Introduction
Muscle injury represents approximately one third of injuries related to sports activity;
it mainly affects the lower limbs and has an important relationship with withdrawal
from sports activities.[1]
[2]
[3] For an adequate diagnosis, we opted for a clinical evaluation, with the use of imaging
tests reserved for diagnostic confirmation, qualification, and quantification of the
lesion.[4]
There are some etiological factors with a well-established association with an increased
risk of muscle injuries. Among them, we can mention age, previous muscle injury, ethnicity,
overload, and imbalance of muscle forces.[5] The therapeutic management of these lesions has not presented substantial changes
over the last few years, and the RICE (rest, ice, compression, and elevation) protocol
is the most widely used treatment.[4]
[6]
[7]
Even after performing an appropriate treatment protocol, the high rate of re-injury
and prolonged absence from sports activities[3]
[8] motivates the constant search for new therapies that can improve the results. Seeking
to fill this space, the use of orthobiologicals has been gaining space in the treatment
of various orthopedic lesions, including muscle injuries.[9] Among the available orthobiologicals, the use of adult mesenchymas stem cells, especially
those derived from adipose tissue, already presents consistent results in terms of
differentiation capacity,[10]
[11] rapid growth,[12] ease of obtaining,[13] good experimental results[14]
[15] and promising clinical results.[16]
[17]
Thus, in the search for alternatives for muscle repair, the present study proposes
to evaluate the hypothesis that muscle healing can be optimized using adipose tissue-derived
stem cells (ADSCs), in an experimental model of muscle injury reproduced in rabbits.
It precisely aims at the histological and macroscopic evaluation of the healing process
of acute lesions of the femoral rectus muscle using ADSCs.
Material and Method
Experimental Design
An experimental study was conducted with 9 pure New Zealand male rabbits, aged 28
to 32 weeks and with approximate weight between 3 and 3.5 kg. The animals were acquired
from a commercial establishment and kept in the development center of experimental
models for biology and medicine throughout the study. During this period, the animal
was kept in an individualized environment, 12/12 hrs dark-light cycle, with food and
water ad libitum. The hind legs of the animals (18 legs) were randomly divided (using specific software
and opaque envelopes) in the study groups according to the intervention to be performed
([Figure 1]). In the group I–control, the hind legs were kept intact, in group II–SHAN, the
experimental lesion was performed without association with additional treatments,
and in group III – ADSCs, the experimental lesion was performed and the addition of
ADSCs to the lesion site, as a treatment intervention ([Figure 1]). The study had its initial version and subsequent reports approved by the ethics
committee for the use of animals (CEUA, in the Portuguese acronym) of our institution
and followed the guidelines for the use and management of animals proposed by our
institution besides meeting the criteria proposed in the animal research: reporting
of in vivo experiments (ARRIVE) guidelines.[18]
Fig. 1 General experimental design. Description: The image represents the general division
of the groups from the first stage of the procedure, when the collection of fat took
place, to the respective evaluations.
Procedures
To perform the experiments, whether fat collection, lesion protocol, or material collection,
the animals were submitted to the following analgesic and anesthetic protocol: To
start the procedures, the animal was submitted to analgesia and preoperative antibiotic
therapy with tramadol (5 mg/kg) and terramycin (50 mg/kg); after 30 minutes anesthesia,
ketatamine (50 mg/kg) and xylazine (10 mg/kg) were started. As a method of postoperative
analgesia, meloxican (0.5 mg/kg) and tramadol (5 mg/kg) were maintained until the
end of the 3rd postoperative day, and these same medications were administered in case of pain or
discomfort of the animal after this period. Evaluations regarding stress, discomfort,
and pain were performed daily at the development center of experimental models for
biology and medicine.
Experimental Model of Acute Muscle Injury
After an anesthetic protocol already presented, the animals with paws belonging to
the group II—SHAN or to the group III—ADSC' were submitted to trichotomy, antisepsis,
asepsis, anterior skin incision in the thigh, dilvulsion by planes, and exposure of
the femoral rectus throughout its extension ([Figure 2A]). Next, partial injury in the 1/3 middle of the femoral rectus was performed ([Figure 2B]), with coldblade, and marking of the extremities ([Figure 2C]) with nylon 6-0 (Nylon 6-0, Shalon, Alto da Boa Vista, GO, Brazil), at approximately
0.5 cm proximal and distal to the lesion.[19]
[20] After performing the procedures and anesthetic recovery, the animal was encouraged
to apply load to the limb, without restrictions.
Fig. 2 Experimental model of muscle injury. Description: (A) Exposure of the recto femoral muscle to its full extent. (B) Experimental lesion in the middle third of the femoral rectus muscle. (C) Marking the extremities of the femoral rectus muscle injury with non-absorbable
point.
ADSCs—Fat Collection to implant ADSCs
For the preparation and implantation of ADSCs, all animals were submitted to abdominal
fat collection 2 weeks before the experimental lesion. To collect fat, the animals
were anesthetized with the same protocol, and then a lower abdominal median incision
was performed, with dissection by planes until aponeurosis of the rectum muscle. Identification
of the left superficial epigastric artery in the inguinal region was performed, and
a fat fragment with weight ranging from 2 +/- 0.5 grams was collected.[10]
[11]
[21]
The fat fragment was transported, in PBS buffer solution, from the collection site
to the laboratory to follow the specific procedures of preparation of ADSCs.
Preparation of ADSCs
The preparation of the cells followed a protocol that had been previously published.[10]
[11]
[21] Briefly, the fat was washed extensively with phosphate buffer saline (PBS), minced,
and enzymatically digested at 37 °C for approximately 30 min using 0.075% collagenase
type IA (Sigma; St. Louis, MO). The digested tissue was filtered using 100 mm strain
to obtain a cell suspension containing the stromal vascular fraction. After centrifugation,
the pellet was resuspended in culture media (CM) consisting of Dulbecco's modified
Eagle's media (DMEM; Mediatech, Herndon, VA), 10% fetal bovine serum (FBS Gibco; Grand
Island, NY), and 1% of a solution of antibiotic/antimycotic (penicillin G 10,000 U/ml,
amphotericin B 25 mg/ml and streptomycin 10,000 mg/ml). Cells were allowed to adhere
to the flask for 24 h, after which fresh media was added. The cells were incubated
at 37 °C and 5% CO2 in CM until they reached semi-confluence. The cellular confluence was avoided to
prevent potential spontaneous differentiation. Culture media was changed every 2–3 days.
Cells were rinsed with PBS and incubated with 1:100 dilution of dialkylcarbocyanine
solution, a fluorescent cell membrane marker, (Vybrant DiI; Molecular Probes, Eugene,
OR) for 30 min at 37 °C in accordance with the manufacturer's protocol. The labeled
cells were harvested with 0.25% trypsin/ 1mM EDTA solution. To perform the autologous
transplantation, cells were suspended to a concentration of 1-2 × 106 labeled cells.
ADSCs Implant
The paws included in group III—ADSCs were initially submitted to the protocol of experimental
muscle injury and then submitted to the application of ADSCs directly on the site
of the lesion.[15] The application occurred through direct visualization with intramuscular infiltration
of the 1-2 × 106 of marked ADSCs.
Muscle Tissue Collection
After the 2-week postintervention period, the animals were anesthetized and then submitted
to painless death through overdose of anesthetics (ketamine 200 mg/kg + xylazine 40 mg/kg
and tramadol 10 mg/kg). For collection, a cutaneous incision was performed according
to the previous route and divulsion by planes until exposure of the previously injured
region in the femoral recto muscle (previously marked with nylon 6-0). Then, the femoral
recto muscle incision was performed in the region between the 6-0 nylon points (site
of muscle injury). The collected material was stored in a formaldehyde solution at
10% to follow the entire histological evaluation protocol.
Histological Analysis
Material Preparation
The muscle fragments were fixed in 10% formaldehyde for 24 hours and dehydrated in
increasing concentrations of ethyl alcohol, diaphanized by xylol and impregnated by
liquid paraffin in a greenhouse, regulated at 60 °C. In sequence, the blocks were
cut into minot microtome, adjusted to 4 μm with a 50 μm distance between the cuts.
The cuts thus obtained were placed on slides previously greased with Mayer albumin
and kept in a regulated oven at 37 °C, for 24 hours, for drying and gluing. After
preparation, the slides were stained with hematoxylin and eosin (H&E) and picrosirius
red techniques.
Quantitative Evaluation of inflammatory process
In view of the existence of inflammatory processes resulting from tissue lesions,
five images of each slide were obtained through an Olympus IX 81 optical microscope
(Olympus Corporation, Shinjuku-ku, Tokyo, Japan) coupled to an Olympus DP72 camera
(Olympus Corporation, Shinjuku-ku, Tokyo, Japan). These images, obtained with an increase
of 40X, were analyzed with the help of ImageJ Software (ImageJ 1.53h, National Institutes
of Health, Bethesda MA, USA).
For analysis, the cells related to the scar inflammatory process were isolated through
the plugin segmentation, thus excluding the nuclei referring to muscle fibers, and then the plugin counter
cell was applied to quantify the number of total cells remaining in each image. The data
obtained were compiled and later separated between the groups (Group II—SHAN or Group
III—ADSCs). At the end, a comparative analysis was performed regarding the effects
of treatment with ADSCs on muscle healing.
Qualitative Assessment of Muscle Healing
Considering the healing process of muscle injury and collagen changes that occur over
time, a qualitative methodology was performed using as reference the muscle healing
process and the respective color changes over this period. For this analysis, we used
the images obtained through the microscope Zeiss AX10 (Zeiss, Jena, Thuringia, Germany)
coupled to a Zeiss AxioCam ICc5 camera (Zeiss, Jena, Thuringia, Germany) of the slides
colored in red picrosirius. In a simplified way, a descriptive analysis was performed
on the proportion of fibers in an advanced healing aspect, that is, with yellow/orange
coloration.
Macroscopic Analysis
The evaluation of the local morphology was performed at the immediate moment of material
collection. In this evaluation, the following aspects were analyzed: changes in color,
solidity, level of fibrosis, presence of infectious signs, and local inflammatory
response.[22] Additionally the images were also documented through Canon photographs (Canon EOS
Rebel T5; Canon, Manaus, AM, Brazil) for further verification and presentation of
results.
Sample Calculation and Statistical Analysis
Considering the pioneering of the study, the number of animals was decided after analysis
of the relevant literature.[15]
[23] The data obtained in the quantitative analysis of the inflammatory response were
tabled and statistically analyzed with the BioStat 2009 program (AnalystSoft Inc.,
Alexandria, VA, USA). First, the data were submitted to the Shapiro-Wilk test to verify
the normality of the groups and after the analysis of variance (ANOVA)/Tukey test
for parametric data, and Kruskal-Wallis/Dunn for non-parametric data, to determine
the significance of the results. The level for rejection of the null hypothesis was
set at 5% (p ≤ 0.05), with an asterisk marking the significant values.
Results
Histological Analysis
The inflammatory process of muscle healing was ongoing in both groups, given the presence
of inflammatory tissue in the sample analyzed. However, the quantitative analysis
showed that the addition of ADSCs is related to a decrease in the number of inflammatory
cells per field in the 2-week evaluation ([Figure 3]). On the quantitative analysis, we noticed a decrease from 164.2 cells in the group
without the addition of ADSCs to 89.62 cells per field in the group with the addition
of ADSCs, representing a 46% decrease in the number of inflammatory cells after the
addition of ADSCs ([Figure 4]). Considering that the control group did not present any evidence of inflammatory
process, it did not enter this quantification.
Fig. 3 Microscopic histology of muscle healing—Inflammatory evaluation. Description: Images
representative of muscle healing in different groups. Group I—Control: Hematoxylin
and eosin (H&E) image with an increase of 40X, representative of the original muscle
tissue, not submitted to injury or intervention protocols, presenting muscle tissue
with habitual appearance. Group II—SHAN: H&E image with an increase of 40X, representative
of the group submitted only to experimental lesion with two weeks of evolution, presenting
a large amount of inflammatory tissue and muscle tissue in the initial phase of healing.
Group III—ADSCs: H&E image with an increase of 40X, representative of the group submitted
to experimental injury and treatment with the use of ADSCs, showing a decrease in
the number of inflammatory cells, and muscle tissue in the early process of healing.
Fig. 4 Graphic image of inflammatory analysis. Description: Images representative of the
average number of cells per group. In blue, the number of cells per field of the group
submitted to treatment with ADSCs and, in orange, of the group not submitted to any
treatment is represented. In this analysis, there is an important decrease in the
number of inflammatory cells after the addition of ADSCs in the treatment of acute
muscle injury.
The picrosirius red technique, under polarization, showed that the treated group had
more orange/yellow fibers, which evidences accumulation of thicker collagen fibers
compatible with the final healing process ([Figure 5]).
Fig. 5 Microscopic histology of muscle healing - muscle evaluation. Description: Images
representing collagen modifications in groups submitted to experimental injury and
treatment with ADSCs. Group II—SHAN: Picrosirius image, representative of the initial
phase of collagen modification with minimal number of fibers colored in orange or
yellow. Group III—ADSCs: Picrosirius image, representative of more advanced phase
of collagen modification with a greater number of fibers colored in orange or yellow.
Macroscopic Analysis
There was no change in the general state of the animal or infectious signs in the
hind legs submitted to experimental injury or intervention with the addition of ADSCs.
The animals were able to walk in the cage in the first postoperative days and at no
time presented modification in the acceptance of the diet or water. In both groups,
the animals' muscle tissue already presented a healing aspect in the final phase,
with remarkable fibrotic changes on its surface. The macroscopic evaluation did not
show any differences between the group submitted and the one not submitted to intervention
([Figure 6]).
Fig. 6 Macroscopic evaluation using ADSCs. Description: Experimental injury - aspect of
the experimental model immediately after muscle injury and marking with nylon 6-0.
Group II—SHAN: Representative image of the macroscopic aspect after 2 weeks postoperatively.
Group III—ADSC: Representative image of the macroscopic aspect of the group submitted
to injury and intervention (addition of ADSCs) after 2 weeks postoperatively.
Discussion
The main findings of the present study refer to the achievement of good histological
results after the use of ADSCs in the treatment of acute muscle injury. These findings
are added to recent studies published by our group, which show promising results for
the use of different orthobiologics, (i) stem cells[15] and (ii) scaffolds.[22] These first studies were able to satisfactorily reproduce well-established results
in other research groups and are functioning as motivators for continuity in the development,
improvement, and use of orthobiologics.
In the experimental model presented, with an acute, cutting lesion of muscle tissue,
we hope that the use of ADSCs will be able to optimize muscle healing through three
mechanisms,[24] (i) production of growth factors, with optimization of angiogenesis and reduction
of pathways that favor cellular apoptosis; (ii) immunosuppressive action by decreasing
activity in T and B lymphocytes; and (iii) induction in the differentiation of fibroblasts
into myocytes.
The choice of an acute injury with early evaluation was motivated by greater functionality
and performance of stem cells in the first days after the intervention. Thus, we tried
to evaluate a probable acceleration in the functional recovery over time, after the
use of ADSCs. In this sense, the great innovation of this work was precisely to present
the first study using ADSCs in the treatment of acute muscle injury in an experimental
model.
Among the limitations of the present study, we can mention the difficulties for sample
calculation, given the pioneering of the study; the use of an experimental model that
is not reproducible in clinical practice, since the scathing lesions are not the most
frequent; and the lack of additional evaluation with other methods, such as biomechanics
and functional evaluation. As prospects for the future, we hope to maintain pioneering
and continue the work with development, production, and evaluation of the most diverse
orthobiologics available.
