Cannabis Sativa Smoke Inhalation Decreases Bone Filling Around Titanium Implants: A Histomorphometric Study in Rats

Abstract

Purpose: 

Although the harmful effect of tobacco smoking on titanium implants has been documented, no studies have investigated the effects of cannabis sativa (marijuana) smoking. Thus, this study investigated whether marijuana smoke influences bone healing around titanium implants.

Materials: 

Thirty Wistar rats were used. After anesthesia, the tibiae surface was exposed and 1 screw-shaped titanium implant was placed bilaterally. The animals were randomly assigned to one of the following groups: control (n = 15) and marijuana smoke inhalation (MSI) 8 min/d (n = 15). Urine samples were obtained to detect the presence of tetra-hidro-cannabinoid. After 60 days, the animals were killed. The degree of bone-to-implant contact and the bone area within the limits of the threads of the implant were measured in the cortical (zone A) and cancellous bone (zone B).

Results: 

Tetra-hidro-cannabinoid in urine was positive only for the rats of MSI group. Intergroup analysis did not indicate differences in zone A-cortical bone (P > 0.01), however, a negative effect of marijuana smoke (MSI group) was observed in zone B-cancellous bone for bone-to-implant contact and bone area (Student's t test, P < 0.01) values.

Conclusions: 

Considering the limitations of the present study, the deleterious impact of cannabis sativa smoke on bone healing may represent a new concern for implant success/failure.

Classic long-term studies have already established the high predictability of osseointegration with the use of titanium implants.^1,2 However, some factors have been related to the failure of the osseointegrated implants that includes some systemic diseases, bone quality, surgical trauma degree, bacterial contamination, and tobacco smoke.^3–7

Cannabis sativa also known as marijuana, one of the most consumed drugs in the world,⁸ has been investigated about its influence in the immunological human function for >25 years.⁹ Marijuana is the most frequently used illicit drug in the United States and in many other countries, particularly among young people. Although marijuana usage rates are lower than those for legal drugs, such as cigarettes and alcohol, they are significantly higher than those for other illicit drugs, such as cocaine and ecstasy.¹⁰

The main psychoactive component in marijuana is the cannabinoid tetra-hidro-cannabinoid (THC, present in marijuana's leaves). It functions by activating specific cell-surface cannabinoid receptors, CB1 and CB2, which are normally engaged by a family of endogenous ligands, the endocannabinoids. Compounds that bind these receptors induce cannabimimetic responses in vivo and in vitro. Recent evidence indicates that cannabinoids might influence vascular inflammatory diseases in several ways such mediating vasodilatation particularly in lipopolysaccharide-induced diseases.¹¹ The cannabinoids can also reduce the immune response and damage the defense of the host against viral, bacterial, and protozoa's infections.¹²

Experimental models have been used for host response evaluation in cultures of cells exposed to THC and in human populations of declared consumers of marijuana. These studies suggest that the cannabinoids can affect the resistance of the host by modifying the primary and secondary immune system, especially the function of lymphocyte T and B, as well as the NK cells and macrophages.¹⁰ Moreover, cannabinoids can act as immunomodulators by modifying the production and function of cytokines in the acute phase of the inflammatory process in the phases Th1 and Th2. Animal and cell experiments have demonstrated that THC exerts complex effects on cellular and humoral immunity. THC was shown to modulate the immune response of T lymphocytes. It suppressed the proliferation of and T helper 2 (Th2) cytokines.¹¹

Considering bone metabolism, the suspect of cannabis effect was confirmed by some molecular studies, which demonstrated a negative impact on bone physiology by cannabinoids.^10,11 Thus, considering the lack of data about the effects of cannabis in bone healing around titanium implants, this study aimed to evaluate in the possible effect of marijuana smoke inhalation (MSI) on bone repair around titanium implants placed in tibias of rats.

Materials and Methods

Sample

Thirty male Wistar rats (300–400 g) were included in the study. The animals were kept in plastic cages with access to food and water ad libitum. Before the surgical procedures all animals were allowed to acclimatize to the laboratory environment for a period of 5 days. The Bahian Science Foundation Animal Care and Use Committee approved the protocol.

Legal and Ethical Aspects

After a lawyer pledge, cannabis sativa sample (1280 kg) was donated to Bahian Science Foundation by the Brazilian Federal Police Department under legal authorization by the State Secretary of Public Security and the State Secretary of Health authorities. As determined by law, all participants of the research were instructed to wear gas protective devices to avoid contamination with the cannabis smoke. Smoke exposure was conducted in an open place on the university campus specifically designated for the research.

Implant Surgery

General anesthesia was obtained by intramuscular administration of ketamine (0.5 mL/kg). Skin was cleansed with iodine surgical soap. An incision of ∼1 cm in length was made and the bone surface of the tibiae surgically exposed by blunt dissection. Under profuse saline irrigation bicortical implant beds were drilled at a rotary speed not exceeding 1500 rpm. One screw-shaped pure titanium implant prototype (4.0 mm in length and 2.2 mm in diameter) was placed until the screw thread had been completely introduced into the bone cortex. Finally, soft tissues were replaced and sutured. Postoperatively, the animals received antibiotic (Pentabiótico, Wyeth-Whitehall Ltd., São Paulo, SP, Brazil) (1 mL/kg), given through a single intramuscular injection.⁴

Experimental Design

After implant surgeries, the animals were randomly assigned to one of the following groups: control (CTRL) group (n = 15) and MSI group, 60 days of intermittent MSI (n = 15). Animals from MSI group were intermittently housed in a device consisted of an acrylic chamber (45 × 25 × 20 cm³) with an air pump and 2 inflow/outflow tubes that work as a smoke-generating apparatus. Briefly, during the 60-day experimental period, 5 animals at a time were housed once a day for 8 minutes in the chamber of exposure to marijuana smoke produced by burning 3 g of dried marijuana leaves placed in a pipe that was puffed into the chamber. Thus, the animals were forced to breathe the smoke that contaminated the air during the 60-day period. The animals of CTRL group were not exposed to the cannabis smoke at any time.

THC Analysis

At day 60 and before killing the animals, urine samples were obtained of each animal from both groups to detect THC levels by placing the rats in metabolic cages. The urine samples were analyzed by SYVA-Rapid test (Roche Diagnostics, Indianapolis, IN) that detects THC traces in urine.

Histometric Evaluation

After 60 days of smoke inhalation, all animals were killed; the tibiae were removed and fixed in 4% neutral formalin for 48 hours. Undecalcified sections were prepared as previously described,¹³ i.e., the blocks were dehydrated by using an ascending series of ethanol (60%–100%) and embedded in glycolmethacrylate (Technovit 7200; Heraeus Kulzer GmbH, Wehrheim, Germany). Subsequently, the sections (20–30 μm) were obtained and stained by 1% toluidine blue staining. The percentage of bone-to-implant contact (BIC) and bone area (BA) within the threads of the implants were obtained by a blinded examiner (Motic Images Advanced 3.0; Motic Inc., Richmond, BC, Canada). The data were arranged separately in cortical (zone A) and cancellous bone (zone B) areas, as previously described.¹⁴

Statistical Analysis

The Kolmogorov-Smirnov test was used to verify the normality of the data and a parametric test were used. An intergroup analysis tested the null hypothesis that there were no differences in bone healing rate in zones A and B (cortical and cancellous bone, respectively) between CTRL and MSI groups. After calculating all the means and standard deviation for each group, the Student's t test was applied to the data. For all of the analysis, a 1% significance level (α = 0.01) was established and data were analyzed using the BioEstat 4.0 software.

Results

The experiment finished with 23 animals instead of 30 (CTRL, n = 15 and MSI, n = 8). Seven rats died during the experiment because of respiratory failure provoked by intermittent cannabis smoke inhalation.

THC in Urine

THC was detected in urine samples from all animals from MSI group (n = 8). Negative THC detection was found in all samples from control animals. Also, according to the Brazilian Research Protocol for Illegal Drugs, all participants of the research that had contact to cannabis smoke had their urine tested. All their samples were negative to THC (Table 1).

Table 1: Tetra-hidro-cannabinoid (THC) Detected in Urine Samples From MSI and CTRL Animals and From 4 Researchers (RES) That Participated in the Cannabis Smoke Exposure

Histometric Data

As shown in Table 2, an intergroup statistical analysis did not indicate differences (P = 0.237) between groups in zone A (cortical BA) for BA (30.5 ± 17.8 μm² vs 24.8 ± 3.0 μm² for CTRL and MSI groups, respectively) and BIC (191.5 ± 96.2 μm vs 213.6 ± 49.5 μm for CTRL and MSI groups, respectively). On the other hand, with respect to the zone B (cancellous BA), data analysis showed that MSI significantly (P = 0.001) decreased bone filling (BA, 23.3 ± 9.1 μm² vs 15.61 ± 7.3 μm² for CTRL and MSI groups, respectively) and BIC (387.27 ± 176.3 μm vs 193.4 ± 84.4 μm for CTRL and MSI groups, respectively) in the treads. Figures 1–3 illustrate the bone healing around the implants for both groups.

Table 2: Intragroup Data (Means ± Standard Deviation) in Zone A (Cortical Bone) and Zone B (Cancellous Bone) for Bone Area (BA/μm²) and Bone-to-Implant Contact (BIC/μm) According to Each Experimental Group

Fig. 1: . Photomicrographs illustrating bone repair in the implant surface representative of control-CTRL (A) and marijuana-MSI (B) groups. Toluidine blue/original magnification = ×4.

Fig. 2.: Photomicrographs illustrating bone repair in the implant surface representative of control-CTRL (A) and marijuana-MSI (B) groups. Toluidine blue/original magnification = ×40.

Fig. 3.: Photomicrographs illustrating bone repair in the implant surface representative of control-CTRL (A) and marijuana-MSI (B) groups. Toluidine blue/original magnification = ×100.

Discussion

The present study demonstrated that MSI had a deleterious impact on cancellous bone healing around titanium implants by reducing bone filling and BIC inside the implant threads whereas no effect was observed in the cortical bone. The negative effects of cigarette smoking on periodontitis and around titanium implants have been demonstrated by the authors^14–16 where cigarette smoking seems to reduce bone capacity to formation and/or increasing resorption. Despite the limitations of the present study, it is the first one, by our knowledge, that demonstrates the negative impact of cannabis sativa smoke on bone healing around titanium implants.

According to Tam et al,¹⁷ in vertebrates, bone mass and shape are determined by continuous remodeling consisting of the concerted and balanced action of osteoclasts, the bone resorbing cells, and osteoblasts, the bone forming cells. First, endocannabinoids are expressed by immune cells and that cannabinoid receptors (CB1 and CB2) at the surface of immune cells are activated after infection or immune stimulation.^18,19 The second general principle is that the main immune targets of cannabinoid-based drugs involve the suppression of cytokines and cell-mediated immunity, through cannabinoid receptor-dependent and -independent mechanisms.¹⁰ The results of the present study clearly demonstrated that marijuana smoke can decrease bone mass around titanium implants in the cancellous zone. As a matter of fact, to explain such phenomenon some observations might be addressed considering the CB1/CB2 receptor system of the cannabinoids.

Bone, especially trabecular, is densely innervated by sympathetic fibers.^20,21 These fibers release norepinephrine, thus potently mediating central signals that restrain bone formation and stimulate bone resorption.²² Because CB1 is expressed in such nerve fibers elsewhere,²³ it was further explored its presence in bone sympathetic nerve fibers. Indeed, immunohistochemical analysis using the sympathetic marker TH²⁴ confirmed the occurrence of a network of TH-positive fibers in the intertrabecular spaces of cancellous bone in mice. The fibers were close to the bone trabeculae with terminal nerve processes penetrating the osteoblast palisades, thus being in intimate proximity to these cells, indicating the presence of CB1 receptors in sympathetic fibers that innervate the trabecular bone.

On the other side, CB2 receptors are located principally in immune cells, among them leukocytes, spleen, and tonsils. Immune cells also express CB1 receptors but there is markedly more mRNA for CB2 than CB1 receptors in the immune system.¹¹ One of the functions of CB receptors in the immune system is modulation of cytokine release.^19,25 Activation of the CB1 receptor produces marijuana-like effects on psyche and circulation, whereas activation of the CB2 receptor does not. CB2 is expressed in most hematopoietic cells, including macrophages, and attenuates immune responses. In line with these reports, it was found that osteoclasts, cells derived from the monocyte-macrophage lineage, have high levels of CB2 mRNA.²⁶ Specific cannabinoids as the CB2 is also characterized by increased activity of trabecular osteoblasts (bone-forming cells), increased osteoclast (the bone-resorbing cell) number, and a markedly decreased number of diaphyseal osteoblast precursors.²⁷ CB2 is expressed in osteoblasts, osteocytes, and osteoclasts.²⁶

Our results were not able to present any disturb in the cortical bone around the implants placed in the rats that inhaled the marijuana smoke. Interestingly, this phenomenon could be explained by the presence of a CB2-specific agonist that does not have any psychotropic effects, which enhances endocortical osteoblast number and activity.²⁶

In our histomorphometric analysis, the cannabis sativa smoke inhalation group (MSI) presented lower values of BA in the threads of the implants when compared with the control group. Marijuana inhalation somehow seems to impair the healing of the cancellous bone probably because of THC effect in inhibiting CB2 expression on osteoblasts and consequently reducing bone formation. In the same way, the reduced BIC values in the MSI group may also suggest that the occurrence of a restraining in the trabecular osteoclastogenesis, apparently because of the inhibition of the proliferation of osteoclast precursors and receptor activator of NF-κB ligand expression in bone marrow-derived osteoblasts/stromal cells.^25,28 Moreover, it was demonstrated that endocannabinoids in gingival tissue can regulate the periodontal inflammation through NF-κB pathway inhibition.²⁹ The endocannabinoid system seems to participate in bone physiology by regulating bone mass, bone loss, and osteoclast activity by signaling via the peripheral CB2 expressed in bone and its effect decreased the bone healing around the implants in the experimental rats from cannabis group.³⁰

As long as it is well established that titanium implant success is a reality and only a few situations, such as cigarette smoking, may cause implant failure, this article is the first in the literature to raise the concern about the consumption of marijuana as a possible factor to explain some implant failures in patients that do not always inform the dentist that they are marijuana users. Nevertheless, further epidemiological and molecular studies are mandatory to confirm such suspect.

Conclusions

Marijuana smoke decreases bone filling around titanium implants by impairing trabecular bone formation in a histometric study in rats. The deleterious impact of cannabis sativa smoke on bone healing may represent a new concern for implant success/failure.

Disclosure

The authors claim to have no financial interest, directly or indirectly, in any entity that is commercially related to the products mentioned in this article.

Acknowledgments

The authors thank The National Council for Scientific and Technological Development (CNPq), a foundation linked to the Ministry of Science and Technology (MCT), for supporting this research (grant 308114/2004-3); Bahian Science Foundation (FBDC) for providing the animal facility; and The Federal Police Department of Brazil, Public Security Secretary and The Health State Secretary authorities for providing legal use of cannabis sativa sample; Dr. Daiane Peruzzo for statistical analysis and Dr. Roberto Vieira for the histomorphometric support.