Implantate

Photoengineering of Bone Repair Processes

Apr 2006, Vol. 24, No. 2: 169-178, Photomedicine and Laser Surgery

Dr. Antonio Luiz B. Pinheiro, D.D.S., Ph.D.

Laser Center, School of Dentistry, Department of Propedêutica and Clínica Integrada, Universidade Federal da Bahia, Canela Salvador, BA, Salvador, Brazil.

Institute for Research and Development, Universidade do Vale do Paraíba, S˜o José dos Campos, SP, Brazil.

Marleny Elizabeth M.M. Gerbi, Ph.D.

Departamento de Prótese e Cirurgia Buco Facial, Centro de Ciências da Saúde, Universidade Federal de Pernambuco, Recife, PE, Brazil.

Objective: This paper aims to report the state of the art with respect to photo engineering of bone repair using laser therapy. Background Data: Laser therapy has been reported as an important tool to positively stimulate bone both in vivo and in vitro. These results indicate that photophysical and photochemical properties of some wavelengths are primarily responsible for the tissue responses. The use of correct and appropriate parameters has been shown to be effective in the promotion of a positive biomodulative effect in healing bone. Methods: A series of papers reporting the effects of laser therapy on bone cells and tissue are presented, and new and promising protocols developed by our group are presented.

Results: The results of our studies and others indicate that bone irradiated mostly with infrared (IR) wavelengths shows increased osteoblastic proliferation, collagen deposition, and bone neo formation when compared to non irradiated bone. Further, the effect of laser therapy is more effective if the treatment is carried out at early stages when high cellular proliferation occurs. Vascular responses to laser therapy were also suggested as one of the possible mechanisms responsible for the positive clinical results observed following laser therapy. It still remains uncertain if bone stimulation by laser light is a general effect or if the isolate stimulation of osteoblasts is possible.

Auf Wunsch senden wir Ihnen die komplett Studie

Khadra M, Kassem N, Haanaes H R, Ellingsen J E, Lyngstadaas S P.

 Enhancement of bone formation in rat calvarial bone defects using low-level laser therapy.

Oral Surg Oral Med Oral Pathol Oral Endod. 2004; 97: 693-700.

The aim of the study by Khadra was to investigate the effect of laser therapy with GaAlAs on titanium implant healing and attachment in bone. This study was performed as an animal trial of 8 weeks duration with a blinded, placebo-controlled design. Two coin-shaped titanium implants with a diameter of 6.25 mm and a height of 1.95 mm were implanted into cortical bone in each proximal tibia of twelve New Zealand rabbits (n=48). The animals were randomly divided into irradiated and control groups. The laser was used immediately after surgery and carried out daily for 10 consecutive days. The animals were killed after 8 weeks of healing. The mechanical strength of the attachment between the bone and 44 titanium implants was evaluated using a tensile pullout test. Histomorphometrical analysis of the four implants left in place from four rabbits was then performed. Energy-dispersive X-ray microanalysis was applied for analyses of calcium and phosphorus on the implant test surface after the tensile test. The mean tensile forces, measured in Newton, of the irradiated implants and controls were 14.35 (SD±4.98) and 10.27 (SD±4.38), respectively, suggesting a gain in functional attachment at 8 weeks following laser.

The histomorphometrical evaluation suggested that the irradiated group had more bone-to-implant contact than the controls. The weight percentages of calcium and phosphorus were significantly higher in the irradiated group when compared to the controls, suggesting that bone maturation processed faster in irradiated

Effect Of Low-Power GaAlAs Laser (660 Nm) On Bone Structure And Cell Activity: An
Experimental Animal Study
Nicola RA, Jorgetti V, Rigau J, Pacheco MT, dos Reis LM, Zangaro RA. Vale of Paraiba
University, Sao Jose dos Campos, SP, Brazil. renatanicolau@hotmail.com, Lasers Med Sci. 2003;18(2):89-94.
Low-level laser therapy (LLLT) is increasingly being used in the regeneration of soft tissue. In the
regeneration of hard tissue, it has already been shown that the biomodulation effect of lasers
repairs bones more quickly. We studied the activity in bone cells after LLLT close to the site of the bone injury. The femurs of 48 rats were perforated (24 in the irradiated group and 24 in the
control group) and the irradiated group was treated with a GaAlAs laser of 660 nm, 10 J/cm2 of
radiant exposure on the 2nd, 4th, 6th and 8th days after surgery (DAS). We carried out
histomorphometry analysis of the bone. We found that activity was higher in the irradiated group
than in the control group: (a) bone volume at 5 DAS (p=0.035); (b) osteoblast surface at 15 DAS
(p=0.0002); (c) mineral apposition rate at 15 and 25 DAS (p=0.0008 and 0.006); (d) osteoclast
surface at 5 DAS and 25 DAS (p=0.049 and p=0.0028); and (e) eroded surface (p=0.0032).

We concluded that LLLT increases the activity in bone cells (resorption and formation) around the site of the repair without changing the bone structure.

Osseointegration Of Endosseous Ceramic Implants After Postoperative Low-Power Laser Stimulation: An In Vivo Comparative Study
Guzzardella GA, Torricelli P, Nicoli-Aldini N, Giardino R.
Department of Experimental Surgery/Codivilla-Putti Research Institute, Rizzoli Orthopaedic Institute, Bologna, Italy. gaetanoantonio.guzzardella@ior.it
Clin Oral Implants Res. 2003 Apr;14(2):226-32.
Stimulation with low-power laser (LPL) can enhance bone repair as reported in experimental
studies on bone defects and fracture healing. Little data exist concerning the use of LPL
postoperative stimulation to improve osseointegration of endosseous implants in orthopaedic and
dental surgery. An in vivo model was used for the present study to evaluate whether Ga-Al-As
(780 nm) LPL stimulation can improve biomaterial osseointegration. After drilling holes, cylindrical implants of hydroxyapatite (HA) were placed into both distal femurs of 12 rabbits. From postoperative day 1 and for 5 consecutive days, the left femurs of all rabbits were submitted to LPL treatment (LPL group) with the following parameters: 300 J/cm2, 1 W, 300 Hz, pulsating emission, 10 min. The right femurs were sham-treated (control group). Three and 6 weeks after implantation, histomorphometric and microhardness measurements were taken. A higher affinity index was observed at the HA-bone interface in the LPL group at 3 (P<0.0005) and 6 weeks (P<0.001); a significant difference in bone microhardness was seen in the LPL group vs. the control group (P<0.01). These results suggest that LPL postoperative treatment enhances the bone-implant interface.

Clin Oral Implants Res. 2004; 15 (3): 325-332.
Khadra M, Ronold H J, Lyngstadaas S P, Ellingsen J E, Haanaes H R.

The histomorphometrical evaluation suggested that the irradiated group had more bone-to-implant contact than the controls. The weight percentages of calcium and phosphorus were significantly higher in the irradiated group when compared to the controls (P=0.037) and (P=0.034), respectively, suggesting that bone maturation processed faster in irradiated bone. These findings suggest that LLLT might have a favourable effect on healing and attachment of titanium implants.

Laser Therapy Plays A Role In Bone Healing
Lasers Surg Med. 1998; 22: 97-102.
Luger et al. studied the effect of HeNe laser on the healing of tibial bone fractures in rats.

63 J (35mW) was given transcutaneously daily over the fracture area. After 4 weeks the tibia was removed and tested at tension up to failure. The maximal load at failure and the structural
stiffness of the tibia were found to be elevated significantly in the irradiated group, whereas the
extension maximal load was reduced. In addition, gross non-union was found in four fractures in
the control group, compared to none in the irradiated group.

Computerized Morphometric Assessment Of The Effect Of Low-Level Laser Therapy On
Bone Repair: An Experimental Animal Study
Silva Júnior AN, Pinheiro AL, Oliveira MG, Weismann R, Ramalho LM, Nicolau RA. J Clin Laser Med Surg. 2002; 20: 83-87
The aim of this study was to evaluate morphometrically the amount of newly formed bone after
GaAlAs laser irradiation of surgical wounds created in the femur of rats. Low-level laser therapy
(LLLT) has been used in several medical specialties because of its biomodulatory effects on
different biological tissues. However, LLLT is still controversial because of contradictory reports.
This is a direct result of the different methodologies used in these works. In this study, 40 Wistar rats were divided into four groups of 10 animals each: group A (12 sessions, 4.8 J/cm2 per session, observation time of 28 days); group C (three sessions, 4.8 J/cm2 per session,
observation time of 7 days). Groups B and D acted as nonirradiated controls. The specimens
were routinely processed to wax and cut at 6-microm thickness and stained with H&E. For
computerized morphometry, Imagelab software was used. RESULTS: Computerized
morphometry showed a significant difference between the areas of mineralized bone in groups C
and D (p = 0.017). There was no difference between groups A and B (28 days; p = 0.383).

Effects Of Visible NIR Low Intensity Laser On Implant Osseointegration In Vivo
Laser Med Surg Abstract issue, 2002: 11.
Blay A, Blay C C, Groth E B et al.
The effects of 680 and 830 nm lasers on osseointegration was studied by Blay. 30 adult rats were divided into three groups; two laser groups and one control. The rats in the two laser groups had pure titanium Frialit-2 implants implanted into each proximal metaphysis of their respective tibias, inserted with a 40 Ncm torque. The initial stability was monitored by means of a resonance frequency analyser. Ten irradiations were performed, 48 hours apart, 4 J/cm2 on two points, starting immediately after surgery. Resonance frequency analysis indicated a significant difference between frequency values at 3 and 6 weeks, as compared to control. At 6 weeks the removal torque in the laser groups was much higher than in the control group.

Bone Repair Of The Periapical Lesions Treated Or Not With Low Intensity Laser
(Wavelenght=904 nm)
Laser Surg Med. Abstract Issue 2002. abstract 303.
Sousa G R, Ribeiro M S, Groth E B.
The effect of bone repair in periapical lesions has been studied by Sousa []. 15 patients with a
total of 18 periapical lesions were divided into two groups. One group received endodontic
treatment and/or periapical surgery. The patients in the other group were submitted to the same
procedure and in addition the lesions were irradiated by GaAs laser, 11 mW, 9 J/cm2. This
therapy was performed during 10 sessions with an interval of 72 hours. Bone regeneration was
evaluated through X-ray examination. The results showed a significant difference between the
laser and the control group in favor of the laser group.

Low-Power Laser Irradiation Improves Histomorphometrical Parameters and Bone Matrix Organization During Tibia Wound Healing In Rats
Garavello-Freitas I, Baranauskas V, Joazeiro PP, Padovani CR, Dal Pai-Silva M,
da Cruz-Hofling MA.
Faculdade de Engenharia Eletrica e Computacao, Departamento de Semicondutores
Instrumentos e Fotonica, Universidade Estadual de Campinas, Av. Albert Einstein N.400, 13 083- 970 Campinas, SP, Brazil.
J Photochem Photobiol B. 2003 May-Jun;70(2):81-9.
The influence of daily energy doses of 0.03, 0.3 and 0.9 J of He-Ne laser irradiation on the repair
of surgically produced tibia damage was investigated in Wistar rats. Laser treatment was initiated 24 h after the trauma and continued daily for 7 or 14 days in two groups of nine rats (n=3 per laser dose and period). Two control groups (n=9 each) with injured tibiae were used. The course of healing was monitored using morphometrical analysis of the trabecular area. The organization of collagen fibers in the bone matrix and the histology of the tissue were evaluated using Picrosirius-polarization method and Masson's trichrome. After 7 days, there was a significant increase in the area of neoformed trabeculae in tibiae irradiated with 0.3 and 0.9 J compared to the controls. At a daily dose of 0.9 J (15 min of irradiation per day) the 7-day group showed a significant increase in trabecular bone growth compared to the 14-day group. However, the laser irradiation at the daily dose of 0.3 J produced no significant decrease in the trabecular area of the 14- day group compared to the 7-day group, but there was significant increase in the trabecular area of the 15- day controls compared to the 8-day controls. Irradiation increased the number of hypertrophic osteoclasts compared to non-irradiated injured tibiae (controls) on days 8 and 15.
The Picrosirius-polarization method revealed bands of parallel collagen fibers (parallel-fibered
bone) at the repair site of 14-day-irradiated tibiae, regardless of the dose. This organization
improved when compared to 7-day-irradiated tibiae and control tibiae. These results show that
low-level laser therapy stimulated the growth of the trabecular area and the concomitant invasion
of osteoclasts during the first week, and hastened the organization of matrix collagen (parallel
alignment of the fibers) in a second phase not seen in control, non-irradiated tibiae at the same
period. The active osteoclasts that invaded the regenerating site were probably responsible for
the decrease in trabecular area by the fourteenth day of irradiation.

Effect Of 830-Nm Laser Light On The Repair Of Bone Defects Grafted With Inorganic
Bovine Bone And Decalcified Cortical Osseous Membrane
Barbos Pinheiro AL, Limeira Junior Fde A, Marquez Gerbi ME, Pedreira Ramalho LM, Marzola C, Carneiro Ponzi EA, Oliveira Soares A, Bandeira De Carvalho LC, Vieira Lima HC, Oliveira Goncalves T. Laser Center, School of Dentistry, Federal University of Bahia, Salvador, Brazil.
albp@ufba.br
J Clin Laser Med Surg. 2003 Dec;21(6):383-8.
OBJECTIVE: The aim of this study was to assess histologically the effect of LLLT (lambda830
nm) on the repair of standardized bone defects on the femur of Wistar albinus rats grafted with
inorganic bovine bone and associated or not to decalcified bovine cortical bone membrane.

BACKGROUND DATA: Bone loss may be a result of several pathologies, trauma or a
consequence of surgical procedures. This led to extensive studies on the process of bone repair
and development of techniques for the correction of bone defects, including the use of several
types of grafts, membranes and the association of both techniques. There is evidence in the
literature of the positive effect of LLLT on the healing of soft tissue wounds. However, its effect on
bone is not completely understood.
MATERIALS AND METHODS: 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 (Genox
+ Gen-derm + LLLT). Bone defects were created at the femur of the animals and were treated
according to the group. The animals of the irradiated groups were irradiated every 48 h during 15
days; the first irradiation was performed immediately after the surgical procedure. The animals
were irradiated transcutaneously in four points around the defect. At each point a dose of 4 J/cm2 was given (phi approximately 0.6 mm, 40 mW) and the total dose per session was 16 J/cm2. The animals were humanely killed 15, 21, and 30 days after surgery. The specimens were routinely
processed to wax, serially cut, and stained with H&E and Picrosirius stains and analyzed under
light microscopy.
RESULTS: The results showed evidence of a more advanced repair on the irradiated groups
when compared to non-irradiated ones. The repair of irradiated groups was characterized by both
increased bone formation and amount of collagen fibers around the graft within the cavity since
the 15th day after surgery, through analysis of the osteoconductive capacity 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
submitted the implantation of graft.

Effects Of Pulse Frequency Of Low-Level Laser Therapy (LLLT) On Bone Nodule
Formation In Rat Calvarial Cells
Ueda Y, Shimizu N.
Department of Orthodontics, Nihon University School of Dentistry at Matsudo Chiba, Japan.
J Clin Laser Med Surg. 2003 Oct;21(5):271-7.
OBJECTIVE: The purpose of this study was to determine the effect of pulse frequencies of lowlevel laser therapy (LLLT) on bone nodule formation in rat calvarial cells in vitro.
BACKGROUND DATA: Various photo-biostimulatory effects of LLLT, including bone formation,
were affected by some irradiation factors such as total energy dose, irradiation phase, laser
spectrum, and power density. However, the effects of pulse frequencies used during laser
irradiation on bone formation have not been elucidated.
MATERIALS AND METHODS: Osteoblast-like cells isolated from fetal rat calvariae were
irradiated once with a low-energy Ga-Al-As laser (830 nm, 500 mW, 0.48-3.84 J/cm2) in four
different irradiation modes: continuous irradiation (CI), and 1-, 2-, and 8-Hz pulsed irradiation (PI-
1, PI-2, PI-8). We then investigated the effects on cellular proliferation, bone nodule formation,
alkaline phosphatase (ALP) activity, and ALP gene expression.
RESULTS: Laser irradiation in all four groups significantly stimulated cellular proliferation,
bone nodule formation, ALP activity, and ALP gene expression, as compared with the nonirradiation group. Notably, PI-1 and -2 irradiation markedly stimulated these factors, when
compared with the CI and PI-8 groups, and PI-2 irradiation was the best approach for bone
nodule formation in the present experimental conditions.

CONCLUSION: Since low-frequency pulsed laser irradiation significantly stimulates bone
formation in vitro, it is most likely that the pulse frequency of LLLT an important factor affecting
biological responses in bone formation.

Effect Of Low-Level Laser Irradiation On Osteoglycin Gene Expression In Osteoblasts
Hamajima S, Hiratsuka K, Kiyama-Kishikawa M, Tagawa T, Kawahara M, Ohta M, Sasahara H,
Abiko Y.
Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Lasers Med Sci. 2003;18(2):78-82.
Many studies have attempted to elucidate the mechanism of the biostimulatory effects of lowlevel laser irradiation (LLLI), but the molecular basis of these effects remains obscure. We
investigated the stimulatory effect of LLLI on bone formation during the early proliferation stage of
cultured osteoblastic cells. A mouse calvaria-derived osteoblastic cell line, MC3T3-E1, was
utilised to perform a cDNA microarray hybridisation to identify genes that induced expression by
LLLI at the early stage. Among those genes that showed at least a twofold increased expression, the osteoglycin/mimecan gene was upregulated 2.3-fold at 2 h after LLLI. Osteoglycin is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix which was previously called the osteoinductive factor. SLRP are abundantly contained in the bone matrix, cartilage cells and connective tissues, and are thought to regulate cell proliferation, differentiation and adhesion in close association with collagen and many other growth factors. We investigated the time-related expression of this gene by LLLI using a reverse transcription polymerase chain reaction (RTPCR)method, and more precisely with a real-time PCR method, and found increases of 1.5-2- fold at 2-4 h after LLLI compared with the non-irradiated controls. These results suggest that the increased expression of the osteoglycin gene by LLLI in the early proliferation stage of cultured osteoblastic cells may play an important role in the stimulation of bone formation in concert with
matrix proteins and growth factors.

Effect Of Low-Power Laser Irradiation On Bony Implant Sites
Dortbudak O, Haas R, Mailath-Pokorny G.
Department of Oral Surgery, Dental School, University of Vienna, Austria.
orhun.doerbudak@univie.ac.at
Clin Oral Implants Res. 2002 Jun;13(3):288-92.
This study was designed to examine the effects of low-energy laser irradiation on osteocytes and bone resorption at bony implant sites. Five male baboons with a mean age of 6.5 years were used in the study. Four holes for accommodating implants were drilled in each iliac crest. Sites on the left side were irradiated with a 100 mW low-energy laser (690 nm) for 1 min (6 Joule) immediately after drilling and insertion of four sandblasted and etched (Frialit-2 Synchro)
implants. Five days later, the bone was removed en bloc and was evaluated
histomorphometrically. The mean osteocyte count per unit area was 109.8 cells in the irradiated
group vs. 94.8 cells in the control group. As intra-individual cell counts varied substantially,
osteocyte viability was used for evaluation. In the irradiated group, viable osteocytes were found
in 41.7% of the lacuna vs. 34.4% in the non-irradiated group. This difference was statistically
significant at P <0.027. The total resorption area, eroded surface, was found to be 24.9% in the
control group vs. 24.6% in the irradiated group. This difference was not statistically significant.
This study showed that osteocyte viability was significantly higher in the samples that were
subjected to laser irradiation immediately after implant site drilling and implant insertion, in
comparison to control sites. This may have positive effects on the integration of implants. The
bone resorption rate, in contrast, was not affected by laser irradiation.

Laser technology in orthopedics: preliminary study on low power laser therapy to improve
the bone-biomaterial interface
Guzzardella GA, Torricelli P, Nicoli Aldini N, Giardino R.
Experimental Surgery Department, Research Institute Codivilla Putti, Bologna, Italy.
gaetanoantonio.guzzardella@ior.it
Int J Artif Organs. 2001 Dec;24(12):898-902.
Low Power Laser (LPL) seems to enhance the healing of bone defects and fractures. The effect
of LPL in other orthopedic areas such as osteointegration of implanted prosthetic bone devices is still unclear. In the present study, 12 rabbits were used to evaluate whether Ga-Al-As (780 nm)
LPL stimulation has positive effects on osteointegration. Hydroxyapatite (HA) cylindrical nails
were drilled into both distal femurs of rabbits. From postoperative day 1 and for 5 consecutive
days, the left femura of all rabbits were given LPL treatment (Laser Group-LG) with the following
parameters: 300 Joule/cm2, 1 Watt, 300 Hertz, pulsating emission, 10 minutes. The right femurawere sham-treated (Control Group-CG). At 4 and 8 weeks after implantation, histologic and histomorphometric investigations evaluated bone-biomaterial-contact. Histomorphometry showed a higher degree of osteointegration at the HA-bone interface in the LG Group at 4 (p < 0.0005) and 8 weeks (p < 0.001). These preliminary positive results seem to support the hypothesis that LPL treatment can be considered a good tool to enhance the bone-implant interface in orthopedic surgery.

Clin Oral Implants Res. 2004 Jun;15(3):325-32.
Low-Level Laser Therapy Stimulates Bone-Implant Interaction: An Experimental Study In Rabbits
Khadra M, Ronold HJ, Lyngstadaas SP, Ellingsen JE, Haanaes HR.
Department of Oral Surgery and Oral Medicine, Faculty of Dentistry, University of Oslo, Oslo, Norway. maawan@odont.uio.no
The aim of the present study was to investigate the effect of low-level laser therapy (LLLT) with a
gallium-aluminium-arsenide (GaAlAs) diode laser device on titanium implant healing and
attachment in bone. This study was performed as an animal trial of 8 weeks duration with a
blinded, placebo-controlled design. Two coin-shaped titanium implants with a diameter of 6.25
mm and a height of 1.95 mm were implanted into cortical bone in each proximal tibia of twelve
New Zealand white female rabbits (n=48). The animals were randomly divided into irradiated and
control groups. The LLLT was used immediately after surgery and carried out daily for 10
consecutive days. The animals were killed after 8 weeks of healing. The mechanical strength of
the attachment between the bone and 44 titanium implants was evaluated using a tensile pullout
test. Histomorphometrical analysis of the four implants left in place from four rabbits was then
performed. Energy-dispersive X-ray microanalysis was applied for analyses of calcium and
phosphorus on the implant test surface after the tensile test. The mean tensile forces, measured
in Newton, of the irradiated implants and controls were 14.35 (SD+/-4.98) and 10.27 (SD+/-4.38),
respectively, suggesting a gain in functional attachment at 8 weeks following LLLT (P=0.013).
The histomorphometrical evaluation suggested that the irradiated group had more bone-toimplant
contact than the controls. The weight percentages of calcium and phosphorus were significantly higher in the irradiated group when compared to the controls (P=0.037) and (P=0.034), respectively, suggesting that bone maturation processed faster in irradiated bone.
These findings suggest that LLLT might have a favourable effect on healing and attachment of
titanium implants.