesthetic machine. Anesthesia was maintained by isoflurane in a mixture of oxygen and air and positive pressure ventilation was applied. An arterial catheter was placed in the hindlimb metatarsal artery for intraoperative monitoring of blood pressure. After zeroing the pressure transducer, it was positioned at the level of the heart. Prior to recovery, ketoprofen 3 mg/kg and buprenorphine 20 µg/kg were administered intravenously. Lidocaine 20 mg/ml 10 ml was infiltrated into the pocket containing the titanium housing. In all pigs, vital signs were monitored using an anesthetic monitor.

Surgical implantation

For acute experiments, an ultrasound echo sonogram was performed to locate the targeted organs. For urinary bladder implantation, the sensor probe was inserted with suprapubic technique, using a T-peel introducer needle (I-Flow corporation, Lake Forest, CA 92630, USA). A similar approach was used for intracardiac implantation. Correct placement was also checked by withdrawal of urine or blood. The sensor probe was externally connected to either the tethered data logger or the MICS/BLE unit.

Two pilot survival experiments were also performed during which the urinary bladder was solely targeted. The first approach was suprapubic, as described earlier. Two skin incisions were performed on the hypogastric (2 cm, midline) and left iliac abdominal (5 cm) regions. A channeling tube was used to create a subcutaneous tunnel between the two incisions. After suprapubic insertion of the sensor probe, the wire and connectors were led subcutaneously to the abdominal iliac incision through the channeling tube. The hypogastric incision was closed. A subcutaneous pocket was created in the lumbar region (rostral to the left middle gluteal muscle), to accommodate the housing. The channeling tube was used to subcutaneously tunnel the sensor probe between the lumbar and the left iliac abdominal incision and connect it to the titanium housing. A silicon rubber mixture (Smooth-On, Inc, Macungie, PA) was applied to the connectors to ensure water resistance. The silicon rubber block and the titanium housing were sterilized by immersion in a Rely + On Perasafe (Puls AS, Norway) solution for 15 min and rinsed with saline before implantation. The wounds were sutured, and local anesthetic was administered subcutaneously. The second approach was inspired from48. To secure the sensor probe in the urinary bladder, a 3-way Foley balloon catheter was attached with suture stitches. A subcutaneous pocket to accommodate the MICS unit was created, as described earlier. A low midline abdominal incision was performed to expose the urinary bladder. The urinary bladder was identified and a small incision of about 5 mm was performed on the ventral bladder wall to insert the catheter and the sensor probe attached to it. The balloon was then inflated with 2 ml sterile saline and the bladder incision was closed using a 3-0 Propylene suture. A channeling probe was used to tunnel the extension cable from the subcutaneous pocket accommodating the housing, through the abdominal wall to connect the MICS unit with the sensor probe. The connection was embedded and sterilized, as explained earlier. The abdominal wall was sutured in 3 layers, the pocket wound was sutured, and local anesthetic was administered subcutaneously.

Spinal cord stimulation

For some acute experiments, the sensor system was used to record bladder pressures while attempting to induce stimulation-driven bladder contractions. Spinal cord stimulation at a sacral level was implemented after performing a sacral laminectomy to expose the L5-S3 spinal cord. Stimulations targeting the sacral micturition center were delivered using a monopolar electrode placed on the dorsal surface of the spinal cord. Trains of biphasic balanced cathodic-first pulses of electrical currents with variable intensity were delivered using a wearable neural stimulator—called STIMEP—designed in compliance with EU directives for active medical devices (90/385, 93/342 and EN 62304 before the application of the new EU regulation MDR2017/745) by the CAMIN team (INRIA/University of Montpellier, France) in association with the Axonic company (Sophia Antipolis, Vallauris, France)36.

Post-operative care

Cephalosporin (25 mg/kg, Zinacef, GSK) was administered at the beginning and by the end of the procedure. Ketoprofen was administered i.m. for 2 days after the surgery. The pigs were monitored, and the wound areas were palpated to check for pain at least twice daily. Rescue analgesia in the form of Buprenorfin was available, but not required.

Termination

For the acute experiments, the pigs were euthanized while in anesthesia with an i.v. injection of potassium chloride. For one survival experiment, one pig was stunned using a captive bolt gun, while for the other, the pig was sedated as described earlier and euthanized with a 10 ml bolus dose (i.v.) of sodium pentobarbital (400 mg/ml Exagon, Richter Pharma).