Analysis of inflammatory cells in osseointegration of CpTi implant radiated by low level laser therapy

Background: Dental implants provide a unique treatment modality for the replacement of a lost dentition .This is accomplished by the insertion of relatively an inert material (a biomaterial) into the soft and hard tissue of the jaws, there by providing support and retention for dental prostheses. Low level laser therapy (LLLT) is an effective tool used to prompt bone repair and remodeling, this has referred to the biostimulation effect of LLLT. The Aim of this study was to evaluate the effects of inflammatory cells on osseointegration of CpTi implant irradiated by low level laser. Materials and Methods: thirty two adult New Zealand white rabbits, received titanium implants were inserted in the tibia. The right side is considered as experimental groups and the left side considered as control groups. Low power diode laser (GaAlAs ) with wave length (904nm) and (5mW)power applicated with the right implants . The sample divided into four groups, eight rabbits were sacrificed at four interval 4days, 1 weeks, 2weeks, and 6weeks respectively. Histological and inflammatory analyses were done for each interval. Results: Histological examination showed acceleration of bone formation and more rapid healing process in the screw implant with laser irradiation than in the control implant .inflammatory analysis showed dramatic decrease with the presence of laser irradiation especially with advancing time. Conclusion: This study illustrated that the inflammatory cells were reduced in osseointegration of dental implant


INTRODUCTION
Dental implants are biocompatible screw like titanium objects that are surgically placed into the mandible or maxilla to replace missing teeth.The mechanism by which an implant is biomechanically accepted by the jaw bone is called osseointegration (1,2) The clinical long-term success of the implants depends on the osseointegration and the adhesion of the soft tissues and epithelium to the titanium surfaces of the implant (3) .Titanium is the most wide spread metal for orthopedic implants intended for bone integration.It represents high fatigue strength comparatively low modulus of elasticity, in respect to other metals, so it is able to support loads and distribute them to bone, limiting stress shielding.Besides titanium is characterized by a thin natural oxide layer on the surface that limits ion release and reactivity, making the surface almost inert and biocompatible (4).
Several treatments have been proposed to improve and accelerate bone formation onto implant surface, among which is low-level laser therapy (LLLT) (5) .LLLT Known as cold laser, soft laser, biostimulation, or photobiomodulation, it basically exposes cell or tissue to laser or lowlevel red or near-infrared (IR) light generated from light-emitting diode.LLLT stimulates or controls cellular function to minimize the extinction of cell or tissue, accelerates the healing of fractures, fast recovery from the damage of soft tissue, nerve, bone, and cartilage, and relieves acute and chronic pain and inflammation (6) .

MATERIALS AND METHODS
All experimental procedures were carried out in accordance with the ethical principles of animal's experimentation.Thirty-two adult male New Zealand weighing 2-3kg were used in this study.Screw CpTi implants were inserted in the right tibia of each animal (experimental side), and left tibia was used as (control side).
Low power diode laser (GaAlAs) with wave length (904nm) and (5mW) power applied with the right screw implants.The animals were divided into four groups, for 4days, 1weeks, 2weeks, and 6weekshealing intervals.
Inflammatory cells counting were done under light microscope, in eight histological sections (stained by hematoxyline and eosin stain) for each healing period and for both experimental and control groups at / magnifying power lens X40.

Histological findings (hematoxylin and eosin stain) At 4 days duration Control group
Screw that implant in the tibia of rabbit shows bone marrow with stromal cells Figure (1).

Experimental group
The histological view of implant site in rabbit tibia treated with the laser irradiation after one week duration shows the bone trabeculae, osteoid tissue formed by active osteoblast Figure (5).
Active bone marrow is indicated by active osteoprogenitor cell, osteoblast cell and by the formation of osteoid tissue Figure (6).

At 2 weeks duration Control group
The histological view of implant site illustrates the apposition of woven bone against the basal bone with many osteoblast cells, osteocyte cell and osteoclast occupying Howship's lacunae Figure (7).

Experimental group
The histological picture of this group shows mature bone formation filling the thread of cortical bone area, the newly bone appear dense with regular distribution of osteocyte cell trapped inside the bone matrix, reversal line separating between old and new bone with the presence of numerous resting lines, Haversian bone lamellae are obviously seen Figure (11).

Inflammatory cells parameter
The result of the present study indicated that the majority of the inflammatory cells parameter was reported at the control group, as well as the first score and decreasing up to the last period of time after treatment in (6 weeks).There was no significant difference between mean values of the control and experimental groups at each healing period and the highest mean values were recorded at the 4 days and 1 week duration, where as after 2and 6 weeks of implantation mean values of scores were clearly less than the first two periods.
In addition to that, the comparisons significant among different periods of times reported a non significant differences at p>0.05 for both control and experimental groups throughout the healing periods (4 days, 1, 2 and 6 weeks).As shown in figure (12) (Table1).The histological findings showed biocompatible and osseoconductive implant surfaces in all groups, with different rates of bone deposition and remodeling.The results showed accelerated growth of bone around implant in groups who were exposed to laser and this may be attributed to the followings: 1-Stimulation with LLLT creates a number of environmental conditions that appear to accelerate the healing of bone in vivo investigations (8) .2-LLLT is an effective tool used to prompt bone repair and modeling post surgery , this has referred to the biostimulation effect of LLLT, it is directly dependent on the dose applied (9,10)   .3-The early bone maturation could be attributed to the stimulation of fibroblast proliferation through the application of LLLT as it has been reported by (11) .4-LLLT-related effects include stimulation of blood flow, recruitment and activation of osteoblasts, osteosynthesis, a decrease in osteoclastic activity and anti-inflammatory action could also be considered as factors that stimulate biomaterial osseointegration.
These findings were in agreement with previous findings (9,(12)(13)(14)(15)(16)(17)(18) .The response of the bone to the trauma through placement the implants by an inflammatory reaction played a great role in fixation of implant at early regenerative process (19) .
Within a few hours after injury, inflammatory cells invade the wound tissue.In addition to the defense functions, inflammatory cells are also an important source of growth factors and cytokines, which initiate the proliferative phase of wound healing (20,21) .At 4 days and 1 week of healing periods there was a large number of inflammatory cells that migrated towered the site of implant while at 2,6 weeks there was marked reduction in the number of inflammatory cells that infiltrated in to the implant sites which indicates earlier acute inflammatory response and more rapid resolution resulting in earlier phase of regeneration ;which agreed with (22)(23)(24) who found that LLLT was able to promote wound healing by reducing inflammation without compromising the proliferation of fibroblasts and keratinocytes.
Goldman, 1987 (25) Walsh, 2003 (26) found that LLLT is supposed to reduce pain, accelerate wound healing and reduce inflammatory processes.Furthermore, it enhances bone remodeling, attenuates pain and modulates the immune system.
The present study suggests for beneficial use of LLLT in practice of dentistry implantation or in other branch related to osseointegration process.

( 1 )
Assistant Professor, Department of Oral Diagnosis, College of Dentistry, University of Baghdad.

Figure 1 :
Figure 1: High magnification view of implant site shows fat cell (FC) in marrow tissue H&E X400.Experimental group Histological view of implant site treated with laser irradiation after 4 days of implantation revealed primitive new bone formation in which future bone is formed as embryonic bone Figure (2).High magnification view of marrow tissue illustrates the presence of fat cells associated with presence of numerous blood islets Figure (3).

Figure 3 :
Figure 3: High magnification of previous figure (2) shows numerous blood in the marrow tissue (arrow) point to blood vessel H&EX400.At one week duration Control groupScrew that implanted in the rabbit tibia shows the formation of osteoid tissue, abundant blood vessels in thread region that follows the screw shape Figure(4).

Figure 4 :
Figure 4: Microphotograph view of bone marrow in thread region in implant site (control) of one week duration shows fat cell (FC) H&EX100.

Figure 6 :
Figure 6: Microphotograph view of implant site treated with laser ir radiation after one week of implantation shows osteoblast cell (OB) and osteoid bone formation (OT) H&E X400.

Figure 7 :
Figure 7: Microphotograph view of implant site after 2 weeks (control) shows formation of woven bone (WB)against basal bone (BB), active formative osteoblast cell (OB), and osteoclast cell (OCL) .H&EX200Experimental groupThe histological view of experimental group with areas of marrow tissue shows the formation of bone trabeculae, osteoblast cell on the surface of the bone, osteocyte cell trapped inside the bone matrix cell.Figure(8, 9)

Figure 8 :
Figure 8: View of implant site of 2 weeks duration treated with laser irradiation showing bone trabeculae (BT) with the osteoblast (OB) and osteocyte trapped inside bone matrix (OC) H&E X400.

Figure 11 :
Figure 11: other figure of mature bone in implant site for 6 weeks duration treated with laser irradiation shows osteocyte cell (OC), reversal line (RL) separated between old bone (OB) and new bone (NB) H&E X400.

Figure 12 : 1 :
Figure 12: Cluster bar chart for mean values of scoring (inflammatory cells outcomes) distributed among different studied sources of variations.