Effect of 830-nm Laser Light on the Repair ofBone Defects Grafted with Inorganic Bovine Bone

and Decalcified Cortical Osseus Membrane

ANTONIO LUIZ BARBOSA PINHEIRO, Ph.D.,1 FRANCISCO DE ASSIS LIMEIRA JÚNIOR, M.Sc.,2MARLENY ELIZABETH MÁRQUEZ GERBI, M.Sc.,2 LUCIANA MARIA PEDREIRA RAMALHO, Ph.D.,3

CLOVIS MARZOLA, Ph.D.,4, ELIZABETH ARRUDA CARNEIRO PONZI, M.Sc.,5ANDRÉ OLIVEIRA SOARES, D.S.,6 LÍVIA CRISTINA BANDEIRA DE CARVALHO, D.S.,6

HELENA CRISTINA VIEIRA LIMA, D.S.,6 and THAIS OLIVEIRA GONÇALVES, D.S.6

ABSTRACT

Objective: The aim of this study was to histologically assess the effect of low-level laser therapy (LLLT)(l830 nm) on the repair of standardized bone defects of the femur of Wistar albinus rats grafted with in-organic bovine bone and associated (or not) with decalcified bovine cortical bone membrane. BackgroundData: Bone loss may be a result of pathology, trauma, or surgical procedure. Extensive studies on the pro-cess of bone repair have been undertaken, and several techniques for the correction of bone defects havebeen proposed. Amongst them is the use of several types of grafts, the use of membranes, and the combina-tion of both techniques. There is evidence in the literature of the positive effect of LLLT on the healing ofsoft tissue wounds. However, its effect on bone healing is not completely understood. Materials and Meth-ods: Five randomized groups were studied: group I (control); group IIA (Gen-ox®); group IIB (Gen-ox® +LLLT); group IIIA (Gen-ox® + Gen-derm®); and group IIIB (Gen-ox® + Gen-derm® + LLLT). Bone defectswere created at the femur and were treated according to the group. The animals of irradiated groups wereirradiated every 48 h for 15 days; the first irradiation was performed immediately after the procedure. Theanimals were irradiated transcutaneuosly at four points around the defect. At each point, a dose of 4 J/cm2

was given (f~0.6 mm, 40 mW), and the total dose per session was 16 J/cm2. The animals were humanelykilled at 15, 21, and 30 days after surgery. The specimens were routinely processed to wax, serially cut,stained with H&E and Picrosirius stains, and analyzed under light microscopy. Results: The results showedmore advanced repair of the irradiated groups when compared to the non-irradiated ones. The repair of theirradiated group was characterized by both increased bone formation and on the amount of collagen fibersaround the graft within the cavity, as early as the 15th day after surgery, considering the osteoconductivecapacity of the Gen-ox® and the increment of the cortical repair in specimens with Gen-derm® membrane.Conclusion: It is concluded that LLLT had a positive effect on the repair of bone defect by graft associatedor not with the use of biological membrane.

301

1Laser Center, School of Dentistry, Federal University of Bahia, Salvador, Brazil.2Integrated UFPB/UFBA Postgraduate Dental Program, School of Dentistry, Federal University of Bahia, Salvador, Brazil.3Department of Diagnostic and Therapeutics, School of Dentistry, Federal University of Bahia, Salvador, Brazil.4Department of Surgery, School of Dentistry, University of São Paulo at Bauru, Bauru, SP, Brazil.5School of Dentistry, Federal University of Pernambuco, Recife, Pernambuco, Brazil.6Dental Student, School of Dentistry, Federal University of Bahia, Salvador, Brazil.

Journal of Clinical Laser Medicine & SurgeryVolume 21, Number 5, 2003© Mary Ann Liebert, Inc.Pp. 301–306

INTRODUCTION

THE USE OF INORGANIC BOVINE BONE is not a new tool toimprove bone repair, as there are reports as early as

1889.1 This material was used previously in the reconstruc-tion of alveolar ridge2,3; in bone loss associated with bothperiodontal and periapical lesions4,5; in bone defects4,6,7; indental sockets4,8,9; as an hemostatic agent1; and in guided tis-sue regeneration (GTR),10 a widely used technique to correctperiodontal bone defects. When it is used to correct bone de-fects of non-periodontal origin, it is called “guided bone re-generation.”11,12 In both techniques, there is an association ofthe graft with the use of membranes.10,13,14 Although the useof low-level light therapy (LLLT) in the biomodulation ofbone repair has been growing steadily and several studieshave demonstrated positive results in the healing of bone tis-sue,15–22 there are no previous reports on the combination ofLLLT and biomaterials. The aim of this study was to assessthe effect of LLLT on the healing of bone defects treated withinorganic bovine bone combined, or not, with decalcified cor-tical osseous membrane.

MATERIALS AND METHODS

Forty-two young adults healthy male and female Wistar Al-binus rats weighing 270–320 g were kept under natural condi-tions of light, humidity, and temperature at the AnimalExperimentation Laboratory, School of Dentistry, Federal Uni-versity of Bahia. The animals were fed with laboratory pelted

diet and had water ad libidum. The animals were divided intofive groups, and each group was divided into three subgroups(Table 1).

Under intraperitoneal general anesthesia (10% chloral hy-drate, 0.4 mL/100 g), animals had the right leg shaved and thefemur exposed. Standardized 3-mm2 cavities were created onthe superior third of the lateral side of the bone. In group I,the periosteum was repositioned and suturing was performedwith catgut and the skin closed with nylon. In groups II, III,IV, and V, the cavities were completely filled with inorganicbovine bone (Gen-ox®, Baumer S/A, Mogi Mirim, SP Brazil).Group IV and V cavities were covered with decalcified corti-cal osseous membrane (Gen-derm®, Baumer S/A, MogiMirim, SP, Brazil). All wounds were routinely sutured as de-scribed previously.

The animals of groups III and V were submitted to sevensessions of LLLT (Thera Lase, DMC Equipamentos/São Car-los, SP Brazil, l830 nm, 40 mW, u~0.60 mm, CW), at 48-hintervals. The irradiation was performed transcutaneuosly,and the first session was performed immediately after sur-gery. A dose of 4 J/cm2 was applied to four points around thedefect, giving a total of 16 J/cm2 per session and a totaltreatment dose of 112 J/cm2. The technique used in thisstudy was based upon previous studies carried out by Pin-heiro et al.23

The animals were humanely killed at 15, 21, and 30 daysafter surgery by an overdose of general anesthetics. Sampleswere taken and kept on 4% buffered paraformaldeyde solutionfor 5 days. The samples were decalcified with 10% nitric acidand routinely stained with H&E and Picrosirius stains.24

302 Pinheiro et al.

TABLE 1. GROUPS AND SUBGROUPS

Group Subgroups N Procedure

I C15/C21/C30 6 ControlII B15/B21/B3O 9 Inorganic bovine boneIII BL15/BL21/BL30 9 Inorganic bovine bone + LLLTIV BM15/BM21/BM30 9 Inorganic bovine bone + decalcified cortical osseous membraneV BML15/BML21/BML30 9 Inorganic bovine bone + decalcified cortical osseous membrane + LLLT

C, control; B, bone; M, membrane; L, laser.

FIG. 1. Photomicrography of control specimens. (A) At day 15, a discrete bone formation between the cortical was observed.(B) At day 21, the deposition of new bone between the cortical is more evident. (C) At day 30, a complete union of the corticaloccurred. H&E, 340.

RESULTS

At day 15, light microscopy showed, on non-treated speci-mens, a cavity filled by bone marrow and a few bone trabecu-lae. The cortical defect was more pronounced at 21 days aftersurgery and nearly closed at day 30 (Fig. 1).

In group IIA (Gen-ox®), the graft nearly filled all the cavityand was involved by collagen fibers at 15 days after surgery.Immature bone trabeculae were observed at the graft surface,but no newly formed bone around the graft could be seen atthis time. Up to day 30 after surgery, the graft particles wereevenly distributed into the defect, and were partial or totally in-volved by newly formed bone. Newly formed bone trabeculaewere seen initiating the repair of the cortical region (Fig. 2).

At 15 days after surgery, specimens of group III showed thatthe graft filled most of the cavity and was also involved eitherby collagen fibers or mineralizing newly formed bone. On thecortical region, some new neoformed bone trabeculae aroundsome particles of the graft and a dense amount of collagenfibers could be observed. At 21 days after the procedure, an in-creased amount of neoformed bone could be seen and the graftwas involved by collagen fibers. At the end of the experimentalperiod, the cavity was still filled by the graft and the area wasdensely filled by collagen fibers (Fig. 3).

When the graft was associated with the membrane, it wasobserved that its particles filled most of the cavity and were in-volved by thin collagen fibers at day 15 and a discrete bone

neoformation and immature trabeculae could also be observedaround the particles at this time. Later, at day 21, the particlesof the graft were involved by dense neoformed bone. At theend of the experimental period, a progressive and evident boneneoformation around the particles of the graft was detectableand most specimens showed a nearly complete cortical repair(Fig. 4).

When LLLT was combined with graft and membrane, ingroup V (Gen-ox® + Gen-derm® + LLLT), several particleswere observed filling the cavity and there was the presence ofcollagen fibers at day 15. At day 21, the cavity remained filledby the graft, whose particles were involved by dense collagenfibers and neoformed bone. A large amount of collagen fibersat the cortical region was also observed. At day 30, there wasmore pronounced bone formation around the particles of thegraft. There was also a dense and well-organized neoformedtrabeculae, and the cortical repair was complete (Fig. 5).

DISCUSSION

It seems clear from our results that the inorganic Gen-ox® isosteoconductive, as all grafted specimens showed bone neofor-mation around the particles. It is interesting to observe thatmost specimens showed the particles of the graft involved bycollagen fibers and filling the center of the cavity at day 15.Later, at day 21, these particles were unevenly distributed and

LLLT and Repair of Bone Defects Grafted with Inorganic Bone 303

FIG. 2. Photomicrography of group II. (A) At day 15, cortical repair was observed, as well as the presence of delicate newlybone formed within the cavity near to the graft surface. At days 21 and 30 (B, C), newly formed bone could be seen around thegraft. H&E, 340 (A); Picrosirius, 340 (B, C).

FIG. 3. Photomicrography of group III. (A) At day 15, complete cortical repair was observed. At days 21 (B) and 30 (C), newlyformed bone could be seen (arrowhead), as well as collagen fibers encircling the graft. H&E, 340 (A); Picrosirius, 340 (B, C).

involved by a variable amount of newly formed bone. Thelevel of bone neoformation did not change much up to day 30in most groups, except for group V, in which bone neoforma-tion was more evident between days 21 and 30.

It is accepted that, although inorganic Gen-ox® has osteo-conductivity, the repair of the defect may be slow because ofthe need of the graft to be reabsorbed, slowing down the pro-cess. In this study, the presence of macrophages and giant cellsaround the particles, as observed previously,2,25 may explainthe reduction in the amount of particles throughout the experi-mental time. On the other hand, a previous report suggestedthat this type of material may also be incorporated into thenewly formed bone.26

The presence of the membrane seemed not to have a definiteinfluence on the repair of the core of the defect, probably dueto the osteoconductivity of inorganic Gen-ox®; however, thecortical repair was more evident when the membrane was used,as seen previously.11 It is clear that the use of the graft preventsthe formation of fibrosis of the lesion, and also protects thecavity4 and acts as a framework for the deposition of neo-formed bone.27

Although the biomodulatory effect of LLLT on bone regen-eration was observed previously under different experimentalconditions and is aligned with our results,15,16,19–21,28–33 othershave found no effect.34–39 This may be due to several aspectsof experimental design. It is important to consider also thesystemic effect of the LLLT,40–43 which was not considered insome previous reports that did not find effects of the LLLT.44,45,48

Other reports used very low doses.35,36 The use of inappropri-

ate wavelengths may also result in negative reports.36,46 Higherwavelengths are more resistant to dispersion than lower onesand penetrate deeply into the skin.47 It was mentioned that632.8-nm laser light penetrates 0.5–1 mm before losing 37% ofits intensity.48 On the other hand, infrared (IR) wavelengthspenetrate 2 mm before losing the some percentile of energy.This is a clear indication of the use of IR laser light on bone tis-sue. However, systemic effect may not be disregarded whenvisible laser light is used.

The presence of large amounts of collagen fibers on irradi-ated subjects was the major finding in our study, and thismay represent an early effect of LLLT on repair. Previousstudies demonstrated increased collagen production follow-ing LLLT.3,40,41,49,50 As collagen is an important componentof the extracellular matrix of the bone, and increased amounts,as seen in our study, may indicate a positive effect of LLLTon the healing of bone, despite the fact that in some speci-mens there was no difference in the amount of newly formedbone between irradiated animals and controls. It is necessaryto consider that the large amount of collagen fibers repre-sents increased bone neoformation after the mineralizationof the matrix. This aspect was clearly observed when the graftwas associated with membrane and LLLT at day 30, when,besides the large amount of collagen fibers, there was thepresence of dense bone trabeculae. The mechanism by whichLLLT interferes in collagen synthesis is not fully under-stood; however, it may be due to alterations on genetic regu-lation or modulation of enzymatic activity involved in themetabolism of collagen.50

304 Pinheiro et al.

FIG. 4. Photomicrography of group IV. At day 15 (A) and 21 (B), newly formed bone could be observed within the cavity. Atday 30 (C), there was integration of the graft with the newly formed bone. H&E, 340 (A, B) and 3200 (C).

FIG. 5. Photomicrography of group V. At days 15 (A) and 21 (B), the graft was encircled by fibrous tissue and bone. At day 30(C), an increased amount of newly formed bone was noticed within the cavity. Picrosirius, 340 (A, B); H&E, 3100 (C).

It is uncertain whether the biomodulation of bone forma-tion is an overall effect on mesenchymal cells or a directstimulation of osteoblasts.22 It is possible that the observedresults on irradiated subjects are due to increased release ofgrowth factors, mainly FGF, which is found in bone tissueand acts on differentiated cells, increasing both cell prolifera-tion and secretion of components of the matrix.51

The doses used in this study are aligned with several previousreports, which suggested that 1–5 J/cm2 is effective in inducingpositive effects in both bone and soft tissues.19,21,23,28,41,43,50,52 It isimportant to note that four points of irradiation were used tofractionate the total dose per session. The points of irradiationaround the defect were chosen because the results of irradiationof the graft and or the presence of the membrane grafted areacould be uncertain. The presence of the particles and or themembrane would difficult the diffusion of light into the tissues.

A total dose per session of 16 J/cm2 is in accordance with theclinical parameters recommended by Pinheiro et al.23 How-ever, some reports have suggested higher doses.16,18,33,41 Theliterature shows that biomodulatory effects are dose depen-dent.33,41,49,53–57. It is also recognized that other factors such asthe phase of cell growth,58,59 and the frequency and number ofsessions21,33 also influence the final results of LLLT.

When groups II and III are compared, it is evident that neo-formation of the bone was similar at day 15. However, at days21 and 30, bone formation was more evident in both the core ofthe cavity and in the cortical region. Although irradiated sub-jects showed a dense deposition of collagen fibers around theparticles on the core of the defect and at the cortical region,the same was not detected on control specimens, especially onthe core of the defect. In groups IIIA and IIIB, osteoconductiv-ity was discretely observed at 15 days after surgery. However,on non-irradiated animals, cortical repair was a constant fea-ture. Although on subgroup BML15, deposition of collagenfibers on the core of the defect was quite different from that ob-served on non-irradiated subjects, no cortical repair was de-tectable. On the other hand, from day 21, bone formation wasequivalent in all groups, but the presence of collagen fiberswas more evident on irradiated ones. A strong effect of LLLTwas noticed at day 30, where very high amounts of collagenfibers were present, and evident signs of the presence of a moremature bone was also observed when compared to non-irradi-ated subjects.

CONCLUSION

It is concluded that LLLT had a positive biomodulatory ef-fect on the repair of bone defects via the implantation of in-organic bovine bone associated (or not) with bovine bonemembrane.

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Address reprint requests to:Antonio Pinheiro, Ph.D.

School of DentistryFederal University of Bahia

Av Araújo Pinho, 62Canela, Salvador, BA, Brazil, 40110–150

E-mail: albp@ufba.br

306 Pinheiro et al.