bone healing in rats submitted to weight-bearing and non-weight-bearing exercises

8/8/2019 Bone Healing in Rats Submitted to Weight-bearing and Non-weight-bearing Exercises

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Bone healing in rats submitted to weight-bearing and

non-weight-bearing exercises

Carlos Romualdo Rueff-Barroso1ABCDEF, Débora Milagres1BCD, Juliana do Valle1B,

Gustavo Casimiro-Lopes2BCD, José Firmino Nogueira-Neto3CD,

José Fernando Cardona Zanier 4CD, Luis Cristóvão Porto1ADEFG

1 Tissue Repair Laboratory, Department of Histology and Embryology, Rio de Janeiro State University, Brazil2 Department of Physiological Sciences, Rio de Janeiro State University, Brazil3 Department of Pathology and Laboratories, Rio de Janeiro State University, Brazil4 Department of Internal Medicine – Radiology Service, Pedro Ernesto University Hospital, Rio de Janeiro State

University, Brazil

Source of support: This study was supported in part by grants from the National Council for Scientific and Technological Development (CNPq) and the Carlos Chagas Foundation (FAPERJ)

Summary

Background: To investigate the effects of low-intensity exercise on bone healing during a short time.

Material/Methods: We made a surgical 1-mm perforation in the upper third medial cortical of the right tibia of 45 male Wistar rats (3 months old; mean weight, 282±34 g). Animals were randomly assigned to a swim-

ming exercise group (SWIM, n=15), a running exercise group (RUN, n= 15), or a no exercise con-trol group (CON, n=15). Treatment sessions (10 minutes/day, 5 days/week) were done for 7, 14,or 21 days. Tibias were removed for radiographic, morphometric, and stereologic analyses. Bloodsamples were obtained for biochemical analyses.

Results: Serum phosphorus levels were higher in animals in the RUN group compared with animals in theSWIM group on the seventh day. On the 14th day, the tibias of the animals in the SWIM and RUNgroups exhibited higher radiopacity in radiographic grades than animals in the CON group. Nodifference in collagen morphometry was verified. On the 21st day, serum alkaline phosphatase lev-els were higher in animals in the CON group than they were in the exercise groups, and animalsin the SWIM and CON groups demonstrated an increase in newly formed bone in comparison toanimals in the RUN group.

Conclusions: At the 14th day of treatment, weight-bearing exercise, assessed by radiography, was found to be ben-eficial for bone healing. Results at the 21st day of treatment further supported the benefits of non-

weight-bearing exercises, showing that weight-bearing exercise may improve bone repair in rats.

key words: bearing exercise • swimming • bone healing • collagen • tissue repair

Full-text PDF: http://www.medscimonit.com/fulltxt.php?ICID=869436

Word count: 2958

Tables: 2

Figures: 3

References: 33

Author’s address: Luis Cristóvão Porto, Laboratório de Reparo Tecidual, Instituto de Biologia Roberto Alcântara Gomes, Universidadedo Estado do Rio de Janeiro. Av Prof Manuel de Abreu 444 3o. andar – 20551-170 Rio de Janeiro, RJ, Brasil,e-mail: [email protected] or [email protected]

Authors’ Contribution:

A Study Design

B Data Collection

C Statistical Analysis

D Data Interpretation

E Manuscript Preparation

F Literature Search

G Funds Collection

Received: 2008.04.07

Accepted: 2008.08.05

Published: 2008.11.01

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Basic Research

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Current Contents/Clinical Medicine • IF(2007)=1.607 • Index Medicus/MEDLINE • EMBASE/Excerpta Medica • Chemical Abstracts • Index Copernicus

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B ACKGROUND

Mechanical loads generated by physical activities play an im-portant role in skeletal development [1,2], as do the forc-es generated through mechanical loading during exercise,

which promote osteogenesis [3]. Weight-bearing during ex-

ercise plays an important role in improving the mechani-cal properties of bone [4–6], and it is commonly indicatedfor improving bone health in postmenopausal women as astrategy for reducing the risk of osteoporosis [5]. Otherwise,swim exercise has been used as an alternative to weight-bear-ing exercise in patients with osteoporosis [7,8] or for elderly persons for whom weight-bearing exercise is difficult. Somebeneficial effects of swim exercise have been demonstratedin clinical (9) and experimental models (10). However, theeffects of weight-bearing and non-weight-bearing exerciseson bone healing remain controversial [11–14].

Bone healing is a well-orchestrated physiological process that leads to proliferation and differentiation of osteoprogenitor

cells for osteoblasts, osteoclasts, chondroblasts, fibroblasts,and endothelial cells. Moreover, it is influenced by mechan-ical, physical, chemical, neural, and endocrine factors andthe general environment and local factors [15].

It is known that bone tissue presents a piezoelectric pro-priety that converts the forces generated by running (im-pact) and swimming (increase of the muscle contraction)exercises into osteogenic stimuli resulting in an increasein bone mineral density [16,17] and altering the serumbiomarkers of bone activity, such as alkaline phosphatase[18,19], calcium [20], and phosphorus [21]. In addition,physical exercises act positively on collagen structure [22].

But there are few studies comparing weight-bearing andnon-weight-bearing exercises and its effects on bone heal-ing processes. For this reason, we used swim and run exer-cises simulating an environment in which we could evalu-ate the responses of bone injury to low-intensity impact andnonimpact exercises.

The purpose of this study was to investigate, during 3 weeks, whether low-intensity exercise might have a beneficial effect on bone healing in rats as assessed by radiographic, histo-logic, and biochemical variables.

M ATERIAL AND METHODS

Experimental animals

Forty-five male Wistar rats (Rattus norvegicus albinus ), 3months old with a mean weight of 282±34 g, were housed, 5per cage, with free access to standard rat chow and tap waterin the animal laboratory facility with a controlled tempera-ture in a 12-hour light/dark cycle. The Ethics Committeefor Experimental Animals Use and Care (CEA) of IBRAG-UERJ approved all the animal procedures (CEA/82/2005);this study adheres to the ACSM animal care standards.

Surgical procedure

To simulate a bone healing environment and to eliminatethe possible confounding factors that are too difficult to con-trol (eg, type of fracture line and stability), we made a per-foration in the upper third of the right tibia using a micro-

drill to produce a uniform lesion as described by Guerinoand associates [12]. Animals were anesthetized with an in-traperitoneal injection of ketamine (0.3 mg/kg) and xyla-

zine (0.2 mg/kg). Under aseptic conditions, a small incision was made in the skin of the rats, and the medial surface of the tibia was exposed. A hole (1 mm in diameter) was thenbored using a dentistry burr. Only 1 surface of the corticaltibia was perforated.

Exercise animal groups

Twenty-four hours after the surgery, the animals were divid-ed into the following groups: CON (control group, n=15),SWIM (swimming exercise group, n=15), and RUN (run-ning exercise group, n=15). They were observed duringthe subsequent 7, 14, or 21 days. Body mass was measured

before surgery and at death. This analysis was expressed asbody weight gain (Table 1). Food intake was measured dai-ly. No forced exercise was imposed on the CON group, nor

was additional weight placed.

Swimming exercise group

Swim exercise occurred in a glass swimming pool (50 cmlength, 40 cm width, and 80 cm depth) filled with water70 cm in depth at 34±2°C. Each animal swam alone for 10minutes, 5 days per week for 7, 14, or 21 days. After swim-ming, their coats were blown dry, and they were put backin their cages. The swimming water was changed every day after the end of the exercises.

Running exercise group

The training protocol for animals in the RUN group wasconducted with a motor-driven running wheel (Insight ® Ribeirão Preto, São Paulo, Brazil) for 10 minutes, 5 daysper week, for 7, 14, or 21 days, similar to the protocol forthe SWIM animals.

Killing, blood sampling, and biochemistry

On the last day of the experiment, after intraperitoneal an-esthesia of the animals with an injection of sodium pento-

barbital (30 mg/kg), blood was collected directly from theright atrium, and a cardiac perfusion with 4% paraformal-dehyde for fixation was done in each animal. The level of serum alkaline phosphatase (ALP) activity was measured

GroupBody Weight (g)

7 days 14 days 21 days

CON 6.2±1.5 30.4±4.4 32.9±2.6

SWIM 7.5±2.6 26.4±2.0 31.4±6.1

RUN 9.5±1.9 29.5±3.7 36.7±7.6

p NS NS NS

Table 1. Body weight gain during the experimental period

CON – control group; SWIM – swimming exercise group;RUN – running exercise group; NS – no signifcance (p>0.05).Data are shown as means ±SEM.

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using the AMP method (2-amino-2-methyl-1-propanol buf-fer). Bone-specific alkaline phosphatase (BAP) activity wasmeasured by heat inactivation at 56°C for 10 minutes and

was determined by the AMP method. Calcium and phospho-rus levels were determined by the methylthymol blue andphosphomolybdate/UV methods, respectively. BioSystems®

(Barcelona, Spain) kits were used for all analyses

Radiographic analyses

The process of bone repair was monitored and analyzed onthe day of death by lateral and anteroposterior radiographs.

A mammography (MAMMOMAT® 3000 Nova Mammography System, Siemens Healthcare Diagnostics, Deerfield, IL, USA)

with kV =25, mAs =20.0, and 18×24 cm x-ray films (HDRC-I,IBF) was used to evaluate the radiopacity at the lesion’s borderand the trabeculae of the newly formed bone. The radiolucent aspect was graded as 0, and the radiopacities of the healingareas were compared by an experienced radiologist without knowledge of the duration and exercise protocol of the ani-

mal; the radiopacities received scores of 1, 2, 3, or 4. We con-sidered low radiopacity as scores 0 plus 1 (worse result) andhigh radiopacity as scores 2, 3, and 4 (better result).

Histology

Right tibias were fixed for 48 hours in 4% paraformalde-hyde and then carefully dissected. Then, the tibias were de-calcified in 10% EDTA for 6 weeks (solution was changed3 times per week) and embedded in paraffin. Sagittal sec-tions (5 μm thick) through the bone defect were mount-ed on slices. The sections were stained with picro-Mallory and picro-Sirius red.

Image analysis system

The image analysis system was composed of a light micro-scope (Zeiss Axioplan 2 – Zeiss, Jena, Germany) and a CCDcolor camera (color view XS – SIS, Münster, Germany) con-nected to a desktop computer. A color, brightness, and con-trast test chart was used to calibrate the monitor. The light microscope power knob was adjusted to keep illuminationconstant at a fixed value. All images were acquired using a20×/0.5 Plan Neofluar objective lens (Zeiss, Jena, Germany).The condenser was adjusted to polarized light microsco-py and matched to the numeric aperture of the objectivelens. A stage micrometer (Objektmikrometer – Zeiss, Jena,

Germany) was used to calibrate the system. The dimen-sions of the microscopic field were 430.8×344.7 μm (width× height). All images (1280×1024 pixels) were acquired af-ter setting up for Köhler illumination on the screen, andall light was directed to the camera. Camera pixel size was6.7×6.7 μm. The exposure time and gamma were set to 100ms and 1. All images were stored as uncompressed files.

Morphometric analyses

For morphometric assessments, histologic sections stainedby picro-Sirius red were viewed under polarized light. Thismethod allows indirect evaluation of the stage of bone ma-

trix organization based on the birefringence of the collagenfibers [23]. Organization of the collagenous fibers could beseen in different colors (greenish, yellowish, or reddish) ac-cording to the increased thickness of the fibers within the

newly formed trabeculae in the damaged area. The imageanalysis software, Image-Pro Plus 4.5 (Media Cybernetics,Bethesda, MD), was used to accurately distinguish the col-ors and measure the total area of each. Fifty fields withineach bone-defect group were analyzed. The measurementsof trabecular bone volume (TBV%) within the injury site

were obtained using a microscope by a point counting sys-tem (test system with 16 points) and ×20 magnification. Inthe perforation area, the grid was superimposed across 50fields per group. Thus, the TBV% was estimated by point counting the newly formed trabeculae bone and the trabec-ular void area: TBV%=P

P(trabecular bone)/P

Twhere, P

T

is the total test points (=16). Only samples from animals tobe killed on the 21st day were analyzed.

Statistical analyses

Data are shown as means ±standard error of the mean.Comparisons among groups for the same day were made

with a 1-way analysis of variance (ANOVA) of Kruskal-Wallis

followed by a post-hoc test of Dunn (for nonparametric data)and 1-way ANOVA followed by a post-hoc test of Newman-Keuls (for parametric data). The radiopacity (low, high) wasanalyzed with a Fisher exact test. Differences were consid-ered significant if values for P were less than or equal to.05.

All analyses and graphs were performed using GraphPadPrism version 5.0 for Windows (GraphPad Software, SanDiego, CA, USA).

RESULTS

Body weight gain was similar among the 3 groups (Table 1). Also, there was no difference in food intake (data not

shown). Figure 1 shows radiographically that the tibias of the exercise groups showed a higher level of radiopacity (P= 0.001) in terms of density at the lesion’s border compared

with animals in the CON group on the 14th day. Moreover,the radiopacity of the trabeculae of newly formed bone in-side the lesion on the 14th day was higher in animals in theRUN group than in animals in the SWIM group (P= 0.01).

Table 2 shows the levels of serum calcium and phosphorusand bone-formation markers. Phosphorus levels on the sev-enth day in the RUN group were slightly higher than refer-ence values [24]. Phosphorus levels in the RUN group werestatistically significantly (P= 0.003) higher compared withthose of the SWIM group. On the 14th day, we observed that

the serum level of calcium was lower in the SWIM groupcompared with the other 2 groups (P= 0.02). On the 21st day,the same finding was noted regarding phosphorus levels inthe SWIM group compared with the CON (P= 0.002) andRUN (P= 0.003) groups. Animals in the CON group showedhigher ALP (P= 0.02; P= 0.05) and BAP (P= 0.004; P= 0.03)

values on the 21st day, when compared with the SWIM andRUN groups, respectively.

Morphometric assessment of the collagen fibers’ birefrin-gence demonstrated that there were no differences in thepercentages of greenish, reddish, or yellowish areas with-in newly formed bone in all the groups of the same week

(Figure 2D).

Trabecular bone volume demonstrated a significant increasein the growth of trabecular bone in the CON (P≤0.001) and

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SWIM (P ≤0.001) groups compared with the RUN animalson the 21st day (Figures 2 and 3).

DISCUSSION

All groups showed similar behavior regarding an increasein body mass, which demonstrates that there were no dif-

ferences in exercise intensity between the RUN and SWIMgroups throughout the experiment. Weight gain of growingrats varies with the intensity of the running exercise; there-fore, body mass can be used as an additional indicator tohelp equalize the energy expenditure and training inten-sities of animals undergoing running and swimming exer-cises [4]. The body masses of the animals in the 2 exercisegroups were matched during the training period.

In contrast with animals in the CON group, animals in theexercise groups exhibited a high radiopacity level at the le-sion’s border. These data may suggest that necrotic bone

was resorbed by osteoclasts, and the deposition of newly

formed bone by osteoblasts in the border of the lesion wasbetter in animals in the exercise groups than it was in ani-mals in the control animals. Moreover, the trabecular radi-opacity was higher in animals in the RUN group compared

with animals in the SWIM group. These data indicate that weight-bearing exercise of running animals, in comparisonto the non-weight-bearing exercise of swimming animals,may have enhanced the bone healing.

The process of mineralization requires a normal plasmaconcentration of calcium, phosphate, vitamin D, and hor-

mones as well as sufficient local vascularization [25,26].On the seventh day, we noted that the phosphorous levelsof animals in the RUN group were slightly above their ref-erence values [24]. These data suggest that an increase inosteoclast activity, necessary for the resorption of necroticbone before new bone formation, may explain the betterresults of animals in the RUN group at the 14th day. An el-evation of serum ALP levels suggests an increase in osteo-blast activity [27]. Osteoblasts secrete large amounts of ALP

when they are actively depositing bone or organic matrix.Because some of this enzyme diffuses into the blood, ALPblood levels are a good index of ongoing bone formation[28]. Although in this model, animals in the CON group

exhibited higher ALP and BAP values when compared withanimals in the SWIM and RUN groups on the 21st day, allof these values were within the normal reference values forrats [24]. Guerino and colleagues [12] investigated ALP val-

Figure 1. Radiopacity o the lesion’s border (A) and o the trabeculae o newly ormed bone (B). Data are shown as means ±SEM.*P <0.05,dierence rom the CON group and **P <0.05 dierence rom the SWIM group.

BA

Day Group ALP (U/I) BAP (U/I) Calcium (mg/dL) Phosphorus(mg/dL)

7

CON 219.8±28.4 206.6±27.4 9.56±0.38 11.7±0.82

SWIM 174.5±9.85 158.5±11.5 9.13±0.33 10.3±0.48

RUN 185.8±31.2 164.5±29.0 10.3±0.39 12.6±0.20**

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CON 201±11.6 177.4±12.1 8.12±0.22 9.8±0.40

SWIM 185.2±19.3 173.6±19.2 7.42±0.14* 9.5±0.50

RUN 228.2±13.3 211.2±13.3 8.25±0.27** 9.0±0.48

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CON 248.8±16.3 215.4±17.2 8.24±0.55 11.3±0.57

SWIM 178.2±9.14* 158.8±9.49* 7.13±0.30 8.8±0.23*RUN 196.6±11.7* 167.6±11.3* 8.90±0.69 10.8±0.46**

Table 2. Calcium, phosphorus, alkaline phosphatase (ALP) and bone-specifc alkaline phosphatase (BAP) levels rom blood o healing bone rats.

CON – control group; SWIM – swimming exercise group; RUN – running exercise group. Data are shown as means ±SEM.* p<0.05, dierence rom CON group o the same day and ** p<0.05 dierence rom SWIM group o the same day.


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ues in rats with a bone defect treated with physical exerciseand ultrasound and could not find an increase in ALP val-ues in trained animals either. These data suggest that theexercise intensity used in our model was not sufficient tostimulate an increase in ALP concentrations. On the oth-

er hand, maybe the size of the experimental site (1 mm di-ameter) was not sufficient to alter serum levels of alkalinephosphatase, which is really a measure of systemic boneturnover, probably affected by the activity levels.

Woven bone, in which the collagen fibrils are irregularly ar-ranged, is formed in response to an emergency need for fast bone formation, such as in bone healing [29]. The extracel-lular matrix, and collagens in particular, plays an important role in force transmission and bone structure maintenance[30]. Turnover of the matrix is influenced by physical activi-ty, whereby both collagen synthesis and the levels of degrad-ing metalloprotease enzymes increase with mechanical load-

ing [31]. Quantitative measurements of the birefringenceof collagenous fibers during the 3-week period of treatment revealed no differences among all groups. These findingssuggest that these low-intensity exercises were not sufficient

to promote enhancement of the production of collagenousfibers detectable by morphometry.

Animals in the RUN group showed a delay of newly formedtrabecular bone volume in comparison with the others whenevaluated by TBV%. Animals in the CON group appearedto lag behind animals in the SWIM group, although the dif-ferences were not statistically significant. These data were inaccordance with the radiographic findings, which showedthat the SWIM group curve assumed an ascending trajectory

from the first to the last day of the experiment. Therefore, we suppose that if the duration of the therapeutic exercis-es had been longer, then possibly the non-weight-bearingexercise animals would exhibit better results than both thecontrol group and the weight-bearing exercise group withrespect to therapeutic bone healing exercise. Moreover, aneffect of raising the daily swimming time or adding extra

weight to the rats’ tails to enhance the muscular contrac-tion strength could also be tested.

Water resistance evoked a strong muscular contraction in an-imals in the SWIM group. Bone tissue responds to repeatedmechanical deformation and muscular contractions, whichleads to an increase in electric pulsed currents in bone via

piezoelectric effects. When mechanical forces through me-chanical loads during exercise are converted into electricalstimuli, specific bone tissue cells can begin the osteogenesisprocess [32]. Bone contains a continuous cytoplasmic net-

work made up of osteocytes, which are all joined by gap junc-tions that undoubtedly represent the morphologic substrate,potentially acting as electric synapses that unite osteocytesin a functional syncytium [29]. Hence, they are the majorresponsive cells within the bone matrix to sense mechanicalloading and translate the strains of mechanical loads intobiochemical signals of resorption and formation related tothe intensity and distribution of strain signals [33]. Our re-search was not intended to investigate the mechanisms and

pathways of bone tissue responses during bone repair as-sociated with exercises [29,33]; rather, it aimed to observeand analyze the positive and negative effects on bone heal-ing resulting from low-exercise intensity.

Figure 2. Photomicrographs o bone healingsections. Newly ormed primary bonecould be demonstrated with picro-Siriusstain in the CON group (A), SWIM group(B), and with picro-Mallory in the RUNgroup (C). Photomicrograph o bone

healing with picro-Sirius staining underpolarized light in the SWIM group (D).Note the presence o osteons (arrow)and the arrangement o collagen matrix;t=newly ormed trabeculae bone, andb=bone marrow compartment. Scale barA–C=100 μm; D=50 μm.

DC

BA

Figure 3. Stereologic analyses. Trabecular bone volume (TBV%) asnewly ormed bone within the injur y site. Data are shownas means ±SEM.*P <0.05, dierence rom the CON groupand **P <0.05 dierence rom the SWIM group.

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The present study focused on demonstrating the positive re-sponses of low-intensity exercises on bone healing. In sum-mary, animals in the RUN group obtained better results onthe 14th day of the experiment in terms of radiopacity at thelesion’s border and the trabeculae of newly formed bone,and on the 21st day, based on stereologic measurements; an-

imals in the SWIM group finished with better bone forma-tion compared with animals in the RUN group. However,animals in the CON group did not differ from animals inthe SWIM group regarding this variable. It is suggested that exercise associated with different phases of recovery couldbe used to promote better bone consolidation.

In summary, bone radiopacity at the border of the injuredsite was higher in animals in the RUN group compared withanimals in the CON group at the 14th day, and radiopacity of newly formed bone was higher in animals in the RUNgroup than in animals in the SWIM group at 14 days. At 21days, radiographic analyses showed no differences amongthe groups. However, at this time, animals in the SWIM and

CON groups were better, because animals in the RUN grouppresented lower trabecular bone volume as newly formedbone at the injured site when compared with animals inthe other groups. Animals in the RUN group also had ele-

vated levels of serum calcium and phosphorus at different time points, which could reflect bone resorption. Additionalstudies, analyzing low-intensity exercise of longer periods of time, would elucidate this hypothesis.

CONCLUSIONS

At the 14th day, weight-bearing exercise, as demonstratedby radiographic analyses, was beneficial for bone healing.

At the 21st

day, the results lent additional support to ben-efits of non-weight-bearing exercises, showing it may im-prove bone repair in rats.

Acknowledgments

The authors are grateful to Egberto Gaspar Moura andCaroline Fernandes Santos for their assistance and for re-

viewing the manuscript and to Vagner Bernardo for his help with the analysis system procedures.

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