LIGHT ACCELERATED IMPLANTOLOGY CLINICAL AND SCIENTIFIC DOSSIER

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LIGHT ACCELERATED IMPLANTOLOGY CLINICAL AND SCIENTIFIC DOSSIER June 2013 Biolux Research Ltd. 825 Powell Street, Suite 230, Vancouver BC, Canada V6A 1H7 Toll Free: 1.888.669.0674 Tel: 1.604.669.0674 Fax: 1.604.608.5558 www.bioluxresearch.com

INDEX 1. IMPLANTOLOGY. PUBLISHED RESEARCH SUMMARIES 1.1 Bouquot J, Brawn P. Combined New Technologies to Improve Dental Implant Success - - Quantitative Ultrasound Evaluation of NIR-LED Photobiomodulation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2008; 106(3):e6 1.2 Bouquot J, Brawn P. Combined New Technologies to Improve Dental Implant Success - - Quantitative Ultrasound Evaluation of NIR-LED Photobiomodulation. Abstracts of the 2008 Annual Meeting of the American Academy of Oral Medicine (2008) 1.3 Brawn P, Kwong-Hing A, Boeriu S and Clokie CM. Accelerated Implant Stability After LED Photobiomodulation. J Dent Res 87(Spec Iss B):2021, 2008 1.4 Brawn P, Kwong-Hing A. A histologic comparison of light emitting diode phototherapytreated hydroxyapatite-grafted extraction sockets. Implant Dent 2007; 16(2):204-11 1.5 Ghuloom M. The Effect of light emitting diode (LED) on the healing of endosseous intraoral implants. MS thesis. University of Toronto, Toronto, 2012. Print. 1.6 Khadra M, Kasem N, and Brawn P. Phototherapy promotes attachment and subsequent proliferation of human osteoblast-like cells. J Dent Res 87(Spec Iss B):3308, 2008 1.7 Kwong Hing A, Brawn P. Accelerated implant stability in indirect sinus lifts with bone grafts using LED phototherapy. FDI World Dental Congress. Shenzhen, China. 2006. 2. SUPPORTING LITERATURE Page 2

1. IMPLANTOLOGY Combined New Technologies to Improve Dental Implant Success -- Quantitative Ultrasound Evaluation of NIR-LED Photobiomodulation. Bouquot J, Brawn P. Abstracts of the 2008 Annual Meeting of the American Academy of Oral Medicine (2008) Background: Dental implants must be placed in healthy bone for successful osteointegration and stability. Low bone density (LBD) and ischemically damaged, desiccated bone both have a poor ability to remodel and are, therefore, contraindications for implants. Readily available diagnostic imaging devices, includ- ing dental radiographs, lack the ability to adequately identify such bone. However, the new technology of through-transmis- sion or quantitative ultrasound (QUS) is specifically cleared by the FDA to safely identify LBD and dehydrated bone and has a very low ( 3%) false positive rate. Near-infrared light emitting diode (NIR-LED) therapy or photobiomodulation has been shown in cultured cells and animal models to stimulate bone healing and production. The present investigation uses QUS to determine the efficacy of in-vivo NIR-LED phototherapy to increase bone density and/or hydration of abnormal alveolar bone. Methods: 68 patients received LED therapy (OsseoPulse, version 1.0, Biolux Research Ltd., Vancouver, Canada; 15 min- utes daily for 3 months) to 294 QUS positive edentulous alveolar sites of LBD/desiccation. Before and after QUS scans were graded blindly by two independent observers (5-point scale: 0 normal bone, 4 most severe), after calibration, and compared using matched pair analysis. Results: After NID-LED photomodulation the average grade improved from 2.43 to 1.33 (44.3% improvement), with 42% of sites returning to completely normal bone and 18.4% returning to grade 1. The mean difference (improvement of bone quality) of 1.11 was very statistically significant (matched pair analysis: Std error 0.06914; t-ratio 15.9896; DF 293; prob less than 0.0001; 95% confidence interval 0.558-1.242). Conclusion: NID-LED therapy seems to hold good potential for improving alveolar bone prior to implant placement, but long-term improvement must be evaluated, as must actual im- plant stability. Page 3

Combined New Technologies to Improve Dental Implant Success - Quantitative Ultrasound Evaluation of NIR-LED Photobiomodulation Bouquot J, Brawn P, Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2008; 106(3):e6 Background: Dental implants must be placed in healthy bone for successful osteointegration and stability. Low bone density (LBD) and ischemically damaged, desiccated bone both have a poor ability to remodel and are, therefore, contraindications for implants. Near-infrared light emitting diode (NIR-LED) therapy or photobiomodulation has been shown in cultured cells and animal models to stimulate bone healing and production. Study: 68 patients received 12 weeks of OsseoPulse therapy to 294 sites of ischemic/low bone density. Bone density/hydration improved by 44%, and 42% of sites returned to completely normal bone. Conclusion: OsseoPulse therapy seems to hold good potential for improving alveolar bone prior to implant placement. Accelerated Implant Stability After LED Photobiomodulation Brawn P, Kwong-Hing A, Boeriu S and Clokie CM, J Dent Res 87(Spec Iss B):2021, 2008 Study: This article examines the effect of OsseoPulse treatment on implant stability, as assessed by resonance frequency analysis. 35 patients had 63 dental implants placed. 23 patients were treated with the OsseoPulse for 21 days. All implants were tested for primary stability with an Osstell Mentor TM at the time of implant placement, 14, 30, 60 and 90 days. Conclusion: Patients treated with the OsseoPulse demonstrated significantly (p<0.05) improved dental implant stability at day 14, day 30 and day 60. There was a 58% reduction in time required to achieve sufficient stability to load implants compared to controls. These results may suggest that OsseoPulse treatments may allow for more rapid integration of dental implants and earlier loading. Page 4

A histologic comparison of light emitting diode phototherapy-treated hydroxyapatitegrafted extraction sockets Brawn P, Kwong-Hing A, Implant Dent 2007; 16(2):204-11 Case Study: After bilateral extraction of periodontally involved lower molars an investigational OsseoPulse TM was used daily for 21 days on the treated side after grafting both sockets with Hydroxyapatite (HA) Osteograf LD300. Bone regeneration of the OsseoPulse TM treated and nontreated socket graft was compared. Histologic evaluations showed enhanced bone formation and faster particle resorption associated with the OsseoPulse TM treated socket graft compared with the untreated socket. Conclusion: In this bilateral case study the accelerated bone healing in the OsseoPulse TM treated HA socket graft may provide faster implant placement compared to untreated treated socket grafts. Page 5

The Effect of light emitting diode (LED) on the healing of endosseous intraoral implants Ghuloom M. MS thesis. University of Toronto, Toronto, 2012. Print. Objective: The objectives of this study are to assess the role of photobiomodulation on the early bone tissue healing period applied to the practice of endosseous dental implants using a randomized clinical trial (RCT), and to assess the role of photobiomodulation on peri-implant alveolar crestal bone in the early healing period of bone tissue. Study: After obtaining ethics approval from the University of Toronto, a total of 72 patients requiring 76 dental implants (35 female and 37 male; mean age 63.5; range 35-100 years) were recruited and consented for inclusion into this trial. Patients received dental implants by a single operator. Implants were randomly assigned to one of two groups, control (n=47), and treatment group (n=29). All patients in the study received dental implant(s.) The treatment group had to apply light emitting diode (LED) delivered by OsseoPulse device to the surgical site, while the control group did not. Patients in the treatment group were educated on how to use the OsseoPulse device and were instructed to apply it to the surgical site preoperatively for 20 minutes, and for additional 10 daily sessions of 20 minutes each postoperatively starting the day of surgery. Implant stability was assessed using the Osstell device which utilizes resonance frequency analysis (RFA) technology and expresses data as Implant Stability Quotient (ISQ) values. ISQ of the implants was measured at time of surgery immediately following implant placement. It was also reassessed on a frequent basis in weeks 1, 2, 4, and 8 post operatively. The radiographs were taken immediately after insertion and at 8 weeks follow-up appointment. Radiographs were analyzed using SigmaScan Pro and the data were analyzed statistically using SPSS and Stata. Conclusion: Data suggest that photobiomodulation by means of LED will allow for a continuous increase in ISQ value and may reduce crestal bone resorption in the early phases of bone healing around dental implants. As expected, there was a continuous decrease in ISQ value in the control group over the first 4 weeks following implant placement. This was followed by an increase at 8 weeks postoperatively. On the other hand, this ISQ decrease was not seen in the treatment group; instead, there was a continuous increase in ISQ values over the follow-up period. A significant change in ISQ values (p < 0.001) was seen between the groups at every follow-up visit in comparison to initial visit. Regression analysis study showed that the primary effect of LED on ISQ values was seen over the first 2 weeks (p < 0.05). The mean crestal bone loss among the control was significantly greater than the treatment group at 8 weeks (p = 0.05). Page 6

Phototherapy promotes attachment and subsequent proliferation of human osteoblastlike cells Khadra M, Kasem N, and Brawn P, J Dent Res 87(Spec Iss B):3308, 2008 Study: The aim of the study was to investigate the effect of OsseoPulse treatment on attachment and proliferation of human osteoblast-like cells cultured on titanium implant material. Cells derived from human mandibular bone were exposed to OsseoPulse and then seeded onto titanium discs. Non-exposed cultures served as controls. Cell proliferation was determined after staining by counting cells under a light microscope. Furthermore, MTT analyses were also performed to determine cellular attachment and proliferation. Conclusion: In this cellular model the attachment and proliferation of human osteoblast-like cells cultured on titanium implant material was significantly enhanced (p <0.05) after OsseoPulse TM treatment compared to controls. Page 7

Accelerated implant stability in indirect sinus lifts with bone grafts using LED phototherapy. Kwong Hing A, Brawn P. FDI World Dental Congress. Shenzhen, China. 2006. Introduction: The stimulating effects of laser phototherapy on bone regeneration and implant osseointegration has been shown in a number of in vitro and animal studies. The effect of light emitting diode (LED) phototherapy on implant stability placed via an indirect sinus lift with simultaneous bone graft as assessed by resonance frequency analysis (RFA) has not been investigated. Materials/Methods: In this case study, the LED based system used was the investigational Biolux extra-oral phototherapy device (Biolux Research Ltd, Vancouver Canada, www.bioluxresearch.com). The LED array operates in the visible red spectrum at a continuous wavelength of 618nm. The device has an integrated alignment system that allows the dentist to position and fix the array on the cheek, directly over the surgical site. The computer controlled power supply is programmed to deliver the prescribed phototherapy regimen every time it is used. Because of the need to treat the site on a daily basis, the device has been designed for home treatments by the patient after instructions in its use. The Biolux device was used daily for 21 days post implant placement unilaterally in conjunction with 2 bilateral implants, indirect sinus lifts with bone grafts utilixing BioOss particularate. The posterior implants were tested for primary stability with an Ostell RFA device at implant placement and then weekly for 4 weeks and then monthly for 2 months prior to the initiation of prosthetic procedure. Results: The LED phototherapy treated implant showed increased RFA readings as indicated with an initial implant stability quotient (ISQ) of 39, at one week 65 at 2 weeks 68 and then plateaued out at 70 for 3 and 4 weeks and at 2 and 3 months. The untreated (control) side demonstrated an ISQ at placement of 41 and then 42 at one week, 45 at two weeks, 52 at 3 weeks, 58 at 4 weeks and then 64 at 2 and 3 months. The implants were restored conventionally with screw retained bridges. Conclusions: The accelerated implant stability in the phototherapy treated implant placed with simultaneous indirect sinus lift and bone graft as measured with an Osstell Mentor device as ISQ, may provide faster implant stability compared to non LED phototherapy treated implants. This may allow shorter treatment times before prostherics can be initiated. Page 8

2. SUPPORTING LITERATURE Orthodontics in the 21st century: a view from across the pond. Kau CH. J Orthod. 2012 Jun;39(2):75-6 Biotechnology in Orthodontics. Kau CH. Dentistry 2012, 2:5 Low-Level Laser Therapy for Implants Without Initial Stability Campanha BP, Gallina C, Geremia T, Drumond Loro RC, Valiati R, Hubler R, Gerhardt de Oliveira M. Photomedicine and Laser Surgery 2009 00(00):1-5 The effects of low level laser irradiation on osteoblastic cells. Coombe AR, Ho CT, Darendeliler MA, Hunter N, Philips JR, Chapple CC, Yum LW. Clin Orthod Res. 2001 Feb;4(1):3-14. Placebo-controlled randomized clinical trial of the effect two different low-level laser therapies (LLLT) intraoral and extraoral on trismus and facial swelling following surgical extraction of the lower third molar. Aras MH, Güngörmüş M, Lasers Med Sci. 2009 May 31. Low Level Laser Irradiation Precondition to Create Friendly Milieu of Infracted Myocardium and Enhance Early Survival of Transplanted Bone Marrow Cells. Zhang H, Hou, JF, Wang W, Wei YJ, Hu S J of Cell and Mol Med 2009 Sep 1. Effects of Low-Level Laser Therapy After Corticision on Tooth Movement and Paradental Remodeling. Kim, S-J, Moon S-U, Kang S-G, Park Y-G. Lasers in Surgery and Medicine 41:524 533 (2009 Phototherapy enhances bone regeneration in direct sinus lifts, Kwong-Hing and Brawn, poster at BIOS 2007 Effects of laser therapy on attachment, proliferation and differentiation of human osteoblast-like cells cultured on titanium implant material, Khadra et al, Biomaterials 2005; 26: 3503-3509 Low-Level laser therapy stimulates bone-implant integration: an experimental study in rabbits, Khadra et al, Clin. Oral Impl. Res., 2004 Determining optimal dose of light emitting diode phototherapy for proliferation of human oral fibroblasts. Khandra and Brawn Absorption measurements of cell monolayers relevant to mechanisms of laser phototherapy: reduction or oxidation of cytochrome c oxidase under laser radiation at 632.8nm. Karu T, Pyatibrat L, Kolyakov SF and Afanasyeva NI: Photomedicine and Laser Surgery, 2008 26:593-599 Page 9

Resonance frequency analysis of implants subjected to immediate or early functional occlusal loading. Successful vs. failing implants. Glauser R, Sennerby L, Meredith N, Rée A, Lundgren AK, Gottlow J, Hämmerle CHF: Clin. Oral Impl., 2004 15:428-434 Effect of low-level laser therapy on bone repair: Histological study in rats. Pretel H, Lizarelli RFZ, and Ramalho LTO: Lasers Surg. Med., 2007 39:788-796 Effect of low-level laser treatment after installation of dental titanium implantimmunohistochemical study of RANKL, RANK, OPG: an experimental study in rats. Kim Y-D, Kim S-S, Hwang D-S, Kim S-G, Kwon Y-H, Shin S-H, Kim U-K, Kim J-R, and Chung I-K: Lasers Surg. Med., 2007 1-10 Mitochondrial signal transduction in accelerated wound and retinal healing by nearinfrared light therapy. Eells JT, Wong-Riley MTT, VerHoeve J, Henry M, Buchman EV, Kane MP, Gould LJ, Das R, Jett M, Hodson BD, Margolis D, Whelan HT: Mitochondrion, 2006 4:559-567 Near infrared light protects cardiomyocytes from hypoxia and reoxygenation injury by a nitric oxide dependant mechanism. Zhang R, et al: J Mol & Cell Card., 2009 46:4-14 Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone- and MPP+ -induced neutrotoxicity. Ying R, Kiang HL, Whelan HT, Ealls JT, Wing-Riley MTT: Brain Reseach, 2008 1243:167-173 Power games. Lane N: Nature, 2006 443: 901-903 Irradiation effect of low-energy laser on alveolar bone after tooth extraction. Takeda Y: Experimental study in rats. Int J Oral Maxillofac Surg 1988, 17:388-391. The effects of the helium-neon laser on postsurgical discomfort: a pilot study. Clokie C, Bentley KC, Head TW: J Can Dent Assoc 1991, 57:584-586. Enhancement of angiogenesis in regenerating gastrocnemius muscle of the toad (Bufo viridis) by low-energy laser irradiation. Bibikova A, Belkin V, Oron U: Anat Embyrol (Berl) 1994, 190: 597-602. Effect of low-energy laser (He-Ne) irradiation on the process of bone repair in the rat tibia. Barushka O, Yaakobi T, Oron U:. Bone 1995, 16:47-55. Promotion of bone repair in the cortical bone of the tibia in rats by low energy laser (He- Ne) irradiation. Yaakobi T, Maltz L, Oron U: Calcif Tissue Int 1996, 59:297-300. Laser modulation of angiogenic factor production by T-lymphocytes. Agaiby AD, Ghali LR, Wilson R, Dyson M: Lasers Surg Med 2000, 26:357-363. Low-energy laser irradiation stimulates bone nodule formation at early stages of cell culture in rat calvarial cells. Ozawa Y, Shimizu N, Kariya G, Abiko Y: Bone 1998, 22:347-354. Page 10

Effects of near-infrared low-level laser irradiation on microcirculation. Maegawa Y, Itoh T, Hosokawa T, Yaegashi K, et al: Lasers Surg Med 2000, 27:427-437. In vitro effects of low-level laser irradiation at 660 nm on peripheral blood lymphocytes. Stadler I, Evans R, Kolb B, Naim JO, et al: Lasers Surg Med 2000, 27:255-261. Impact of low level laser irradiation on infarct size in the rat following myocardial infarction. Ad N, Oron U: Int J Cardiol 2001, 80:109-116. Comparison of the low level laser therapy effects on cultured human gingival fibroblast proliferation using different irradiance and same fluence. Almeida-Lopes L, Rigau J, Zangaro RA, Guidugli-Neto J, et al: Lasers Surg Med 2001, 29:179-184. Improvement of macromolecular clearance via lymph flow in hamster gingiva by lowpower carbon dioxide laser-irradiation. Shimotoyodome A, Okajima M, Kobayashi H, Tokismitsu I, et al: Lasers Surg Med 2001, 29:442-447 Effect of NASA light-emitting diode irradiation on wound healing. Whelan HT, Smits RL Jr, Buchman EV, Whelan NT, et al: J Clin Laser Med Surg 2001, 19:305-314. Laser stimulation on bone defect healing: an in vitro study. Guzzardella GA, Fini M, Torricelli P, Giavaresi G, et al: Lasers Med Surg 2002, 17:216-230. Histology study of the Effect of Laser Therapy on Bone Repair. Blaya DS, Guimaraes MB, Pozza DH, Weber JBB, do Oliviera MG, J Cont. Dent. Pract. 2008 Sept.1, 9(6):1-8. Low level 809-nm diode laser-induced in vitro stimulation of the proliferation of human gingival fibroblasts. Kreisler M, Christoffers AB, Al-Haj H, Willershausen B, et al: Lasers Surg Med 2002, 30:365-369. Effect of low-power laser irradiation on cell growth and procollagen synthesis of cultured fibroblasts. Pereira AN, Eduardo Cde P, Matson E, Marques MM: Lasers Surg Med 2002, 31:263-267. Computerized morphometric assessment of the effect of low-level laser therapy on bone repair: an experimental animal study. Silva Junior AN, Pinheiro AL, Oliveira MG, Weismann R, et al: J Clin Laser Med Surg 2002, 20:83-87. 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, et al: Lasers Med Sci 2003, 18:89-94. High-intensity pulsed laser irradiation accelerates bone formation in metaphyseal trabecular bone in rat femur. Ninomiya T, Miyamoto Y, Ito T, Yamashita A, et al: Bone Miner Metab 2003, 21:67-73. Effect of 830-nm laser light on the repair of bone defects grafted with inorganic bovine bone and decalcified cortical osseous membrane. Pinheiro AL, Limeira Junior Fde A, Gerbi ME, Ramalho LM, et al: J Clin Laser Med Surg 2003, 21:301-306. Page 11

Direct stimulatory effect of low-intensity 670 nm laser irradiation on human endothelial cell proliferation. Schindl A, Merwald H, Schindl L, Kaun C, Wojta J: Br. J Dermatol 2003, 148:224-336. Therapeutic photobiomodulation for methanol-induced retinal toxicity. Eells JT, Henry MM, Summerfelt P, Wong-Riley MT, Buchmann EV, Kane M, Whelan NT, Whelan HT. Proc Natl Acad Sci U S A. 2003 Mar 18;100 (6):3439-44 Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation. Vinck EM, Cagnie BJ, Cornelissen MJ, Declercq HA, Cambier DC. Lasers Med Sci. 2003; 18 (2):95-9. Light-emitting diode treatment reverses the effect of TTX on cytochrome oxidase in neurons. Wong-Riley MT, Bai X, Buchmann E, Whelan HT. Neuroreport. 2001 Oct 8; 12 (14):3033-7. Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice. Whelan HT, Buchmann EV, Dhokalia A, Kane MP, Whelan NT, Wong-Riley MT, Eells JT, Gould LJ, Hammamieh R, Das R, Jett M. J Clin Laser Med Surg. 2003 Apr; 21 (2):67-74. Page 12