Photofunctionalization of Dental Implant Surfaces - A Histomorphometric Animal Study

ABSTRACT

Aims:

To compare and evaluate the degree of osseointegration of UV-treated (photo functionalized) and non-treated dental implants surface coated with Calcium phosphate using the Resorbable Blast Media (RBM) technique in an animal model.

Settings and Design:

Evaluative-Animal study design.

Materials and Methods:

Six titanium dental implants of diameter 3.2 mm and length of 8 mm with Calcium phosphate coated surface using RBM or resorbable blast media technology (Implant Genesis: Genesis Normo Implant system) were placed epicrestally into the proximal femoral condyle of New Zealand white female rabbits such that each animal received two implants. Before implantation, one out of the two dental implants was photo functionalized with intense UV light for 15 minutes. After twelve weeks of healing, the animals were euthanized and the harvested specimens were analyzed using histomorphometric light microscopy to assess two parameters bone-implant contact and bone volume density.

Statistical Analysis Used:

SPSS version 23. P less than 0.05 is considered statistically significant. Tests used ANOVA followed by Tukey post hoc test.

Results:

All six dental implants were osseointegrated. The overall mean bone-implant contact area (BIC) was 57.76% for non-UV treated whereas 88.4367% for UV-treated dental implants. The overall mean bone volume density (BVD) was 32.2333% for non-UV treated whereas 67.7533% for UV-treated dental implants.

Conclusion:

Significant effects were observed on the osseointegration of dental titanium implants within twelve weeks after UV photo functionalization. The UV photo functionalization of dental titanium implants in the current study significantly altered the BIC and bone density on osseointegration when observed over twelve weeks.

INTRODUCTION

Titanium dental implants enhance the overall quality of oral health by improving masticatory function[1] and speech.[2] Replacing missing teeth with dental titanium implants renders a positive impact on the quality of oral health[3,4] compared to conventional removable dental prostheses.[4,5] However, a protracted healing time of at least six months is recommended for the dental implant for appropriate bone-implant osseointegration, and subsequent loading is a cause of concern for successful and safe restoration.[6]

Osseointegration refers to a direct bone-to-metal interface without the interposition of non-bone tissue. Osseointegration was first defined as direct contact between living bone and the surface of a load-carrying implant at the histological level by Brånemark in 1983.

It is important to note that the recently discovered phenomenon of Biological Ageing is marked by altered biological characteristics present with reduced bone-implant contact values. According to Att W. and Ogawa T., this means that , longer the shelf life, more aging occurs, which in turn leads to reduced BIC values.[7,8] Studies by Aita et al., 2009 and Att and Ogawa, 2012 report that, four-week-old titanium surfaces needed twice the healing time to achieve similar osseointegration levels and exhibited less BIC than newly prepared titanium surfaces.[4,7]

Recently reported time dependent reduction in biological capabilities of titanium since processing, elucidates the phenomenon defined as biological aging of titanium. BIOLOGICAL AGEING is the degradation of the physiochemical properties of the implant surface with time.[9]

The TiO of manufactured implants may lose some ability for bioactive integration with the bone after a storage time of as little as 2 weeks, during which time there is a degradation of bioactivity. However, the bioactivity can be regained with exposure to UV. This non–surface-altering conditioning is known as photo functionalization. UV photo functionalization is defined as the phenomenon of titanium surface modification after intense UV treatment, including the change in the physicochemical properties and the improvement in biological features. The titanium surface is energy conditioned by UV radiation. This imparts slight positive surface energy and hydrophilicity to the titanium dental implant surface. This conditioning renews biological activity lost after a shelf life of as little as 2 weeks. UV radiation has chemical and biological effects on the osseous-implant interface. Photofunctionalization for as little as 15 minutes accelerates healing and increases bone-to-implant contact. The most effective time exposure and UV wavelength require identification to produce a surface most conducive for osseointegration.[10,11]

The human studies by Funato and Ogawa, 2013, Funato et al., 2013; and Suzuki et al., 2013 measured only the implant stability quotient (ISQ) and not the histomorphometry. Implant healing time for investigation was four weeks and the implants were placed into compact bone with submerged healing in the aforementioned studies.[4]

This pilot study undertakes the feasibility of improvising cost-effective calcium phosphate-coated dental implants by photo functionalisation causing super osseointegration with reduced healing time and better success rate. Therefore, this study aimed to evaluate the effect of UV photo functionalization of RBM-coated titanium dental implants on osseointegration for 12 weeks, in an animal model and site of implantation with bone formation rates comparable to the human mandible. The null hypothesis was that UV photo functionalisation of calcium phosphate-coated RBM treated implant surfaces will not exhibit a significant difference in the levels of osseointegration in UV treated than in non-UV treated dental implants studied in a rabbit femur model.

MATERIALS AND METHODS

Surgical procedures

Three New Zealand white male rabbits (Oryctolagus cuniculus) healthy large animals, weighing between 3.0-3.5 kg and ideally 8-9 months of age were used for the study. The study procedures were following the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India; Consent for conducting the animal study was obtained from CPCSEA after approval of the Institutional Animal Ethics Committee (IAEC) Ethical approval for the study was obtained from the Institutional Review Board. Three New Zealand White rabbits as per the inclusion criteria were bought from a licensed breeder (Small Animals Breeding Section, Kerala Veterinary and Animal Sciences University (KVASU), Mannuthy, Thrissur, Kerala). The rabbits were acclimatized and accustomed to handling 4 weeks before the study. Their body weights were monitored daily, for comparison with body weight post-surgery, as an aid in evaluating recovery. The rabbits were trained to eat from a syringe in case supplemented feeding would be necessary post-surgery. Before the surgery, the fur on the ears of the rabbits was clipped, to enable vessel catheterization at the time of surgery.

Before the surgical step, the proximal femur area skin was shaved and washed with betadine and the pre-operative antibiotic Enrofloxacin 5 mg/kg BID was delivered subcutaneously. Following conventional modalities of general anesthesia (35 mg/kg ketamine and 3 mg/kg xylazine) and local anesthesia (lidocaine hydrochloride diluted to 0.5%, not exceeding 7 mg/kg total dose, SC or intra-incisional) surgical site preparation for implant placement was performed. Each rabbit received 2 dental implants (3.2 mm diameter and 8 mm length with Calcium phosphate coated surface using RBM or resorbable blast media technology) in the proximal femoral condyle according to the manufacturer’s instructions (Implant Genesis: Genesis Normo Implant system) [Figure 1]. Before implant placement, one out of the two implants was photo functionalized for 15 minutes using a UV chamber (Genesis Laminar Flow-240-260 nm). Periosteum and fascia were sutured after placing the coverscrews of the implants. Post-operative pain control was achieved with subcutaneous administration of 0.05 mg/kg of Buprenorphine every 12 hours and Carprofen 2 mg/kg every 24 hours for 3-5 days. The rabbits were closely monitored for signs of pain (not eating, abnormal body posture, reluctance to move, or aggression) and additional pain medication was given if necessary. The appetite of the animals was closely monitored and food supplements were given when required.

Figure 1.

Implant Genesis: Genesis Normo Implant system

Specimen preparation, light microscopy, and histomorphometric analysis

After 12 weeks of healing, the rabbits were sacrificed with intravenous administration of thiopental sodium through the auricular vein. Each set of implants was surgically removed en bloc with an adjacent bone collar and immediately fixed in 4% neutral formaldehyde. The specimens were then embedded in light-curing resin. Undecalcified ground sections were cut and prepared using a hard microtome, that is, Linear Precision Saw Microtome (Accutome 100, Struers, Denmark). Specimens were then ground to an approximate thickness of (70-100 micrometer thickness) and stained with Hot Stevenel’s blue and van Gieson’s Picrofuchsin and histological views were assessed under a light microscope [Figure 2]. All light microscopic calculations were done with a ><10 objective and ><10 eyepieces. The percentage of bone-to-implant contact (BIC) and Bone Volume Density (BVD) was calculated with appropriate software (Image J software) for image analysis. [Figures 3, 4a, and b]. The percentage of BIC in all of the threads at the bone cortex and intramedullary area were calculated. The calculation of the BIC area was performed as a percentage of the implant surface area where the bone could have been in direct contact with the implant (overall bone area) and the area where bone deposited on the implant (measured bone area). The BIC was calculated: BIC = measured bone/overall bone area × 100%).

Figure 2.

Specimens ground to an approximate thickness of (70-100 micrometer thickness) and stained with Hot Stevenel’s blue and van Gieson’s Picrofuchsin

Figure 3.

Bone-Implant interface when not treated with UV light

Figure 4.

(a) Histological section of implant not treated with UV light (b) Histological section of UV treated implant surface

Statistical analysis

Data were statistically analyzed using SPSS for Windows (Version 23.0; SPSS Inc., Chicago, USA). The data for overall implant surface area, BIC in mm, and BIC in percentage were distributed normally. Parametric tests were employed. All tests were performed at a confidence level of 95%.

RESULTS

Histomorphometric image analysis using Image J software evaluated the two major parameters of the study, namely Bone Implant Contact (BIC) and Bone Volume Density (BVD). The mean values of BIC and BVD obtained for UV treated dental implants (88.43% mean BIC and 67.75% mean BVD) were found to be greater than non treated dental implants (57.76% mean BIC and 32.23% mean BVD) [Tables 1, 2 and Figure 5]. The results of this study suggest that there is a significant difference in the BIC and BVD of UV treated dental implants than non UV treated dental implants after 12 weeks. A summary of the data and the statistical analysis can be found in the Tables below [Tables 1 and 2].

Table 1.

Descriptive statistics

	Group	N	Mean	Std. Deviation	Std. Error Mean
Bone implant contact	treated	3	88.4367	3.52959	2.03781
	untreated	3	57.7600	1 45836	.84198
Bone volume density	treated	3	67.7533	5.13873	2.96685
	untreated	3	32.2333	2.89194	1 66966

Table 2.

Unpaired T test

	t	df	Significance. (2- tailed)	Mean Difference	Std. Error Difference	95% Confidence Interval of the Difference
						Lower	Upper
Bone implant contact	13.913	2.6 64	.001	30.676 67	2.204 91	23.13 055	38.222 78
Bone volume density	10.434	3.1 51	.002	35.520 00	3.404 40	24.97 420	46.065 80

Figure 5.

New bone formation at bone implant interface after photofunctionalisation

DISCUSSION

New Zealand white rabbits (Oryctolagus cuniculus) weighing 3.0-3.5 kg were used in this experimental animal study to evaluate the levels of osseointegration in UV-treated and non-treated dental implants. The histomorphometric study was based on the assessment of two major parameters namely Bone Implant Contact (BIC) and Bone Volume Density (BVD) using Image J software. Both non-UV treated (control group) and UV-treated dental implants (experimental group), were surgically placed in the proximal part of the femur bone of the rabbit model and after 12 weeks, the animals were euthanized to harvest the femur bone implanted with the control and experimental dental implants for histomorphometric analysis.

The rationale for why animal usage is necessary for the study can be brought to light with the following literature. That is, in vitro tests demonstrate the response of individual cell lines/primary cells but not the tissue response to materials.[12] The selection of rabbits as an experimental model in this study has ample support from the available literature. Rabbits are big enough to place multiple implants which is not possible in rats.[13] Also, the size and temperament of rabbits make them easy to handle.[13] The rabbit is also convenient in that it reaches skeletal maturity shortly after sexual maturity at around 6 months of age.[14]

The site of implantation was chosen to be the femoral condyle as it is been reported that the ideal location for the same is the proximal femoral condyle. It has both cortical and cancellous bone.Also, the bone at this place gives adequate space for the implantation and therefore a wider and longer implant can be used. Commonly available implant dimensions like 6 mm diameter and 8 mm long implant can be placed with ease by this approach in comparison with the recommended norm of 2 mm diameter and 6 mm length.Ajayi and Shawulu et al., 2012 investigated the morphology and morphometry of the bones of the thigh, crus, and foot of ten New Zealand white adult rabbits to outline the peculiarities of the species and attempted to establish a morphofunctional paradigm.[15] According to the results of the study, the mean values of femur parameters measured are: Width of the proximal femur on the right side was 1.539 (±0.131) and on the left side was 1.583 (±0.111); the Length of the femur on the right side was 8.230 (±0.271) and 8.230 (±0.263) on the left side. Taking into account the above-mentioned dimensional considerations and the mean values of rabbit femur parameters presented in the literature, the present study uses a dental implant of 3 mm diameter and 8 mm length.

Genesis Normo Tapered Implants (3.2 mm diameter and 8 mm length) with Ca phosphate coated surface were used in the study. Implant Genesis applies the RBM (Resorbable Blast Media) technique to induce the sub micro topography. The material used for the RBM process is Calcium Phosphate, which is a highly resorbable and biocompatible material. The usage of Calcium Phosphate as the blasting material also eliminates the need of using strong acids for the removal of blasting material remnants. Resorbable Blast Media (RBM) surfaces treated dental implants had advantages because surface area increases as particles are blasted on the surface, and the degree of osseointegration increases.

As reviewed by Elkhidir and Cheng in 2017, light wavelengths visible to the human eyes range from 400 to 700 nm.[16] Ultraviolet (UV) light on the other hand can be either UVA (320 400 nm), UVB (290 320 nm), or UVC (10 290 nm). The range used in biological investigations is 200 400 nm; UVA light can remove hydrocarbons by inducing TiO2 photocatalysis. UVC irradiation is considered superior in reducing surface carbon levels, improving hydrophilicity, and enhancing protein adsorption and cell function. Similar observations were given by Flanagan,[11] therefore, photo functionalisation of the dental implants used in the present study was carried out using a UV chamber (Genesis Laminar Flow) delivering UV light of wavelength 240 260 nm within the referred range of biological investigation. As per the available literature, 12 minutes of exposure of dental implants to UV light was used in the study.[17]

To analyze undecalcified sections of bone and teeth, Donath and Breuner (1982) developed a sawing and grinding technique that enabled the preparation of thin bone/implant sections.[18] These are mostly examined after staining at a thickness of 20 30 μm. This method serves as the basis for the assessment of bone implant contact.[19]

The mean values of BIC and BVD in the case of UV photo functionalized dental implants significantly increased after 12 weeks time period offered for osseointegration.

CONCLUSION

The null hypothesis was rejected. The UV photo functionalization of dental titanium implants in the current study significantly altered the BIC and bone density on osseointegration when observed for a period of twelve weeks.

Compliance with ethics guidelines

All institutional and national guidelines for the care and use of laboratory animals were followed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.