Effects of infrared laser on the bone repair assessed by x-ray microtomography (μct) and histomorphometry

Effects of infrared laser on the bone repair assessed by x-ray microtomography (µct) and histomorphometry

Alessandra Rossi Paolilloabc, Fernanda Rossi Paolillob, Alessandro M. Hakme da Silvac,d, Rodrigo Bezerra de Menezes Reiffe, Vanderlei Salvador Bagnatob, José Marcos Alvesc

aDepartment of Occupational Therapy, Federal University of São Carlos (UFSCar), Rodovia

Washington Luiz, Km. 235, CEP 13565-905, São Carlos, SP, Brazil bOptics Group from São Carlos Institute of Physics (IFSC), University of São Paulo (USP), Av.

Trabalhador Sãocarlense, 400, CEP 13560-970, São Carlos, SP, Brazil cBioengineering Program, University of São Paulo (USP), Av. Trabalhador Sãocarlense, 400, CEP

13560-970, São Carlos, SP, Brazil dDepartment of Physics, Federal University of São Carlos (UFSCar), Rodovia Washington Luiz,

Km. 235, CEP 13565-905, São Carlos, SP, Brazil eDepartment of Medicine, Federal University of São Carlos (UFSCar), Rodovia Washington Luiz,

Km. 235, CEP 13565-905, São Carlos, SP, Brazil

ABSTRACT

The bone fracture is important public health problems. The lasertherapy is used to accelerate tissue healing. Regarding diagnosis, few methods are validated to follow the evolution of bone microarchitecture. The aim of this study was to evaluate the effects of lasertherapy on bone repair with x-ray microtomography (µCT) and histomorphometry. A transverse rat tibia osteotomy with a Kirchner wire and a 2mm width polymeric spacer beads were used to produce a delayed bone union. Twelve rats were divided into two groups: (i) Control Group: untreated fracture and; (ii) Laser Group: fracture treated with laser. Twelve sessions of treatment (808nm laser, 100mW, 125J/cm², 50seconds) were performed. The µCT scanner parameters were: 100kV, 100µA, Al+Cu filter and 9.92µm resolution. A volume of interest (VOI) was chosen with 300 sections above and below the central region of the fracture, totaling 601sections with a 5.96mm. The softwares CT-Analyzer, NRecon and Mimics were used for 2D and 3D analysis. A histomorphometry analysis was also performed. The connectivity (Conn) showed significant increase for Laser Group than Control Group (32371±20689 vs 17216±9467, p˂0.05). There was no significant difference for bone volume (59±19mm3 vs 47± 8mm3) and histomorfometric data [Laser and Control Groups showed greater amount of cartilaginous (0.19±0.05% vs 0.11±0.09%) and fibrotic (0.21±0.12% vs 0.09±0.11%) tissues]. The negative effect was presence of the cartilaginous and fibrotic tissues which may be related to the Kirchner wire and the non-absorption of the polymeric that may have influenced negatively the light distribution through the bone. However, the positive effect was greater bone connectivity, indicating improvement in bone microarchitecture.

Keywords: bone, lasertherapy, x-ray microtomography, histomorphometry

1. INTRODUCTION Fracture with a non union or delayed-union are important public health problems. The lasertherapy is used to accelerate tissue healing due to enhanced mitochondrial function, gene modulation, anti-inflammatory effect and improvement of microcirculation.1,2 Regarding diagnosis, few methods are validated to appreciate and follow the evolution of bone microarchitecture during a fracture repair. Histomorphometry is usually performed for analysis of bone repair. However, the x-ray microtomography (µCT) is a modern tool that allows 3D quantitative analysis and 2D and 3D reconstructions.3

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1.1 Bone Tissue

The bone is formed by cortical and trabecular bony tissues. Osteoclasts and osteoblasts are the main cells present in these tissues. Bone remodeling occur due to local variations of strains, to hormonal and metabolic changes during the life. There are a phase of resorption of a piece of bone by the osteoclasts action and, another phase, where the osteoblasts deposit bone matrix.4 The bone healing is characterized by formation of fracture hematoma, fibrocartilagenous (soft) callus and bony (hard) callus.

1.2 Lasertherapy

Lasertherapy is an application of low-power monochromatic or quasimonochromatic and coherent electromagnetic radiation in the red and near infrared spectrum (~600-1000 nm).

Several in vitro5 experiments and animal6 studies have shown that lasertherapy may photostimulate bone healing with increased blood supply or neovascularization which may promote regenerative and proliferative biological processes. Moreover, lasertherapy may improve collagen fiber organization, increase bone marrow cellularity and induce active osteoclasts.6

However, few studies are performed with humans. Clinical trials with lasertherapy were carried out in the field of dentistry to investigate, for example, orthodontic tooth movement7 and dental implant.8 The light interaction with biological tissues in animals, mainly bone tissue, is particularly interesting to perform future clinical trials.

1.3 Microtomography (µct)

Qualitative and quantitative analysis of the bone microstructure can be performed by X-ray computed microtomography (μCT). Bone quality is monitored clinically by measurement of bone mineral density (BMD), but this is not sufficient to completely identify bone fragility and risk of fracture in patients9. The measurement of microstructural parameters thorough three dimensional morphometric analysis (3D) should be incorporated to increase the determination of fracture risk and to improve the diagnosis of osteoporosis.

The morphometric analysis of a trabecular bone sample is a measure of the microstructural parameters and can be performed by conventional histomorphometry10 or by X-ray Microtomography (μCT) using a x-ray radiation source with parallel and monochromatic beam (synchrotron radiation)11 or a radiation source with conical and polychromatic field (benchtop scanners)12. The hardware for μCT conical field began to be developed in the 90’s13,14.

There are several steps for the reconstruction of cross-sections and morphometric analysis of a trabecular bone sample by μCT. The morphometric analysis by μCT is performed using the reconstruction of the 2D cross-sections of the sample (2D analysis) and the 3D reconstruction of the sample (3D analysis), as described by Hildebrand et al. (1999)15.

The 2D parameters are calculated using stereological techniques and assuming that bone sample has a microstructure composed of plates or rods. The equations were originally developed for conventional histomorphometry and continue to be used in 2D μCT analysis. The algorithm calculates the input parameters (bone surface area - BS, bone volume – BV, tissue sample volume - TV which is the bone volume plus the pore volume) based on the number of pixels across the images histogram (levels of gray scale in the image) where white pixels represent bone and black pixels the pores.

The 3D morphometric analysis is not based on a structural model of plates or rods. All the 3D measurements of parameters, including the primaries parameters (bone surface - BS); bone volume - BV); tissue volume - TV), are performed directly from the reconstruction of the bone tissue object. The BS measurement is calculated using the method of “Marching Cubes” by placing triangles on the bone surface sample16. The (BV) measurement is calculated using tetrahedrons built in triangle’s surface17. The TV is determined by a counting of voxels. There is an important morphometric parameter called connectivity (Conn) that was defined as a measure of the degree that a structure is multiply connected. This means the maximum number of connections that can be broken before the structure is separated into two parts. The connectivity measurement depends on trabecular bone pattern factor parameter (Tb.Pf)18. The basic idea in the creation of Tb.Pf parameter was the fact that all patterns or structures can be described by their relation between concave and convex surfaces. Simplified, that means a lot of convex structures indicate a badly connected pattern. A lot of concave structures is the result of a well connected bone pattern19.





2. METODOLOGY 2.1 Animal Groups

Twelve Wistar rats (body mass ∼450g) supplied by a local breeding facility (University of São Paulo) were kept in a collective cage with food and water ad libitum, on a 12:12h light–dark cycle at 23 ± 1 °C. All animal procedures were performed according to the protocol of the “Guide for the Care and Use of Laboratory Animals”20. This study was approved by the Ethics Committee of the Federal University of São Carlos (UFSCar) in São Carlos, Brazil (number 050/2011). The animals were divided into two groups: Control Group: fracture untreated; Laser Group: fracture treated with laser.

2.2 Bone Fracture Model

The Wistar rats were anaesthetized using an intraperitoneal injection of xylazine 2% (12mg/Kg) and ketamine (80mg/Kg). A transverse rat tibia osteotomy with a Kirchner wire and 2mm width polymeric spacer beads were used to delay union in the fracture (Figure 1) as previously described by Milles et al.21, who have demonstrated that an inexpensive, technically straightforward model can be used to create a range of outcomes from normal healing to impaired healing, to non-unions.

Figure 1. Bone fracture with delay union evidenced by photographic and x ray images.

2.3 Lasertherapy

The parameters of Laser (Twin laser, MMOptics, São Carlos, SP, Brazil) were: 808nm, 100mW, 125J/cm², 50seconds and continuous mode. The treatment with infrared laser (Figure 2) started 5 weeks after the surgery following a sequence of 5 days on and 2 days off until 12 sessions were reached as previously described by Lirani-Galvão et al.22

Figure 2. Infrared laser (808nm, 100mW, 125J/cm2) applied on the fracture of tibia in rats Wistar (contact mode at a 90º angle with the skin).





2.4 Microtomography (µct) and Histomorphometry

The µCT analysis was carried out with the SkyScan scanner (SkyScan 1172, SkyScan NV, Aartselaar, Belgium) using the scanning parameters 100 kV, 100 µA, Al+Cu filter and 9.92 µm resolution. The volume of interest (VOI) was the 300 sections above and below the central region of the fracture, totaling 601 sections or 5.96 mm (height). The softwares CT-Analyzer (Bruker, Belgium), and Mimics (Materialise, Belgium) were used to perform 2D and 3D morphometric analysis respectively. An example of the image processing with the Materialize software can be seen in the Figure 3 that shows cross sectional views of the fracture site and a rendering of it. After, the bone specimens were included in paraffin blocks and their sections stained with Masson trichrome for histomorphometric analysis.

Figure 3. X-ray microtomographic image processing.

2.5 Statistical Analysis

Results are expressed as mean ± standard deviation. The data analysis was done using Statistica for Windows release 7 software (Statsoft, Tulsa, Ok). The one-way analysis of variance (one-way ANOVA) was performed. Significant results of one-way ANOVA were subjected to a post-hoc analysis (Tukey) for intergroup comparisons.

3. RESULTS The connectivity (Conn) showed significant increase for the Laser Group than the Control Group (32371±20689 vs 17216±9467, p˂0.05).

There was no significant difference for bone volume (59±19mm3 vs 47± 8mm3). A greater amount of cartilaginous (0.19±0.05% vs 0.11±0.09%) and fibrotic (0.21±0.12% vs 0.09±0.11%) tissues were found through the histomorfometric data for the Laser and Control Groups, respectively.

The connectivity and fribrotic data can be seen in the Figures 3 and 4, respectively.





Figure 3. Connectivity: the Laser Group showed increased bone connectivity as evidenced by µCT analysis.

Figure 4. Fibrotic tissue: the Control and Laser Groups showed greater amount of fibrotic tissue as evidenced by histomorphometry.

4. DISCUSSION The investigation of light interaction with biological tissue is important when target tissue is the bony tissue because fractures are considered a public health problem. The current study showed that the presence of the cartilaginous and fibrotic tissues is a negative effect in the fracture healing treated by lasertherapy which may be related to the Kirchner wire and the non-absorption of the polymeric spacer in the fracture site may have influenced negatively the light distribution through the bone tissue.

However, the current study showed that the positive effect of lasertherapy was the increase in bone connectivity that indicates an improvement in bone microarchitecture. Bossini et al.23 showed that infrared laser improves bone repair in the tibia of osteoporotic rats with increased in the newly formed bone as well as the fibrovascularization and





angiogenesis. In addition, Tim et al.24 founded an acceleration in the deposition and organization of newly formed bone and activated expression of osteogenic factors (RUNX-2 and BMP-9 genes).

The protocol for lasertherapy may lead to differences in the therapeutic results. We used single point with high dose (100 mW and 125J/cm2), similarly to the studies of Bossini et al.23 and Tim et al.24 (single point, 100mW and 120J/cm2). However, these authors performed a bone defect in the tibia of rats and, in the current study, we performed a complete fracture with Kirchner wire and spacer. In this context, the metal material for fracture stabilization or the polymeric spacer for induction of delay union may have influenced negatively to light distribution through the bone.

Then, we suggest that the lasertherapy with lower doses and various points of laser irradiation around the fracture site should be investigated to improve the bone repair. In the studies of Lopes et al.,25,26 an infrared laser was applied on four points around the fracture with 40mW and 4 J/cm2 per point associated with bone morphogenetic proteins, and guided bone regeneration to improve fracture repair. These authors25,26 concluded that the laser irradiation protocol associated with other techniques was effective in improving bone healing on the fractured bone.

Future studies should investigate the synergic effects of different techniques to promote the osteogenesis in fracture healing.

5. CONCLUSION The current study showed the effects of infrared laser on the bone repair assessed by x-ray microtomography (µct) and histomorphometry. The negative effect was presence of the cartilaginous and fibrotic tissues which may be related to the Kirchner wire and the non-absorption of the polymeric that may have influenced negatively the light distribution through the bone. However, the positive effect was greater bone connectivity as evidenced by µCT analysis, indicating improvement in bone microarchitecture. In addition, the µCT is a powerful diagnosis technique that allows 3D quantitative analysis and 2D and 3D reconstructions of the bone repair.

ACKNOWLEDGEMENTS

We would like to thank the São Paulo Research Foundation (FAPESP) - grant no. 2013/07276-1 and 2013/14001-9, the National Council for Scientific and Technological Development (CNPq) - grant no. 573587/2008. The authors also acknowledge scientific contributions and helpful advice from Nelson Ferreira da Silva Junior.

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Effects of infrared laser on the bone repair assessed by x-ray microtomography (μct) and histomorphometry

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