This in-vivo study was performed in a rat model, where safety and performance when executing microvascular anastomoses with Symani was assessed. Anastomosis patency rates, histological outcomes and execution times were evaluated in this study.
The robotic system
The Symani Surgical System (Fig. 1a) is a robotic device specifically designed for microsurgical open procedures. The flexible platform uses the principles of teleoperation to provide surgeons with high precision in manipulation of very small anatomy such as vessels, nerves, and ducts. The system consists of a master console controlling the end-effectors including robotic arms on which articulated microinstruments are mounted. The robotic arms may be positioned in any anatomical region.
The Master Console is an ergonomic chair equipped with surgeon-controlled manipulators along with a footswitch. The surgeon can directly move the manipulators in the same manner as she or he would with manual instrumentation scaling down this motion to the arms and then to the robotic microinstruments. On the robotic arms of the system, articulated microinstruments (7 degrees of freedom) are installed and moved above any anatomical region. The system features motion scaling ranging between 7 × to 20 × and includes tremor filtration to increase surgical precision.
Users
Three microsurgeons performed the arterial and venous anastomoses, they had previous microsurgery experience from 8 to 12 years. These surgeons contributed extensively to bench testing of Symani and received the training program prior to executing the anastomoses in this study. Hence, they are considered fully proficient in using the system safely and effectively.
Animal model
This study was performed in rat femoral vessels, which are a common target in microvascular anastomosis training and research model21,22. The same protocol was used in the two centers where the study was carried out: the Laboratory Animal Housing Center (CeSAL), University of Florence (Florence, Italy), and the Jesús Usón Minimally Invasive Surgery Center (CCMIJU, Cáceres, Spain). Microsurgical anastomoses on femoral veins were performed on 22 Wistar rats (Rattus Norvegicus) with a minimum weight of 350 g. Among them, 9 rats were in a preclinical study approved in 2018 by the Italian Ministry of Health and carried out at CeSAL; while 13 rats were in a preclinical study approved in 2019 by the Ethical Committee of Animal Experimentation of the CCMIJU, where the study was performed. Microsurgical anastomoses on femoral arteries were performed on 34 Sprague Dawley rats (Rattus Norvegicus) with a minimum weight of 350 g. This study was approved in 2019 by the Italian Ministry of Health and carried out at the CeSAL. All methods were carried out in accordance with the specific guidelines and regulations in force in Spain and Italy, as well as in compliance with European legislation on animal welfare and protection of animals used for scientific purposes. The authors complied with the ARRIVE guidelines.
Study design for vein and artery anastomoses
The Symani robot was placed close to the surgical table with microinstruments directed toward the rat femoral vessels (Fig. 1b). In microsurgery it is common in clinical practice to work with a microscope face-to-face: on one side of the microscope the lead surgeon (who in this study was the one who manipulated the robot during the robotic procedure) and on the opposite side of the microscope the assistant surgeon that supports during the procedure. Both the surgeon and the assistant used the microscope. The anastomoses were performed on femoral veins and arteries, the execution time was evaluated, and patency was assessed immediately after the procedure and also after one week. Histopathological characteristics of vessels were evaluated after animal sacrifice. Both the vein and artery studies were two-armed controlled trials to allow direct and unbiased comparison of the Symani-aided microanastomosis with the manual technique. Both the left and right femoral vessels of the animal were sutured. A manual (M) anastomosis was performed on one vessel, and on the other side, a robotic (R) anastomosis was performed. The treatment (M or R) was alternated with respect to the side of the animal: left or right vessel and temporal execution (M-R: first M then R; or R-M: first R then M).
Before surgery, the rats were prepared by a veterinarian with intraperitoneal anesthesia using Xylazine (5–10 mg/kg) and Zoletil100 (20–40 mg/kg) at CeSAL and with inhalatory anesthesia using Sevoflurane at CCMIJU. Rats were carefully placed in a supine position over an animal warming pad and prepared for surgery. A trichotomy of the groin areas was performed, followed by disinfection with povidone-iodine and chlorhexidine and administration of prophylactic broad-spectrum antibiotic to prevent potential post-operative infections. After that, the surgeon proceeded with skin incision and vessel preparation by using standard microsurgical instruments. Once the femoral vein or artery was exposed and isolated, a colored background was placed behind the vessel for contrast. The diameter of the vessel was measured with a digital caliber and secured with a double microvascular clamp. Then, the vessels were cut in the middle, and end-to-end microvascular anastomoses were completed using 10-0 nylon sutures (S&T AG, Neuhausen, Switzerland). After the execution of the two anastomoses (left/right rat groin, one robotic, the other manual), the skin incisions were closed with 3-0 prolene sutures (Ethicon, Pennsylvania, United States). After skin closure, the animals were monitored for physiological recovery from anesthesia and housed individually with daily animal welfare assessment.
Patency evaluation
Patency is a primary endpoint of vein and artery studies as the restoration of vascular flow is directly related to the quality and precision of the anastomosis. Indeed, traumatic tissue handling or lack of precision in suture placement may cause a thrombotic cascade through leak or hemodynamic turbulence. The Acland’s “milking test” was used to assess anastomotic patency23,24. Patency outcome was binary (patent or not patent) based on surgeon’s judgment after the Acland’s milking test. Patency evaluation was performed immediately after anastomosis (T0) for both veins and arteries, with re-evaluation after one week for veins (T1W) and after two weeks for (T2W) for arteries, as complete re-endothelialization of intraluminal suture is faster in the vein than the artery18. The exploration of the anastomotic site was performed with the same method of analgesia, anesthesia, and incision as that at the time of intervention.
Histological analysis
Histological analysis of the anastomosis site was conducted on vessel tissue harvested at the last patency evaluation (T1W or T2W for veins and arteries, respectively) just prior to sacrifice the animal. The tissue was collected in a closed-circuit disposable container pre-filled with 4% formaldehyde solution. The histological evaluation was performed by AnaPath Services GmbH (Liestal, Switzerland). The sections were stained with hematoxylin and eosin, as well as with Masson’s trichrome. A semi-quantitative histopathological analysis was performed for each vein and artery segment on the stained sections according to an adapted ISO 10993-6:2016(E) scoring system (Supplementary Table 1), including the following parameters: vessel reaction, vessel inflammation, inflammatory/host reaction at suture site, total average score (vessel reaction + vessel inflammation + tissue reaction at the suture site). In the analysis, a score difference of 0.0–2.9 indicates no or minimal host reaction, 3.0–8.9 indicates a slight host reaction, and 9.0–15.0 indicates a moderate host reaction while a score ≥ 15.1 indicates a severe host reaction compared to a reference material.
A Principal Component Analysis (PCA) was carried out using histological data with the aim to detect if there were different histological profiles in between all robotic and manual anastomosis carried out in all veins and arteries. PCA allows analyzing data sets that contain a variety of characteristics per observation; this allows data to be interpreted as a set while conserving joint information. PCA is used to reduce the dimensionality of the data set and allow simplified visualization of this multidimensional data. This analysis takes all the values of the histological variables as input and combines them to evaluate the degree of separation among data points in a low-dimensionality scatter plot. The three main histological variables were endothelial loss, fibrin/platelet thrombus, and intimal proliferation. We have used the first principal two components (PC1 and PC2) to plot the data in two dimensions and visually identify the closely related groups of data points: those related to robotic intervention and those related to manual intervention.
Execution time
The time to perform single sutures, as well as the time to complete anastomoses, was recorded for vein and artery procedures as well as robotic and manual techniques. Mean values were calculated for each user. Manual and robotic anastomoses also were assessed separately.
Statistical analysis
Statistical analyses were performed using GraphPad Prism software version 8.0 (GraphPad Software, San Diego, CA, USA). All data are expressed as the mean ± standard deviations. A Chi-squared test on patency outcomes (two-sided, confidence interval 95%) was performed to compare manual and robotic procedures. Time data are expressed as the mean ± standard deviations, and differences between means were analyzed by Student’s t-test; p-values < 0.05 were considered to indicate statistically significant differences between groups. Logarithmic regression of suture time was used for the graphical representation but further analysis was not performed. PCA was used to analyze differences between robotic and manual procedures by using confidence ellipses and their distance to emphasize groups and their separation on the scatter plots. PCA was carried out using the free software PAST (PAleontological STatistics) version 3.