PEEK 3D printed lightweight dorsal skin fold chambers reduce chamber-related animal distress

The dorsal skinfold chamber is a major model for the repetitive examination of vascular changes and inflammation. However, the dorsal skinfold chamber causes considerable stress to test animals. Therefore, the model is not only criticized by animal welfare groups. This criticism is understandable, because many improved models have already been released, but the classic titanium chamber is mainly used17,23,24,27,28,29. For the generalization of plastic chambers, it lacked on the one hand a simple manufacturing protocol and on the other hand a proof of superiority in the reduction of distress.

Here we present a simple model 3D printed in PEEK. We show that the PEEK chamber reduces distress and prolongs peak observation time.

Surgical requirements and the quality of intravital microscopy are equivalent in PEEK and titanium chambers. When implanting the PEEK port, penetration of the skinfold with screws can be omitted, due to the light weight of the port. In titanium chambers, the screws cause penetrating holes at the base of the skinfold, which are associated with chamber tilt. The PEEK chamber is secured by tear-resistant sutures, which the test animal cannot reopen (FibreWire, Arthrex, Munich, Germany). These sutures cause less trauma than previously used screws. After the implantation of the chamber, the experiments proceed without difference with the titanium chambers. In particular, there are no differences in the quality of repetitive intravital fluorescence microscopy.

The most common complication of dorsal skinfold chamber experiments is lateral tilting of the chamber during the second week.17. By week 3, 50% of the titanium chambers tilt to a >90° position, causing the animal to be immobilized. In all PEEK chambers, the tilt remained below 90° for three weeks. The only PEEK chamber that had a 50° inclination the second week remained constant the following week. This is clearly due to the reduced weight. In contrast, the titanium chambers continued to tilt over time. Therefore, the stable position of the PEEK chambers could extend the maximum duration of future experiments to four or five weeks.

Besides a decrease in tilt during the third week, the use of PEEK chambers also reduced the distress of the experimental animals during the postoperative period. Postoperative weight loss is significantly higher in animals in the titanium chamber and does not return to baseline values ​​within three weeks. With PEEK chambers, on the other hand, the test animals reach their initial weight as early as the second week. Consistent with our data, previous research has described up to 15% postoperative weight loss for titanium chambers and decreased weight loss for non-metallic dorsal skinfold chambers30,31,32.

This difference in stress was confirmed by FCM measurement, a non-invasive measure of adrenocortical activity.33. FCMs increased significantly after implantation of a titanium port while a slight increase in FCMs was observed for PEEK ports. Therefore, postoperative stress was mainly related to the titanium chamber and not to the surgery itself. In the middle and late phase, FCMs returned to baseline values ​​in both groups. Design of future titanium chamber experiments should consider increased postoperative stress as a potential bias34.

In contrast to FCMs and changes in body weight, mice in the two groups did not differ in burrowing activity or distress score. The distress score remained at a low level after the operation. The postoperative increase of 7/66 points in the distress score was statistically significant. However, the values ​​remained in the lower range of the score.

To our knowledge, this study is the first to investigate the distress of laboratory animals with dorsal skinfold chambers. Despite the lack of animal distress data, many chamber improvements have already been published. These improvements were supposed to reduce animal distress and enable MRI imaging. A simple development is a smaller titanium frame with an equally large viewing window29. These small titanium chambers are marketed in the United States (SM100 small back kit, APJ Trading Co., Ventura, CA, USA). Schreiter et al. describe the advantages of a self-designed small titanium chamber: no postoperative recovery period was required, younger animals could be used, and their stress was believed to be reduced1. However, titanium chambers are not MRI compatible and screw fixation is required. Additionally, titanium is difficult to process and cannot be manufactured in life science facilities.

Innovative developments are plastic chambers, which have been used for years in Japan and the USA. The first Duracon plastic chamber was described in 2003 by Ushiyama et al..17. The post illustrated reduced lean and a supposed reduction in distress, due to the lightweight Duracon material. However, quantification of tilt and distress was not performed. Additionally, these early plastic chambers continued to use screw fixing17.35. These screws penetrate the skin and cause large wounds at the base of the chamber.

Another weight reduction was achieved by using thermoplastic PEEK. PEEK is characterized by high flexural modulus (3738 MPa) and tensile strength (100 MPa) compared to Duracon (2500 MPa, 87 MPa) and acrylic glass (3210 MPa, 75 MPa)36. PEEK can therefore resist the bite of rodents. In addition, PEEK can be manufactured with additive manufacturing processes, which translates into other advantages such as a high degree of geometric freedom, low production costs and flexibility regarding the production of single parts or in one piece.37.

The first lightweight PEEK chambers were introduced by Gaustad et al. and Seynhaeve et al.23.28. The weight of the PEEK chamber was as low as 1g and 1.1g, respectively. The chambers were fixed with sutures (Gaustad) or small screws (Seyhaeve). Mice fitted with the chambers showed full capacity for movement, climbed and gained weight like mice without chambers. We observed similar positive effects for PEEK chambers (body weight, climbing, mobility). Additionally, we verified reduced distress using a standardized protocol. We repeatedly measured chamber tilt and found that the PEEK chambers significantly reduced tilt during the third week of the experiment. Lightweight chambers with reduced lateral tilt allow for increased observation times of up to a month27. This is particularly relevant for research on biomaterials and oncology: observation times of the dorsal skinfold chamber of three to five weeks could allow the study of the long-term integration of biomaterials (fibrosis, formation of giant cells, vascularization of implants)38. In oncology, longer observation times could significantly improve the model, since tumor cell growth already occupies a large part of the current maximum observation time.39.

The reduction in inclination was significant, although the method of measurement had limitations, as the position of the chamber depends on the position of the body. The measurement was performed on anesthetized animals in a vertical position with all feet on the ground. However, when positioning the animals, slight deviations of the angles occurred.

Another limitation of the study is that the standardized distress score does not focus on immobilization of test animals. However, the obstacle to free movement, due to the inclination and the weight of the chamber, probably represents the main restriction for laboratory animals. Electronic recording of animal mobility through tracking systems or recording of time spent climbing in the cage could increase the power of stress analysis. However, the use of standardized distress scores allows comparisons with previous experiments.

The small number (n = 6) of test animals can be considered as another limitation of the study. However, the PEEK chamber was significantly superior in key outcomes, such as tilt and weight loss. Therefore, no additional laboratory animals had to be included. Another limitation related to the study design is that the PEEK and titanium chambers have different sizes. Therefore, all conclusions are related to the design (size, weight), but not to the material (PEEK vs titanium). Low and lightweight titanium chambers can also reduce animal distress compared to standard large titanium chambers. Still, we consider PEEK to be a more suitable material because it increases availability, enables imaging, and reduces costs. These main advantages make a lightweight titanium group obsolete.

Conclusion

In experiments with dorsal skinfold chambers, animals are particularly stressed by conventional titanium chambers. This system needs to be reviewed, within the framework of the 3Rs. Despite the development of smaller and lighter chambers, most dorsal skinfold chamber experiments in recent years have continued to use titanium chambers. We have shown that lighter chambers can significantly reduce animal distress and even extend the maximum duration of the experiment. PEEK chambers are particularly suitable for this purpose: they are autoclavable, stable enough to resist rodents, inexpensive and widely available thanks to 3D printing.

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