Skeletal muscle has remarkable regenerative capacity in most minor injuries induced by mechanical laceration, overstretching, and toxins. However, volumetric muscle loss (VML) injury, a large volume of muscle loss beyond the self-repair capacity, causes functional disability and morphological deformities. This study investigated the effects of myofiber injection into a decellularized extracellular matrix (ECM) and resistance training (RT) on skeletal muscle regeneration following VML injury.
6-months-old male Fischer CDF rats and 2-months-old F344-Tg (UBC-EGFP) rats (myofiber donors) were used in this study. Approximately 20% of the mass of the lateral gastrocnemius (LGAS) was excised and replaced by ECM of similar dimensions. Thirty myofibers were injected into the injured region seven days post-injury. Ladder climbing (RT) was allowed 10 days post-defect surgery, and the rats were subjected to ladder climbing with a weight every third day for 6 weeks.
After 56 days of recovery and exercise training, the cross-sectional area (CSA) of intact muscle in the EXE group (5,104±92 μm2) increased significantly compared to that in the ECM (4,657±79 μm2) group. The number of blood vessels larger than 20 μm in diameter, capillaries excluded, showed a significant difference between the ECM+EXE (34.25±4.2) and ECM (21.75±3.89) groups. A significant reduction of fibrosis in the ECM+EXE (44.50±1.6%) group was observed compared to the ECM (69.25±1.9%) and ECM+FIB+EXE (63.00±1.7%) groups. Moreover, the small muscle fiber area within the transplanted ECM was significantly larger in the ECM+EXE (1.37±0.03 mm2) than in both the ECM (0.49±0.01 mm2) and ECM+FIB (0.62±0.01 mm2) groups.
These data suggest that ECM transplantation with RT effectively repairs VML by enhancing hypertrophy, angiogenesis, and myofiber infiltration throughout the entire ECM.
Skeletal muscles, a bundle of multinucleated and contractile fibers, play a crucial role in the locomotion and structural support of the body [
The extracellular matrix (ECM) is a three-dimensional structure that is consisted of glycoproteins, heparin sulfate proteoglycans, glycosami-noglycans, and type IV collagen [
Satellite cells (SCs), adult skeletal muscle stem cells, are located between basal lamina and sarcolemma and are mitotically quiescent. Quiescent SCs are characterized by paired box 7, Pax7, which are crucial in SC development and lineage determination [
Resistance training (RT) heavily influences the functional properties of skeletal muscle by modifying the structure, mass, and type of myofibers [
Vascularization is an inevitable concern in tissue engineering and tissue regeneration to provide nutrients and oxygen efficiently [
Therefore, this study aimed to determine the efficiency of skeletal muscle regeneration with supporting components, blood vessels, and nerves in the middle region of transplanted ECM via single fiber injection with RT.
Male fischer 344 rats (Charles River Laboratories; Wilmington, MA), about 400 g of weight, were used in this study. F344-Tg (UBC-EGFP) was used for a myofiber donor. We used the rat to quantify regenerated myofibers from the donor SCs. Donor myofibers express the enhanced green fluorescent protein gene under the control of the human ubiquitin-C promoter. Animals were housed in a 12-hour light/dark cycle room and given adlibitum access to food and water. A total of 32 rats were used for the study. They were randomly assigned to one of four groups (n=8). Functional recovery was evaluated after 56 days from the initial defect surgery. All experimental procedures were conducted under Institutional Animal Care and Use Committee (IACUC) guidelines (AUP-2015-00008).
Fat and connective tissues were excised from muscles and cut into small pieces. Deionized water (dH2 O) was injected throughout the whole muscles, and the muscles were placed in dH2 O for 2 hours to allow cellular swelling and rupture. Phosphate buffered saline (PBS) was injected throughout the entire muscles, and the muscles were placed in PBS for 2 hours. 0.25% Trypsin-EDTA (Gibco® Life Technologies; Burlington, ON, CA, USA) was injected throughout the whole muscles, and the muscles were placed in an oven for 45 minutes at 37°C. The tissues were then placed in glycerol (Fisher; Pittsburgh, PA, USA), disodium ethylenediaminetetraacetate dehydrate (EDTA) (Bio-Rad Laboratories; Her-cules, CA, USA), deoxycholic acid (Fisher; Pittsburgh, PA, USA), and so-dium dodecyl sulfate (SDS) (Sigma-Aldrich; St. Louis, MO, USA) solution for 48 hours. The tissues were placed in the electrophoresis machine with Tris base (Fisher; Pittsburgh, PA, USA), Glycine (Fisher; Pittsburgh, PA, USA), and SDS (Sigma-Aldrich; St. Louis, MO, USA) solution at a constant voltage of 60 V for 24 hours. 2% SDS was injected through the muscles, and the muscles were placed in SDS for 6 hours. The electrophoresis process was repeated until all cellular materials were eliminated. The ECM was rinsed in dH2 O until the water looked clear. The ECM was placed in sterile PBS (Invitrogen; Carlsbad, CA, USA) 1% antibiotic-antimycotic (Sigma-Aldrich; St. Louis, MO) (1% AA) overnight on a shaker. Next, the ECM was placed in 70% ethanol for 4 hours, subsequently placed in PBS 1% AA, and exposed to ultraviolet light overnight. Decellularized ECMs were stored at 4°C in sterile PBS with 1% AA until use in implantation.
Decellularization procedures using an electrophoresis system. (A) Fat and connective tissues were excised from the porcine muscle and cut into small pieces. (B) The tissues were placed in the electrophoresis machine with tris base, glycine, and SDS solution at a constant voltage of 60 V for 24 hours. (C) The electrophoresis process was repeated until all cellular materials were eliminated and the tissues became transparent. (D) The decellularized ECMs were placed in sterile PBS and exposed to ultraviolet light overnight until they were used for implantation.
Prior to VML surgery, a total of 32 animals were randomly assigned to one of four experimental groups: implantation of the ECM (ECM), implantation of the ECM followed by myofiber injection (ECM+FIB), implantation of the ECM followed by RT (ECM+EXE), and implantation of the ECM followed by myofiber injection and RT (ECM+ FIB+EXE). All animals underwent removal of about 20% of muscle mass from the lateral gastrocnemius (LGAS). A piece of the exact dimensions of ECM was transplanted immediately, and myofibers were injected seven days later, as described by Collins [
Defect surgery and RT protocol. (A) Description of intact lateral gastrocnemius (LGAS). (B) About 20% of muscle mass from the LGAS was excised. (C) A piece of the exact dimensions of ECM was transplanted immediately at the injury site. (D) The ECM was sutured tightly by using a modified Kessler stitch. The suture was also a marker for a border of the defected muscle. (E) GFP expressing myofibers were injected 7 days after defect surgery. (F) RT started at 10 days post defect surgery. The rats climbed a ladder every third day for 6 weeks. (G) Description of experimental design.
Extensor digitorum longus (EDL) was excised from a donor rat and immediately digested by filtered 1.5% Collagenase type I in DMEM for 2 hours at 37°C and 5% CO2. EDL isolation protocol is well optimized; age, sex, different muscles, or conditions should be considered to isolate single myofibers. Since the purpose of single myofiber injection was just delivering SCs, we used EDL instead of LGAS. During digestion, EDL was gently shaken every 30 minutes. When single myofiber was detached from the EDL, EDL and single fibers were transferred to a pre-warmed Petri dish with DMEM. One hundred single myofibers were isolated by pipetting from each EDLs.
The animals underwent 7 days of recovery to mitigate inflammation and increase the injected myofibers' survival ratio. The original stitches were removed to expose and visualize the transplanted ECM. GFP-expressing myofibers were isolated from F344-Tg (UBC-EGFP). 30 myofibers were injected evenly through the entire injury area using a 28.5- gauge needle with saline (SAL). After fiber injection, the biceps femoris and skin were sutured as previously described.
Exercise treatment groups, ECM+EXE and ECM+FIB-EXE, began RT at 10 days post defect surgery. This time allowed ECM adhesion with the injury site and force transmission across the defect region [
The LGAS was sectioned into 10 μm thickness perpendicular to the LGAS orientation with a Leica CM1900 cryostat microtome (Leica Microsystems; Wetzlar, Germany) at −20°C. Hematoxylin and eosin (H&E) (Thermo Fisher Scientific) staining was performed to identify intact and regenerated fibers. Masson’ s trichrome (Polysicence) was used to distinguish between myofiber and fibrous connective tissues. The sections were mounted with a permount mounting medium (Fisher Scientific; Waltham, MA). The sections were visualized with a Nikon Diaphot microscope with an Optronix Microfire digital camera. Histological quantification of H&E was performed on each level with the 20× objective lens.
A series of 10 μm cross-sections were taken from the top, middle, and bottom regions of the LGAS muscle. In preparation for immunofluorescent identification, sections were fixed in acetone for 3 minutes. Afterward, they were washed in 1X PBS and blocked with 5% normal donkey serum in PBS containing 1% bovine serum albumin (BSA). Sections were first incubated with primary antibodies against PECAM-1 (1:20, mouse monoclonal) and neurofilament 200 (1:200, rabbit polyclonal). PECAM-1 was detected with donkey anti‐ mouse IgGTRITC fluorescein (1:100, λ=546 nm), and neurofilament 200 was detected with anti‐ rabbit‐ Alexa 488 (1:100, λ=495 nm). Finally, all sections were counter-stained with DAPI (1:1,000, λ=425 nm) to identify nuclei. Following a final wash in PBS, the sections were mounted in Permount mounting medium (Fisher Scientific; Waltham, MA, USA).
Immunofluorescence was visualized with a Leica DM LB2 fluorescence microscope and photographed with a Leica DFC340FX digital camera (Leica Microsystems; Wetzlar, Germany). At each level and within each region of the ECM, the number of nerves and vessels was counted using Image J. A percentage of regenerated skeletal muscle and fibrotic tissue area in the ECM area was calculated. A blood vessel was only counted if its lumen was greater than 20 μm in diameter.
The data were presented as mean and standard deviation (mean±SD). One-way analysis of variance (ANOVA) was used for the analysis of group samples. Comparisons between the groups were made using Tukey’ s post hoc tests if it is significantly. Statistical significance was defined as
The average mass of the excised muscle was 176±16 mg wet weight, approximately 20% of the total mass in the LGAS. The defect mass was approximated by initial body weight. The overall morphology of the VML LGAS was flattened compared to the LGAS from a contralateral limb. In previous work using homologous ECM transplantation, the morphology of the LGAS was well maintained following 56 days of recovery [
Small myofiber density in the middle region of VML. Myofiber ingrowth was observed in the regenerating muscle area. Representative images of (A) ECM, (B) ECM+FIB, (C) ECM+EXE, and (D) ECM+FIB+EXE. Newly regenerating muscle small muscle fibers were stained red, and the fibrotic tissue area was stained blue. (E) Tissues were stained by Masson's trichome method, and regenerating small muscle fiber area was measured (n=4 in a group). ∗∗∗ and ∗ indicate values that are different between the two groups. ∗∗∗
CSA of uninjured muscle in the middle region of VML. Intact muscle hypertrophy was observed in the ECM+EXE. Representative images of (A) ECM, (B) ECM+FIB, (C) ECM+EXE, and (D) ECM+FIB+EXE. (E and F) Tissues were stained with hematoxylin and eosin, and CSA was measured (n=4 ∗15 sections in a group) ∗∗∗ indicates values that are different between the two groups. ∗∗∗
Neurogenesis and angiogenesis within the transplanted ECM were measured by immunohistochemical analysis. Neurofilament staining revealed the presence of innervation at the border of regenerating area filled with central nuclei myofibers (
Neurogenesis at the border between the intact and injured region of VML. Innervation was observed at the border of regenerating area filled with central nuclei myofibers. Representative images of (A) ECM, (B) ECM+FIB, (C) ECM+EXE, and (D) ECM+FIB+EXE. Neurons were identified by neurofilament 200 (GFP), and nuclei were stained by DAPI (blue) staining (n=4 ∗15 sections in a group). (E) The total number of nerves was counted, and (F and G) nerve CSA was measured.
Angiogenesis in the middle region of VML. Vascularization was observed in the regenerating region. Representative images of (A) ECM, (B) ECM+FIB, (C) ECM+EXE, and (D) ECM+FIB+EXE. Blood vessels were identified by PECAM-1 (TRITC), and nuclei were stained by DAPI (blue) staining. (E and F) The total number of blood vessels larger than 20 um in diameter was counted in the regenerating region (E) (n=4 ∗15 sections in a group), and the entire region (F) (n=4 ∗15 sections in a group). ∗indicate values that are different between two groups. ∗
Fibrous connective tissues were measured by Masson’ s trichrome staining. A significant attenuation of connective tissue area in the ECM+EXE (44.50±1.6%) was observed compared to ECM (69.25±1.9%) and ECM+FIB+EXE (63.00±1.7%) (
Fibrosis in the middle region of VML. Fibrosis was observed in the regenerating region. Representative images of (A) ECM, (B) ECM+FIB, (C) ECM+EXE, (D) ECM+FIB+EXE, and (E) contralateral LGAS. (F) Fibrous connective tissues were measured by Masson's trichrome staining (Blue) (n=4 in a group). ∗∗∗ and ∗ indicate values that are different between the two groups. ∗∗∗
The large volume of skeletal muscle loss results in functional deficits, cosmetic flaws, and a permanent handicap. Moreover, associated psychological distress ensues from functional deficits and cosmetic flows accompanied by traumatic injuries. Current therapies improve myofiber infiltration and functional properties but fail to prevent fibrosis and improve angiogenesis and myofiber infiltration into the middle region of the ECM. Therefore, developing a therapy that can improve myofiber infiltration through entire regions of the transplanted ECM is imperative.
This study tested the combined effects of myofiber injection and RT on a VML injury. The results showed only the ECM+EXE positively affected skeletal muscle regeneration after 8 weeks of recovery. The ECM+EXE group significantly increased 15.7% mass recovery compared to the ECM group. RT increased muscle protein synthesis ratio, muscle mass, and contractile properties. RT by ladder-climbing stimulated several muscle groups, including the soleus, plantaris, and gastrocnemius. Ladder climbing training resulted in a 5-26% increase in muscle mass [
Moreover, muscle hypertrophy induced by 6 weeks of ladder climbing in VML injured LGAS increased by 11% of the mass and 16% of tetanic force [
The transmission of nerve impulses causes skeletal muscle contraction. Innervation is vital in functional morphological skeletal muscle maintenance. Since denervation results in adverse effects on the muscle, it is essential that regenerating muscle fibers should accompany innervation in a VML injury. RT not only increases CSA and force generation but also affects neural plasticity on skeletal muscle. A 12-weeks of high RT slowly attenuated neuromuscular diseases [
Blood vessels supply nutrients and oxygen to muscle tissues. In previous research in our lab, transplantation of ECM alone or with mesenchymal stem cells increased blood vessel density, not in the middle region. It means that the transplanted ECM can be a scaffold for angiogenesis. In this study, the ECM+EXE contained a significantly higher number of blood vessels than the ECM in the regenerating and entire areas. Resistance and endurance training triggered angiogenesis and vascularization [
The remarkable finding in this study was that the ECM+EXE prevented fibrosis and promoted myogenesis in the middle region of the ECM. A repair of VML injury with only ECM implantation resulted in an incomplete recovery associated with functional loss and insufficient myofiber infiltration into the middle region of ECM [
The combined treatments of RT and single fiber injection did not have an additive effect. Even though a significant difference was not found between the ECM+EXE and ECM+EXE+INJ, the ECM+EXE recovered more LGAS muscle mass. The ECM+EXE recovered 87.6% of the LGAS mass, while the EXE+INJ groups recovered only 75.2% of the LGAS mass. After 6 weeks of RT, the ECM+EXE group climbed the ladder with 227% body mass, while the EXE+INJ group climbed the ladder with 187% body mass. The ECM+EXE significantly lifted more weight compared to the ECM+EXE+INJ. EXE+INJ receives surgeries twice, a defect surgery and a surgery for fiber injection, which may cause additional damage. Also, long-term RT needs to be examined because 56 days of recovery was not long enough to complete muscle regeneration in a 20% mass of severe VML injury. Small myofibers were still growing in the entire region of the ECM after 56 days of recovery.
In summary, the study proved the therapeutic potential for a continuous RT following ECM implantation. RT significantly affected histological and morphological recovery over ECM implantation alone. Different levels of RT intensity could affect skeletal muscle regeneration and functional properties. Donor single myofibers failed to survive and fuse to myofiber efficiently in severe VML. Myofiber injection into Prevascularized ECM is necessary to improve the survival ratio of donor stem cells.
The authors have no conflicts of interest to disclose.
Conceptualization: K Lee; Data curation: K Hong; Formal analysis: K Lee, K Hong; Funding acquisition: K Hong, W Park; Methodology: K Lee, W Park; Project administration: K Lee, W Park; Visualization: K Lee, K Hong; Writing-original draft: K Lee; Writing-review & editing: K Lee, K Hong, W Park.