Laser biomodulation in bone implants: a Raman spectral study

Bone, 2013

Introduction: Radiotherapy to the appendicular skeleton can cause an increased risk of developing catastrophic fractures with delayed bone healing or non-union, and may subsequently require multiple procedures and amputation. Biomechanical studies suggest that irradiated bone is more brittle, but the cause is unclear and cannot be explained by changes to bone structure or quantity, suggesting that there are crucial changes in irradiated bone material properties. Raman spectroscopy provides a means to assess the chemical properties of the mineral and matrix constituents of bone, which could help explain post-radiation embrittlement. In this study we use a murine tibial model with focal irradiation and perform Raman spectroscopy to test the hypothesis that changes in bone chemistry following irradiation is consistent with reduced bone quality and persists in the long term after irradiation. Methods: Female BALB/F mice aged 12 weeks were subjected to unilateral, localized hindlimb irradiation in 4 daily 5 Gy fractions (4 × 5 Gy) totaling 20 Gy, and were euthanized at 1, 4, 8, 12, and 26 weeks postirradiation (n = 6/group). The irradiated (right) and non-irradiated contralateral control (left) tibiae were explanted and assessed by non-polarized and polarized Raman spectroscopy over the proximal cortical bone surface. Raman parameters used included the mineral/matrix ratio, mineral crystallinity, carbonate/phosphate ratio, collagen cross-link ratio, and depolarization ratio. Results: Significantly increased collagen cross-link ratio and decreased depolarization ratio of matrix were evident at 1 week after irradiation and this persisted through 26 weeks. A similar significant decrease was observed for depolarization ratio of mineral at all time points except 8 and 26 weeks. At 4 weeks after irradiation there was a significantly increased mineral/matrix ratio, increased mineral crystallinity, and decreased carbonate/ phosphate ratio compared to controls. However, at 12 weeks after irradiation these parameters had moved in the opposite direction, resulting in a significantly decreased mineral/matrix ratio, decreased crystallinity and increased carbonate/phosphate ratio compared to controls. At 26 weeks, mineral/matrix, crystallinity and carbonate/phosphate ratios had returned to normal. Discussion: In this mouse model, Raman spectroscopy reports both bone mineral and collagen cross-link radiation-induced abnormalities that are evident as early as one week after irradiation and persists for 26 weeks. The picture is one of extensive damage, after which there is an attempt at remodeling. We hypothesize that pathological cross-links formed by radiation damage to collagen are poorly resorbed during the altered remodeling process, so that new tissue is formed on a defective scaffold, resulting in increased bone brittleness.

Photomedicine and Laser Surgery, 2007

The aim of this study was to assess, through Raman spectroscopy, the incorporation of calcium hydroxyapatite (CHA; ~960 cm -1 ), and scanning electron microscopy (SEM), the bone quality on the healing bone around dental implants after laser photobiomodulation (830 nm). Background Data: Laser photobiomodulation has been successfully used to improve bone quality around dental implants, allowing early wearing of prostheses. Methods: Fourteen rabbits received a titanium implant on the tibia; eight of them were irradiated with 830 nm laser (seven sessions at 48-h intervals, 21.5 J/cm 2 per point, 10 mW, ~0.0028 cm 2 , 86 J per session), and six acted as control. The animals were sacrificed 15, 30, and 45 days after surgery. Specimens were routinely prepared for Raman spectroscopy and SEM. Eight readings were taken on the bone around the implant. Results: The results showed significant differences on the concentration of CHA on irradiated and control specimens at both 30 and 45 days after surgery ( p < 0.001). Conclusion: It is concluded that infrared laser photobiomodulation does improve bone healing, and this may be safely assessed by Raman spectroscopy or SEM.

LASER THERAPY, 2000

Tissue healing is a complex process that involves local and systemic responses. The use of Low Level Laser Therapy (LLLT) for wound healing has been shown to be effective in modulating both local and systemic response. Usually the healing process of bone is slower than that of soft tissues. The effects of LLLT on bone are still controversial as previous reports show different results. This paper reports recent observations on the effect of LLLT on bone healing. The amount of newly formed bone after 830nm laser irradiation of surgical wounds created in the femur of rats was evaluated morphometricaly. Forty Wistar rats were divided into four groups: group A (12 sessions, 4.8J/cm 2 per session, 28 days); group C (three sessions, 4.8J/cm 2 per session, seven days). Groups B and D acted as non-irradiated controls. Forty eight hours after the surgery, the defects of the laser groups were irradiated transcutaneously with a CW 40mW 830nm diode laser, (f~1mm) with a total dose of 4.8J/cm 2. Irradiation was performed three times a week. Computerized morphometry showed a statistically significant difference between the areas of mineralized bone in groups C and D (p=0.017). There was no significant difference between groups A and B (28 days) (p=0.383). In a second investigation, we determined the effects of LLLT on bone healing after the insertion of implants. It is known that dental implants need four and six months period for fixation on the maxillae and on the mandible before receiving loading. Ten male and female dogs were divided into two groups of five animals that received the implant. Two animals of each group acted as controls. The animals were sacrificed 45 and 60 days after surgery. The animals were irradiated three times a week for two weeks in a contact mode with a CW 40mW 830nm diode laser, (f ~1mm) with a total dose per session of 4.8J/cm 2 and a dose per point of 1.2J/cm 2. The results of the SEM study showed better bone healing after irradiation with the 830nm diode laser. These findings suggest that, under the experimental conditions of the investigation, the use of LLLT at 830nm significantly improves bone healing at early stages. It is concluded that LLLT may increase bone repair at early stages of healing.

Lasers in medical science, 2014

The aim of this study was to evaluate bone repair in anemic and non-anemic rats submitted or not to laser phototherapy and hydroxyapatite graft. Animals were divided in eight groups of five animals: Clot; Laser; Graft; Graft + Laser; iron deficiency anemia (IDA) + Clot; IDA + Laser; IDA + graft; IDA + graft + Laser. When appropriate irradiation with infrared laser was done during 15 days at a 48-h interval. Animals were killed at day 30; samples were analyzed by Raman spectroscopy. Three shifts were studied and statistically analyzed: ~960, ~1,070, and ~1,454 cm−1. Graft + laser showed highest ~960 peak was statistically different from all other healthy groups. No statistical difference was found between Clot and IDA + Clot in any shift. The IDA + Graft and IDA + Graft + Laser groups had low mean peak values for shifts ~960, ~1,070, and ~1,454 cm−1. The results in this study indicate that using hydroxyapatite (HA) and laser irradiation in healthy subjects is favorable to mineral deposition and bone maturation, this being of importance for some groups at risk, such as astronauts. In iron deficiency anemia cases, the use of graft, associated or not to laser irradiation, resulted in low collagen and low carbonate and phosphate HA.

International Congress Series, 2003

The aim of this study was to assess histologically the effect of LLLT (λ 830 nm) on the repair of standardized bone defects on the femur of Wistar albinus rats which were grafted with inorganic bovine bone Gen-ox ® . Three randomized groups were studied: group I (control, n=6); group II (Gen-ox ® , n=9) and group III (Gen-ox ® + LLLT, n=9). The animals were irradiated every 48 h during 15 days; the first irradiation was performed immediately after the procedure. The animals were irradiated transcutaneuosly at four points around the defect. At each point a dose of 4 J/cm 2 was given (Ø~0.6 mm, 40 mW) and the total dose per session was 16 J/cm 2 . The animals were killed by an overdose of general anesthetic 15, 21 and 30 days after surgery. The specimens were routinely processed by embedding in paraffin, serially cut and stained with H&E and Picrosirius and analyzed under light microscopy. The results showed evidence of a more advanced repair in the irradiated group when compared to the non-irradiated groups. The repair of the irradiated group was characterized by both increased bone formation and amount of collagen fibers around the graft within the cavity from the 15th day after surgery, also considering the osteoconductive capacity of the Gen-ox ® . We conclude that LLLT had a positive effect on the repair of bone defects implanted with inorganic bovine bone.

Mechanisms for Low-Light Therapy VII, 2012

We studied peaks of calcium hydroxyapatite-CHA on defects grafted with MTA, treated or not with Light Emitting Diode-LED or IR Laser. 54 rats were divided in 6 groups each subdivided into 3 subgroups (15,21,30d). LED (λ850 ± 10nm) or IR Laser (λ850 nm) was applied over (LED) or in 4 points around the defect at 48 h intervals for 15 days. Raman readings were taken at the surface of the defect. The smaller overall intensity of the peak was found in Group MTA + Laser (1510.2 ± 274.1) and the highest on Group LED (2322 ± 715). There were no statistically significant differences between non-irradiated subjects on regards the CHA peaks. On the other hand, there were statistically significant differences between the Group Clot and LED, Clot and Laser, and Clot and MTA + Laser (p =0.01, p = 0.02, p = 0.003). There were no significant differences between Group MTA and MTA + LED (p=0.2) but significant differences were seen between Groups MTA and MTA + Laser (p=0.01). Significant differences were also observed between Groups LED and Laser (p <0.001) and between Groups MTA + LED and MTA + Laser (p=0.009). MTA, due to its characteristics, seemed to be directly affected by the light. However, the use of either phototherapy positively affected bone healing similarly as observed on different studies using other biomaterials. The overall analysis of our results indicated that the use of either light source resulted in a better, more advanced, and of quality bone repair.

Journal of analytical & bioanalytical techniques, 2015

Quality and alterations in the biochemical composition of bones used for dental implantation after radiotherapy in cancer patients is always a critical and debatable factor. Clinically the irradiated bone is similar to control bone. The aim of this study was to verify any compositional alterations in human mandible bone after irradiation using Raman microspectroscopy. A total of 36 bone biopsies (21-control, 4-cancer and 11-irradiated) were investigated. Data acquisition points were determined under histopathological supervision. Both mineral and matrix constituents were analyzed by computing area associated with of phosphate (958 cm-1), carbonate (1070 cm-1) and matrix (amide I) bands. Unpaired Student's t-test was employed to measure level of significance. Absolute mineral contents (phosphate and carbonate) were highest in cancerous specimens. Spectral profile and band-intensity calculations suggest proximity of irradiated specimens with control specimens. Significant differences in both matrix and mineral contents were observed when control/irradiated samples were compared against cancerous specimens. However, no significant differences were observed between control and irradiated groups. Irradiated bone is similar to control and cause of implant loss could be related to osteocytes of the surrounding tissue.

The use of laser for bone cutting can be more advantageous than the use of drill. However, for a safe clinical application, it is necessary to know the effects of laser irradiation on bone tissues. In this study, the Fourier Transform Infrared spectroscopy (FTIR) was used to verify the molecular and compositional changes promoted by laser irradiation on bone tissue. Bone slabs were obtained from rabbit's tibia and analyzed using ATR-FTIR. After the initial analysis, the samples were irradiated using a pulsed Er,Cr:YSGG laser (2780nm), and analyzed one more time. In order to verify changes due to laser irradiation, the area under phosphate (1300-900cm -1 ), amides (1680-1200cm -1 ), water (3600-2400cm -1 ), and carbonate (around 870cm -1 and between 1600-1300cm -1 ) bands were calculated, and normalized by phosphate band area (1300-900cm -1 ). It was observed that Er,Cr:YSGG irradiation promoted a significant decrease in the content of water and amides I and III at irradiated bone, evidencing that laser procedure caused an evaporation of the organic content and changed the collagen structure, suggesting that these changes may interfere with the healing process. In this way, these changes should be considered in a clinical application of laser irradiation in surgeries.

Brazilian Dental Journal, 2013

Use of biomaterials and light on bone grafts has been widely reported. This work assessed the influence of low-level laser therapy (LLLT) on bone volume (BV) and bone implant contact (BIC) interface around implants inserted in blocks of bovine or autologous bone grafts (autografts), irradiated or not, in rabbit femurs. Twenty-four adult rabbits were divided in 8 groups: AG: autograft; XG: xenograft; AG/L: autograft + laser; XG/L: xenograft + laser; AG/I: autograft + titanium (Ti) implant; XG/I: xenograft + Ti implant; AG/I/L: autograft + Ti implant + laser; and XG/I/L: xenograft + Ti implant + laser. The animals received the Ti implant after incorporation of the grafts. The laser parameters in the groups AG/L and XG/L were λ=780 nm, 70 mW, CW, 21.5 J/cm 2 , while in the groups AG/I/L and XG/I/L the following parameters were used: λ=780 nm, 70 mW, 0.5 cm 2 (spot), 4 J/cm 2 per point (4), 16 J/cm 2 per session, 48 h interval × 12 sessions, CW, contact mode. LLLT was repeated every oth...

Lasers in medical science, 2012

We studied peaks of calcium hydroxyapatite (CHA) and protein and lipid CH groups in defects grafted with mineral trioxide aggregate (MTA) treated or not with LED irradiation, bone morphogenetic proteins and guided bone regeneration. A total of 90 rats were divided into ten groups each of which was subdivided into three subgroups (evaluated at 15, 21 and 30 days after surgery). Defects were irradiated with LED light (wavelength 850 ± 10 nm) at 48-h intervals for 15 days. Raman readings were taken at the surface of the defects. There were no statistically significant differences in the CHA peaks among the nonirradiated defects at any of the experimental time-points. On the other hand, there were significant differences between the defects filled with blood clot and the irradiated defects at all time-points (p < 0.001, p = 0.02, p < 0.001). There were significant differences between the mean peak CHA in nonirradiated defects at all the experimental time-points (p < 0.01). The mean peak of the defects filled with blood clot was significantly different from that of the defects filled with MTA (p < 0.001). There were significant differences between the defects filled with blood clot and the irradiated defects (p < 0.001). The results of this study using Raman spectral analysis indicate that infrared LED light irradiation improves the deposition of CHA in healing bone grafted or not with MTA.