Alltogether

i An update to this article is included at the end
Materials Science & Engineering C 101 (2019) 64–75
Contents lists available at ScienceDirect
Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec
Cannabidiol-loaded microspheres incorporated into osteoconductive T

scaffold enhance mesenchymal stem cell recruitment and regeneration of
critical-sized bone defects
⁎
Amir Kamalia,b, Ahmad Oryana, ,1, Samaneh Hosseinib, Mohammad Hossein Ghanianc,
⁎⁎
Maryam Alizadehc, Mohamadreza Baghaban Eslaminejadb, ,2, Hossein Baharvandb
a
Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
b
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
c
Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
A R T I C LE I N FO A B S T R A C T
Keywords: Recruitment of mesenchymal stem cells (MSCs) to an injury site and their differentiation into the desired cell
Cannabidiol lineage are implicated in deficient bone regeneration. To date, there is no ideal structure that provides these
MSCs migration conditions for bone regeneration. In the current study, we aim to develop a novel scaffold that induces MSC
Controlled release migration towards the defect site, followed by their differentiation into an osteogenic lineage. We have fabri-
Bone regeneration
cated a gelatin/nano-hydroxyapatite (G/nHAp) scaffold that delivered cannabidiol (CBD)-loaded poly (lactic-co-
Tissue engineering
glycolic acid) (PLGA) microspheres to critical size radial bone defects in a rat model. The fabricated scaffolds
were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM), and then analyzed for
porosity and degradation rate. The release profile of CBD from the PLGA microsphere and CBD-PLGA-G/nHAp
scaffold was analyzed by fluorescence spectroscopy. We performed an in vitro assessment of the effects of CBD
on cellular behaviors of viability and osteogenic differentiation. Radiological evaluation, histomorphometry, and
immunohistochemistry (IHC) analysis of all defects in the scaffold and control groups were conducted following
transplantation into the radial bone defects. An in vitro migration assay showed that CBD considerably increased
MSCs migration. qRT-PCR results showed upregulated expression of osteogenic markers in the presence of CBD.
Histological and immunohistochemical findings confirmed new bone formation and reconstruction of the defect
at 4 and 12 week post-surgery (WPS) in the CBD-PLGA-G/nHAp group. Immunofluorescent analysis revealed
enhanced migration of MSCs into the defect areas in the CBD-PLGA-G/nHAp group in vivo. Based on the results
of the current study, we concluded that CBD improved bone healing and showed a critical role for MSC mi-
gration in the bone regeneration process.
1. Introduction exogenous cells with biomaterials to improve the healing process of

large-sized defects [4,5].
Bone healing is a complicated biological process that consists of an MSCs have the capability to differentiate into an osteoblastic cell
initial inflammatory response followed by recruitment and differ- lineage. They release cytokines which modulate the inflammatory re-
entiation of mesenchymal stem cells (MSCs) [1,2]. Incomplete response and induce production of various growth factors [3]. However,
generation of critical-sized bone defects are principally related to the limitations to the in vivo use of exogenous MSCs for regeneration of
inadequate migration of MSCs into the defect site and inability of the critical-sized bone defects include poor proliferation and osteogenic
migrated MSCs to fully differentiate into osteogenic precursor cells [3]. diffrentation [6]. Hence, development of a novel scaffold that can re-
Numerous bone substitutes have been developed that combine cruit MSCs to an injury site and allow them to differentiate into
⁎
Corresponding author.
⁎⁎
Correspondence to: M. B. Eslaminejad, Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology
and Technology, Tehran, Iran.
E-mail addresses: Oryan@shirazu.ac.ir (A. Oryan), Eslami@royaninstitute.org (M. Baghaban Eslaminejad).
1
P.O. Box 1731, Shiraz 71345, Iran.
2
P.O.Box: 16635-148, Tehran, Iran.
https://doi.org/10.1016/j.msec.2019.03.070
Received 3 February 2018; Received in revised form 5 March 2019; Accepted 21 March 2019
Available online 24 March 2019
0928-4931/ © 2019 Elsevier B.V. All rights reserved.
A. Kamali, et al. Materials Science & Engineering C 101 (2019) 64–75
Fig. 1. Schematic representation of various groups and subsequent treatment protocols; Sc: scaffold, Se: segmental defect, nHAp: nano-hydroxyapatite, CBD: can-
nabidiol, PLGA: poly (lactic-co-glycolic acid).
osteogenic lineages would be an alternative strategy to exogenous osteogenic processes [23]. Schmuhl et al. have shown that CBD en-
MSCs. hanced migration of MSCs via activation of P42/44 MAPK, and subse-
Stromal cell-derived factor-1 (SDF-1) is a chemokine protein en- quently induced MSC differentiation into an osteoblastic lineage under
coded by the C-X-C motif chemokine ligand 12 (CXCL12) gene. Binding in vitro conditions [14]. Systemic delivery via an intraperitoneal in-
of SDF-1 to its main receptor [C-X-C chemokine receptor type 4 jection of CBD on bone healing was examined in a rat fracture model
(CXCR4)] triggers migration of MSCs and other progenitor cells into the [19]. In this study, the researchers observed that CBD resulted in
injury site [7]. SDF-1 has been combined with scaffolds or loaded into fracture healing and improved biomechanical properties of the fracture
microspheres to facilitate migration of MSCs into large bone defects callus by enhanced PLOD1 gene expression, which encodes an enzyme
[8–10]. Because SDF-1 is susceptible to degradation, denaturation, and involved in collagen crosslinking [19].
inactivation [11,12], bioactive molecules have been introduced as al- In the current study, we fabricated a novel scaffold to recruit MSCs
ternatives [13]. Cannabidiol (CBD) is a bioactive molecule that can into large bone defects by using CBD-loaded microspheres incorporated
increase in vitro MSC migration [14]. CBD is one of the known 113 into an osteoconductive scaffold. Next, we analyzed the short- and long-
active cannabinoid compounds identified in cannabis, a phytocanna- term osteogenic activities of CBD under in vitro and in vivo conditions
binoid [15]. The pharmacological effects of cannabinoid compounds by real-time PCR, histopathology, histomorphometry, im-
include analgesia, muscle relaxation, immunosuppression, anti-in- munohistochemistry (IHC), and micro-computed tomography (micro-
flammatory, anti-allergic, neuroprotection, and antineoplastic effects CT) scan.
[16]. Numerous clinical trials have been conducted with cannabinoid
compounds as treatment for Dravet syndrome and epilepsy [17,18]. 2. Materials and methods
According to research, the cannabinoid ligands efficiently improved
ovariectomy-induced bone loss and enhanced fracture healing [19,20]. 2.1. Materials
CBD receptors (CB1 and CB2) are important mediators of the tissue
healing process [21,22]. Most of the cannabinoid impacts on the bone CBD was obtained from Tocris Company (Tocris, Bio-Techne
and skeletal system are attributed to these receptors and their ligands. Corporation, USA). Poly (lactic-co-glycolic acid) (PLGA, 50:50,
In particular, CB2 signaling has a regulatory effect on several pro- Resomer® RG 503H, inherent viscosity: 0.32–0.44 dl/g), polyvinyl
65
Fig. 2. SEM micrographs of the CBD-free G/nHAp (A, a), CBD-PLGA microspheres (B, b) and CBD-PLGA-G/nHAp (C, c) scaffolds prepared by the freeze-drying
method. The G/nHAp scaffold show a homogeneous porous structure.
alcohol (PVA, mw 30,000–70,000), Tween 20, and dichloromethane 2.2.2. Fabrication of the CBD-PLGA microspheres
(DCM) were purchased from Sigma-Aldrich (USA). Gelatin (Gel-bovine We used a single emulsion (O/W) solvent evaporation method to
skin, type B, isoelectric point ~5) was purchased from Sigma-Aldrich prepare PLGA microspheres that contained CBD. Briefly, 30 mg of
(St. Louis, MO, USA). N-hydroxysuccinimide (NHS, 97%) was obtained polymer and 3 mg of CBD were dissolved in 2 ml of DCM. This oil phase
from the Aldrich Chemical (Milwaukee, WI, USA). 1-Ethyl-3-(3-di- was added to 5 ml of an aqueous solution of 1% w/v PVA that contained
methylaminopropyl) carbodiimide hydrochloride (EDC) was purchased Tween 20, and homogenized for 3 min at 7000 rpm (Heidolph Silent
from Sigma-Aldrich (Milano, Italy). Crusher M, Germany) in an ice bath. The emulsion was immediately
added to 100 ml of an aqueous solution of PVA (0.1% w/v). The ex-
traction step was followed by stirring for 3 h at 300 rpm at room tem-
2.2. Methods perature to allow the organic solvent to evaporate and for solidification
of the microspheres. The microspheres were collected by centrifugation
2.2.1. Scaffold preparation for 20 min at 9000 rpm and washed 3 times with DW to remove the
In order to fabricate the G/nHAp scaffold, we added 0.1 g nHAp to residual surfactants. The microspheres were frozen at −20 °C, lyophi-
20 ml of 5% w/v gelatin solution. The solution was stirred for 12 h at lized, and stored at −20 °C until use.
37 °C. The resultant gel was maintained at −20 °C for 24 h, and then
freeze-dried. EDC and NHS (in a 2:1 ratio) were dissolved in acetone 2.2.3. Fabrication of the blank and CBD-PLGA microsphere incorporated
[90% v/v in distilled water (DW)] and then added the freeze-dried gel scaffolds
to chemically crosslink the scaffolds. The scaffolds were washed in DW, The CBD-PLGA microsphere incorporated scaffold (CBD-PLGA-G/
freeze-dried, and sterilized in 70% ethanol and UV light for subsequent nHAp) was prepared using a previously described post-seeding tech-
experiments. nique [24]. Briefly, 1 mg of CBD-PLGA was suspended in 1 ml DW. We
66
Fig. 3. Characterization of bioscaffolds; A) in vitro release of CBD from PLGA microsphere and CBD-PLGA-G/nHAp scaffold in PBS during 25 days. B)
Biodegradability of G/nHAp scaffold. C) Effects of the scaffolds on mRNA expression of the OCN, ALP, and ColI genes on day 21.
Fig. 4. Macroscopic and diagnostic imaging (X-ray and Micro CT) of the radial bone defects 4 and 12 week post-surgery (micro-CT scan data obtained only at 12 week
post-surgery).
Table 1
Macroscopic scores of the healed defects at 12 weeks.
Type of evaluation Untreated defect (1) Median (min- Autograft (2) Median (min- G/nHAp (3) Median (min- CBD-PLGA-G/nHAp (4) Median (min- Pa
max) max) max) max)
Macroscopic 1 (0-1) 3 (3-3) 1 (1-1) 2 (2-3) 0.003
G: gelatin, nHAp: nano hydroxyl apatite, CBD: cannabidiol, PLGA: poly (lactic-co-glycolic acid) microsphere.
Complete union (+3 score), presence of cartilage (+2 score), presence of soft tissue (+1 score), nonunion (0 score).
P < 0.01 (1 vs. 2 and 4), (3 vs. 2); P < 0.05 (3 vs. 4).
a
Kruskal–Wallis non-parametric ANOVA.
67
Table 2
Results obtained from radiographical evaluations of the regenerated defects at 4 and 12 weeks.
Post-operative weeks Untreated defect (1) Median (min- Autograft (2) Median (min- G/nHAp (3) Median (min- CBD-PLGA-G/nHAp (4) Median (min- Pa
max) max) max) max)
4 0 (0-0) 4 (5-6)b 2 (1-2)d 3 (3-5)f 0.002

12 1 (0-1) 8 (7-9)c 4 (3-5)e n (6-7)g 0.003
b,c
P < 0.01 (2 vs. 1) and b,cP < 0.05 (2 vs. 3); d,eP < 0.05 (3 vs. 1); f,gP < 0.05 (4 vs. 1 and 3).
a
Kruskal–Wallis non-parametric ANOVA.
Table 3 load cell.

Micro-CT scan analysis of bone defect sites.
Group BV (Mean ± SD) TV (Mean ± SD) BV/TV (%) 2.2.7. Scanning electron microscopy (SEM)
Untreated defect 0.34 ± 0.06 2.93 ± 0.43 11
We used a scanning electron microscope (Crossbeam®, 1540XB by
Autograft 1.41 ± 0.44 1.48 ± 0.56 95⁎⁎ Zeiss) to examine the surface and internal 3D architecture of the fab-
G/nHAp 0.67 ± 0.05 1.83 ± 0.18 36⁎ ricated scaffolds. Pore sizes were measured in a minimum of 100 pores
CBD-PLGA-G/nHAp 1.38 ± 0.82 1.49 ± 0.66 92⁎⁎ per scaffold. We used scanning electron microscopy (SEM) to determine
the mean particle size and microsphere distribution on the scaffold.
BV = bone volume (mm3), TV = total volume (mm3), No. of slices for evalua-
tion = 200, slice thickness = 6 μm. *,**: indicates treatment group versus un-
treated group. G: gelatin, nHAp: nano hydroxyl apatite, CBD: cannabidiol, 2.2.8. In vitro release of CBD from the PLGA microspheres and CBD-PLGA-
PLGA: poly (lactic-co-glycolic acid) microsphere.
⁎⁎
G/nHAp
P < 0.001.
⁎ We prepared a suspension of microspheres with a final concentra-
P < 0.01.
tion of 1 mg/ml in phosphate buffer saline (PBS, pH 7.4), which was
subsequently incubated at 37 °C under constant agitation at 50 rpm.
dropped 500 μl of the suspension onto one side of the G/nHAp scaffold
The released medium was entirely withdrawn at pre-determined time
and 500 μl of the suspension onto another side of the scaffold. Finally,
points followed by the addition of fresh PBS. The release of CBD from
the microsphere-scaffold composite was lyophilized and stored at
the CBD-PLGA-G/nHAp scaffold was carried out by immersing the
−20 °C.
scaffolds in PBS at 37 °C. The release medium was collected at the same
time points as the microspheres. The concentration of CBD in the su-
2.2.4. Evaluation of porosity
pernatants was determined by fluorescence spectroscopy at an excita-
We used the liquid displacement method to evaluate porosity of the
tion wavelength of 280 nm and an emission wavelength of 307 nm. The
fabricated scaffolds [25]. Briefly, the porous scaffolds were immersed in
release profile was determined based on the following equation:
a graduated cylinder that had an initial volume of absolute ethanol (V1)
and incubated for 5 min. The total volume of ethanol and the sample m
was recorded as V2. V3 was defined as the residual level of absolute Released drug(%) = ⎡ t ⎤ × 100
⎢ m0 ⎦
⎣ ⎥
ethanol after removal of the scaffold. We calculated the porosity of the
scaffolds according to the following equation: where, mt was the mass of released CBD at each time interval and m0
(V − V3) was the total mass of CBD in the microspheres. The total drug content of
P% = 1 × 100 microspheres was measured by dissolving the desired amount of mi-
(V2 − V3)
crospheres in dichloromethane followed by extraction of CBD with an
Each scaffold was analyzed in triplicate. equal volume of water. The encapsulation efficiency was calculated
based on the following equation:
2.2.5. Biodegradation analysis
Biodegradability of the G/nHAp scaffold was determined according m
Encapsulation efficiency (%) = ⎡ m ⎤ × 100
to a previously described protocol [26]. Briefly, the scaffolds ⎢
⎣ mf ⎥
⎦
(1.5 × 1.5 cm) were immersed in a simulated body fluid (SBF) solution
of 0.2 ml of SBF/mm3 of the scaffold at 37 °C. We removed the samples where, mm was the measured mass of drug in microspheres and mf was
from the SBF at different time points (3, 7, 14, 21, and 28 days) and the mass of drug consumed for fabrication of the microspheres.
washed them with DW. All samples were freeze-dried and analyzed for
changes in morphology and weight loss. We calculated the biode- 2.2.9. Quantitative RT-PCR (qRT-PCR) analysis
gradation rate of the G/nHAp scaffold according to the following We seeded the MSCs onto the scaffolds and allowed to incubate for
equation: 21 days. Total RNAwas extracted from the cells using the RNeasy Micro
D − D2 ⎤ Kit (Qiagen, 74004). cDNA was synthesized by using the Revert Aid
D% = ⎡ 1 × 100 First Strand cDNA Synthesis Kit (Fermentas, Sankt Leon-Rot, Germany,
⎣ D1 ⎥
⎢ ⎦
k1632) according to the manufacturer's instructions. The qRT-PCR re-
where, D1 was the original scaffold weight and D2 represented the action was performed with the SYBR Green Master Mix (Origene,
weight of the freeze-dried sample after its removal from SBF. We ana- Rockville, MD, USA) using a real-time PCR system (Applied Biosystems
lyzed 3 independent samples for each group. Life Technologies, Inc., ABI StepOnePlus) and analyzed with StepOne
software (Applied Biosystems, version 2.1). Relative quantification was
2.2.6. Mechanical analysis of the fabricated scaffold performed by the comparative CT method (2−ΔΔCt method), where a
The compressive strength and modulus of the composites were number of target genes were normalized to an endogenous control
measured at room temperature. We tested the specimen (cylindrical (GAPDH) and relative to a calibrator group (control group-2D culture
disk: 6 mm thickness × 12 mm diameter) with a universal tensile ma- flask). All reactions were performed in duplicate and we collected all of
chine (Santam, IRI) at a crosshead speed of 1 mm/min with a 1000 N the samples from 3 biological replicates. Table S1 lists the primers.
68
Fig. 5. Histopathological sections from the radial bone defects in rats at 4 week post-operation. There are still some remnants of the CBD-free G/nHAp scaffold, while
the CBD-PLGA-G/nHAp scaffolds were completely degraded and almost replaced with new tissues including fibrous, cartilage and bone tissues. Minimum healing of
the defect site was seen in the untreated defect group. LACT: loose areolar connective tissue; FT: fibrous connective tissue; OB: old bone; BM: bone marrow; BV: blood
vessels, G: Gap; NB: new bone formation, BG: Bone graft, Ch: chondrocyte; HC: hyaline cartilage; WB: woven bone; SC: scaffold residue; O: osteoblast; Os: osteon, FB:
fibroblast, Stained with H&E.
2.2.10. Animals and surgical procedures 2.2.11. Gross morphology

A total of 40 adult male Wistar rats (200–250 g) were purchased We performed macroscopic evaluation for the presence of re-
from Razi Institute, Karaj, Iran. All animals were anesthetized by in- generation of the radial bone defects in each group. The results were
tramuscular (IM) injections of ketamine hydrochloride (2 mg/kg) and blindly scored, as follows: 0 (non-union defects without instability), +1
Xylazine (1 mg/kg). Next, we made a bilateral 3 cm incision over the (incomplete union with presence of fibrous connective tissue within the
rats' forearms and, subsequently, 5 mm of the radius diaphysis was defect), +2 (incomplete union with presence of cartilage within the
ostectomized with an electrical bone saw (Strong Co., Seoul, South defect), and + 3 (complete union with presence of the bridging bone)
Korea) under normal saline irrigation. The ulnar bones were left intact [27].
as a natural protector of the defect site. The bone defects (20 defects/
group) were untreated or treated with either an autograft (CBD-free G/ 2.2.12. Radiological evaluation
nHAp scaffold) or CBD-PLGA-G/nHAp in the defect areas Plain lateral X-rays (35 kV, 1.5 mA for 3 s) were taken of each radial
(2 × 2 × 5 mm3) as seen in Fig. 1. The animals received 1 mg/kg me- bone at 4 and 12 WPS. Each radiograph was scored according to a
loxicam as post-operative analgesia and antibiotic therapy of 10 mg/kg previously described scoring system to evaluate the healing process of
enrofloxacin. This experiment was approved by the local Ethics Com- the radial bone defect [28].
mittee of “Regulations for using animals in scientific procedures” in the
school of Veterinary Medicine, Shiraz University, Shiraz, Iran. The an- 2.2.13. Micro-computed tomography (micro-CT)
imals were euthanized 1, 4, and 12 week post-surgery (WPS). Their The harvested radial bone samples were assessed by a Scanco micro-
radial bones were harvested and used for subsequent experiments. CT 35 scanner (Scanco, Wangen-Brüttisellen, Switzerland) at 70 kVP,
69
Fig. 6. Histopathological sections from the radial bone defects in rats 12 week post-operation. The CBD-free G/nHAp was degraded over time and replaced by new
tissues (fibrous connective tissue, cartilage, and osseous tissue). The maximum similarity to the autograft was seen in the CBD-PLGA-G/nHAp group, in which the
defect site was completely filled with new bone and cartilage tissue. FT: fibrous connective tissue; OB: old bone; BM: bone marrow; NB: new bone formation, BG:
Bone graft, CT: connective tissue; OC: osteocyte; HC: hyaline cartilage; CB: compact bone; MB: mature bone, SC: scaffold residue; Os: osteon, Stained with H&E.
114 μA for 800 ms. We calculated bone volume (BV), total volume (TV), 2.2.15. Immunohistochemistry (IHC) analysis
and the BV/TV ratio (%) based on the data obtained from the micro-CT IHC was performed to detect osteogenic and angiogenic differ-
scans. entiation of the recruited cells at the defect site (12 WPS). Histological
slides were incubated with citrate buffer (Dako, Glostrup, Denmark) at
60 °C for heat-induced epitope retrieval and blocked with 1% hydrogen
2.2.14. Histopathologic and histomorphometric studies peroxide/methanol (Sigma-Aldrich, St Louis, MO, USA) for 30 min at
The bone tissues from all groups were fixed in 10% neutral buffered room temperature. Subsequently, they were incubated overnight at 4 °C
formalin (NBF) solution for 48 h and then decalcified with 14% EDTA with primary antibodies OCN (ab13420, Abcam, MA, USA), OPN
(pH 7.4) for 28 days. The decalcified bone samples were subsequently (ab8448, Abcam, MA, USA), and collagen type I (ColI, sc-59772, Santa
embedded in paraffin and cut into 5 μm sections. Then, they were Cruz Biotechnology, CA, USA). The color reaction was developed with
stained with hematoxylin and eosin (H&E). The histological sections ready-to-use 3,3′-diaminobenzidine (Dako Liquid DAB) color solution.
were examined by a light microscope (Olympus BX51; Olympus, Tokyo, The slides were counterstained with hematoxylin and visualized by a
Japan) and Evaluated by an independent pathologist who was blinded light microscope (Olympus BX51; Olympus, Tokyo, Japan).
to the study groups. We evaluated the migration of MSCs into the defect area. We did
Immunofluorescence labeling of the bone sections (1 WPS) overnight at
70
Table 4
Histomorphometric results of the defect sites 12 week post-surgery.
Value Untreated defect (1) (Mean ± SD) Autograft (2) (Mean ± SD) G/nHAp (3) (Mean ± SD) CBD-PLGA-G/nHAp (4) (Mean ± SD) Pa
Fibrocyte + fibroblastb 165.1 ± 44.2 7.5 ± 1.5 54.4 ± 5.4 9.4 ± 2.3 0.002
Chondroblast + chondrocytec 3.1 ± 0.7 18.2 ± 4.8 35.4 ± 6.9 13.5 ± 2.6 0.001
Osteoblast + osteocyted 5.2 ± 1.6 171.3 ± 15.5 58.1 ± 7.3 184.8 ± 17.6 0.000
Osteoclaste 0.00 4.4 ± 1.2 1.3 ± 0.5 3.1 ± 1.7 0.001
Blood vesselsf 22.4 ± 8.1 7.3 ± 2.9 16.8 ± 5.2 9.4 ± 2.6 0.000
Osteong 0.00 11.7 ± 2.5 1.3 ± 0.9 8.6 ± 1.5 0.001
a
One-way ANOVA followed by Tukey post-hoc test.
b
P < 0.01 (1 vs. 2 and 4); P < 0.05 (3 vs. 2 and 4), (1 vs. 3).
c
P < 0.01 (1 vs. 2, 3 and 4); P < 0.05 (3 vs. 2 and 4).
d
P < 0.01 (1 vs. 2 and 4); P < 0.05 (3 vs. 2 and 4), (1 vs. 3).
e
P < 0.05 (3 vs. 2 and 4), (1 vs. and 2, 3 and 4).
f
P < 0.01 (1 vs. 2 and 4); P < 0.05 (3 vs. 2 and 4), (1 vs. 3).
g
P < 0.05 (3 vs. 2 and 4), (1 vs. and 2, 3 and 4).
4 °C with rat CD90 (ab225, Abcam) and CD29 (NBP2-16974, Novus Fig. S4 shows the mechanical strength of the fabricated scaffolds.
Biologicals, San Diego, CA, USA) antibodies. After 3 washes with PBS, The compressive modulus results for the scaffolds were: 1.98 ± 0.06
the sections were incubated with donkey anti-mouse AlexaFluor 488 (CBD-free G/nHAp) and 2.17 ± 0.48 (CBD-PLGA-G/nHAp).
(Abcam) and goat anti-mouse FITC (Abcam) secondary antibodies for
1 h at room temperature. The labeled sections were then washed and 3.2. Cell viability assay
mounted with 100 mM glycine in PBS. The sections were imaged on a
fluorescence microscope (Olympus BX51; Olympus, Tokyo, Japan). Isolated cells were characterized to cofirm their mesenchymal
phentotype (Fig. S5). We conducted the MTT assay at 1, 3, and 7 days to
2.2.16. Statistical analysis confirm MSC viability in the presence of different concentrations of
The quantitative data were analyzed by one-way ANOVA with CBD (Fig. S6). Based on the MTT results, we did not find any changes in
subsequent Tukey post-hoc tests. Non-parametric ANOVA and the cell viability in the presence of different concentrations of CBD.
Kruskal-Wallis test were used for statistical analysis of the qualitative
data obtained from the scored values. If the differences were significant 3.3. In vitro release of CBD
(P < 0.05), then the data were analyzed by the Mann-Whitney U test.
All statistical analyses were assessed with GraphPad Prism software, Fig. 3B shows the in vitro release profile of CBD from the PLGA
version 6.00 (Graphpad Prism, Inc., San Diego, CA, USA). microspheres and CBD-PLGA-G/nHAp scaffold over 25 days. The en-
capsulation efficiency of CBD in the microspheres was 70.12 ± 4.46%.
The cumulative release profile showed a continuous release of CBD
3. Results
from both the PLGA microspheres and CBD-PLGA-G/nHAp scaffold.
However, there was a burst release during the first 48 h from the PLGA
3.1. Characterization of the scaffolds and CBD-PLGA microsphere
microspheres while the CBD release rate was considerably reduced by
incorporation into the scaffold (P < 0.05). By 25 days, 71.25 ± 3.28%
SEM micrographs of the CBD-free G/nHAp revealed proper inter-
of the total CBD was released from the PLGA microspheres. After
connectivity and large-sized pores in all fabricated scaffolds (Fig. 2A).
25 days, 44.37 ± 4.15% of the CBD was released from the scaffolds.
The CBD-free G/nHAp and CBD-PLGA-G/nHAp scaffolds showed the
porosity of 85.3 ± 3.4% and 89.5 ± 2.1%, respectively. The mean
pore size for CBD-free G/nHAp and CBD-PLGA-G/nHAp was 3.4. qRT-PCR analysis
345.3 ± 9.8 μm and 353 ± 7.2 μm. XRD SEM patterns of the G/nHAp
scaffold showed that the angles of diffraction peaks for the Ca-P groups We used RT-PCR analysis to assess the level of osteogenic-related
were similar to HA. Analysis of the XRD patterns revealed the presence genes expressed by the MSCs seeded onto the fabricated scaffolds (CBD-
of crystalline phases consistent with phases listed in the ICDD database free G/nHAp, CBD-PLGA-G/nHAp) after 21 days (Fig. 3C). CBD-PLGA-
(Fig. S1). G/nHAp showed a higher expression level of OCN compared to the
The presence of CBD in the PLGA microspheres was confirmed by CBD-free G/nHAp and control (2D cell culture) groups (P < 0.05).
fluorescence microscopy (Fig. S2). The PLGA microspheres did not emit MSCs cultured in the CBD-free G/nHAp expressed a higher level of ALP
fluorescence; however, the CBD-PLGA had a sharp fluorescence in- compared to the other groups (P < 0.01). ColI had more expression in
tensity. Fig. 2B and C show SEM micrographs of the CBD-PLGA mi- the CBD-PLGA-G/nHAp scaffold followed by the CBD-free G/nHAp
crospheres and their distribution within the G/nHAp scaffold, respec- scaffold in comparison to the control (P < 0.05).
tively. The microsphere surfaces were quite smooth with no observed
pores. The microspheres had a mean size of 11.6 ± 1.3 μm. The CBD- 3.5. Assessment of bone formation by gross morphology, radiology, and
PLGA microspheres were uniformly distributed in the porous scaffold micro-CT scan
and had infiltrated into the scaffold (Fig. S3). There were no significant
differences observed in the microspheres before and after incorporation We evaluated gross views of the harvested radial bones after 4 and
into the scaffold. 12 WPS (Fig. 4), and macroscopically scored them based on the newly
We immersed the G/nHAp scaffold in SBF solution at different time formed tissues at 12 WPS (Table 1). The defects treated with the CBD-
points to evaluate the biodegradation rates as percent of scaffold free G/nHAp group were replaced by fibrous and cartilaginous-like
(Fig. 3A). The rates at different time points were: 4.4 ± 1.7% (3 days), tissues. The CBD-PLGA-G/nHAp was completely filled with bony-like
7.2 ± 0.8% (7 days), 18.6 ± 3.2% (14 days), 26.5 ± 2.4% (21 days), tissue, which was similar to the autograft. In contrast, the defect site in
and 30.7 ± 3.1% (28 days). the untreated group remained either empty or only filled with fibrous
71
A. Kamali, et al.
72
Fig. 7. Immunostaining of the injured area in different treatment groups for bone regeneration and angiogenesis. Immunohistochemical analysis of the ColI, OCN, and OPN genes was used to determine the osteogenesis
and angiogenesis in the samples. The brown color represents positive staining for ColI, OCN, and OPN. **P < 0.01, *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
Materials Science & Engineering C 101 (2019) 64–75
Fig. 8. Migration of the MSCs was also examined by immunostaining method. CBD treatment led to the enhanced recruitment of CD90+/CD29+ cells to the defect
site, **P < 0.01, *P < 0.05.
connective tissue. The macroscopic scores were higher in both the CBD- We conducted IHC analysis to evaluate Col I, OCN, and OPN os-
PLGA-G/nHAp scaffold and autograft groups compared to the CBD-free teogenic related markers (Fig. 7). IHC results showed increased ColI
G/nHAp scaffolds (P < 0.01). No significant differences existed be- protein expression in the autograft and CBD-PLGA-G/nHAp groups
tween the autograft and CBD-PLGA-G/nHAp groups in terms of mac- compared to the other groups (P < 0.05). Both OPN and OCN highly
roscopic union scores (P > 0.05). expressed in the autograft and CBD-PLGA-G/nHAp groups (P < 0.05).
Fig. 4 and Table 2 show the radiographs and related scores from the There were no OCN and OPN protein expressions detected in the un-
experimental groups. The X-ray scores revealed superior new bone treated defect. To confirm recruitment to the defect area in response to
formation and unions in the CBD-PLGA-G/nHAp and autograft groups local delivery of CBD, the bone tissue sections were stained for the MSC
compared to the CBD-free G/nHAp scaffolds and untreated groups at 4 markers CD90 (green) and CD29 (red) as shown in Fig. 8 [27]. The 2
and 12 WPS (P < 0.05). We observed more significant bone union in markers showed highly localized MSCs in the defect site. The CBD-
the CBD-free G/nHAp group compared to the untreated group PLGA-G/nHAp treated animals had higher numbers of MSCs compared
(P < 0.05). The bone gap was radiopaque in both CBD-PLGA-G/nHAp to the CBD-free G/nHAp group.
and autograft groups 12 WPS in contrast to the CBD-free G/nHAp and Table S2 lists the parameters of ultimate load (N), stress (N/mm2),
untreated defect, which were almost radiolucent with a range of opa- strain (%) and stiffness (N/mm) obtained from the biomechanical
cities. testing. The autograft and CBD-PLGA-G/nHAp groups showed the
We conducted micro-CT scans to characterize the newly-formed highest ultimate load, stress, and stiffness compared to the other groups
tissues in the 3D images at 12 WPS (Fig. 4, Table 3). The BV/TV ratio, (P < 0.01), whereas the untreated defects showed the greatest strain
as an index of new bone formation, was significantly higher in the CBD- (P < 0.05).
PLGA-G/nHAp (92%) and autograft (95%) groups followed by the CBD-
free G/nHAp (36%) scaffold and untreated defect (11%) groups
(P < 0.05). 4. Discussion
Cannabinoids supposedly possess healing properties that are medi-

3.6. Histopathologic, histomorphometric, and immunohistochemical ated through recruitment of regenerative cells into injury sites
findings [14,29,30]. The contribution of cannabinoids in bone reconstruction
and their pharmacological effects on stem cell migration is un-
Histological analysis of the radial bones from the experimental determined. We have selected the G/nHAp porous scaffold as an os-
groups was performed at 4 (Fig. 5) and 12 (Fig. 6) WPS. There was a teoconductive vehicle for local delivery of CBD to explore its bone
higher degree of new bone formation and cartilaginous tissue in the healing potential in a preclinical setting.
CBD-PLGA-G/nHAp group, which was similar to the autograft. Histo- Analysis of physical properties of fabricated scaffolds revealed that
pathological analysis showed that autograft integrated to one side of incorporation of CBD-PLGA in G/nHAp scaffold led to a slight change in
the old bone after 4 weeks. Integration to both sides occurred at 12 the scaffold microstructure (i.e. porosity and pore size) which was not
WPS. The remnants of the CBD-free G/nHAp scaffold were present in statistically significant. Similarly, a comparison of CBD-PLGA-G/nHAp
the injury site and surrounded by fibrous connective tissue at 4 WPS. and G/nHAp scaffolds confirmed the comparable mechanical strength
After 12 weeks, these defects were filled with fibrocartilage and carti- in the CBD-PLGA-G/nHAp scaffold according to the compressive stress-
laginous tissues. In contrast, the bone gap was filled by a loose areolar strain curve. Here, we used post-seeding method for incorporation of
connective tissue in the untreated group at 4 WPS, which was subse- CBD-PLGA into G-nHAp Scaffold. Accordingly, we expect to see no
quently replaced by fibrovascular tissue at 12 WPS. significant changes in the mechanical and structural properties of G-
Table 4 shows histomorphometric results of newly formed osseous, nHAp scaffold by using this method. These findings are in agreement
cartilaginous, and fibrous tissues at 12 WPS. The osseous and cartila- with Zhang et al. study that represented incorporation of BMP2-loaded
ginous tissues showed the highest density in the autograft and CBD- PLGA microspheres into the porous nanofibrous scaffold via post-
PLGA-G/nHAp groups (P < 0.05) followed by the CBD-free G/nHAp seeding technique did not change the mechanical properties of original
group. In contrast, untreated groups had significantly more dense fi- scaffold [31].
brous connective tissues and immature granulation tissues (number of The in vitro release profile of CBD showed that incorporation of
fibrocytes, fibroblasts, newly-formed blood vessels, and density of col- CBD-PLGA into the G/nHAp scaffold decreased CBD release over
lagen fibers; P < 0.01). 25 days. Therefore, we assumed that incorporation of CBD-PLGA into
73
the G/nHAp scaffold controlled the CBD release in a sustainable of the defect area in this group. Another factor that might affect bone
manner. We assessed CBD biocompatibility under in vitro conditions. mechanical properties is the quality of the collagenous matrix. It has
Based on the MTT results, different concentrations of CBD caused no been shown that CBD increased expression of PLOD1, which indirectly
significant changes in cell viability. A previous study reported that increased the collagen crosslink ratio [19]. This ratio is an indicator of
concentrations of 9 μM and greater of CBD caused significant increase collagen maturity [39] considered extremely important for the me-
in cell viability relative to the control [32]. An in vitro migration assay chanical properties of repaired bone.
showed that CBD significantly induced MSC migration (Fig. S7), which Overall, the present study showed that CBD was an effective small
was confirmed by in vivo immunostaining. Schmuhl et al. reported the molecule for stimulation of MSCs migration and their differentiation
effect of CBD on MSC migration at the 3 μM concentration [14], which into osteogenic lineages, which rendered the osteoinductivity for os-
was similar to our study (~3.2 μM). Regarding the role of cannabinoids teoconductive (e.g., G/nHAp) scaffolds. Controlled delivery of CBD
in cellular migration, endocannabinoids such as 2-arachidonoyl gly- could be utilized in the future in commercial bone tissue scaffolds to
cerol and arachidonoyl cyclopropylamide were found to exert a pro- increase bone healing, particularly in critical-sized bone defects.
migratory impact on microglial cells via activation of “abnormal-can- Further investigations would be required to examine the optimal CBD
nabidiol-sensitive receptors” [33,34]. Another study showed that N- concentration in a controlled delivery system for bone regeneration.
arachidonoyl serine (an endocannabinoid-like substance) was pro-mi-
gratory on human dermal microvascular cells [35]. These findings re- 5. Conclusion
vealed a critical role for cannabinoids and their receptors on migration
of different cell lineages. We developed and evaluated of a novel scaffold for healing of large
The impact of CBD on osteogenic differentiation of MSCs was also bone defects. The results indicated that this scaffold was reliable and
investigated in vitro. In the current study, qRT-PCR analysis of three had acceptable therapeutic efficacy. We observed migration of the BM-
osteogenic markers indicated that the 21 day culture of MSCs on CBD- MSCs towards the injury site and their differentiation into osteoblasts,
free G/nHAp and CBD-PLGA-G/nHAp scaffolds increased osteogenic which indicated an appropriate biocompatibility and osteoinductivity
activity of MSCs. Our findings agreed with previous studies that as- of the fabricated scaffold. Controlled-release of CBD enhanced MSC
sessed CBD osteogenic activity [14]. The low expression of ALP in the recruitment and bone reconstruction, which were confirmed by various
CBD-PLGA-G/nHAp group might be related to earlier initiation of the analyses. Based upon the in vitro and in vivo results, the CBD-PLGA-G/
mineralization processs. Of note, ALP is one of the earliest markers of nHAp scaffold could be introduced as a promising alternative to con-
osteoblastic cell differentiation. ALP shows a maximal expression level ventional treatments. The results of the present study provided first-
on day 7, which declines over time, with initiation of the mineralization time in vivo evidence for induction of MSCs recruitment and their os-
process [36]. In our study, we have observed that ALP expression de- teogenic differentiation by controlled local delivery of CBD.
clined at day 21, whereas there was increased expression of the late
genes, OCN and COL1. Although the CBD-free G/nHAp scaffold could Acknowledgment
trigger the differentiation of MSCs into an osteogenic lineage, this
process was slower than in the CBD-PLGA-G/nHAp scaffold. This work was supported by the Veterinary School of Medicine,
Our histopathological and histomorphometric findings combined Shiraz University, Shiraz, Iran, Royan Institute, Tehran, Iran, and the
with imaging techniques (micro-CT and radiology) showed a specific Iran National Science Foundation (INSF), Tehran, Iran (grant number
CBD-induced enhancement in new bone formation via sustained release 96006039).
of CBD. The CBD-PLGA-G/nHAp treated group significantly improved
bone healing compared to the CBD-free G/nHAp group. In our study, Conflict of interest
the bone healing process occurred through endochondral ossification,
which was mainly related to the regenerative effect of CBD. An in vivo The authors declare that they have no conflicts of interest.
experimental study evaluated the effect of Cannabis sativa (marijuana)
smoke inhalation on bone healing and reported inhibition of the early Appendix A. Supplementary data
stages (30 days) of bone regeneration around titanium implants [37].
Another study, in agreement with our findings, suggested the positive Supplementary data to this article can be found online at https://
effects of CBD on fracture healing during the late stage of healing (after doi.org/10.1016/j.msec.2019.03.070.
6 weeks). These controversial results of the cannabinoids impact on
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75
Update
Volume 126, Issue , July 2021, Page
DOI: https://doi.org/10.1016/j.msec.2021.112179
Materials Science & Engineering C 126 (2021) 112179
Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/msec
Corrigendum
Corrigendum to “Cannabidiol-loaded microspheres incorporated into

osteoconductive scaffold enhance mesenchymal stem cell recruitment and
regeneration of critical-sized bone defects” [Mater. Sci. Eng. C. 101C (2019)
pages 64–75]
Amir Kamali a, Ahmad Oryan a, *, Samaneh Hosseini b, Mohammad Hossein Ghanian c,
Maryam Alizadeh c, Mohamadreza Baghaban Eslaminejad b, Hossein Baharvand b
a
Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
b
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
c
Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
The authors regret that recently, a Pubpeer note has highlighted images. However, these corrected figures did not change any results,
similarities between two images in our paper entitled “Cannabidiol- discussion, conclusion, references, etc. of our original manuscript.
loaded Microspheres Incorporated into Osteoconductive Scaffold We deeply apologize for this unintentional mistake, which occurred
Enhance Mesenchymal Stem Cell Recruitment and Regeneration of during manuscript preparation, and would like to request a correction,
Critical-sized Bone Defects”, volume 101, pages 64–75 (2019) published providing new images for Figs. 4 and 7 to avoid any potential ambigu
in Materials Science & Engineering: C with panels in another paper by ities in the future.
the same research group. Indeed, image similarities in microscopy im The new Figs. 4 and 7 are provided below.
ages shown in Figs. 4 and 7 seem to have been occurred. Fig. 4:
We run an investigation to elucidate the origin of these irregularities.
It appeared that this overlapping is due to mislabelling of the same
images stored in different places.
Following a search in our archives, we have found the original im
ages and would like to publish a corrective note to show the correct
DOI of original article: https://doi.org/10.1016/j.msec.2019.03.070.

* Corresponding author.
E-mail address: Oryan@shirazu.ac.ir (A. Oryan).
https://doi.org/10.1016/j.msec.2021.112179
Available online 11 May 2021

0928-4931/© 2021 Elsevier B.V. All rights reserved.
A. Kamali et al. Materials Science & Engineering C 126 (2021) 112179
Fig. 7:
The authors would like to apologize for any inconvenience caused.
2
dentistry journal
Review
Cannabidiol in Dentistry: A Scoping Review
Carla David 1,2 , Alejandro Elizalde-Hernández 2 , Andressa S. Barboza 2 , Gabriela C. Cardoso 2 ,
Mateus B. F. Santos 2 and Rafael R. Moraes 2, *
1 Biopathological Research Group, Faculty of Dentistry (GIBFO), University of the Andes Mérida,
Mérida 5101, Venezuela
2 Graduate Program in Dentistry, Universidade Federal de Pelotas, Pelotas 96015-560, Brazil
* Correspondence: moraesrr@gmail.com
Abstract: Cannabidiol (CBD) has been gaining increased attention in contemporary society but
seems to have been little explored in dentistry. This scoping review mapped the scientific and
technological scenarios related to the use of CBD in dentistry. Peer-reviewed publications were
searched in five international databases, patents were searched in five technological platforms. In
total, 11 articles and 13 patents involving CBD in dentistry-related applications were included. The
countries contributing to most articles were Brazil (27.3%) and USA (18.2%). The studies involved
experiments on animals (63.6%) and/or using bacteria or cells (36.4%), and no clinical study was
found. Three different applications of CBD were observed: periodontal therapy (45.4%), aid for bone
regeneration (27.3%), and general use in oral therapies (27.3%). Patent inventors were based in China
(53.8%) or USA (46.2%). The patent claims were mainly compositions for oral care, tooth whitening,
injury repair, antifungal, anti-inflammatory, and analgesic effects. A total of 76.9% of the patents were
filed in association with a company. In general, research suggests that CBD has promising biological
properties for applications in dentistry, whereas patents indicate that the current interest of industry
relies on compositions for oral care. There appears to be extensive room available for research and
technological applications of CBD in dentistry.
Citation: David, C.;
Elizalde-Hernández, A.; Barboza, Keywords: plants; cannabinoids; cannabis; dental medicine; patents
A.S.; Cardoso, G.C.; Santos, M.B.F.;
Moraes, R.R. Cannabidiol in
Dentistry: A Scoping Review. Dent. J.
2022, 10, 193. https://doi.org/ 1. Introduction
10.3390/dj10100193
Endocannabinoids are naturally occurring molecules in mammals produced in the en-
Academic Editor: Gianrico Spagnuolo docannabinoid system [1,2], which interact with endogenous substances (e.g., anandamide
and 2-arachidonoylglycerol), phytocannabinoids (e.g., cannabidiol, tetrahydrocannabinol),
Received: 16 September 2022
Accepted: 11 October 2022
or synthetic cannabinoid analogs [3]. Cannabinoids can bind to specific CB1 and CB2
Published: 17 October 2022
receptors found in plasma membranes, nerve fibers (CB1), and tissue cells (CB2) [1]. CB1
receptors in the brain are predominantly presynaptic and responsible for regulating mem-
Publisher’s Note: MDPI stays neutral ory, mood, sleep, appetite, and pain by releasing neurotransmitters. CB1 receptors are also
with regard to jurisdictional claims in
present at lower concentrations in peripheral tissues, including cardiac, testicular, muscular,
published maps and institutional affil-
hepatic, pancreatic, and adipose tissues. CB2 receptors are probably responsible for the
iations.
immunomodulatory and anti-inflammatory effects of cannabinoids and are expressed in
the spleen and hematopoietic cells [2].
Cannabidiol (CBD) is a compound without psychoactive activity derived from Cannabis
Copyright: © 2022 by the authors.
sativa plant [4]. CBD has been investigated more frequently and to a greater extent when
Licensee MDPI, Basel, Switzerland. compared with other cannabinoids in the contemporary literature [4], and has shown po-
This article is an open access article tential as a therapeutic agent in various physiopathological conditions or disease states [4].
distributed under the terms and Therapeutic uses of CBD have been linked to its anti-inflammatory [5], antioxidant [2], and
conditions of the Creative Commons analgesic [6] properties, including varied applications such as in bone tissue cell differenti-
Attribution (CC BY) license (https:// ation [3,7–9], neuroprotection, antiepileptic, anxiolytic, and anti-cancer agents [10]. CBD
creativecommons.org/licenses/by/ may also be used for health benefits that are not yet thoroughly studied, including stress
4.0/).
Dent. J. 2022, 10, 193. https://doi.org/10.3390/dj10100193 https://www.mdpi.com/journal/dentistry

Dent. J. 2022, 10, 193 2 of 14
relief, relaxation, and sleep improvement. The product can be used in edible, tincture, and
vape modes.
Increasing research and development of CBD and its medical applications have been
undertaken due to financial support by various private and governmental organizations in
the past years. The global CBD market is expected to reach approximately USD 30 billion
by 2025 [11]. CBD has a variety of potential uses in dentistry and oral medicine. Research
targeting the therapeutic possibilities of CBD has been developed relative to its use in the
oral cavity [12] and expression of endocannabinoid receptors in tooth, periodontal, and
bone tissues [13,14], for instance. Therefore, it would appear to be promising to explore the
possibilities for future research on the use of CBD, its compounds, or their synthetic analogs
for dental applications. The aim of this scoping review was to map the contemporary
scientific and technological scenarios related to the use and applications of CBD in dentistry
at present.
2. Materials and Methods

This scoping review has been reported according to the Preferred Reporting Items for
Systematic Reviews and Meta-Analysis Extension for Scoping Reviews guideline [15]. The
study protocol was based on the framework proposed by Peters et al. (2015) [16], according
to The Joana Briggs Institute [17], and is available at https://osf.io/yk65c/ (accessed on
10 October 2022). For the mapping, the following parameters were selected: (i) Population—
articles and technological products; (ii) intervention—use of CBD or its synthetic analogs;
(iii) comparison: other substances or treatments that may have been tested (if applicable);
(iv) dental and oral medicine applications, (v): articles or patents. One general question
guided the review: What scientific applications and technological products based on CBD
or its synthetic analogs are being used in dentistry at present?
2.1. Eligibility Criteria

The inclusion criteria consisted of in vitro or in vivo studies and patents that have
evaluated or reported the use of CBD for dental applications or subjects (e.g., periodontol-
ogy, oral surgery). Studies that evaluated the use of CBD alone or associated with other
substances (e.g., vitamins) or biomaterials in dentistry were included. In addition, studies
that evaluated other synthetic analogs (HU-308, HUF-101, fluoro-cannabidiol, HU-320,
CBD-DMH-7-oic acid, deoxy-CBD, 11-hydroxy-CBD or O-1602) were also included. The
search was restricted to documents published in English without restriction on date. Case
reports, case series, pilot studies, opinion articles, letters, and conference abstracts were
excluded due to the high risk of bias associated to these articles. Reviews were excluded to
concentrate the analysis to original articles. Studies on the use or smoke of cannabis plants
and other cannabinoids also were excluded. Patents including CBD were excluded when
its application or scope of patents was not related to dentistry or oral medicine.
2.2. Information Sources and Search

The searches in literature databases were performed by two independent reviewers
(CD and AE-H) and patent tools (CD and AB), with no starting date and continuing
through to December 2021. The research for articles was carried out in five international
databases: PubMed/MEDLINE, Scopus, Web of Science, Embase, and Cochrane Library.
The search strategy was based on the MeSH terms of PubMed and the specific terms of
the other databases, using the search strategies presented in Table 1. The reviewers also
hand-searched the reference lists of the included articles to identify additional manuscripts.
In addition, a search and analysis of patent applications was conducted via the following
databases—United States Patent and Trademark Office (USPTO), Google Patents, World
Intellectual Property Organization (WIPO), Espacenet (European Patent Office, EPO), and
Questel Orbit (Paris, France), which contains patent data on over 90 authorities. The search
strategy is described in Table 1. Furthermore, a patent search was carried out using the
following International Patent Classification codes: A61K-6/00 (preparations to dentistry),
Dent. J. 2022, 10, 193 3 of 14
A61K-8/97 (derived to algae, fungi, lichens or plants; from derivatives thereof). The
searches were conducted in December 2021.
Table 1. Search strategy used in the different databases.
(THC) OR (Tetrahydrocannab) OR (Dronabin) OR (Cannab) OR (Phytocannab) OR (Marijuana) OR

(Synthetic cannab) OR (Cannabidiol) AND (Bone and Bones [Mesh] OR (Bone and Bones) OR (Bones
and Bone Tissue) OR (Bones and Bone) OR (Bone Tissue) OR (Bone Tissues) OR (Tissue, Bone) OR
(Tissues, Bone) OR (Bony Apophyses) OR (Apophyses, Bony) OR (Bony Apophysis) AND (Dentistry)
PubMed/MEDLINE
OR (Implants, Dental) OR (Dental Implant) OR (Implant, Dental) OR (dental Pain) OR
(anti-inflammatory) OR (Pulp, Dental) OR (Pulps, Dental) OR (Dental Pulps) OR (Regeneration,
Periodontal Guided Tissue) OR (Guided Periodontal Tissue Regeneration) OR (Periodontal Guided
Tissue Regeneration) OR (Bone reparation)
ALL (“THC” OR “Tetrahydrocannab” OR “Dronabin” OR “Cannab” OR “Phytocannab” OR
“Synthetic cannab” OR “Cannabidiol” AND (“Bone and Bones” OR “Bone and Bones” OR “Bones
and Bone Tissue” OR “Bones and Bone” OR “Bone Tissue” OR “Bone Tissues” OR “Tissue, Bone” OR
“Tissues, Bone” OR “Bony Apophyses” OR “Apophyses, Bony” OR “Bony Apophysis”) AND
Scopus
(“Dentistry” OR “Implants, Dental” OR “Dental Implant” OR “Implant, Dental” OR “dental Pain”
OR “anti-inflammatory” OR “Pulp, Dental” OR “Pulps, Dental” OR “Dental Pulps” OR
“Regeneration, Periodontal Guided Tissue” OR “Guided Periodontal Tissue Regeneration” OR
“Periodontal Guided Tissue Regeneration” OR “Bone reparation”)
TS = (THC) OR (Tetrahydrocannab *) OR (Dronabin *) OR (Cannab *) OR (Phytocannab *) OR
(Marijuana *) OR (Synthetic cannab *) AND (Dentistry) OR (Implants, Dental) OR (Dental Implant)
Web of Science OR (Implant, Dental) OR (dental Pain) OR (anti inflamatory) OR (Pulp, Dental) OR (Pulps, Dental)
OR (Dental Pulps) OR (Regeneration, Periodontal Guided Tissue) OR (Guided Periodontal Tissue
Regeneration) OR (Periodontal Guided Tissue Regeneration) OR (Bone reparation)
(Synthetic cannab) OR (Cannabidiol) AND (Bone and Bones) [Mesh] OR (Bone and Bones) OR (Bones
Cochrane Library
OR (Implants, Dental) OR (Dental Implant) OR (Implant, Dental) OR (dental Pain) OR
(anti-inflammatory) OR (Pulp, Dental) OR (Pulps, Dental) OR (Dental Pulps) OR (Regeneration,
Periodontal Guided Tissue) OR (Guided Periodontal Tissue Regeneration) OR (Periodontal Guided
Tissue Regeneration) OR (Bone reparation)
(Synthetic cannab) OR (Cannabidiol) AND (Bone and Bones) [Mesh] OR (Bone and Bones) OR (Bones
Embase
OR (Implants, Dental) OR (Dental Implant) OR (Implant, Dental) OR (dental Pain) OR (anti
inflamatory) OR (Pulp, Dental) OR (Pulps, Dental) OR (Dental Pulps) OR (Regeneration, Periodontal
Guided Tissue) OR (Guided Periodontal Tissue Regeneration) OR (Periodontal Guided Tissue
Regeneration) OR (Bone reparation)
THC OR Tetrahydrocannab OR Dronabin OR Cannab OR Endocannab OR Phytocannab OR
Marijuana OR Synthetic cannab OR Cannabidiol OR Endocannabinoide AND “Bone and Bones OR
Bones and Bone Tissue OR Bones and Bone OR Bone Tissue OR Bone Tissues OR Tissue, Bone OR
Tissues, Bone OR “Bony Apophyses OR Apophyses, Bony OR Bony Apophysis OR Dentistry OR
Orbit
Implants, Dental OR Dental Implant OR Implant, Dental OR dental Pain OR anti-inflammatory OR
Pulp, Dental OR Pulps, Dental OR Dental Pulps OR Regeneration, Periodontal Guided Tissue OR
Guided Periodontal Tissue Regeneration OR Periodontal Guided Tissue Regeneration or
Bone reparation
Google patents
Bone reparation
Dent. J. 2022, 10, 193 4 of 14
Table 1. Cont.

USPTO
Bone reparation
WIPO
Bone reparation
Espacenet
Bone reparation
*: The asterisk is used for searching terms with other variations at the end of the word.
2.3. Selection of Sources of Evidence

All records identified were imported into the EndNote program (EndNote X9; Thom-
son Reuters, New York, NY, USA). The independent researchers identified articles and
patents by first analyzing titles and abstracts for relevance and eligibility criteria using
the online system Rayyan QCRI (Hamad Bin Khalifa University, Doha, Qatar). Retrieved
records were classified as included, excluded, or uncertain. Full-text versions of the in-
cluded and uncertain records were selected for further eligibility screening. Discrepancies
in a screening of titles/abstracts and full texts were resolved by discussion. In case of
disagreement, the opinion of a third reviewer (RRM) was sought.
2.4. Data Charting, Data Items, and Analysis

We created two spreadsheets in the Microsoft Office Excel 2013 software (Microsoft
Corporation, Redmond, Washington, DC, USA) for data extraction from articles and patents.
The spreadsheets were pilot tested by three reviewers (CD, AB, RRM) to reach a consensus
on which data to collect and how. Two independent reviewers (CD, AB) extracted the main
relevant data, specifically focusing on the outcomes in dentistry, and influence of CBD on
the dental applications. The following items of data were collected for articles: year, first
author, journal, study design, country of the corresponding author, CBD dosage, route of
administration, CBD presentation, manufacturers, applications (e.g., periodontal therapy,
aid for bone regeneration, oral therapy), and sponsors of the studies. From patents, the
following data were collected: patent numbers, year, title, inventors, country of inventors,
main claims, and company. The mapping of research findings and patents was carried out
considering the application of CBD in different dental subjects.
3. Results
A total of 2312 unique articles and 989 patents were identified (Figure 1) with 2294 arti-
cles and 974 patents excluded based on title and/or abstracts. From the 18 full-text articles
From patents, the following data were collected: patent numbers, year, title, inventors,
country of inventors, main claims, and company. The mapping of research findings and
patents was carried out considering the application of CBD in different dental subjects.
3. Results
Dent. J. 2022, 10, 193 5 of 14
A total of 2312 unique articles and 989 patents were identified (Figure 1) with 2294
articles and 974 patents excluded based on title and/or abstracts. From the 18 full-text ar-
ticles and 15 patents assessed for eligibility, 11 studies and 13 patents were included in
and 15 patents assessed for eligibility, 11 studies and 13 patents were included in the
the qualitative synthesis, making up a total of 24 documents included in this review.
qualitative synthesis, making up a total of 24 documents included in this review.
Figure 1. Flowchart
Figure 1. Flowchart of
of the
the selection
selection process
process of
of articles
articles and
and patents.
patents.
3.1. Characteristics of Studies
3.1. Characteristics of Studies
Characteristics of the 11 original studies included are presented in Table 2. The studies
were Characteristics
published betweenof the2009
11 original
and 2020studies included
in various are presented
journals, in Table
the majority 2. Thecould
of which stud-
ies were
not publishedas
be categorized between 2009 and 2020
being dedicated in variousdental
to publishing journals, the majority
science only. Theofcountries
which could
that
not be categorized as being dedicated to publishing dental science only. The
contributed most of the articles were Brazil (27.3%) and USA (18.2%). The studies involved countries that
contributed most of the articles were Brazil (27.3%) and USA (18.2%). The studies
experiments in animals (63.6%) and/or using bacteria or cells (36.4%), no clinical study involved
experiments
was found. Tenin studies
animalsinvestigated
(63.6%) and/or
CBD using bacteria
and one or cells (36.4%),
study investigated no clinical
its derivative study
HU-308.
was found. Ten studies investigated CBD and one study investigated
CBD dosages used across the studies varied widely, with 3–10 mg/kg being the most its derivative HU-
308. CBDrange
frequent dosages used across
of dosage the studies
in animal studies,varied widely, with 3–10
with intraperitoneal mg/kg being the
administration, most
whereas
frequent
direct rangeofofCBD
contact dosagewasin animal
the modestudies, with intraperitoneal
of administration administration,
in cell studies. whereas
The presentation of
direct contact of CBD was the mode of administration in cell studies. The
CBD was mainly in the form of a powder dissolved in saline (54.5%). The most frequent presentation of
CBD was mainly in the form of a powder dissolved in saline (54.5%).
manufacturers of CBD were from the USA (36.4%) and Germany (27.3%). Three different The most frequent
manufacturers
applications of CBD
of CBD were fromwere
in dentistry the USA (36.4%)
observed, and Germany
namely periodontal(27.3%). Three
therapy different
(45.4%), aid
for bone regeneration processes in oral surgery (27.3%), and a general use in oral therapies
(27.3%). Sponsors of the studies were either federal funding agencies, governmental
authorities, or universities. We could not detect any studies that were sponsored by
private companies.
Dent. J. 2022, 10, 193 6 of 14
Table 2. Characteristics of the original articles included, n = 11.
First
Study CBD or
Author, Journal Country Dosage Administration Presentation Manufacturer Main Application Sponsor
Type Analog
Year
Int. CBD powder dissolved in
Napimoga, THC Pharm, CAPES and
Immunophar- Brazil Animal CBD 1 mg/kg Injected i.p 2% polysorbate 20/Tween Periodontal therapy
2009 [18] Germany UNIUBE, Brazil
macol. 80- in saline
CBD powder dissolved in
Klein, THC Pharm, CAPES and PUCRS,
Phytother. Res. Brazil Animal CBD 5–10 mg/kg Injected i.p 2% polysorbate 20/Tween Oral therapy
2018 [19] Germany Brazil
80- in saline
Kogan, J. Bone Miner. CBD powder dissolved in MAX-IV laboratory, Aid for bone NIH, USA and Israel
Israel Animal CBD 5 mg/kg Injected i.p
2015 [8] Res. ethanol/emulphor/saline Sweden regeneration Anti-Drug Authority
Shiraz University,
Mater. Sci. Eng. CBD powder dissolved in Royan Institute, and
Kamali, Tocris company, Aid for bone
C Mater. Biol. Iran Animal CBD 30 mg/kg Direct contact 2% polysorbate 20/Tween the Iran National
2019 [7] USA regeneration
Appl. 80- in saline Science Foundation,
Iran
Cuba, J. Clin. Pharm. CBD powder dissolved in THC Pharm, CAPES and PUCRS,
Brazil Animal CBD 3, 10, 30 mg/kg Injected i.p Oral therapy
2017 [20] Ther. Tween 80- in saline Germany Brazil
Greenhouse
Scionti, Health Ministry,
Front. Physiol. Italy Animal CBD 10 mg/kg NS Pure CBD (>99%) cultivation at Periodontal therapy
2016 [21] Italy
CREA-CIN, Italy
Gu, Animal 10 mg/kg NS NS Cayman Chemical
Front. Immunol. USA CBD Periodontal therapy NIDCR, USA
2019 [22] Cells 0.1–1.0 µg/mL Direct contact NS Co., USA
University of
Ossola, HU-308 powder dissolved Buenos Aires and
J. Periodontol. Argentina Animal HU-308 200 µL per tooth Topical Tocris, USA Periodontal therapy
2016 [23] 100% ethanol/saline CONICET,
Argentina
Rawal, J. Periodontal. CBD powder dissolved in University of
USA Cells CBD 0.01–30 µM Direct contact Sigma-Aldrich, USA Periodontal therapy
2012 [24] Res. methanol Tennesse, USA
Stahl,
Cureus Belgium Cells/bacteria CBD NS Direct contact NS NS Oral therapy NS
2020 [25]
Petrescu, Aid for bone
Medicina Romania Cells CBD 0.75 µM Direct contact NS NS CNCS, Romania
2020 [26] regeneration
CBD: cannabidiol; i.p: intraperitoneal; NS: not specified; CAPES: Coordination for the Improvement of Higher Education Personnel; UNIUBE: University of Uberaba; PUCRS: Pontifical
Catholic University of Rio Grande do Sul; NIH: National Institutes of Health; NIDCR: National Institute of Dental and Craniofacial Research, USA; CONICET: National Scientific and
Technical Research Council; CNCS: National Council of Scientific Research.
Dent. J. 2022, 10, 193 7 of 14
3.2. Characteristics of Patents

Characteristics of the 13 patents included are shown in Table 3 [27–39]. The inventors
were based in only two countries: China (53.8%) and USA (46.2%). One patent was
published in 2014 and the others between 2017 and 2020. The main claims presented in
the patents involved compositions for oral care (e.g., toothpaste, mouthwash, dental floss),
tooth whitening ability, injury repair potential, antifungal and anti-inflammatory agents,
analgesic effects, and even treatment of viral infections. One group of inventors and one
company from China were the inventors of four patents included. A total of 76.9% of the
patents were filed in association with a company.
Table 3. Characteristics of the patents included, n = 13.
Patent
Country Year Title Main Claims Inventors Company
Number(s)
US20190076349
US10172786 Oral care George
Toothpaste, tooth
EP3233021 composition Anastassov, Axim
USA 2014 powder, or mouthwash
EP3233021 comprising Lekhram Biotechnologies
solution
WO2016/100516 cannabinoids Changoer
US20160166498
Zhang Ke, Tan
Toothpaste, dentifrice,
Xin, Yu
Use of the tooth mouthwash, gel, gum Han Yi
CN109939012 China 2017 Zhaohui, Lian
whitening CBD or film-forming agent Biotechnology
Meng, Chang
for tooth whitening
Tanran, Jin Qian
Composition Zhang Ke, Tan
comprising a Xin, Yu
Toothpaste with oral Han Yi
CN109939011 China 2017 cannabinoid Zhaohui, Lian
injury repair potential Biotechnology
toothpaste and its Meng, Chang
preparation method Tanran, Jin Qian
Toothpaste,
mouthwash, chewing
Oral care
WO2019/055420 gum, lozenge, coated
USA 2017 formulations and Gerald Curatola NS
US20190076343 interdental device, and
methods for use
coated dental floss as
oral care products
Cannabis and
derivatives thereof
for the treatment of
Composition for
WO2019030762A2 pain and
treating dental pulp in-
EP3664795A2 USA 2018 inflammation related Veronica Stahl CannIBite
flammation/infection
US20200222361A1 with dental pulp and
and bone defects
bone regeneration
related to dental jaw
bone defects
A kind of toothpaste Zhang Ke, Tan
and preparation Xin, Yu
Toothpaste with oral Han Yi
CN109939011A China 2019 method thereof Zhaohui, Lian
injury repair potential Biotechnology
containing Meng, Chang
cannabinoid Tanran, Jin Qian
Zhang Ke, Tan
Application of the Xin, Yu
Tooth whitening Han Yi
CN109939012A China 2019 cannabidiol in tooth Zhaohui, Lian
product Biotechnology
whitening Meng, Chang
Tanran, Jin Qian
Dent. J. 2022, 10, 193 8 of 14
Table 3. Cont.
Patent
Country Year Title Main Claims Inventors Company
Number(s)
A kind of toothpaste
and preparation Luan Yunpeng,
method thereof with Zheng
CN109925246A China 2019 Toothpaste NS
sterilization, Shuangqing, Li
anti-inflammatory, Zhipeng
and analgesic efficacy
Cannabinoid-infused
Dental dressing pouch David Tumey,
US20200163746A1 USA 2019 post-operative dental NS
for an oral surgical site Harry Panahi
dressing
Buzzelet
Dental care cannabis
Device for topical Dana Zeitouni, Development
WO2019239357A1 USA 2019 device and use
administration Aharon M. Eyal and
thereof
Technologies
Xu Haiyan, He
Toothpaste with Wei, Xia
Jiangxi
Cannabinoid- antifungal, Meilian, He
Caoshanhu
CN111671698A China 2019 containing functional anti-inflammatory, Zhijiang, Kite
Oral Care
toothpaste analgesic, and Xiao, Xiao
Products
neuroprotector effects Hong, Xie
Lingyu
Chen Qi, Xiao
Toothpaste, dentifrice,
Fengsha, Gull
Oral care mouthwash, oral spray, Yunnan
Lianmeng,
CN111973480A China 2019 composition and oral patch, chewing Hanmeng
Chang Tanran,
application thereof gum or buccal tablet Pharmaceutical
Li Ruyan, Li
for oral hygiene
Qingzhong
Compositions for
Maria Crisler,
preventing and Composition for Shaman
WO2020257588A1 USA 2020 Emma Diponio,
treating viral treating viral infections Naturals
Philip Kennedy
infections
Abbreviations: i.p: intraperitoneal, NS: not specified.
3.3. Synthesis of Results and Summary of Evidence

The main applications of CBD and its synthetic analogs in dentistry are illustrated
in Figure 2. The in vitro and in vivo studies included in this review reported that CBD
may have beneficial effects as an adjunct to periodontal therapy. When HU-308, a CBD-
derivative drug, was tested, the study demonstrated that the CB2 receptor played a role
in the control of periodontal damage and the oral alterations it causes in the alveolar
bone, gingival tissue, and salivary function [23]. CBD showed anti-inflammatory and
anti-bone resorption properties [23]. Modulation of host responses by CBD was described
as an interesting and alternative approach to conventional periodontal therapy [18,24].
Furthermore, CBD could promote fibrotic gingival enlargement, increase in the production
of gingival fibroblasts and production of healing growth factors. It also led to a decrease in
the production and activity of metalloproteinases [24]. The possibility was also observed
that CBD may have antimicrobial properties and be effective in reducing the colony count of
bacterial strains of dental plaque, which could control and reduce inflammatory periodontal
diseases of bacterial origin [22,25].
Dent. J. 2022, 10, 193 9 of 14
Dent. J. 2022, 10, x FOR PEER REVIEW 9 of 14
Figure 2. Applications of CBD in dentistry according to the reviewed literature. (1) CBD could re-
Figure 2. Applications of CBD in dentistry according to the reviewed literature. (1) CBD could
duce colonies of periodontal pathogens, showing antimicrobial properties. (2) In soft tissues, CBD
reduce coloniesCB2
could activate of periodontal
receptors inpathogens, showing
fibroblasts and antimicrobial
lead to properties.
gingival fibrosis (2) In soft
by increasing tissues, CBD
the production
could activate
of gingival CB2 receptors
fibroblasts in fibroblasts
via stimulation and lead
of growth to gingival
factors fibrosis
and decrease inby increasing
matrix the production
metalloproteinases.
ofSimultaneously,
gingival fibroblasts via stimulation
CBD could of growththe
inhibit or modulate factors and decrease
production in matrix
of cytokines, metalloproteinases.
chemokines, and pro-
inflammatory growth
Simultaneously, CBD factors, interfering
could inhibit with the migration
or modulate of macrophages
the production and neutrophils,
of cytokines, chemokines, gen-
and
erating anti-inflammatory,
pro-inflammatory antioxidant,
growth factors, and analgesic
interfering with the potentials.
migration Potential treatments
of macrophages andinclude ul-
neutrophils,
cers, mucositis, and as an adjunct in periodontal diseases. (3) In bone, CBD may promote the stim-
generating anti-inflammatory, antioxidant, and analgesic potentials. Potential treatments include
ulation of mesenchymal cells via p42/44 mitogen-activated protein kinases (MAPK) toward the le-
ulcers, mucositis, and as an adjunct in periodontal diseases. (3) In bone, CBD may promote the
sion site and its differentiation into osteoblasts.
stimulation of mesenchymal cells via p42/44 mitogen-activated protein kinases (MAPK) toward the
lesion The
site and its differentiationproperties
anti-inflammatory into osteoblasts.
of CBD also made it a suitable alternative for the
prevention and treatment of oral mucositis, by reducing the inflammatory process and the
The anti-inflammatory properties of CBD also made it a suitable alternative for the
severity of lesions [9,19]. CBD was shown to enhance the healing process of common ul-
prevention and treatment of oral mucositis, by reducing the inflammatory process and
cers by reducing the number of colony counts of bacterial strains in dental plaque when
the severity of lesions [9,19]. CBD was shown to enhance the healing process of common
compared with well-established synthetic oral care products [25]. Furthermore, CBD
ulcers by reducing the number of colony counts of bacterial strains in dental plaque when
alone was shown to improve fracture healing by promoting the stimulation of mesenchy-
compared with well-established synthetic oral care products [25]. Furthermore, CBD alone
mal cells via activation of p42/44 receptors at the lesion site and stimulating their differ-
was shown to improve
entiation into fracture
osteoblasts, healing
which by promoting
indicated the stimulation
biocompatibility of mesenchymal
and osteoinductivity cells
[6,7].
via activation of
Furthermore, p42/44 receptors
mesenchymal at the
stem cells lesionfrom
isolated site dental
and stimulating their
tissues were differentiation
capable of differ-
into osteoblasts, which indicated biocompatibility and osteoinductivity [6,7].
entiating into osteoblasts when exposed to a low CBD dose, and a better expression Furthermore,
of
mesenchymal
bone proteins stem cells isolated
was observed [26]. from dental tissues were capable of differentiating into
osteoblasts when exposed to a low CBD dose, and a better expression of bone proteins was
observed [26].
4. Discussion
This scoping review mapped the available literature and patents on the use of CBD
4. Discussion
for dentistry-related applications. Most of the peer-reviewed literature suggested that the
This scoping review mapped the available literature and patents on the use of CBD
positive effects of CBD could be attributed to its anti-inflammatory, analgesic, antimicro-
for dentistry-related applications. Most of the peer-reviewed literature suggested that the
bial, biological, and osteoinductive properties. The patents also seemed to rely on these
positive effects of CBD could be attributed to its anti-inflammatory, analgesic, antimicrobial,
properties to offer technological applications of CBD in dentistry. The dental subjects
biological, and osteoinductive properties. The patents also seemed to rely on these prop-
mainly involved in the literature and patents reviewed were periodontology, oral medi-
erties to offer technological applications of CBD in dentistry. The dental subjects mainly
cine, and oral surgery. These subjects, the potential technological applications of CBD, and
involved in the literature and patents reviewed were periodontology, oral medicine, and
future research challenges on the topic are discussed as follows.
oral surgery. These subjects, the potential technological applications of CBD, and future
research challenges on the topic are discussed as follows.
Dent. J. 2022, 10, 193 10 of 14
4.1. Periodontal Therapy

CBD has anti-inflammatory properties and was shown to be capable of reducing
alveolar bone loss in induced periodontitis [18,25]. Pharmacologically, the selective activa-
tion of CBD at CB2 receptors allows therapeutic, analgesic, or anti-inflammatory effects,
while they prevent the secondary effects resulting from the activation of CB1 receptors [5].
These anti-inflammatory properties manifested themselves in a similar way to that of other
endogenous cannabinoids, including anandamide. Specifically, the studies demonstrated
that anandamide is present and regulated during the progression of periodontal disease
and is involved in the suppression of pro-inflammatory mediators. Moreover, it is possibly
physiologically involved in the protection of periodontal tissues against excess inflamma-
tion [18]. Furthermore, it can block NT-kB, a regulator of the immune and inflammatory
response, normally activated by endotoxins from bacterial lipopolysaccharides, and is also
capable of reducing the production of inflammatory mediators such as interleukins [5].
Effects also have been shown to involve the inhibition or modulation of the production of
cytokines, chemokines, and pro-inflammatory growth factors, along with interference in
macrophage and neutrophil migration and reduction of oxidative and nitrosative stress [2].
The anti-inflammatory potential of CBD, similar to that of endocannabinoids, has directly
or indirectly demonstrated similar behavior at cannabinoid receptors, which could have
interesting clinical implications. However, the results of another study suggested that
CBD may promote increased gingival fibrosis, thereby increasing the production of gin-
gival fibroblasts, transformative growth factor b and fibronectin with decrease in matrix
metalloproteinase production and activity [24]. Low CBD concentrations have increased
transforming growth factor beta levels by as much as 40% at 24 h, suggesting that CBD may
promote fibrosis in this way [24]. Moreover, CBD may increase the levels of anandamide,
which may promote fibrosis via CB1 or other receptors [24].
4.2. Oral Medicine

These anti-inflammatory and analgesic properties of CBD have been reported to be
dose-responsive, without ideal doses having been established for a possible antioxidant
and anti-inflammatory action [9,21,26]. This combination of anti-inflammatory, antioxidant,
and analgesic properties of CBD may render a more potent action than classical antioxi-
dants, which could generate a change in the production of pro-inflammatory mediators
responsible for inflammatory pathologies such as oral mucositis [9]. This could make it
possible to use of CBD as a potential therapy for the treatment of the symptoms of mucositis
as CBD resulted in favorable epithelial changes in ulcer lesions in vivo. An important point
capable of influencing this tissue response is the possible ability of CBD to induce effects
on keratinocytes without showing side effects. Despite its anti-inflammatory effect, CBD
did not accelerate wound healing [19]. Therefore, the influence of CBD on keratinocytes
continues to be controversial and further studies are needed to investigate its effects and
identify the mechanisms of action.
In an in vivo study [23], the synthetic analog HU-308 caused a reduction in alve-
olar bone loss and inflammatory mediators in gingival tissues, which are increased by
lipopolysaccharide-induced periodontitis in the absence of treatment. The main effect is
local and produced by activation of the CB2 receptors, stimulating the differentiation of
osteoblast cells and mitigating osteoclastogenesis. This suggests that the signaling of the
CB2 receptor produces blockade of bone loss through a direct action on bone cells and
simultaneously by inhibiting the expression of pro-reabsorption cytokines [23].
CBD showed effective antimicrobial properties in the reduction of colonies of bacterial
strains against oral bacteria and two biofilms [22]. High doses of CBD suppressed the
growth of Porphyromonas gingivalis and Filifactor alocis, which are key components of
subgingival microbiota [22]. Dental plaque is a balanced, biofilm-organized structure
that contains bacteria responsible for various oral diseases such as gingivitis, periodontitis,
and dental caries. The majority of the microorganisms which dental biofilms are composed
of are Gram-positive bacteria and could be susceptible to the action of CBD, which showed a
Dent. J. 2022, 10, 193 11 of 14
greater reduction in bacterial colonies when compared with other oral hygiene products [25].
However, the efficacy of CBD could vary from one individual to another due to the microbial
diversity of oral biofilms [25].
4.3. Adjuncts to Oral Surgery and Traumatology

CBD has shown favorable biological and osteoinductive properties. When used alone
or in combination with other substances, CBD was sufficiently effective and reliable to
produce cell migration and bone differentiation [6,7,26] and exerted an impact on the
pro-migratory activity of microglial cells through the activation of the endocannabinoid
system [25]. Bone cells express cannabinoid receptors and endocannabinoid metabolizing
enzymes, and cannabinoid receptors are specifically expressed in skeletal sympathetic nerve
endings. Moreover, cannabinoids play an important role in regulation and remodeling
of bone mass [7]. Furthermore, studies have shown that CBD increased the expression of
PLOD1 gene [6,7,26], which indirectly increases the cross-linking ratio of collagen, showing
an indicator of collagen maturity and potential for inducing bone protein expression
and enhancing mineralization. Both actions may improve neobone formation and the
biomechanical properties of bone tissue. However, there are still questions to be answered
about the selective mechanisms of the receptors in the healing and regeneration of bone.
4.4. The Scientific and Technological Scenario

In line with the recent literature on the use of CBD in dentistry, our findings also re-
vealed an increasing interest in technology and appropriation relative to dental applications
of CBD in recent years. Despite the increasing progress in filing these patents, only one
patent [31] dating back to 2018 appeared simultaneously with publication of the results in a
scientific study [24] in 2020. The patents deposited with the application of CBD in dentistry
were mostly related to products for oral care. This area seems to offer space for exploration
of CBD due to the multiplicity of potential dentistry-related applications and the increasing
interest in natural products [40,41]. The incentive to the industrial exploitation of CBD
in dentistry through the consolidation of policies to stimulate scientific and technological
projects could result in economic growth based on research [42]. In spite of the challenges,
today CBD and its synthetic analogs are seen as promising compounds in multiple areas,
especially because of the low cost of these phyto-derivatives [43].
4.5. Limitations
A number of limitations relative to research and technological applications of CBD
in dentistry can be cited. One limitation is the absence of clear regulations governing the
supervision of the quality of CBD [40], which could lead to varied efficiency of CBD for
dental applications across origins of its supply and manufacturers. The constituents of plant-
derived natural products vary greatly, often due to variation in environmental conditions.
Consequently, the quality and concentration of natural products depend on their geographic
location, vegetation, and extraction conditions [44]. Therefore, they may contain different
chemical constituents that promote their therapeutic activities. These aspects leave room
for research concerning the effect of different constituents of CBD-containing solutions for
dental applications. Another limitation highlighted in this review was the heterogeneity
of analytical techniques used for characterizing the products. Variations in control of the
source of these materials, their storage, production process, and contamination could all be
relevant factors influencing quality of products and their therapeutic results [45].
4.6. Future Challenges

The scientific articles reviewed indicated challenges to the current clinical use of CBD
in dentistry and a potentially extensive field for research and products using CBD and its
synthetic analogs, including:
• Studying of the behavior, dosage, and mechanisms of action of CDB used in dentistry-
related clinical conditions;
Dent. J. 2022, 10, 193 12 of 14
• Acceptability of CBD-based treatments among dental patients;

• Standardization of the methods used for extraction of CBD and standardization of
in vitro and in vivo tests with this compound [46];
• Evaluation of the cytocompatibility of CBD in order to develop safe products;
• More basic studies are needed to increase safety of CBD for use in dental patients;
• Since CBD has been approved for use in patients in many countries, clinical studies
with patients are also needed in dentistry, which means that the available evidence is
of low quality, low certainty, and prone to risk of bias.
5. Conclusions
Cannabidiol has been gaining increasing attention in contemporary science and society
but seems to have been little explored in dentistry-related research and patents to date.
Dental research evidence suggests that CBD has anti-inflammatory, analgesic, antimicrobial,
biological, and osteoinductive properties for potential periodontal, oral surgery, and oral
medicine applications. Patents available indicate that at present, the interest of indus-
try relies on compositions for oral care products including toothpaste, mouthwash, and
dental floss. There appears to be extensive room available for research and technological
applications of CBD in dentistry, especially as far as the lack of clinical trials is concerned.
Author Contributions: Conceptualization, C.D., A.S.B., M.B.F.S. and R.R.M.; methodology, C.D.,
A.E.-H., A.S.B., G.C.C.; validation, C.D., A.E.-H., A.S.B., G.C.C., and R.R.M.; investigation, C.D; and
R.R.M. data curation, C.D. and R.R.M..; writing—original draft preparation, C.D., A.E.-H. and R.R.M.;
writing—review and editing, C.D., A.E.-H., A.S.B., G.C.C., M.B.F.S., R.R.M. All authors have read
and agreed to the published version of the manuscript.
Funding: This study was partially supported by Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior—(CAPES), Brazil—Finance Code 001.
Data Availability Statement: The data presented in this study are openly available in Open Science
Framework at https://doi.org/10.17605/OSF.IO/YK65C (accessed on 10 October 2022).
Conflicts of Interest: The authors declare no conflict of interest.
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Current Osteoporosis Reports (2020) 18:433–438
https://doi.org/10.1007/s11914-020-00607-1
BONE AND JOINT PAIN (P MANTYH AND T SCHNITZER, SECTION EDITORS)
The Cannabinoids Effect on Bone Formation and Bone Healing

Bitya Raphael-Mizrahi 1 & Yankel Gabet 1
Published online: 23 July 2020

# Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Purpose of Review Here, we overview the latest findings from studies investigating the skeletal endocannabinoid (EC) system
and its involvement in bone formation and resorption.
Recent Findings The endocannabinoid system consists of endogenous ligands, receptors, and enzymes. The main cannabinoids
found in the cannabis plant are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Cannabinoid receptors CB1 and CB2
are expressed in bone and regulate bone homeostasis in rodents and humans. CBD treatment was shown to enhance fracture
healing in rats. Recent studies in mice indicate that strain, age, and sex differences dictate the skeletal outcome of the EC
activation.
Summary CBD treatment was shown to enhance bone healing, but needs validation in clinical trials. While research shows that
EC activity protects against bone loss, studies on CB1 and CB2 agonists in bone regeneration models are lacking. Whether
modulating the EC system would affect bone repair remains therefore an open question worth investigating.
Keywords Skeleton . Bone . Osteoporosis . Fracture . Cannabis . Cannabinoid . Endocannabinoid . CBD . CB1 receptor . CB2
receptor
Introduction endocannabinoid main endogenous ligands are N-

arachidonoylethanolamine (anandamide, AEA) and 2-
A variety of therapies including opioids are frequently used to arachidonoylglycerol (2-AG) [5, 6] that are hydrolyzed by fatty
manage severe pain in both cancer and non-cancer patients. acid amid hydrolase (FAAH) and monoacylglycerol lipase
Opioids were recently found to inhibit healthy bone remodel- (MAGL) respectfully [7]. The main cannabinoid receptors are
ing and promote bone loss and fractures [1, 2]. While opioid CB1 and CB2; both are G protein–coupled receptor (GPCR)
therapy maybe the most potent analgesic option for cancer class A, seven transmembrane domain [8]. While these receptors
patients, it is widely known that patients may develop a life- are expressed in low levels in a wide range of tissues, high levels
long dependence for this narcotic substance. Thus, it is impor- of CB1 expression are found in the central and peripheral ner-
tant to replace opioid treatment with an alternative pain vous system and high levels of CB2 expression are found in the
reliving remedy with no deleterious effects and perhaps even skeletal and immune system [9, 10]. In fact, CB1 is one of the
with beneficial actions on bone homeostasis and healing. most abundant GPCR expressed in the nervous system [11, 12].
The identification of the psychoactive ingredient of cannabis/ The discovery of the skeletal EC system in 2005 revealed
marijuana, Δ9-tetrahydrocannabinol, THC [3], leads to the dis- its dominant role influencing bone remodeling in health and
covery of the endocannabinoid (EC) system. The EC system is a disease [13, 14]. The EC system effect on skeletal biology was
complicated endocrine system consists of ligands, receptors, and implied from a number of interesting studies. First, it was
biosynthesizing and biodegrading enzymes [4]. The indicated that leptin negatively regulates bone formation, bone
mass, and central production of 2-AG [15]. Secondly, findings
This article is part of the Topical Collection on Bone and Joint Pain show that bone formation and central production of 2-AG are
increased in cases of traumatic brain injury [16–18]. These
* Bitya Raphael-Mizrahi skeletal endocannabinoid connections lead to many more
bityar@tau.ac.il studies investigating the skeletal EC system and the EC activ-
ity in bone throughout life. Indeed, the EC system has an
1
Department of Anatomy and Anthropology, Sackler Faculty of important role in the regulation of bone mass and skeletal
Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel remodeling in animals [19, 20] and humans [21].
434 Curr Osteoporos Rep (2020) 18:433–438
THC and cannabidiol (CBD) are the main pharmacological CB1

active compounds synthesized in cannabis plants. The affinity
in which THC binds to CB1 and CB2 is much higher than the The skeletal effect of CB1 was found to be age- and strain-
affinity of the EC, AEA, and 2-AG [6, 8, 22]. Despite its dependent [14, 20, 30, 32, 33, 41]. In early reports, findings
resemblance to THC at the molecular level, CBD is a weak showed that CB1−/− female mice on a CD1 (a.k.a. ICR) back-
antagonist at both CB1 and CB2 [23, 24]. The mechanism of ground (CB1−/−/CD1) displayed a high peak bone mass and
actions of CBD is not well understood, yet, it is well accepted are protected against ovariectomy (OVX)-induced bone loss
that unlike THC, CBD is not psychoactive [25]. In this review, [14]. These effects were related to the osteoclasts insufficiency
we will focus on the skeletal effects of cannabinoids that are in the CB1−/−/CD1 mice. However, aged CB1−/−/CD1 mice
also well-established analgesics. To date, there is a lack of showed an increased age-related bone loss, partially attributed
solid evidence on the potential effect of cannabis on bone to impaired osteoblast proliferation and differentiation [33].
strength and bone healing. Two groups published conflicted This is in agreement with a report in rats, showing that CB1
findings on the link between cannabis use and bone mineral antagonists increased bone mass in young animals and aggra-
density in humans. One study linked excessive cannabis use vated osteoporotic bone loss in older animals [41]. Together,
with low BMD [26•], while the other showed no association these reports suggest that CB1 impairs bone accrual in young
between cannabis use and bone health [27•]. Because of the but has bone protective actions in aged rodents. Interestingly,
critical differences in the molecular composition of different young CB1−/− mice on a C57BL/6J background (CB1−/
−
cannabis strains, and the plethora of synthetic agonists, we /C57BL) displayed a low peak bone mass [32] in both young
will summarize here the specific roles of CB1 and CB2, as and aged animals. This suggests a CB1 strain–dependent phe-
well as CBD in bone formation and fracture healing. notype in young but not in aged animals, where CB1-
deprivation displayed a reduction in bone formation in all
strains [14, 30, 33, 39, 41]. CB1 has also been attributed a
role in the stimulation of bone formation following head trau-
Bone EC System ma. In mouse models, mild traumatic brain injury resulted in
increased bone formation mediated by CB1 in both the
It is well established that the main EC ligands, AEA and calvaria and the femur [32, 42].
2-AG, are present in the blood circulation [28]. However, Whether the role of CB1 in bone also has a sex-bias remains
the concentrations of these endocannabinoids in the bone unclear. Both male and female CB1−/−/CD1 showed similar
and brain tissue are significantly higher, suggesting a phenotypes at all ages [33]. However, studies on CB1−/
−
local EC production within the bones [19, 29]. Indeed, /C57BL reported results from female animals only [14, 30].
both osteoblasts (bone forming cells, OB) and osteoclasts Further studies are required to determine whether CB1 has a
(bone resorbing cells, OC) produce AEA and 2-AG sex-specific skeletal effect in strains other than CD1.
in vitro [30]. Moreover, OB, OC, osteocytes (bone cells The mechanism of action of CB1 signaling in bone remod-
derived from OB), and chondrocytes (cartilage cells) ex- eling is still controversial. As mentioned, expression levels of
press both the CB1 and CB2 receptors [30–34]. In vitro CB1 in bone cells is very low [10, 14, 38]. Nonetheless, OB
activation of CB2 increases osteoblast proliferation [34] CB1 levels increase with age, implying that the upregulation
and reduces osteoclast numbers [35]. The bone-related of CB1 is protective against age-related bone loss and osteo-
CB1 receptor is expressed in the skeletal sympathetic porosis [33]. The indirect approach proposes that the CB1
neurons and is located in the neuron terminals, in prox- regulation of OB activity is mediated by the negative control
imity to the bone cells, negatively regulating norepineph- on noradrenaline release from sympathetic nerve terminals
rine production and/or release [30]. located near the OB, alleviating the noradrenaline inhibition
Plant-derived cannabinoids, synthetic cannabinoids, of OB function and bone formation [30]. Although it is pos-
and the EC agonists AEA and 2-AG bind to CB1 and sible that the effect of CB1 is both direct and indirect, still, the
CB2 at different binding affinity and selectivity. The exact mechanism of the CB1 action on bone formation is yet
relative activation of CB1 versus CB2 in each cell type to be clarified.
will dictate the overall skeletal outcome [36, 37]. For the
sake of clarity, we will discriminate between the effect of
CB1 and CB2 activation reported using genetically mod- CB2
ified animal models. Studies on CB1−/− mice and CB2−/−
mice indicated that both CB1 and CB2 have a skeletal Studies on CB2 revealed that CB2−/− animals have an age-,
role [10, 30, 33, 34, 38–40]. These effects are dependent strain-, and sex-dependent skeletal phenotype. Young CB2−/−
on other factors such as genetic background, sex, age, mice on a C57BL/6J background (CB2−/−/C57BL) displayed
and hormonal status. normal peak bone mass followed by an increased age-related
Curr Osteoporos Rep (2020) 18:433–438 435
bone loss later in life [10]. The aging male and female CB2−/ signaling in the trabecular bone and the increased bone for-
−
/C57BL showed an increase in bone resorption and bone mation results from the coupling between osteoclasts and os-
formation resulting in a high bone turnover with a negative teoblasts [51]. In the cortical bone also, CB2 agonists stimu-
balance outcome [10, 33, 40], similarly to human postmeno- lated endosteal bone formation [10]. Although the skeletal
pausal osteoporosis [43]. This increased age-related bone loss CB2 effect in vivo may be mediated by non-skeletal cells
phenotype was the first spontaneous phenotype reported for (e.g., monocytes and must cells that release NO in response
CB2−/− mice and is in line with reports from human showing to ECs, which may in turn affect bone formation [52, 53]),
that CB2 polymorphism is associated with osteoporosis and several in vitro studies on isolated OB cultures indicate that
bone strength [21, 44]. Several studies demonstrate that the CB2 signaling stimulates OB proliferation, differentiation,
CB2 skeletal effect is strain-dependent as well. In resemblance and osteogenic activity [10, 30, 34, 40]. While studies are in
to CB1, findings indicate that CB2−/− female mice on CD1 agreement regarding the direct action of CB2 in OB, studies
background (CB2−/−/CD1) and CB2−/−/C57BL have a differ- investigating the direct CB2 effect on murine and human OC
ent skeletal phenotype at young age and similar in aged mice. have reached contradicting results. Some studies concluded
The young CB2−/−/CD1 displayed a high bone mass with that CB2 signaling increases OC differentiation [14, 39],
slow bone turnover relative to WT controls [45]. However, while others in murine and human OC showed evidence that
CB2 knockout in aging CD1 mice resulted in bone loss due to CB2 signaling inhibits osteoclastogenesis [10, 50, 54].
accelerated bone remodeling like the aging CB2−/−/C57BL Although these contradictions are not yet settled, the latter
mice. Comprehensive transcriptomic data produced in a fol- results are in line with all the findings that show increased
lowing study shows that CB2 deficiency has different effects bone resorption in CB2−/− mice [10, 40].
on the genetic profile between the two strains, suggesting that In light of the strain differences observed in mice, it is of
the CB2 deficiency–related skeletal phenotype is strain- utmost importance to elucidate the role of CB2 in humans.
specific [46]. Interestingly, a more recent study showed that While the CB2 exact pathways remains unclear, a substantial
a CB1 and CB2 combined deficiency, on CD1 background number of studies in different human populations indicate that
(CB1−/-CB2−/−/CD1), prevented age-related bone loss by CB2 is strongly associated with bone mineral density [21, 44,
inhibiting osteoclast formation. In this study, young CB1−/ 55–57]. Polymorphism in CNR2, caused from a non-
-
CB2−/−/CD1 presented a high peak bone mass similar to mice conservative missense mutation in the CNR2 sequence
of the same age with a single receptor deficiency, CB1−/−/CD1 (Gly63Arg), effects CB2 expression and activity and is
or CB2−/−/CD1. Additionally, aged CB1−/-CB2−/−/CD1 were strongly associated with osteoporosis and bone strength in
partially protected against age-related bone loss, unlike mice humans [21, 44]. This exact gene polymorphism is also asso-
with a single cannabinoid receptor deficiency that have an ciated with reduced endocannabinoid-modulation of the im-
accelerated age-related bone loss [47•]. mune system and is linked to autoimmunity in human
Interactions between CB2 signaling and sex hormones are Caucasians [58]. These findings are in agreement with the
not entirely understood. For instance, CB2 deficiency in the results from the aforementioned studies in C57BL/6J mice
CD1 strain did not show a skeletal effect in male mice [45]. supporting the bone protective activities of CB2.
Some studies suggested that CB2−/− female mice are partly Apart from the age-related low bone mass phenotype in
protected from osteoporosis in the OVX-induced bone loss CB2−/− mice, it is noteworthy to mention that CB2−/− mice
model [39, 40]. Other studies showed that stimulation of also display significantly longer femurs and an increase in
CB2 signaling with the selective CB2 agonists in OVX mice length of the vertebral bodies when comparing with CB1−/−
prevents OVX-induced bone loss [10, 48]. Although these or WT mice [31, 59]. This indicates that CB2 has a role in the
studies were not conducted on the same mouse strains, we regulation and attenuation of bone elongation in growing an-
may speculate that the skeletal actions of sex hormones de- imals as well.
pend on the presence of CB2, but the skeletal actions of CB2 Together, all findings demonstrate that both CB1 and CB2
activation are independent of sex hormones. On the other receptors have different but important roles in the skeletal
hand, evidence shows that estrogens can modulate EC ligands metabolism. However, we could find no studies on the thera-
in rats as well as CB2 expression in rats and humans [49, 50]. peutic potential of CB1 or CB2 selective agonists/antagonists
More studies are required to better understand the interactions in models of bone healing and bone regeneration. One study
of CB2 and sex hormones in skeletal homeostasis. tested THC, a CB1/CB2 agonist with a 16-fold higher affinity
As in the case of CB1, more research is required to deter- for CB1, in fracture healing in rats. In this single report, THC
mine the exact actions of CB2 on bone formation and resorp- had no significant effect on the structural and biomechanical
tion. Separate studies reported that both CB2 agonists and properties of the fracture callus [60••]. Further studies are
CB2 knockout result in increased bone formation rate [10, warranted to determine the therapeutic potential or possible
40]. The increased bone turnover rate and bone loss in aging deleterious effects of CB1/CB2 agonists/antagonists in bone
CB2−/− mice suggests that bone resorption is inhibited by CB2 healing and regeneration.
CBD The experimental evidence from animals and humans

strongly suggests that CB1 and CB2 are bone protective.
There is a dearth of studies on the potential use of CBD in Studies in mice determined that CB2 activation stimulates
bone homeostasis, fracture healing, and regeneration. A recent bone formation and inhibits bone resorption. A number of
study tested the effect of CBD on spinal cord injury (SCI)– studies in humans indicate that CB2 is protective against os-
induced bone loss in rats [61]. In this study, researchers found teoporosis and may prevent bone fractures. To date, there are
that CBD administration not only attenuated the sublesional no reports on the therapeutic potential of CB1 or CB2 agonists
cancellous bone loss but also enhanced the mechanical prop- and antagonists in bone healing and regeneration. Whether
erties of the femurs in SCI rats. Notably, CBD treatment in modulating the EC system would affect bone repair remains
SHAM rats had no significant effect [61], suggesting that therefore an open question that is worth investigating.
CBD specifically prevented the bone detrimental effects of The main non-psychoactive component of cannabis, CBD,
SCI. We generated unpublished data confirming CBD has had no significant effect in normal bone homeostasis but it
no effect on bone homeostasis in mice. Mice treated with was found to be beneficial in enhancing healing and recovery
5 mg/Kg/day of CBD were analyzed at 12 weeks of age and following bone injury.
showed no difference in the trabecular (trabecular bone frac- Taken together, there is growing evidence that the EC sys-
tion, BV/TV = 6.62% ± 2.39 and 6.92% ± 2.60 in treated vs tem holds important roles in skeletal homeostasis throughout
non-treated mice, p = 0.94) and cortical (cortical thickness: life. Cannabinoid based therapies targeting the EC receptors
146 ± 12 μm and 146 ± 14 μm in treated vs non-treated mice, may be used for treating and preventing age-related and hor-
p = 0.97) femoral bone parameters. On the other hand, CBD mone deficiency–related bone loss and osteoporosis. Also, the
may have a therapeutic effect in bone repair. One study inves- polymorphism in CNR2 can be used as an early diagnostic
tigated the effect of CBD alone or in combination with THC in tool to detect genetic-predisposition to osteoporosis in
a fracture healing model in rats [60••]. Their results indicated humans. Further experimental and clinical studies are warrant-
that CBD alone significantly improved the mechanical prop- ed to establish the putative therapeutic benefit of EC modula-
erties of the fracture callus although it did not increase its tors and phyto-cannabinoids for the treatment of skeletal inju-
volume or mineral content. Treatment with THC alone and ries and regeneration.
in combination with CBD did not improve but also did not
impair bone healing. This CBD-induced increase in callus Compliance with Ethical Standards
strength and toughness was associated with the increased ex-
pression of procollagen-lysine 2-oxoglutarate 5-dioxygenase Conflict of Interest B.R-M. and Y.G. declare no conflict of interest
related to this study.
(PLOD1), a collagen cross-linking enzyme, in cultured prima-
ry osteoblasts treated with CBD [60••]. These results revealed
Human and Animal Rights and Informed Consent This article does not
a specific action of CBD in enhancing fracture healing in long contain any studies with human or animal subjects performed by any of
bones by enhancing the biomechanical quality of the newly the authors.
formed bone. A more recent study confirmed that CBD en-
hances healing and improved biomechanical properties in a rat
model of critical-sized defect in long bones [62••]. In this References
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215. tional claims in published maps and institutional affiliations.
Hindawi Publishing Corporation
Stem Cells International
Volume 2013, Article ID 796715, 8 pages
http://dx.doi.org/10.1155/2013/796715
Research Article
The Cannabinoid Receptor Type 1 Is Essential for
Mesenchymal Stem Cell Survival and Differentiation:
Implications for Bone Health
Aoife Gowran,1 Katey McKayed,1,2 and Veronica A. Campbell1,2

1
Discipline of Physiology, School of Medicine, Trinity Biomedical Sciences Institute, University of Dublin,
Trinity College, Dublin 2, Ireland
2
School of Engineering, Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, University of Dublin,
Trinity College, Dublin 2, Ireland
Correspondence should be addressed to Aoife Gowran; gowrana@tcd.ie
Received 30 November 2012; Revised 30 May 2013; Accepted 4 June 2013
Academic Editor: Pranela Rameshwar
Copyright © 2013 Aoife Gowran et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Significant loss of bone due to trauma, underlying metabolic disease, or lack of repair due to old age surpasses the body’s endogenous
bone repair mechanisms. Mesenchymal stem cells (MSCs) are adult stem cells which may represent an ideal cell type for use in cell-
based tissue engineered bone regeneration strategies. The body’s endocannabinoid system has been identified as a central regulator
of bone metabolism. The aim of the study was to elucidate the role of the cannabinoid receptor type 1 in the differentiation and
survival of MSCs. We show that the cannabinoid receptor type 1 has a prosurvival function during acute cell stress. Additionally,
we show that the phytocannabinoid, Δ9 -Tetrahydrocannabinol, has a negative impact on MSC survival and osteogenesis. Overall,
these results show the potential for the modulation of the cannabinoid system in cell-based tissue engineered bone regeneration
strategies whilst highlighting cannabis use as a potential cause for concern in the management of orthopaedic patients.
1. Introduction anandamide and 2-arachidonoylglycerol, and their degrada-

Mesenchymal stem cells (MSCs) are multipotent adult stem tive enzymes fatty acid amide hydrolase and monoacylglyc-
cells present in the bone marrow which can differentiate along erol lipase, respectively. In addition, exogenous cannabinoids
several lineages, for example, bone, cartilage, and tendon such as the bioactive lipids isolated from the Cannabis sativa
[1]. Musculoskeletal repair relies on a series of orchestrated plant and synthetic cannabinoids are currently used thera-
events that direct the differentiation of MSCs to its progeny, peutically for a number of diseases such as multiple scle-
for example, osteoblasts, chondrocytes, and tenocytes. MSCs rosis [7]. However, phytocannabinoids have a dual toxicity
represent an ideal cell population for use in tissue engineering profile with the psychoactive component of cannabis, Δ9 -
and regenerative medicine due to their ease of isolation, Tetrahydrocannabinol (Δ9 -THC), inducing cell death in a
multipotency, lack of immunogenicity, and immunosuppres- number of cell types [8–11]. Δ9 -THC is a partial agonist of
sive effects [2]. Tissue engineering aims to learn how to the CB1 and CB2 receptors but displays higher efficacy at CB1
induce, modulate and control the differentiation process of over CB2 where it has reported antagonist activity [12].
MSCs in order to provide therapeutics for musculoskeletal The endocannabinoid system is an important regulator of
diseases [3]. We have recently shown that the osteogenic bone mass maintenance. In 2005, Idris et al. reported that CB1
and chondrogenic differentiation process may be controlled receptor inactivation resulted in increased bone mass and
by specific growth factors [4], hypoxia [5], and biophysical protected against ovariectomy-induced bone loss, an in vivo
stimulation [6]. model of osteoporosis [13]. Further investigation of the skel-
The endocannabinoid system is comprised of two G pro- etal phenotype of CB1 knock-out mice has demonstrated that
tein-coupled receptors, CB1 and CB2 , the endogenous ligands animals display increased bone mass at 3 months of age,
2 Stem Cells International
due to reduced osteoclast activity, but develop age-related receptor antagonist/inverse agonist SR141716 was a kind gift
osteoporosis by 12 months, due to enhanced adipocyte dif- form Dr. David Finn at The National University of Ireland,
ferentiation [14]. CB2 receptor agonists increase bone mass by Galway (original source: The National Institute of Mental
enhancing osteoblast numbers and activity, inhibiting the Health’s Chemical Synthesis and Drug Supply Program).
proliferation of osteoclasts and stimulating fibroblastic SR141716 was stored as a 10 mM stock solution in DMSO at
colony formation by bone marrow cells [15, 16]. Furthermore, −20∘ C and diluted to a final concentration of 1 𝜇M in culture
CB2 regulates bone loss during periods of increased bone tur- media. Δ9 -THC was obtained from Sigma-Aldrich Company
nover also involving the regulation of osteoclast function [17]. Ltd. and held under license granted by the Irish Department
The aim of the present study was to elucidate the role of Health and Children. Δ9 -THC was stored as a 80 mM
of the cannabinoid system in the survival and differentia- stock solution in ethanol at −20∘ C and diluted to a final
tion of culture-expanded MSCs in the presence of known concentration of 1 𝜇M in culture media.
osteogenic factors: dexamethasone, 𝛽-glycerophosphate, and
ascorbic acid. The results demonstrate that the CB1 receptor 2.3. RNA Isolation. Total RNA was isolated from MSCs using
is upregulated during osteogenic differentiation of MSCs a NucleoSpin total RNA isolation kit (Macherey-Nagel Inc.,
and is essential for the survival of differentiated MSCs. Germany) following the manufacturer’s instructions. This
We also show that the psychoactive phytocannabinoid, Δ9 - protocol included a DNase step in order to remove any
Tetrahydrocannabinol, has a negative impact on MSC sur- genomic DNA contamination. Total RNA concentrations
vival and osteogenesis. were determined by spectrophotometry (NanoDrop Tech-
nologies, USA) and stored at −80∘ C until required for cDNA
2. Materials and Methods synthesis.
2.1. Culture of Mesenchymal Stem Cells. Three-month-old 2.4. cDNA Synthesis. Total RNA concentrations were adju-
Wistar rats (250–300 g) were obtained from the Bioresources sted to a standard concentration prior to cDNA synthesis.
Unit, University of Dublin, Trinity College. Animals were cDNA was generated from 0.5–1 𝜇g total RNA using High
sacrificed by CO2 asphyxiation and cervical dislocation in Capacity cDNA Archive kit (Applied Biosystems, Germany)
accordance with European guidelines (86/609/EEC). The following the manufacturer’s instructions. The resultant
femur and tibia were dissected free and placed in ster- cDNA was stored at −20∘ C until required for real time PCR.
ile prewarmed supplemented Dulbecco’s modified Eagle’s
medium (s-DMEM; Sigma-Aldrich, UK). Supplements were
10% foetal bovine serum; 100 U/mL penicillin/streptomycin; 2.5. Real-Time PCR. Real-time PCR was performed using
2 mM GlutaMAX; 1 mM L-glutamine; and 1% nonessential Taqman Gene Expression Assays (Applied Biosystems, Ger-
amino acids (Invitrogen, Scotland). The femur and tibia were many) on an ABI Prism 7300 instrument (Applied Biosys-
cut at both epiphyses, and bone marrow was flushed into tems, Germany). The assay IDs for the genes examined were
a 50 mL tube using 5 mL s-DMEM and a 25-gauge needle. as follows: CB1 receptor (Rn00562880 m1), CB2 receptor
The suspension was centrifuged (650 × g) for 5 minutes (Rn01637601 m1), osteocalcin (Rn00566386 g1), and 𝛽-actin
at 20∘ C, resuspended in 10 mL of s-DMEM, and passed (4352340E). Gene expression was calculated relative to the
sequentially through 16-, 18-, and 20-gauge needles. The endogenous control (𝛽-actin) and to the control samples to
suspension was passed through a 40 𝜇m nylon mesh into a give a relative quantification (RQ) value.
sterile Petri dish and incubated in a humidified atmosphere
(95% air and 5% CO2 ) at 37∘ C for 30 min. The supernatant 2.6. Cell Viability Assay. Cell viability was determined by
was removed and split between two T75 flasks. Culture media quantifying the enzymatic conversion of cell permeable
was replaced following 24 hours to remove nonadherent cells. calcein AM (Invitrogen, Scotland) to a fluorescent product
Cells were passaged upon reaching 80–90% confluency to by active intracellular esterases. Briefly, MSCs were grown on
a maximum of 4 passages. The medium was replaced every sterile 96 well plates (6 × 103 cells per well) and treated as
3 to 4 days. To induce osteogenesis, cells were treated with indicated in each experiment. Calcein AM solution (2 𝜇M
osteogenic factors (OF): 100 nM dexamethasone, 10 mM 𝛽- in PBS) was applied to each well and incubated in a
glycerophosphate and 50 𝜇M ascorbic acid for the indicated humidified atmosphere (95% air and 5% CO2 ) at 37∘ C for
time period (2–5 weeks). These cells are referred to as 1 hour. Following incubation calcein fluorescence at 530 nm
differentiated cells, whilst cells maintained in regular culture was determined using a microplate reader heated to 37∘ C
medium are referred to as undifferentiated cells. Addition- (Synergy HT, BioTek Instruments, USA).
ally, the differentiation capacity of MSCs was investigated
and verified using previously described methods for the 2.7. Immunofluorescent Staining for Active Caspase-3 and
induction and detection of osteogenesis, chondrogenesis Apoptotic Nuclei Determination. Following drug treatment,
[4], and adipogenesis [18] in bone marrow derived MSCs MSCs were fixed in 100% methanol for 5 minutes at −20∘ C,
(see Figure 1 in Supplementary Material available online at permeabilised with 0.2% Triton-X100 for 10 minutes, and
http://dx.doi.org/10.1155/2013/796715). washed in 3 changes of PBS at room temperature (RT).
MSCs were blocked with 30% goat serum overnight at 4∘ C
2.2. Drug Treatments. MSCs were incubated with drugs or (Vector Laboratories, USA). Caspase-3 was labelled with a
vehicle for the time indicated in each experiment. The CB1 rabbit antiactive caspase-3 (1 : 1000 in 30% blocking buffer;
Stem Cells International 3
Promega, England) for 1 hour at RT. Labelled protein was differentiated MSCs were deprived of serum in the presence
detected with goat anti-rabbit secondary antibody conjugated or absence of the CB1 receptor antagonist/inverse agonist,
to biotin (1 : 1500 in 30% blocking buffer; Vector Laboratories, SR141716 (SR1; 1 𝜇M), and cell viability was measured by
USA) for 1 hour at RT. MSCs were then incubated with monitoring the metabolism of calcein AM. In undifferen-
avidin-conjugated FITC (1 : 500; Sigma-Aldrich, England) tiated MSCs, fluorescent intensity at 530 nm, a marker of
for 1 hour at RT. Nuclei were stained with Hoechst 33258 cellular metabolism and viability, was 5.62 ± 0.56 (×104 RFU
(1 : 500; Invitrogen, Scotland) for 15 minutes at RT. Coverslips at 530 nm, mean ± SEM), and this was significantly reduced
were mounted with mounting medium (Vector Laboratories, to 1.6 ± 0.69 following serum withdrawal for 24 hours
USA). Incorporated fluorophores were examined with a (𝑃 < 0.001, 1-way ANOVA and Newman-Keuls, 𝑛 = 5;
confocal microscope (Carl Zeiss, Germany) using appropri- Figure 1(b)). In contrast, when differentiated MSCs were
ate excitation wavelengths and filter sets. The number of exposed to serum withdrawal fluorescence was unaffected,
abnormal apoptotic nuclei was determined (by a blinded indicating that the differentiated MSCs were able to withstand
counter) from 10 random fields of view for each treatment serum withdrawal. However, in the presence of SR141716
group with the 𝑛 number indicated in each experiment. (SR1; 1 𝜇M; 24 hours) the differentiated MSCs were unable
to survive following serum withdrawal indicating that the
2.8. Extracellular Matrix Mineralization Quantification. The increased levels of CB1 receptor present in differentiated
specific marker of mineralized bone, hydroxyapatite, was MSCs are essential for survival. Treatment of differentiated
quantified using a commercially available assay kit (Lonza, MSCs with SR1 alone had no effect on MSC cell viability
Switzerland) following the manufacturer’s instructions. indicating that SR1 treatment was not toxic.
Briefly, MSCs were grown on sterile 96 well plates (13 × 103 In addition, we monitored cell death by assessing the
cells per well) and treated as indicated in each experiment. percentage of apoptotic nuclei and the expression of the active
Following treatment, MSCs were washed in PBS (×2) and form of the proapoptotic protein, caspase-3 (Figures 1(c) and
then fixed in 100% ethanol for 20 minutes at RT. MSCs 1(d)). In undifferentiated MSCs, serum withdrawal signifi-
were incubated with fluorescent staining reagent specific for cantly increased the percentage of apoptotic nuclei from 14 ±
hydroxyapatite for 30 minutes at RT. MSCs were washed 2% to 47 ± 3% (mean ± SEM; 𝑃 < 0.001, 1-way ANOVA and
in diluted wash buffer (×3), and fluorescence was read at Newman-Keuls, 𝑛 = 4; Figure 1(c)) and also increased the
518 nm using a spectrophotometer (Labsystems, Finland). expression of active caspase-3 (Figure 1(d)(ii)). However, in
In some experiments, MSCs were grown on glass coverslips differentiated MSCs serum withdrawal evoked significantly
and stained with the fluorescent staining reagent specific less apoptosis (14 ± 1% apoptotic nuclei, mean ± SEM; 𝑃 <
for hydroxyapatite. Nuclei were stained with Hoechst 0.001, 1-way ANOVA and Newman-Keuls, 𝑛 = 4; Figure 1(c)).
33258. Labelled hydroxyapatite and nuclei were visualized In the presence of SR141716 the apoptotic effect of serum
with a confocal microscope (Carl Zeiss, Germany) using withdrawal was restored (43 ± 3% apoptotic nuclei) in the
appropriate excitation wavelengths and filter sets. differentiated MSCs. These results provide evidence that the
CB1 receptor in differentiated MSCs is essential for survival
following an insult such as serum withdrawal.
2.9. Statistical Analysis. Data are reported as the mean ±
SEM of the number of experiments indicated in each case.
ANOVA followed by a Student Newman-Keuls post hoc test 3.2. Δ9 -THC Negatively Impacts on MSC Viability and
was used to determine the statistical significance between Osteogenic Potential. Given that we have shown an essential
groups. For comparisons between relevant treatments, an role for the CB1 receptor in the survival of MSCs during
unpaired Student’s 𝑡-test was performed. stressful stimulus (serum withdrawal) we therefore sought
to elucidate if exogenous cannabinoids could interfere with
MSC viability and differentiation capacity. Hence, we moni-
3. Results tored the effect of exogenous phytocannabinoid Δ9 -THC on
3.1. Increased CB1 Receptor Expression Is Responsible for the viability and osteogenic capacity of MSCs.
MSC Survival during Osteogenesis. As MSCs underwent The effect of the Δ9 -THC on the viability of MSCs was
osteogenic differentiation, a significant increase in CB1 recep- determined by assessing the ability of undifferentiated and
tor mRNA expression was observed after 2 weeks of dif- differentiated MSCs treated with Δ9 -THC to metabolise
ferentiation (6.15 ± 1.28; RQ value, mean ± SEM) com- calcein AM. Treatment with Δ9 -THC (1 𝜇M, 2 weeks) sig-
pared to undifferentiated MSCs (0.36 ± 0.17; RQ value, nificantly reduced undifferentiated MSC metabolic activity
mean ± SEM; 𝑃 = 0.002, Student’s unpaired 𝑡-test, 𝑛 = 5; from 3.68 ± 0.83 (×104 RFU at 530 nm, mean ± SEM) to
Figure 1(a)). No change in CB2 receptor mRNA expression 0.88 ± 0.15 (𝑃 < 0.05, 1-way ANOVA and Newman-
was observed between undifferentiated and differentiated Keuls, 𝑛 = 5; Figure 2(a)). In differentiated MSCs treatment
MSCs (supplemental Figure 2). with Δ9 -THC (1 𝜇M, 2 weeks) induced a significant decrease
Since an induction of CB1 receptor mRNA was evident in MSC metabolic activity (𝑃 < 0.05, 1-way ANOVA
in MSCs undergoing osteogenic differentiation, we sought and Newman-Keuls, 𝑛 = 5; Figure 2(a)). Additionally,
to identify whether the induction of the CB1 receptor was treatment of undifferentiated and differentiated MSCs with
pertinent in the control of any aspect of MSC function and Δ9 -THC (1 𝜇M, 2 weeks) evoked a significant increase in
focused our attention on cell survival. Undifferentiated and the % of apoptotic nuclei (𝑃 < 0.001, 1-way ANOVA and
8 100
∗∗
80
CB1 mRNA expression (RQ)
Apoptotic nuclei (%)

60
∗∗∗ £££
4
40
2 20 +++ +++
0
0
Undifferentiated Differentiated
Undifferentiated MSCs MSCs MSCs
Differentiated MSCs Con Serum withdrawal + SR1 (1 𝜇M)
Serum withdrawal
(a) (c)
8
++
++
Calcein fluorescent intensity
6
(×104 RFU at 530 nm)
∗∗
2 (i) (ii)
££
0
MSCs MSCs
Con
Serum withdrawal
SR1 (1 𝜇M)
Serum withdrawal + SR1 (1 𝜇M) (iii) (iv)
(b) (d)
Figure 1: The CB1 receptor is increased during early osteogenesis and is essential for the survival of differentiated MSCs. (a) Differentiated
MSCs displayed a significant increase in CB1 receptor mRNA expression after 2 weeks of differentiation compared to undifferentiated MSCs
(∗∗ 𝑃 = 0.002, Student’s unpaired 𝑡-test, 𝑛 = 5). (b) Serum withdrawal significantly reduced the metabolic function of undifferentiated MSCs
(Con; ∗∗ 𝑃 < 0.01, 1-way ANOVA and Newman-Keuls, 𝑛 = 5). In differentiated MSCs, serum withdrawal had no effect on metabolic function,
and serum deprived differentiated MSCs displayed significantly greater metabolic function compared to serum deprived undifferentiated
MSCs (++ 𝑃 < 0.01, 1-way ANOVA and Newman-Keuls, 𝑛 = 5). Treatment of differentiated MSCs with SR141716 (SR1, 1 𝜇M; 24 hours) blocked
the ability of differentiated MSCs to survive serum withdrawal (££ 𝑃 < 0.01, 1-way ANOVA and Newman-Keuls, 𝑛 = 5). (c) Serum withdrawal
induced a significant increase in the numbers of undifferentiated MSCs displaying apoptotic nuclei (Con; ∗∗∗ 𝑃 < 0.001, 1-way ANOVA
and Newman-Keuls, 𝑛 = 4) compared to undifferentiated MSCs maintained with serum. Differentiated MSCs survived serum withdrawal
compared to serum deprived undifferentiated MSCs (+++ 𝑃 < 0.001, 1-way ANOVA and Newman-Keuls, 𝑛 = 4). Treatment of differentiated
MSCs with SR1 blocked the ability of differentiated MSCs to survive serum withdrawal compared to serum deprived differentiated MSCs
(£££ 𝑃 < 0.001, 1-way ANOVA and Newman-Keuls, 𝑛 = 4). (d) Representative images of caspase-3 activity in undifferentiated MSCs exposed
to control (i) and (ii) serum withdrawal conditions and caspase-3 activity in differentiated MSCs exposed to control (iii) and serum withdrawal
in the presence of SR1 (iv).
Calcein fluorescent intensity (RFU at 530 nm)
60000 100
80
Apoptotic nuclei (%)

40000
60 ++
40 ∗∗∗
+
20000
∗ 20
0 0
Undifferentiated Differentiated Undifferentiated Differentiated
MSCs MSCs MSCs MSCs
Con Con
Δ9 -THC (1 𝜇M) Δ9 -THC (1 𝜇M)
(a) (b)
15
Hydroxyapatite deposition (RFU at 518 nm)
10
∗∗∗
(i) (ii)
5
+++
0
MSCs MSCs
Con
(iii) (iv) Δ9 -THC (1 𝜇M)
(c) (d)
Figure 2: Δ9 -THC negatively affects MSC viability and inhibits MSC osteogenesis. (a) Treatment of undifferentiated MSCs with Δ9 -THC
(1 𝜇M) significantly reduced viability compared to control undifferentiated MSCs (Con; ∗ 𝑃 < 0.05, 1-way ANOVA and Newman-Keuls,
𝑛 = 5). Also, differentiation of MSCs in the presence of Δ9 -THC significantly decreased viability compared to control differentiated MSCs
(Con; + 𝑃 < 0.05, 1-way ANOVA and Newman-Keuls, 𝑛 = 5). (b) Treatment of undifferentiated MSCs with Δ9 -THC induced a significant
increase in the percentage of apoptotic nuclei compared to control MSCs (Con; ∗∗∗ 𝑃 < 0.001, 1-way ANOVA and Newman-Keuls, 𝑛 = 6).
Also, differentiation of MSCs in the presence of Δ9 -THC significantly increased the percentage of apoptotic nuclei compared to control
differentiated MSCs (++ 𝑃 < 0.001, 1-way ANOVA and Newman-Keuls, 𝑛 = 6). (c) Representative images of cells stained for active caspase-3
in control undifferentiated MSCs (i), undifferentiated MSCs treated with Δ9 -THC (ii), control differentiated MSCs (iii), and differentiated
MSCs in the presence of Δ9 -THC (iv). (d) Differentiation of MSCs in the presence of Δ9 -THC (1 𝜇M) significantly decreased hydroxyapatite
deposits compared to control differentiated MSCs (+++ 𝑃 < 0.001, 1-way ANOVA and Newman-Keuls, 𝑛 = 6).
Newman-Keuls, 𝑛 = 6; Figure 2(b)) and caspase-3 activity Newman-Keuls, 𝑛 = 6; Figure 2(d)). However, MSCs dif-
(Figure 2(c)). ferentiated with OF in the presence of Δ9 -THC had reduced
The effect of the Δ9 -THC on the differentiation of MSCs osteogenic potential (2.30 ± 0.87, RFU at 518 nm, mean ±
was determined by monitoring hydroxyapatite deposits SEM; 𝑃 < 0.001 1-way ANOVA and Newman-Keuls, 𝑛 = 6;
in undifferentiated and differentiated MSCs. Deposits of Figure 2(d)). These results indicate that the phytocannabi-
hydroxyapatite were significantly increased from 1.71 ± 0.07 noid Δ9 -THC has a negative effect on osteogenesis by decreas-
(RFU at 518 nm, mean ± SEM) to 8.30 ± 0.57 in MSCs ing the survival of both undifferentiated and differentiated
differentiated with OF (𝑃 < 0.001, 1-way ANOVA and MSCs.
4. Discussion the activation of ERK [28]. Furthermore, we have previously

shown that activation of the cannabinoid system enhances
The aim of this study was to examine the role of the CB1 the survival, migration, and chondrogenic differentiation
receptor during the osteogenic differentiation of MSCs har- of MSCs, which are the three key points that determine
vested from adult Wistar rats. The results demonstrate that the success of cell-based tissue-engineered repair strategies
the CB1 receptor is increased during MSC osteogenic dif- [29]. Interestingly, Idris et al. [13] suggest that normal bone
ferentiation and is essential for the survival of differentiated formation in CB1 receptor knock-out mice can be maintained
MSCs during the acute insult of serum withdrawal. We by alternative signalling pathways; however, with increasing
also show that the exogenous phytocannabinoid, Δ9 -THC, age these compensatory mechanisms fail leading to decreased
reduced MSC survival and differentiation potential of MSCs. bone formation. Furthermore, the physiological upregulation
Substantial loss of bone due to trauma, tumour ressection, of the CB1 receptor with age has been proposed to protect
metabolic bone disease or lack of bone repair due to ageing against the development of osteoporosis [13]. Results from
may require intervention to restore a positive balance to bone our experiments using SR141716 show that the CB1 receptor
metabolism [19]. MSCs represent an ideal adult stem cell for is necessary for MSC survival following an acute insult, yet
the use in bone repair since strategies for bone regeneration long term (3–5 weeks) CB1 receptor antagonism results in
(osteogenesis, osteoinduction, osteoconduction, and osteo- increased osteogenesis (supplemental Figure 3) indicating
promotion) all fundamentally rely on MSCs [20]. We have a temporal effect of the CB1 receptor on MSC function.
observed that MSCs produce osteocalcin and extracellular This novel temporal response may reflect a dual role for the
hydroxyapatite deposits (supplemental Figures 1 and 3) con- CB1 receptor in MSC physiology: firstly being essential for
firming the potential of isolated MSCs to become bone form- survival during stress which is of relevance to the inhospitable
ing cells suitable for use in bone tissue engineering strategies environment present around areas of bone healing and
in accordance with previously established criteria [21, 22]. secondly acting as a brake on osteogenesis, reflective of endo-
The CB1 and CB2 receptors are G-protein coupled receptors cannabinoids having an inhibitory role during osteogenesis.
which are currently being assessed, along with the putative The osteogenic effect of long-term CB1 receptor antagonism
cannabinoid receptor GPR55, as potential modulators of that we observed may be due to enhanced signalling through
bone mass [23, 24]. It has been previously established that the CB2 receptor, since CB2 receptor signalling leads to
MSCs express CB1 receptors [12, 14, 15], however, we are expansion of the preosteoblastic pool and increased numbers
the first to show a functional increase of the CB1 receptor of osteoblastic colony formation [14, 15]. Furthermore, CB2
during osteogenesis. We did not observe any increase in receptor activation attenuates bone loss in an animal model
CB2 receptor expression (supplemental Figure 2); however, of bone cancer metastases using sarcoma cells [30]. Further
this may be due to the time point analysed (2 weeks) as studies utilizing CB1 and CB2 knock-out animals will be
expression of the CB2 receptor has previously been found necessary to dissect out the exact role of both receptors and
to be expressed after 3 weeks of osteogenic differentiation to corroborate our findings. Alternatively SR141716 may be
in murine bone marrow-derived primary stromal cells [15]. signalling through other receptors such as PPAR-𝛾 [31].
We have also shown that the CB1 receptor has a functional
role in the survival of differentiated MSCs exposed to an Our results also demonstrate that Δ9 -THC prevents
acute insult (serum withdrawal), which is an in vitro model of osteogenesis and induces cell death in both undifferentiated
the environment surrounding bone fractures or orthopaedic and differentiated MSCs. These findings may provide a
implants. Our results indicate that the CB1 receptor is molecular explanation for the results of Nogueira-Filho and
required for MSC survival during the early stages of MSC coworkers [32] who showed reduced cancellous bone healing
osteogenesis. Successful fracture repair and bone healing around titanium implants, due to a reduction in bone filling
around orthopaedic implants rely on favourable biological in rats subjected to cannabis smoke inhalation. In contrast,
and mechanical environments in addition to the recruitment the nonpsychoactive component of cannabis, cannabidiol has
and differentiation of MSCs. However, in certain circum- been shown to reduce bone resorption during experimental
stances the local environment can be actively inhospitable periodontitis in rats due to the reduction in proinflamma-
to infiltrating MSCs resulting in the failure of bone healing tory mediators [33]. It has been reported that Δ9 -THC is
[20, 25]. The CB1 receptor has been demonstrated to be a mitochondrial inhibitor [34], an effect that may inhibit
cytoprotective in many cell types [26, 27]. In our study we the survival of MSCs and osteoblasts since mitochondrial
show that differentiated MSCs have increased CB1 receptor function determines the viability and osteogenic potency of
and display the ability to survive an acute insult (serum these cells [35]. These reports further emphasise the rele-
withdrawal) compared to undifferentiated MSCs. Interest- vance of our observations that Δ9 -THC exposure increases
ingly, Cudaback and coworkers [28] have demonstrated numbers of apoptotic nuclei and induced the expression
that increased cannabinoid receptor expression changes the of active caspase-3 (a proapoptotic downstream signalling
coupling of these receptors to specific kinase pathways and protease involved in the mitochondrial intrinsic pathway of
the efficacy by which cannabinoid receptor ligands induce apoptosis) in undifferentiated and differentiated MSCs. Thus,
the activation of these pathways. Furthermore, they showed Δ9 -THC exposure may lead to a decreased ability of MSCs
that increased CB1 receptor expression enhanced the efficacy to differentiate into their mature bone forming progeny due
of cannabinoids to regulate the prosurvival AKT pathway to a lack of cell viability at early stages of osteogenesis which
whilst low levels of CB1 receptor expression lead only to could inturn impact upon the osteogenic potential of MSCs
(supplemental Figure 4). Furthermore we conclude that this [2] A. I. Caplan, “Adult mesenchymal stem cells for tissue engineer-
effect is specific to a long-term treatment with Δ9 -THC ing versus regenerative medicine,” Journal of Cellular Physiol-
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[4] E. Farrell, F. J. O’Brien, P. Doyle et al., “A collagen-glycosa-
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minoglycan scaffold supports adult rat mesenchymal stem
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already been clearly established that tobacco and alcohol “Hypoxia promotes chondrogenesis in rat mesenchymal stem
consumption negatively impacts on bone health [36]. cells: a role for AKT and hypoxia-inducible factor (HIF)-1𝛼,”
In summary, we have obtained additional insights into Journal of Cellular Physiology, vol. 216, no. 3, pp. 708–715, 2008.
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maintenance by investigating the cannabinoid system during parison of the involvement of p38, ERK1/2 and PI3K in growth
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potential and immunomodulatory profile of MSCs [37, 38]. In of lysosomal permeability by delta 9-tetrahydrocannabinol
view of this, our results demonstrate the potential application in rat cortical neurones: implications for neurodegeneration,”
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in vitro and in vivo physiological functions representing [10] A. Greenhough, H. A. Patsos, A. C. Williams, and C. Paraskeva,
an intriguing avenue for further research. We also provide “The cannabinoid delta(9)-tetrahydrocannabinol inhibits RAS-
MAPK and PI3K-AKT survival signalling and induces BAD-
evidence that the phytocannabinoid Δ9 -THC has a negative mediated apoptosis in colorectal cancer cells,” International
impact on MSC osteogenesis and survival. This may be a Journal of Cancer, vol. 121, pp. 2172–2180, 2007.
relevant factor which should be considered as a potential
[11] M. M. Caffarel, D. Sarrio, J. Palacios, M. Guzman, and C.
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Abbreviations [12] A. C. Howlett, F. Barth, T. I. Bonner et al., “International union
of pharmacology. XXVII. Classification of cannabinoid recep-
MSCs: Mesenchymal stem cells
tors,” Pharmacological Reviews, vol. 54, pp. 161–202, 2002.
OF: Osteogenic factors
[13] A. I. Idris, R. J. van’t Hof, I. R. Greig et al., “Regulation of bone
CB: Cannabinoid
mass, bone loss and osteoclast activity by cannabinoid recep-
SW: Serum withdrawal. tors,” Nature Medicine, vol. 11, pp. 774–779, 2005.
[14] A. I. Idris, A. Sophocleous, E. Landao-Bassonga et al., “Cannabi-
Conflict of Interests noid receptor type 1 protects against age- related osteoporosis by
regulating osteoblast and adipocyte differentiation in marrow
The authors of the paper do not have a direct financial relation
stromal cells,” Cell Metabolism, vol. 10, no. 2, pp. 139–147, 2009.
with any commercial bodies mentioned in the paper.
[15] O. Ofek, M. Karsak, N. Leclerc et al., “Peripheral cannabinoid
receptor, CB2, regulates bone mass,” Proceedings of the National
Acknowledgment Academy of Sciences of the United States of America, vol. 103, no.
3, pp. 696–701, 2006.
This research was funded by Science Foundation Ireland [16] A. Scutt and E. M. Williamson, “Cannabinoids stimulate fibrob-
(07/RFP/BIMF126). lastic colony formation by bone marrow cells indirectly via CB2
receptors,” Calcified Tissue International, vol. 80, no. 1, pp. 50–
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i An update to this article is included at the end

Materials Science & Engineering C 101 (2019) 64–75

Contents lists available at ScienceDirect

Materials Science & Engineering C

Cannabidiol-loaded microspheres incorporated into osteoconductive T

1. Introduction exogenous cells with biomaterials to improve the healing process of

Macroscopic 1 (0-1) 3 (3-3) 1 (1-1) 2 (2-3) 0.003

4 0 (0-0) 4 (5-6)b 2 (1-2)d 3 (3-5)f 0.002

Table 3 load cell.

2.2.10. Animals and surgical procedures 2.2.11. Gross morphology

Cannabinoids supposedly possess healing properties that are medi-

Contents lists available at ScienceDirect

Materials Science & Engineering C

Corrigendum to “Cannabidiol-loaded microspheres incorporated into

DOI of original article: https://doi.org/10.1016/j.msec.2019.03.070.

Available online 11 May 2021

Dent. J. 2022, 10, 193. https://doi.org/10.3390/dj10100193 https://www.mdpi.com/journal/dentistry

2. Materials and Methods

2.1. Eligibility Criteria

2.2. Information Sources and Search

Table 1. Search strategy used in the different databases.

(THC) OR (Tetrahydrocannab) OR (Dronabin) OR (Cannab) OR (Phytocannab) OR (Marijuana) OR

THC OR Tetrahydrocannab OR Dronabin OR Cannab OR Endocannab OR Phytocannab OR

2.3. Selection of Sources of Evidence

2.4. Data Charting, Data Items, and Analysis

Table 2. Characteristics of the original articles included, n = 11.

3.2. Characteristics of Patents

Table 3. Characteristics of the patents included, n = 13.

3.3. Synthesis of Results and Summary of Evidence

4.1. Periodontal Therapy

4.2. Oral Medicine

4.3. Adjuncts to Oral Surgery and Traumatology

4.4. The Scientific and Technological Scenario

4.6. Future Challenges

• Acceptability of CBD-based treatments among dental patients;

BONE AND JOINT PAIN (P MANTYH AND T SCHNITZER, SECTION EDITORS)

The Cannabinoids Effect on Bone Formation and Bone Healing

Published online: 23 July 2020

Introduction endocannabinoid main endogenous ligands are N-

THC and cannabidiol (CBD) are the main pharmacological CB1

CBD The experimental evidence from animals and humans

Aoife Gowran,1 Katey McKayed,1,2 and Veronica A. Campbell1,2

Correspondence should be addressed to Aoife Gowran; gowrana@tcd.ie

Received 30 November 2012; Revised 30 May 2013; Accepted 4 June 2013

Academic Editor: Pranela Rameshwar

1. Introduction anandamide and 2-arachidonoylglycerol, and their degrada-

Apoptotic nuclei (%)

Apoptotic nuclei (%)

4. Discussion the activation of ERK [28]. Furthermore, we have previously

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