Photobiomodulation—Original Research

Photobiomodulation of Matrix Metalloproteinasesin Rat Calcaneal Tendons

Julio Fernandes de Jesus, MSc,1 Diva Denelle Spadacci-Morena, PhD,1 Nayra Deise dos Anjos Rabelo, PhD,2

Carlos Eduardo Pinfildi, PhD,3 Thiago Yukio Fukuda, PhD,4 and Helio Plapler, PhD5

Abstract

Objective: The main objective was to verify the modulatory effects of MMP-1, MMP-3, and MMP-13 levels onthe partially injured calcaneal tendons of rat exposure to photobiomodulation.Background: Photobiomodulation has been shown to have anti-inflammatory and regenerative effects ontendon injuries. However, there is still uncertainty regarding the beneficial effects in matrix metalloproteinase(MMP) levels, especially MMP-1, -3, and -13.Materials and methods: Sixty-five male Wistar rats were used. Sixty were submitted to a direct trauma on thecalcaneal tendons and were randomly distributed into the following six groups: LASER 1, 3, and 7 (10 partiallyinjured calcaneal tendons in each group treated with photobiomodulation for 1, 3, and 7 days, respectively) andSham 1, 3, and 7 (same injury, with simulated photobiomodulation). The remaining five animals were allocatedto the normal group (no injury or treatment procedure). The 780 nm low-level laser was applied with 70 mW ofmean power and 17.5 J/cm2 of fluency for 10 sec, once a day. The tendons were surgically removed andanalyzed for MMP-1, MMP-3, and MMP-13 through immunohistochemistry.Results: MMP-3 levels remained close to normal in all experimental groups ( p > 0.05); however, reductions( p < 0.05) in MMP-1 and MMP-13 levels were detected in the groups submitted to one, three, and seven lowlevel laser therapy applications.Conclusions: The photobiomodulation protocol was able to reduce MMP-1 and MMP-13 levels in injuredcalcaneal tendons.

Keywords: calcaneal tendon, matrix metalloproteinases, inflammation, photobiomodulation, tendinopathy

Introduction

The acute and degenerative calcaneal tendon injuriesdemand deeper understanding and greater precision in

the measurement of various aspects of associated dysfunc-tion.1 This injury presents a significant burden in clinics,representing 45% of musculoskeletal lesions.2–4 There areconditions that make this repair process slow and difficult,one of which is the presence of several pro-inflammatory anddegenerative factors in injured tendon tissue,5–8 such asmatrix metalloproteinases (MMPs), that are attracted andactivated by other pro-inflammatory factors and may furtheraggravate the injuries and perpetuate the changes in the in-jured tendon.8–14

MMPs are a family of zinc- and calcium-dependent en-dopeptidases active at a neutral pH. It has important func-tions in the maintenance, remodeling, and degradation of thetendon matrix and its activity is inhibited by tissue inhibitorsof metalloproteinases (TIMP).15 Further, the synthesis ofMMPs may change substantially in the presence of inflam-matory processes in injured tendons. This can be the caseespecially in the presence of primary pro-inflammatoryfactors seen in the early stages of calcaneal tendon injuries,which stimulate the increase in MMPs’ synthesis.12,16–20

In addition, MMP-1 and MMP-13 levels are upregulatedin injured tendon tissue, especially those submitted to shearstress21; therefore, MMP-3 may play a role in the normalmaintenance and remodeling of tendons.15 The presence of

1Laboratory of Pathophysiology, Instituto Butantan, Sao Paulo, Brazil.2Rehabilitation Sciences Program, Universidade Nove de Julho–UNINOVE, Sao Paulo, Brazil.3Human Movement of Science Department, Physical Therapy Course, Universidade Federal de Sao Paulo, UNIFESP, Sao Paulo, Brazil.4Knee and Hip Rehabilitation, Instituto Trata, Sao Paulo, Brazil.5Surgery Department, Universidade Federal de Sao Paulo-UNIFESP, Sao Paulo, Brazil.

Photobiomodulation, Photomedicine, and Laser SurgeryVolume 37, Number 7, 2019ª Mary Ann Liebert, Inc.Pp. 421–427DOI: 10.1089/photob.2019.4633

421

Dow

nloa

ded

by 1

89.2

.86.

163

from

ww

w.li

eber

tpub

.com

at 0

8/06

/19.

For

per

sona

l use

onl

y.

MMP-1, MMP-3, and MMP-13 also promotes degradationof type I and III collagen, the main collagen types that makeup the calcaneal tendon. The uncontrolled presence of theseagents can make the injured tendon become functionallyweakened and susceptible to ruptures.10,12 Thus, it becomesimportant to control these MMPs to improve the repairprocess of calcaneal tendon injuries.

There are some reports showing that photobiomodulationcontrols the presence of primary pro-inflammatory factors,8

stimulates collagen proliferation and realignment,7,14 andelicits angiogenesis22 in tendon injury models. However,there is still a lack of research investigating the photo-biomodulation anti-inflammatory mechanisms at this ex-perimental model on the control of secondary pro-inflammatory factors. Thus, the objective of this study is tofurther clarify the modulatory effects of photobiomodulationon the MMP-1, MMP-3, and MMP-13 levels. This studywill provide information that demonstrates possible speci-ficities on the use of this resource for the repair of calcanealtendon injuries.

Materials and Methods

The Research Ethics Committee of Universidade Federalde Sao Paulo evaluated and approved the present study,under protocol no. 0074/2011.

A total of 65 male rats (Rattus norvegicus)—lineage:Wistar; var. albinus, order: Rodentia, class: Mammalia, age12 weeks, and body mass 270–300 g, were used. The ani-mals were housed in standard polypropylene cages (five percage), in 12-h light/12-h dark cycles, temperature around20�C, humidity of 65%, and water and food ad libitum.

Injury

Sixty animals were randomly anesthetized with intraper-itoneal injection of ketamine hydrochloride (100 mg/kg) andxylazine hydrochloride (50 mg/kg).

After anesthesia, the areas corresponding to the calcanealtendon of the right hind paw of the animal were shaved. Theanimal was then positioned at the base of a small guillotinedevice designed to induce direct trauma injury to the cal-caneal tendons with the right hind paw positioned at thedesignated site of the equipment; a dorsiflexion of the anklewas exerted until the dorsal region touched the base of theequipment. Finally, a weight of 186 g was released from aheight of 20 cm above the central part of the calcanealtendon of each animal, corresponding to an energy potentialof 364.9 mJ at the time of the trauma.7,8,14,23,24 The fiveremaining animals were not exposed to any procedure.

Groups

After the partial injury to the calcaneal tendon, the 60animals were randomly allocated into the following sixgroups: LASER 1, 3, and 7 (10 partially injured calcanealtendons in each group treated with photobiomodulation for1, 3, and 7 days, respectively) and Sham 1, 3, and 7 (sameinjury, with simulated photobiomodulation).

The remaining five animals (10 tendons) were allocated tothe normal group (no injury or treatment procedure).

Photobiomodulation

A low-level laser device (model: Twin Laser/MMOptics�–Sao Carlos, Sao Paulo, Brazil) with active semiconductor(AsGaAl) was used in this study. Table 1 presents the pho-tobiomodulation parameters.

The animals received daily exposures of photobiomodu-lation (a single application per animal), in a continuous way,with fluence equal to 17.5 J/cm2, for 10 sec and total finalenergy of 0.7 J. The protocol started 1 min after the injurywas induced. The animals were stabilized in a foam cageduring the photobiomodulation applications, and the laserirradiations were employed using the contact technique cov-ering all calcaneal tendon injured sites.14 The animals weretreated for 1 day (LASER 1), 3 consecutive days (LASER 3),and seven consecutive days (LASER 7).8,14,24 The animalsof Sham groups (1, 3, and 7) received simulated photo-biomodulation applications with the device switched off;however, the equipment remained in contact with the injuredarea following the same procedures as the LASER groups.

Euthanasia

The animals of LASER/Sham groups were euthanized(anesthetic overdose) 24 h after the last photobiomodulationexposure with the exception of normal group animals thatwere euthanized on the last day of the experiment (eighthday).

Sample procedures

The calcaneal tendons of both hind paws of the normalgroup were collected, along with the 60 tendons (right hindpaw) of the animals belonging to the other groups. Therefore,70 (65 animals) calcaneal tendons were surgically removedand immediately washed in 0.9% saline and then fixed in4% paraformaldehyde in 0.1 M Millonig buffer (pH 7.2–7.4)for 12 h.

After fixation, the tendons were dehydrated, diaphanized,and embedded in paraffin. The sections were made withlongitudinal cuts obtained in a microtome (model: RM2155;Leica) with a thickness of five microns (5 lm) for the im-munohistochemical technique. For the adhesion of the cutsreferring to the immunohistochemical analyses, the slideswere dipped in silane (3-aminopropyl-triethoxysilane; Sigma).

Table 1. Photobiomodulation

Dosimetric Parameters

Photobiomodulation parameters

Light source AsGaAl (LASERdiode)

Wavelength (nm) 780Mean power (mP), mW 70Beam area, cm2 0.04Power density (DP), W/cm2 1.75Energy density (DE), J/cm2 17.5Length of each treatment

session, sec10

Dose of each treatment session, J 0.7Cumulative dose (LASER 1, 3,

and 7, respectively), J0.7/2.1/4.9

422 FERNANDES DE JESUS ET AL.

Dow

nloa

ded

by 1

89.2

.86.

163

from

ww

w.li

eber

tpub

.com

at 0

8/06

/19.

For

per

sona

l use

onl

y.

Therefore, for each animal studied, four nonconsecutivesections were obtained and randomly allocated—one forhematoxylin–eosin (H&E) staining and three for immuno-histochemistry (MMP-1, MMP-3, MMP-13).

Immunohistochemistry

The slides were dewaxed and exposed to specific anti-body kits for MMP-1 (monoclonal anti-MMP-1, brand:ABCAM-AB52915, dilution 1:200), MMP-3 (polyclonalanti-MMP-3, brand: ABCAM-AB38929, dilution 1:50), andMMP-13 (polyclonal anti-MMP-13, brand: ABCAM-AB39012,dilution 1:100).

Antigen retrieval was performed with porcine Trypsin inphosphate-buffered saline (PBS, code: T7409; Sigma-Aldrich). Blocking of endogenous peroxidase was per-formed with 10v hydrogen peroxide (3%), then washed intap water and deionized water, and left in phosphate bufferpH 7.4 (PBS). The nonspecific proteins were blocked byimmersing the slides in casein (Synth, Sao Paulo, Brazil)diluted in phosphate buffer pH 7.4.

The slides were incubated with the primary antibodiesand specific diluent (Spring Bioscience, Pleasanton, CA) for24 h at 4�C and subsequently incubated with the secondaryantibody Reveal (Spring Biosciences) for 45 min at 37�C.After that procedure, the slides were revealed in diamino-benzidine (Spring Biosciences) for 10 min at room temper-ature. Finally, the slides were counterstained with Harrishematoxylin (Merck, Darmstadt, Germany) and mountedwith Entellan (Merck).

The photobiomodulation anti-inflammatory response wasanalyzed in three calcaneal tendons’ microscopic fields(proximal: the region just under the myotendinous junc-tion, intermediate: the area between these two regions, anddistal: the region just above the osteotendinous junction).Those microscopic images were previously obtained at400 · magnification (objective of 40 · ) with a camera(DFC420; Leica Microsystems, Switzerland) coupled to aDMLB microscope (Leica Microsystems). After that, thepositive cells for MMP-1, MMP-3, and MMP-13—markedin brown shades—were quantified with image software LASversion 4.5.0 (Leica Microsystems).

Statistical analysis

The data normality distribution has been verified usingthe Shapiro–Wilk test and homogeneity of variances byLevene test. The three tendon regions (proximal, interme-diate, and distal) were compared using one-way analysis ofvariance, if the F statistic was significant; Bonferroni’s post-test was applied.

The dependent variables of interest in this study wereMMP-1, MMP-3, and MMP-13. The independent variables(groups) were transformed into dummy variables. It wasconsidered for simple regression model the forced input intoa single block the independent variables. This regressionmodel was performed to predict possible photobiomodula-tion influences on MMP levels.

The level of significance was set at 5% ( p < 0.05), and theStatistical Package for Social Sciences (SPSS, version 20.0)was used for all analyses.

Ta

ble

2.

MM

P-1

,M

MP

-3,

an

dM

MP

-13

Po

sitiv

eC

ells

(M

ea

n–

SD

)o

fth

eT

hree

Ten

do

nR

eg

io

ns

An

aly

zed

(P

ro

xim

al,

In

term

ed

ia

te,

an

dD

ista

l)

MM

P-1

MM

P-3

MM

P-1

3

Pro

xim

al

Inte

rmed

iate

Dis

tal

Mea

n–

SD

pP

roxi

ma

lIn

term

edia

teD

ista

lM

ean

–S

Dp

Pro

xim

al

Inte

rmed

iate

Dis

tal

Mea

n–

SD

p

No

rmal

18

.9–

5.6

15

.7–

6.0

19

.9–

6.9

18

.2–

3.7

>0.0

57

.7–

4.2

7.6

–2

.76

.5–

4.1

7.2

–3

.3>0

.05

23

.7–

9.6

22

.1–

8.3

23

.3–

7.3

23

.3–

7.3

>0.0

5S

ham

11

8.4

–1

1.4

11

.6–

6.2

14

.6–

6.4

14

.9–

6.8

>0.0

56

.4–

5.4

5.9

–2

.87

.5–

3.7

6.6

–2

.9>0

.05

14

.5–

6.3

14

.2–

4.2

14

.2–

6.1

14

.3–

6.8

>0.0

5L

AS

ER

19

.7–

4.6

11

.5–

5.3

9.8

–3

.11

0.3

–2

.6>0

.05

5.2

–2

.55

.3–

2.9

6.4

–2

.85

.5–

2.1

>0.0

51

2.8

–7

.51

2.0

–8

.11

1.6

–4

.71

2.1

–5

.9>0

.05

Sh

am3

17

.1–

7.6

19

.1–

6.1

15

.6–

3.3

17

.2–

4.2

>0.0

56

.5–

3.5

6.8

–3

.66

.2–

3.9

6.5

–3

.2>0

.05

22

.0–

7.1

22

.3–

3.5

23

.4–

6.4

22

.5–

3.2

>0.0

5L

AS

ER

31

5.6

–7

.71

4.8

–6

.01

4.2

–4

.91

4.8

–3

.5>0

.05

4.2

–2

.95

.6–

1.9

4.8

–3

.74

.9–

2.0

>0.0

51

9.4

–1

2.1

14

.8–

7.0

16

.9–

6.4

17

.0–

7.7

>0.0

5S

ham

71

2.9

–4

.61

1.7

–5

.41

4.8

–3

.81

3.1

–3

.0>0

.05

4.4

–2

.26

.3–

4.3

6.6

–4

.45

.7–

2.9

>0.0

51

3.7

–4

.21

1.8

–5

.21

1.0

–3

.41

2.1

–1

.8>0

.05

LA

SE

R7

13

.0–

4.6

10

.3–

2.5

10

.3–

4.6

11

.3–

2.3

>0.0

54

.2–

2.4

6.6

–2

.66

.5–

3.4

5.8

–1

.9>0

.05

12

.6–

4.3

14

.2–

4.6

11

.3–

4.5

12

.7–

.6>0

.05

MM

P,

mat

rix

met

allo

pro

tein

ase.

PHOTOBIOMODULATION OF TENDONS’ MMP LEVELS 423

Dow

nloa

ded

by 1

89.2

.86.

163

from

ww

w.li

eber

tpub

.com

at 0

8/06

/19.

For

per

sona

l use

onl

y.

Results

There were no differences ( p > 0.05) between the threetendon regions analyzed (proximal, intermediate, and distal)for MMP-1, MMP-3, and MMP-13 mean number of positivecells (Table 2), and the regression model proved to be ef-ficient ( p < 0.05) to predict the outcome for MMP-1 andMMP-13, but not for MMP-3 ( p > 0.05).

Although the regression model did not prove to be effi-cient to predict MMP-3 outcomes, the experimental LASER3 group presented lower levels of MMP-3 than the normalgroup ( p < 0.05). However, the other experimental groups(LASER 1, LASER 7, and Sham 1, 3, and 7) did not presentmodulations at MMP-3 levels when the means of the ana-lyzed areas were compared with those of the normal group( p > 0.05). Differences were identified between the experi-mental groups and the normal group ( p < 0.05) for MMP-1and MMP-13 levels. LASER 1, LASER 3, LASER 7, andSham 7 had lower MMP-1 levels than the normal group, andLASER 1, LASER 3, LASER 7, Sham 1, and Sham 7 hadlower MMP-13 levels than the normal group (Table 3).

Figure 1 shows photomicrography of tendon morphology(H&E) and immunohistochemistry positive cells (b) forMMP, both acquired with 40 · objective lens.

Discussion

The main objective of this animal controlled trial was toevaluate the photobiomodulation of MMP-1, MMP-3, andMMP-13 levels in injured calcaneal tendons of rats. Themost significant findings of this study were the non-modulation of MMP-3 levels and reductions in MMP-1 andMMP-13 levels in the groups submitted to one, three, andseven photobiomodulation applications.

The application of photobiomodulation to optimize therepair process of the calcaneal tendon is very promisingbecause this treatment modality has the ability to use themodulatory pathway to control inflammatory processes.8,10,24

This ability to control inflammatory processes elucidatesthe relevance of using this treatment modality because thepresence of primary pro-inflammatory factors8,10,15,21 caninduce the increase in MMPs’ levels. In addition, the un-controlled presence of MMP-1, MMP-3, and MMP-13 tendsto increase the degradation of extracellular matrix,25 as wellas of the calcaneal tendon collagen, fragilizing and ham-pering its repair process.15,21,26–28

Unlike other studies on the same topic,10,12 there was adecrease in MMP-1 and MMP-13 levels in the experimental

groups in the current study, and MMP-3 levels showed nomodulations. A previous experimental study by our researchgroup,8 with the same design as the current study, showedthat 780 nm low-level laser has the capacity to modulate IL-1b, COX-2, and PGE2 levels when the same treatmentprotocol was implemented. The use of the same design interms of dose and groups may explain the present results,given that these primary pro-inflammatory factors can trig-ger the increase in MMPs’ levels.15,21

Although the inflammatory process may perpetuate andaggravate the calcaneus tendon injury,29,30 most of theselesions are caused by repetitive mechanical stress in thetendon and consequently morphological changes.1,31 Be-cause of this and differently from previous studies, we choseto use a well-established injury model that mimicked thischaracteristic.5,7,8,14,24 It is possible that these other forms ofinjury (chemical: collagenase10,12,22 and carrageenan11,25)were more sensitive to cause modulation of MMPs’ levels.Further, MMP-3 is more active in the maintenance ofhealthy tendons and in later phases of the repair process(remodeling phases). These phases are in contrast to thosepromoted by the experimental model times of the currentstudy,15,21 which focused on the inflammatory phase of therepair process (1, 3, and 7 days) and which may haveinfluenced the nonmodulation of the levels of this enzyme.

The Sham groups presented some differences in theirMMP-1 and MMP-13 levels. These results may be causedby the mechanical stresses imposed by the rat’s walkingpattern. There are reports in the literature showing increasedMMP-1 levels in tendinopathic tissue submitted to shearstress, unlike the mechanical overload imposed by the rat’swalking pattern. Instead, cyclic strain overload (similar tothe rat’s mechanical walking pattern) causes a reduction inMMP-1 levels and may cause an upregulation of MMP-13.15

The interaction between photobiomodulation and thedifferent pathways of tissue repair processes is dependent onseveral factors related to wavelength, mean power, dose,and frequency of treatment.32,33 This can be observed inprevious studies that used photobiomodulation with l�=810 nm10,12 and 830 nm,11,25 mean power of 100 mW10,12

and 40 mW,11,25 and final energy varying between 1 and3 J.10–12,25 Although the dosage adjustments and the treat-ment regimens of these studies differed from those of thepresent study, reductions in MMP-13 levels were also de-tected, as well as reductions in COX-2, TNF-a, MMP-2,MMP-3, and MMP-9. Casalechi et al.22 used photo-biomodulation with l�= 780 nm, mean power of 22 mW,

Table 3. The Regression Results for MMP-1, MMP-3, and MMP-13

MMP-1 MMP-3 MMP-13

B 95% CI p B 95% CI p B 95% CI p

Constant 18.31 15.79 to 20.83 0.00 7.73 5.98 to 9.48 0.00 23.03 19.70 to 26.36 0.00Sham 1 vs. normal -3.39 -7.04 to 0.26 0.06 -1.11 -3.60 to 1.33 0.36 -8.73 -13.44 to -4.02 0.00LASER 1 vs. normal -7.98 -11.54 to -4.42 0.00 -2.23 -4.70 to 0.23 0.07 -10.89 -15.61 to -6.18 0.00Sham 3 vs. normal -0.71 -4.27 to 2.84 0.68 -1.94 -4.56 to 0.67 0.14 -0.46 -5.18 to 4.24 0.84LASER 3 vs. normal -3.44 -7.00 to 0.11 0.05 -2.89 -5.52 to -0.27 0.03 -5.99 -10.71 to -1.28 0.01Sham 7 vs. normal -5.31 -8.87 to -1.75 0.00 -1.96 -4.43 to 0.50 0.11 -10.86 -15.57 to -6.15 0.00LASER 7 vs. normal -6.94 -10.60 to -3.28 0.00 -1.91 -4.45 to 0.62 0.13 -10.28 -15.27 to -5.28 0.00

424 FERNANDES DE JESUS ET AL.

Dow

nloa

ded

by 1

89.2

.86.

163

from

ww

w.li

eber

tpub

.com

at 0

8/06

/19.

For

per

sona

l use

onl

y.

E = 1.54 J, and one, three, four, and seven applications andfound similar results to those of the present study, that is,modulations in MMP-1 and MMP-13 levels.

Another study shows specific effects of different wave-lengths and dosage adjustments of photobiomodulation forthe most diverse factors seen in an injury and consequentrepair process of the calcaneal tendon.34 It is possible thatthe reduction in MMPs’ levels after photobiomodulation wasdirectly and indirectly influenced by the treatment regimenused.35 This is due to the fact that previous studies dem-onstrated interactions between the same photobiomodulationprotocol applied in this study (l�= 780 nm) and the TIMP36

and other studies published by our group showing modula-tion of primary inflammatory factor levels.8 These effectsseem to be achieved by the modulatory pathway.

Although currently photobiomodulatory protocol haspresented positive effects for the control of MMPs’ levels,

new experimental studies involving low level laser therapyeffects in all phases of the calcaneal tendon repair process(inflammatory, proliferative, and remodeling phases) areneeded.

Conclusions

The photobiomodulation protocol applied in this animalmodel was able to reduce MMP-1 and MMP-13 levels ininjured calcaneal tendons in the early stages of injury andthe prechronic phase.

Acknowledgments

The authors of this study thank the Sao Paulo ResearchFoundation (FAPESP) for supporting and funding our re-search proposal (2017/09658-0) and Magna AparecidaMaltauro Soares for the technical support.

FIG. 1. Photomicrographyof tendon morphology (H&E)and immunohistochemistrypositive cells (b) for MMPs;both acquired with 40 ·objective lens. H&E,hematoxylin-eosin; MMP,matrix metalloproteinase.

PHOTOBIOMODULATION OF TENDONS’ MMP LEVELS 425

Dow

nloa

ded

by 1

89.2

.86.

163

from

ww

w.li

eber

tpub

.com

at 0

8/06

/19.

For

per

sona

l use

onl

y.

Author Disclosure Statement

Julio Fernandes de Jesus and Diva Denelle Spadacci-Morena would like to declare that they had financial supportby Sao Paulo Research Foundation (FAPESP)—researchproposal 2017/09658-0—to acquire the necessary immuno-histochemistry kits to realize this research.

References

1. Cook JL, Rio E, Purdam CR, Docking SI. Revisiting thecontinuum model of tendon pathology: what is its merit inclinical practice and research? Br J Sports Med 2016;50:1187–1191.

2. Perucca Orfei C, Lovati AB, Vigano M, et al. Dose-relatedand time-dependent development of collagenase-inducedtendinopathy in rats. PLoS One 2016;11:e0161590.

3. Cassel M, Baur H, Hirschmuller A, Carlsohn A, Frohlich K,Mayer F. Prevalence of Achilles and patellar tendinopathyand their association to intratendinous changes in adoles-cent athletes. Scand J Med Sci Sports 2015;25:e310–e318.

4. Cassel M, Risch L, Intziegianni K, et al. Incidence ofAchilles and patellar tendinopathy in adolescent elite ath-letes. Int J Sports Med 2018;39:726–732.

5. Oliveira FS, Pinfildi CE, Parizoto NA, et al. Effect of lowlevel laser therapy (830 nm) with different therapy regimeson the process of tissue repair in partial lesion calcaneoustendon. Lasers Surg Med 2009;41:271–276.

6. Doral MN, Alam M, Bozkurt M, et al. Functional anatomyof the Achilles tendon. Knee Surg Sports Traumatol Ar-throsc 2010;18:638–643.

7. Wood VT, Pinfildi CE, Neves MA, Parizoto NA, HochmanB, Ferreira LM. Collagen changes and realignment inducedby low-level laser therapy and low-intensity ultrasound inthe calcaneal tendon. Lasers Surg Med 2010;42:559–565.

8. de Jesus JF, Spadacci-Morena DD, dos Anjos Rabelo ND,Pinfildi CE, Fukuda TY, Plapler H. Low-level laser therapyin IL-1beta, COX-2, and PGE2 modulation in partially in-jured Achilles tendon. Lasers Med Sci 2015;30:153–158.

9. Fredberg U, Stengaard-Pedersen K. Chronic tendinopathytissue pathology, pain mechanisms, and etiology with aspecial focus on inflammation. Scand J Med Sci Sports2008;18:3–15.

10. Marcos RL, Leal-Junior EC, Arnold G, et al. Low-levellaser therapy in collagenase-induced Achilles tendinitis inrats: analyses of biochemical and biomechanical aspects.J Orthop Res 2012;30:1945–1951.

11. Guerra Fda R, Vieira CP, Almeida MS, Oliveira LP, de AroAA, Pimentel ER. LLLT improves tendon healing throughincrease of MMP activity and collagen synthesis. LasersMed Sci 2013;28:1281–1288.

12. Marcos RL, Arnold G, Magnenet V, Rahouadj R, Magda-lou J, Lopes-Martins RA. Biomechanical and biochemicalprotective effect of low-level laser therapy for Achillestendinitis. J Mech Behav Biomed Mater 2014;29:272–285.

13. de Almeida P, Tomazoni SS, Frigo L, et al. What is the besttreatment to decrease pro-inflammatory cytokine release inacute skeletal muscle injury induced by trauma in rats: low-level laser therapy, diclofenac, or cryotherapy? Lasers MedSci 2014;29:653–658.

14. de Jesus JF, Spadacci-Morena DD, Rabelo ND, Pinfildi CE,Fukuda TY, Plapler H. Low-level laser therapy on tissuerepair of partially injured achilles tendon in rats. PhotomedLaser Surg 2014;32:345–350.

15. Magra M, Maffulli N. Matrix metalloproteases: a role inoveruse tendinopathies. Br J Sports Med 2005;39:789–791.

16. Archambault J, Tsuzaki M, Herzog W, Banes AJ. Stretchand interleukin-1beta induce matrix metalloproteinases inrabbit tendon cells in vitro. J Orthop Res 2002;20:36–39.

17. Kjaer M. Role of extracellular matrix in adaptation oftendon and skeletal muscle to mechanical loading. PhysiolRev 2004;84:649–698.

18. Asundi KR, King KB, Rempel DM. Evaluation of geneexpression through qRT-PCR in cyclically loaded ten-dons: an in vivo model. Eur J Appl Physiol 2008;102:265–270.

19. Riley G. Tendinopathy—from basic science to treatment.Nat Clin Pract Rheumatol 2008;4:82–89.

20. Hosaka YZ, Uratsuji T, Ueda H, Uehara M, Takehana K.Comparative study of the properties of tendinocytes de-rived from three different sites in the equine superficialdigital flexor tendon. Biomed Res 2010;31:35–44.

21. Magra M, Maffulli N. Molecular events in tendinopathy: arole for metalloproteases. Foot Ankle Clin 2005;10:267–277.

22. Casalechi HL, Leal-Junior EC, Xavier M, et al. Low-levellaser therapy in experimental model of collagenase-inducedtendinitis in rats: effects in acute and chronic inflammatoryphases. Lasers Med Sci 2013;28:989–995.

23. Costa MS, Pinfildi CE, Gomes HC, et al. Effect of low-level laser therapy with output power of 30 mW and 60 mWin the viability of a random skin flap. Photomed Laser Surg2010;28:57–61.

24. de Jesus JF, Spadacci-Morena DD, Dos Anjos Rabelo ND,Pinfildi CE, Fukuda TY, Plapler H. Low-level laser therapy(780 nm) on VEGF modulation at partially injured Achillestendon. Photomed Laser Surg 2016;34:331–335.

25. Da Re Guerra F, Vieira CP, Marques PP, Oliveira LP,Pimentel ER. Low level laser therapy accelerates the ex-tracellular matrix reorganization of inflamed tendon. TissueCell 2017;49:483–488.

26. Fu SC, Chan BP, Wang W, Pau HM, Chan KM, Rolf CG.Increased expression of matrix metalloproteinase 1(MMP1) in 11 patients with patellar tendinosis. Acta Or-thop Scand 2002;73:658–662.

27. de Araujo Munhoz FB, Baroneza JE, Godoy-Santos A,et al. Posterior tibial tendinopathy associated with matrixmetalloproteinase 13 promoter genotype and haplotype.J Gene Med 2016;18:325–330.

28. Gibbon A, Hobbs H, van der Merwe W, et al. The MMP3gene in musculoskeletal soft tissue injury risk profiling: astudy in two independent sample groups. J Sports Sci 2017;35:655–662.

29. Rees JD, Stride M, Scott A. Tendons—time to revisit in-flammation. Br J Sports Med 2014;48:1553–1557.

30. Abate M, Silbernagel KG, Siljeholm C, et al. Pathogenesisof tendinopathies: inflammation or degeneration? ArthritisRes Ther 2009;11:235.

31. Cook JL, Purdam CR. Is tendon pathology a continuum? Apathology model to explain the clinical presentation ofload-induced tendinopathy. Br J Sports Med 2009;43:409–416.

32. Karu T. Photobiology of low-power laser effects. HealthPhys 1989;56:691–704.

33. Carrinho PM, Renno AC, Koeke P, Salate AC, ParizottoNA, Vidal BC. Comparative study using 685-nm and 830-nm lasers in the tissue repair of tenotomized tendons in themouse. Photomed Laser Surg 2006;24:754–758.

426 FERNANDES DE JESUS ET AL.

Dow

nloa

ded

by 1

89.2

.86.

163

from

ww

w.li

eber

tpub

.com

at 0

8/06

/19.

For

per

sona

l use

onl

y.

34. Marques AC, Albertini R, Serra AJ, et al. Photo-biomodulation therapy on collagen type I and III, vascularendothelial growth factor, and metalloproteinase in exper-imentally induced tendinopathy in aged rats. Lasers MedSci 2016;31:1915–1923.

35. Tumilty S, Mani R, Baxter GD. Photobiomodulation andeccentric exercise for Achilles tendinopathy: a randomizedcontrolled trial. Lasers Med Sci 2016;31:127–135.

36. Gavish L, Perez L, Gertz SD. Low-level laser irradiationmodulates matrix metalloproteinase activity and gene ex-pression in porcine aortic smooth muscle cells. Lasers SurgMed 2006;38:779–786.

Address correspondence to:Diva Denelle Spadacci-Morena, PhD

Laboratory of PathophysiologyInstituto Butantan

Sao Paulo, SP 05504-900Brazil

E-mail: diva.morena@butantan.gov.br

Received: February 6, 2019.Accepted after revision: March 24, 2019.

Published online: June 11, 2019.

PHOTOBIOMODULATION OF TENDONS’ MMP LEVELS 427

Dow

nloa

ded

by 1

89.2

.86.

163

from

ww

w.li

eber

tpub

.com

at 0

8/06

/19.

For

per

sona

l use

onl

y.