Low‐level laser and adipose‐derived stem cells altered remodelling genes expression and improved collagen reorganization during tendon repair

Abstract Objectives The cellular therapy using adipose‐derived mesenchymal stem cells (ASCs) aims to improve tendon healing, considering that repaired tendons often result in a less resistant tissue. Our objective was to evaluate the effects of the ASCs combination with a low‐level laser (LLL), an effective photobiostimulation for the healing processes. Materials and methods Rats calcaneal tendons were divided into five groups: normal (NT), transected (T), transected and ASCs (SC) or LLL (L), or with ASCs and LLL (SCL). Results All treated groups presented higher expression of Dcn and greater organization of collagen fibres. In comparison with T, LLL also up‐regulated Gdf5 gene expression, ASCs up‐regulated the expression of Tnmd, and the association of LLL and ASCs down‐regulated the expression of Scx. No differences were observed for the expression of Il1b, Timp2, Tgfb1, Lox, Mmp2, Mmp8 and Mmp9, neither in the quantification of hydroxyproline, TNF‐α, PCNA and in the protein level of Tnmd. A higher amount of IL‐10 was detected in SC, L and SCL compared to T, and higher amount of collagen I and III was observed in SC compared to SCL. Conclusions Transplanted ASCs migrated to the transected region, and all treatments altered the remodelling genes expression. The LLL was the most effective in the collagen reorganization, followed by its combination with ASCs. Further investigations are needed to elucidate the molecular mechanisms involved in the LLL and ASCs combination during initial phases of tendon repair.


| INTRODUC TI ON
The calcaneus tendon is one of the most affected tendons by ruptures, and its recovery after an injury turns to be a challenge in the current clinical practice. 1 The calcaneus tendon may present reduced mechanical resistance in the elderly, after prolonged immobilization and after different types of injuries arising from tendinopathies, sports practices and daily activities, due to changes in the organization and composition of its extracellular matrix (ECM). 2 During tendon repair, the formation of fibrous tissue occurs, less organized and with reduced biomechanical resistance, leading to the loss of part of the functions compared to the native tissue. 3 In certain regions of the tendons, especially where there is accumulation of specific growth factors, there are mesenchymal stem cells (MSCs) native to the tissue. These cells decrease with age, 4 and factors such as obesity, diabetes and hormonal changes also influence their proliferative and differentiation capacity. 5 There is a growing search for methodologies that aim to stimulate the proliferation and differentiation of native tendon MSCs during the repair process. 5,6 Researches about the treatment of tendon injuries using adipose-derived mesenchymal stem cells (ASCs) show a better organization of collagen bundles, as well as improved biomechanics and gait on treated animals. 6 The photobiostimulation promoted by the low-level laser (LLL) has attracted the attention of researchers in recent years, already being used in clinical practice for the treatment of tendinous and post-operative injuries, accelerating the repair process, with the improvement of the tissue reorganization. 7 Low-level laser therapy promotes tissue repair mainly due to its anti-inflammatory effect, but also has a mitogenic potential. 8 Thus, the combination of LLL and ASCs in vivo could induce a greater proliferation of these cells in the injured region, considering small the percentage of ASCs that survive at the injury site after application. 9 Although some in vitro studies demonstrate the efficiency of LLL treatment combined with MSCs, 8,[10][11][12] the literature is scarce considering the association of both therapies in vivo. Therefore, the objective of the present study was to analyse the effects of the association of LLL (808 nm) to cell therapy with ASCs, especially on remodelling genes and on the recovery of the collagen fibres, at the 14th day of the calcaneus tendon repair.

| Isolation of ASC and cell culture
The procedure was done according to Yang et al 13  Dissociated tissue was filtered using cell strainers (40 μm) and the inactivation of collagenase was then done by the addition of equal volume of Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), followed by centrifugation at 417 g for 10 minutes. The suspending portion containing lipid droplets was discarded and the pellet was resuspended in DMEM (containing 50 mg/L penicillin and 50 mg/L streptomycin) with 15% FBS and transferred to 75 cm 2 bottle, maintained at 37°C with 5% CO 2 until the 5-6th passage (5/6P).

| Flow cytometry
Adipose-derived mesenchymal stem cells at 6P/7P were trypsinized and centrifuged at 1800 rpm for 10 minutes and counted using the

| Experimental groups
A total of 90 male Wistar rats (105-day-old) kept at a constant temperature (23 ± 2°C) and humidity (55%) under a 12/12 hours light/ dark cycle, with free access to food and water, were divided into five

| Protocol for partial transection of the CT and application of ASCs and/or LLL
The animals were anesthetized with intraperitoneal injection of Ketamine (90 mg/Kg) and Xylazine (12 mg/Kg), and the right lower paws submitted to antisepsis and trichotomy. A transverse partial transection (approximately 2/3 of the extent of the injury in depth) was performed in the proximal tendon region located at a distance of 4 mm from its insertion in the calcaneus bone ( Figure 1). [17][18][19] Approximately 4.5 × 10 5 ASCs (5-6P) were resuspended in 30 μL of DMPBS Flush and transplanted in the transected region (TR) of tendons in the SC and SCL groups, using a pipette. In tendons of T and L groups, only 30 μL of DMPBS Flush was applied. Then, the skin was

| Polarization microscopy: birefringence measurements
After fixation, image analyses of the tendons (n = 3) were evaluated to detect differences in morphology based on the aggregation and organization of the collagen bundles, which reflect the variation of birefringence intensity. Birefringence properties were studied using an Olympus BX53 polarizing microscope and an image analyzer (Life Science Imaging Software, Version

| Dosage of hydroxyproline
The TR of tendons (n = 4) was cut and immersed in acetone for 48 hours, also followed by a solution containing chloroform: ethanol

| Real-time PCR Array
The was used as calibrator sample, and for each target gene, the 2 −∆∆CT method was used to calculate the relative expression level (fold change). The results were represented as the relative gene expression in comparison with the calibrator sample that is equal to 1.

| Statistical analysis
For biochemical and molecular analyses, data from different experimental groups were analysed by the analysis of variance (ANOVA), followed by the Tukey test (P < 0.05). The Mann-Whitney U test (P < 0.05) was used only for analysis of the birefringence measurements, also using the GraphPad Prism ® (GraphPad Software, La Jolla, CA, USA), version 3.0. All results were presented as the mean and standard deviation.

| In vitro adipogenic and chondrogenic differentiation of ASCs and positive ASCs markers
ASCs (6-7P) cultures stained with Sudan IV and Alizarin Red SD exhibited red-intracellular lipid droplets and calcification spots, respectively (Figure 2A,B). Flow cytometry showed high expression of CD105 markers and CD90, and low expression of CD34 ( Figure 2C-E).

| Cell migration assay
On day 14, GFP-ASCs were quantified in the TR of tendons. It was observed a tendency to increase of the ASCs in tendons of SCL group in comparison with SC ( Figure 3).

| Genes expression analysis
There was lower expression of Scleraxis (Scx) in the SCL group, higher expression of Tenomodulin (Tnmd) in the SC group and of Growth differentiation factor 5 (Gdf5) in the L group, compared to the T group. The expression of Decorin (Dcn) was increased in all treated groups, SC, SCL and L respectively, compared to the T group ( Figure 4).

| Pro-and anti-inflammatory cytokines quantification
The graph for TNF-α and IL-10 demonstrated a significant increase in IL-10 concentration in the SC group compared to all other groups.
Regarding TNF-α, there were no significant differences between the groups ( Figure 5A).

| Total collagen quantification
No significant difference was observed in the quantification of hydroxyproline between the analysed groups ( Figure 5B).

| Collagen I and III, PCNA and Tnmd quantification
The amount of collagen I was lower in the SCL than in the T and SC groups. For collagen III, the SC group had a higher amount when compared to T and SCL. The L group showed a significant increase compared to only the SCL group. The amount of caspase-3 was decreased in the L group in relation to SCL. No differences were observed in the amounts of PCNA (Proliferating Cell Nuclear Antigen) and Tnmd between the different groups ( Figure 6).

| Collagen fibres organization measurement
Images of tendons analysed under polarization microscopy showed differences in the collagen organization in the TR of all groups ( Figure 7). The birefringence measurements ( Figure 7K) showed higher values in the L group, followed by SCL, SC and T groups. In the images of HE-stained tendons ( Figure 7A-E), the general organization of TR was observed, with higher cellularity in the transected groups compared to the N group ( Figure 7D).

| D ISCUSS I ON
In the present study, the cell migration assay showed the presence of ASCs-GFP in the TR on the 14th day after their application to the tendon, proving the incorporation of the ASCs into the tissue during the repair process. A tendency towards higher fluorescence intensity of the ASCs-GFP was observed in the SCL group compared to the SC. In general, a small percentage of ASCs survives at the injury site after application. 9 A previous study of our group has shown the presence of transplanted ASCs on 3rd and 14th days after tendon transection, probably in response to the intense initial inflammatory process. 19 During the tendon repair, TGF-β1, 26 Il-10 27 and scleraxis 28,29 are responsible for the stimulation of collagen synthesis. No differences were observed in the expression of Tgfb1 among groups, but the higher concentration of IL-10 in the SC group corroborates the Our results do not demonstrate differences in the amount of TNF-α in the different groups, although the literature describes the anti-inflammatory role of LLL 38 and ASCs. 39 Regarding the F I G U R E 5 A, ELISA for the analysis of cytokines IL-10 and TNF-α (pg/mL). Significant differences between groups marked with the same letter (P < 0.05). B, Hydroxyproline concentration (mg/g tissue) of tendon RT: there was no significant difference between the experimental groups expression of Il1b, another proinflammatory cytokine, 40 it was observed a marked tendency to increase in the SC and L groups, corroborating with tendency to increase of Mmp8 gene expression in the same groups. Il1b has been identified as potent inducers of matrix metalloproteinases (MMPs). 41 The L group presented greater expression of Gdf5, a gene in- and Lox, genes responsible for the control of ECM degradation and remodelling. However, the literature demonstrates the effect of LLL and ASCs on these enzymes in a protein level. 7,30,44 Low-level laser-treated group presented a larger organization of the fibres, followed by the SCL, SC and T groups. Studies using LLL or ASCs in tendon repair also demonstrated their role in the reorganization of the collagen matrix 6,7,19 corroborating with our results.
Possibly, the association of treatments was less effective because both therapies present anti-inflammatory effects. 7,8,39 After lesion, the release of several growth factors and cytokines during the inflammation process is responsible for activation of genes responsible for the matrix remodelling. 17,19,41,45 This hypothesis, although speculative, can be considered a limitation of the present study, in which no signalling pathways related to inflammation were analysed at the beginning of the repair process.
According to our data, transplanted ASCs migrated to the tran-