*Article* **Microsurgical Transplantation of Pedicled Muscles in an Isolation Chamber—A Novel Approach to Engineering Muscle Constructs via Perfusion-Decellularization**

**Aijia Cai 1,\* , Zengming Zheng <sup>1</sup> , Wibke Müller-Seubert <sup>1</sup> , Jonas Biggemann <sup>2</sup> , Tobias Fey 2,3 , Justus P. Beier <sup>4</sup> , Raymund E. Horch <sup>1</sup> , Benjamin Frieß 1,† and Andreas Arkudas 1,†**


**Abstract:** Decellularized whole muscle constructs represent an ideal scaffold for muscle tissue engineering means as they retain the network and proteins of the extracellular matrix of skeletal muscle tissue. The presence of a vascular pedicle enables a more efficient perfusion-based decellularization protocol and allows for subsequent recellularization and transplantation of the muscle construct in vivo. The goal of this study was to create a baseline for transplantation of decellularized whole muscle constructs by establishing an animal model for investigating a complete native muscle isolated on its pedicle in terms of vascularization and functionality. The left medial gastrocnemius muscles of 5 male Lewis rats were prepared and raised from their beds for in situ muscle stimulation. The stimulation protocol included twitches, tetanic stimulation, fatigue testing, and stretching of the muscles. Peak force, maximum rate of contraction and relaxation, time to maximum contraction and relaxation, and maximum contraction and relaxation rate were determined. Afterwards, muscles were explanted and transplanted heterotopically in syngeneic rats in an isolation chamber by microvascular anastomosis. After 2 weeks, transplanted gastrocnemius muscles were exposed and stimulated again followed by intravascular perfusion with a contrast agent for µCT analysis. Muscle constructs were then paraffin embedded for immunohistological staining. Peak twitch and tetanic force values all decreased significantly after muscle transplantation while fatigue index and passive stretch properties did not differ between the two groups. Vascular analysis revealed retained perfused vessels most of which were in a smaller radius range of up to 20 µm and 45 µm. In this study, a novel rat model of heterotopic microvascular muscle transplantation in an isolation chamber was established. With the assessment of in situ muscle contraction properties as well as vessel distribution after 2 weeks of transplantation, this model serves as a base for future studies including the transplantation of perfusion-decellularized muscle constructs.

**Keywords:** muscle transplantation; rat gastrocnemius; in situ stimulation; muscle contraction; perfusion-decellularization

**Citation:** Cai, A.; Zheng, Z.; Müller-Seubert, W.; Biggemann, J.; Fey, T.; Beier, J.P.; Horch, R.E.; Frieß, B.; Arkudas, A. Microsurgical Transplantation of Pedicled Muscles in an Isolation Chamber—A Novel Approach to Engineering Muscle Constructs via Perfusion-Decellularization. *J. Pers. Med.* **2022**, *12*, 442. https://doi.org/10.3390/ jpm12030442

Academic Editor: Stephanie Duguez

Received: 17 January 2022 Accepted: 9 March 2022 Published: 11 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Tissue engineering of skeletal muscle holds great promise for the treatment of volumetric muscle loss. It can help to circumvent substantial donor site morbidity, resulting from donor tissue transfer, including free autologous muscle flaps [1–3]. Common tissue engineering approaches have attempted to create three-dimensional (3D) constructs by combining scaffolds with stem cells and growth factors [3,4]. However, the production of biocompatible and stable scaffolds can be time consuming and expensive and cell adherence, invasion, and differentiation can be demanding in this synthetic 3D environment [5,6].

Decellularized extracellular matrix scaffolds have been a popular platform for regenerating skeletal muscle, as they contain structural proteins and molecules of skeletal muscle tissue, which is difficult to mimic by artificial means [6,7]. Established protocols have mostly applied diffusion techniques on skeletal muscle so far, although perfusiondecellularization through a vascular pedicle seems more beneficial by retaining the complex 3D architecture of the native tissue while effectively removing all cells [8]. The presence of a vascular pedicle enables subsequent transplantation of the decellularized and possibly recellularized construct in vivo.

Critical size defects necessitate volumetric tissue constructs for reconstruction, which on the other hand depend on adequate blood supply for survival [9]. Thus, a functional blood vessel network is a prerequisite for the growth of tissues and organs in vivo. The arteriovenous (AV) loop model contains a vein graft anastomosed to an artery and a vein, creating a loop that is transferred to an enclosed implantation chamber [10]. This creates an isolated microenvironment, which is connected to the living organism only by means of the vascular axis. Any disturbing influences from the surroundings are shielded off and the tissue with its vascularization can be analyzed in the controlled environment.

Perfusion-decellularization of a whole muscle via its main vascular pedicle, subsequent recellularization of the skeletal muscle matrix, and transplantation of the whole construct in vivo have the potential to generate new functional muscle tissue. An isolation chamber allows for analysis of the newly generated and vascularized tissue independent of external factors similar to the AV-loop model. Prior to establishing such a model, baseline values concerning vascularization and functionality of such transplanted muscle constructs are needed.

The principal contribution of this work is a novel in vivo animal model for investigating a transplanted complete native muscle isolated on its pedicle in terms of vascularization and functionality.

#### **2. Methods**

#### *2.1. Experimental Animals*

Animal experiments were carried out following the German regulations for the care of laboratory animals at all times. Experiments were approved by the local Animal Care Committee (approval number RUF 55.2.2-2532.2-658-48).

Gastrocnemius muscles were taken from the left hindlimb of 5 male Lewis donor rats (Charles River Laboratories, Sulzfeld, Germany) and transplanted into the left hindlimb of 5 syngenic recipient rats.

All animals were anesthetized during the surgical procedures through the administration of gaseous isoflurane (op-pharma, Burgdorf, Germany) at concentrations between 1.5–5% under spontaneous breathing. For analgesia, animals received intravenous meloxicam (2 mg/kg) and butorphanol (1.5 mg/kg), whereas butorphanol was substituted every 2 h.

#### *2.2. Muscle Preparation*

Donor animals were placed in the supine position on a heating plate. Dissection through the adductor muscles of the left hind limb was carried out under aseptic conditions until the medial gastrocnemius muscle was reached and freed from all muscular and tendon attachments. All vessels branching off the muscle except for the popliteal artery and vein, representing the main pedicle, were ligated. Popliteal vessels were tracked proximally up to

their origin from the femoral vessels until the gastrocnemius muscle was solely attached in situ via its vascular pedicle and the tibial nerve (Figure 1). The distal tendon was then tied with a 4-0 Vicryl suture (Ethicon, Somerville, NJ, USA) and connected to a servomotor lever arm (model 305C Dual-Mode Lever Arm System; Aurora Scientific Inc, Aurora, Ontario, Canada), while the proximal tendon was fixed in a clamp on a self-constructed metal frame, placed under the heating plate (Figure 2). After stimulation of the gastrocnemius, the rat receiving the gastrocnemius muscle as a transplant was surgically prepared (see Section 2.4 for details). tracked proximally up to their origin from the femoral vessels until the gastrocnemius muscle was solely attached in situ via its vascular pedicle and the tibial nerve (Figure 1). The distal tendon was then tied with a 4-0 Vicryl suture (Ethicon, Somerville, NJ, USA) and connected to a servomotor lever arm (model 305C Dual-Mode Lever Arm System; Aurora Scientific Inc, Aurora, Ontario, Canada), while the proximal tendon was fixed in a clamp on a self-constructed metal frame, placed under the heating plate (Figure 2). After stimulation of the gastrocnemius, the rat receiving the gastrocnemius muscle as a transplant was surgically prepared (see Section 2.4 for details). lar and tendon attachments. All vessels branching off the muscle except for the popliteal artery and vein, representing the main pedicle, were ligated. Popliteal vessels were tracked proximally up to their origin from the femoral vessels until the gastrocnemius muscle was solely attached in situ via its vascular pedicle and the tibial nerve (Figure 1). The distal tendon was then tied with a 4-0 Vicryl suture (Ethicon, Somerville, NJ, USA) and connected to a servomotor lever arm (model 305C Dual-Mode Lever Arm System; Aurora Scientific Inc, Aurora, Ontario, Canada), while the proximal tendon was fixed in a clamp on a self-constructed metal frame, placed under the heating plate (Figure 2). After stimulation of the gastrocnemius, the rat receiving the gastrocnemius muscle as a trans-

conditions until the medial gastrocnemius muscle was reached and freed from all muscular and tendon attachments. All vessels branching off the muscle except for the popliteal artery and vein, representing the main pedicle, were ligated. Popliteal vessels were

conditions until the medial gastrocnemius muscle was reached and freed from all muscu-

*J. Pers. Med.* **2022**, *12*, x FOR PEER REVIEW 3 of 16

*J. Pers. Med.* **2022**, *12*, x FOR PEER REVIEW 3 of 16

**Figure 1.** After surgical preparation, the gastrocnemius muscle was attached in situ via its vascular pedicle (arrow) and the tibial nerve (white arrowhead). **Figure 1.** After surgical preparation, the gastrocnemius muscle was attached in situ via its vascular pedicle (arrow) and the tibial nerve (white arrowhead). **Figure 1.** After surgical preparation, the gastrocnemius muscle was attached in situ via its vascular pedicle (arrow) and the tibial nerve (white arrowhead).

**Figure 2.** For electrical stimulation, the muscle was connected to a servomotor lever arm distally and fixed with a clamp proximally. Electrodes ware placed near the entry of the tibial nerve. **Figure 2.** For electrical stimulation, the muscle was connected to a servomotor lever arm distally and fixed with a clamp proximally. Electrodes ware placed near the entry of the tibial nerve. **Figure 2.** For electrical stimulation, the muscle was connected to a servomotor lever arm distally and fixed with a clamp proximally. Electrodes ware placed near the entry of the tibial nerve.

**.** 

#### *2.3. Muscle Stimulation 2.3. Muscle Stimulation*

*2.3. Muscle Stimulation*  Prior to transplantation and perfusion, the gastrocnemius muscle was stimulated via electrodes placed into the muscle close to where the tibial nerve enters the muscle. A Prior to transplantation and perfusion, the gastrocnemius muscle was stimulated via electrodes placed into the muscle close to where the tibial nerve enters the muscle. A Prior to transplantation and perfusion, the gastrocnemius muscle was stimulated via electrodes placed into the muscle close to where the tibial nerve enters the muscle. A stimulation and recording system (150 A and 615A Dynamic Muscle Control and High-Throughput Analysis software suite; Aurora Scientific Inc., Aurora, ON, Canada), was used to stimulate the muscle after optimal length was determined as described by Tamayo et al. [11]. For twitches, the muscle was stimulated every 3 s with 1 ms pulses

for 10 repetitions. The maximum force (i.e., peak force), maximum rate of contraction and relaxation, time to maximum contraction and relaxation, and maximum contraction and relaxation rate were measured. This was followed by 80 Hz, 100 Hz, and 120 Hz tetanic stimulation. Peak force, maximum contraction rate, and time to maximum contraction were assessed. For fatigue testing, a 150 Hz burst of stimulation was applied to the muscle every 3 s for 7.5 min. The fatigue index was determined as the ratio between the maximum and minimum force difference and the maximum force. A break of at least 1 min was taken between each measurement. For passive tension properties, the muscle was stretched to 110% of its original length in 1% steps. During each stretch, contractile forces to gain the length of the muscle were measured.
