uncoupled angiogenesis and osteogenesis in nicotine-compromised bone healing

ORIGINAL ARTICLE JJBMR
Uncoupled Angiogenesis and Osteogenesis inNicotine-Compromised Bone Healing
Li Ma,1 Li Wu Zheng ,1 Mai Har Sham,2 and Lim Kwong Cheung1

1Oral and Maxillofacial Surgery, Faculty of Dentistry, University of Hong Kong, Hong Kong, China2Department of Biochemistry, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

ABSTRACTNicotine is the main chemical component responsible for tobacco addiction. This study aimed to evaluate the influence of nicotine on

angiogenesis and osteogenesis and the associated expression of angiogenic and osteogenic mediators during bone healing. Forty-eight

adult New Zealand White rabbits were randomly assigned to a nicotine group and a control group. Nicotine pellets (1.5 g, 60-day time

release) or placebo pellets were implanted in the neck subcutaneous tissue. The nicotine or placebo exposure time for all the animals was

7 weeks. Unilateral mandibular distraction osteogenesis was performed. Eight animals in each group were euthanized on day 5, day 11 of

active distraction, and week 1 of consolidation, respectively. The mandibular samples were subjected to radiographic, histologic,

immunohistochemical, and real-time reverse-transcriptase polymerase chain reaction examinations. Nicotine exposure upregulated the

expression of hypoxia inducible factor 1a and vascular endothelial growth factor and enhanced angiogenesis but inhibited the

expression of bone morphogenetic protein 2 and impaired bone healing. The results indicate that nicotine decouples angiogenesis and

osteogenesis in this rabbit model of distraction osteogenesis, and the enhanced angiogenesis cannot compensate for the adverse effects

of nicotine on bone healing. � 2010 American Society for Bone and Mineral Research.

KEY WORDS: NICOTINE; BONE HEALING; ANGIOGENESIS; OSTEOGENESIS; BONE MORPHOGENETIC PROTEIN 2; VASCULAR ENDOTHELIAL GROWTH

FACTOR; HYPOXIA INDUCIBLE FACTOR 1a

Introduction

Nicotine is the main chemical component responsible for

tobacco addition.(1) It is of the highest importance among

the potentially toxic substances in tobacco products.(1–3) Studies

showed that nicotine delays bone healing, but the molecular

mechanisms remains unclear.(2,4–8) Recently, we have developed

a nicotine-induced rabbit model of mandibular distraction

osteogenesis and confirmed the positive correlation between

the blood nicotine concentration and compromised bone

healing.(4,5) The molecular mechanism of nicotine-compromised

bone healing could be explored conveniently with this animal

model.

Distraction osteogenesis is a controlled surgical procedure

that initiates a regenerative process. It applies mechanical strain

to enhance the biologic responses in the injured tissues to create

new bone. Distraction osteogenesis shares many features of

embryonic growth, fetal growth, and neonatal limb develop-

ment, as well as fracture repair.(9,10) Compared with bone

fracture, in which the molecular signaling lasts only for a few

days, the signaling in distraction regeneration is magnified and

Received in original form April 15, 2009; revised form November 17, 2009; accepte

Address correspondence to: Lim Kwong Cheung, BDS, PhD, Oral and Maxillofacial Su

E-mail: [email protected]

Journal of Bone and Mineral Research, Vol. 25, No. 6, June 2010, pp 1305–1313

DOI: 10.1002/jbmr.19

� 2010 American Society for Bone and Mineral Research

prolonged as long as the mechanical traction is active. The

molecular signaling cascade induced by the mechanical strain

plays a key regulatory role in translating traction forces into a

biologic response of bone cells.

Angiogenic and osteogenic factors play an important role in

bone healing and regeneration. Vascular endothelial growth

factor (VEGF) is a potent angiogenic mediator inducing

proliferation and migration of endothelial cells. Moreover, it

has been shown to promote chemotaxis(11) and differentiation of

osteoblasts.(12,13) VEGF can interact synergistically with bone

morphogenetic protein (BMP) to promote skeletal development

and bone healing by enhancing cell recruitment, prolonging cell

survival, and increasing angiogenesis.(14) BMPs are the most

potent osteogenic growth factors inducing the osteogenic

differentiation of mesenchymal stem cells.(15–17) BMP acts as an

important regulator that stimulates production of VEGF in

osteoblasts.(18–20) Hypoxia is the most potent stimulus for VEGF

expression.(21,22) Hypoxia-inducible factor 1a (HIF-1a), a central

regulator of hypoxia adaptation in vertebrates, plays a key role in

development, physiology, and disease(23) and activates down-

stream hypoxia-responsive genes such as VEGF.(24–28) We

d December 29, 2009. Published online January 14, 2010.

rgery, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong SAR, China.

1305

hypothesized that nicotine exposure affects angiogenesis and

osteogenesis by altering the gene expression of angiogenic and

osteogenic factors in bone regeneration. In this study, we

assessed angiogenesis, osteogenesis, and the expression of

HIF-1a, VEGF, and BMP-2 in the nicotine-induced rabbit model

of mandibular distraction. Nicotine decouples angiogenesis and

osteogenesis in this experimental model. Enhanced angiogen-

esis cannot compensate for the adverse effects of nicotine on

bone healing.

Materials and Methods

Animal care

The rabbits were kept in a dedicated animal holding facility under

veterinary supervision in the Laboratory Animal Unit of Li Ka Shing

Faculty of Medicine, University of Hong Kong. The animal

experiment was approved by the Committee on the Use of Live

Animals for TeachingandResearchof theUniversity ofHongKong.

Nicotine implantation

Forty-eight male adult New ZealandWhite rabbits (9 months old,

3.4 to 4.0 kg) were randomly assigned to a nicotine group and a

control group (n¼ 24 for each group). Then 1.5-g, 60-day time-

release nicotine pellets or placebo pellets (Innovative Research of

America, Sarasota, FL) were implanted in the neck subcutaneous

tissue of the rabbits. The total nicotine exposure time was 7

weeks, and the animals were exposed to nicotine for at least 4

weeks before mandibular osteotomy (Fig. 1).

Osteotomy and distraction procedures

After nicotine implantation, a standard procedure of mandibular

osteotomy and distraction used in our previous study(5) was

performed. Briefly, the animals were given a preoperative dose of

antibiotic and analgesic. After anesthesia, the skin was incised

along the ventral border on one side of the mandibular body.

A straight-body osteotomy was made immediately cranial

(anterior) to the first premolar root. A custom-made bone-borne

distractor was placed along a plane perpendicular to the

osteotomy and fixed by 2-mm-diameter titanium screws. The

Fig. 1. Time line of nicotine exposure and distraction osteogenesis. The

time of euthanasia: on day 5 (A), on day 11 (B), and on day 18 (C)

respectively, after the commencement of active distraction. Nicotine

exposure: 7 weeks; latency period: 3 days; active distraction: 11 days.

1306 Journal of Bone and Mineral Research

periosteum, muscle, and skin were repositioned and closed with

3–0 sutures. Each animal remained under close observation by a

veterinary technician until it regained consciousness. Post-

operative antibiotic and analgesics were administered. The

clinical condition, weight, and food consumption of the animals

were monitored. After 3 days of latency, the distractor was

activated at 0.9mm per day. Eight animals in each group were

euthanized with an overdose of pentobarbital sodium on day 5

(middle of active distraction), day 11 (end of active distraction),

and day 18 (week 1 of consolidation), respectively, after the

commencement of active distraction. Three of the eight animals

were subjected to radiographic, histologic, and immunohisto-

chemical examinations, and the other five were subjected to

mRNA expression analysis.

Plain radiography

The mandibular samples were harvested and fixed in 10%

neutral phosphate-buffered paraformaldehyde. Each specimen

was placed on an occlusal film with the lingual side touching the

film. Plain radiography was performed by an Orthoralix 9200 X-

ray machine (Gendex, Des Plaines, IL) under a standard

conditions of 50 kV and 16mA.

Micro-computed tomography (mCT)

After plain radiographic examination, the distracted regenerate

and 2 to 5mm of neighboring host bone were harvested. The

specimenswere subjected toquantitativeexaminationbyamCT20

system (Scano Medical AG, Bassersdorf, Switzerland) using a

standard protocol described in our previous study.(5) Each

harvested specimen was placed in a 17-mm-diameter sample

holderwith the sagittal planeof themandibular regeneratevertical

to theX-ray tube.Theserial scanned imagesofeachspecimenwere

inspected on the computer. Oneach scanned image, the total area

of the distraction regenerate was defined as the region of interest

(ROI). The bone volume fraction (the ratio between bone volume

and total volume, BV/TV) of the ROI on each sectionwas calculated

individually, andameanvalueofBV/TV for the total regeneratewas

obtained.

Histologic examination

After mCT examination, the specimens were decalcified in a

solution of 14.5% ethylenediaminetetraacetic acid (EDTA) buffer

(pH 7.2) at room temperature. The specimens were dehydrated

and embedded in paraffin. Axial sections of 5mm in thickness

were cut with a microtome and stained with hematoxylin and

eosin for light microscopy.

Immunohistochemical staining

The sectionswere incubatedwith primary goat antibodies against

type IV collagen (Col IV, Southern Biotech, Birmingham,AB), BMP-2

(Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), VEGF (Santa

CruzBiotechnology, Inc.), andprimarymouseantibodyagainstHIF-

1a (Abcam, Cambridge, MA) and CD31 (Abcam) overnight at 48C.The antibodies have been confirmed to recognize the rabbit-

specific signals in previous studies.(5,29–32) For negative controls,

the primary antibodies were omitted. Goat and mouse ABC

MA ET AL.

staining system kits (Santa Cruz Biotechnology, Inc.) were used to

detect the reaction. The sections were counterstained with

hematoxylin and observed using a computer-assisted image-

analyzing system (Eclipse LV100POL and DS-Ri1, Nikon, Melville,

NY) with morphometric software (NIS-Elememts AR 3.0, Nikon).

Col IV is a component of the basal lamina of vessels. Its staining

allows the proper identification of blood vessels by immuno-

histochemical analysis.(5,33,34) The intensity of staining by CD31 is

far less than that by Col IV. It dose not always stain all endothelial

cells making up a vessel.(34) CD31 may be a suitable marker to

combine with Col IV for the estimation of blood vessels.(34) To

evaluate the neovessel density (NVD), the distraction regenerate

on each slide was divided into six areas (three rows and two

columns for the sections on day 5 of active distraction) or nine

areas (three rows and three columns for the sections on day 11

of active distraction and week 1 of consolidation) at �1

magnification of the objective lens. The vessels in the center of

these areas were counted at �5 magnification of the objective

lens. According to a standard technique described previously,

any single brown-stained cell or cluster of endothelial cells that

was clearly separated from adjacent vessels, histiocytes, and

other connective tissue elements was considered a vessel, and

the branching structures were counted as a single vessel unless

there was a discontinuity in the structure.(5,33) NVD was

calculated by vessel number per observation area.

Real-time reverse-transcriptase polymerase chainreaction (RT-PCR)

The distraction regenerate samples were harvested before the

animals were sacrificed. Under anesthesia, the skin and muscle

were incised and elevated to expose the distraction regenerate.

The regenerate tissue was removed and homogenized

using Mikro-11 Dismembrator U (Braun Biotech International,

Melsungen, Germany). Total RNA was isolated with an RNeasy

Tissue Midi Kit (Qiagen, Hilden, Germany).

cDNA was synthesized using the Superscript first-strand

synthesis system (Invitrogen, Carlsbad, CA). The primers were

VEGF forward: 5’-TCCAGGAGTACCCTGATGAGA-3’; VEGF reverse:

5’-CCCTGGTGAGGTTTGATCC-3’ (157 base pairs; GenBank Acces-

sion Number AF022179); BMP-2 forward: 5’-CACTTGGAGGA-

GAAGCAAGG-3’; BMP-2 reverse: 5’-GCTGTTTGTGTTTCGCTTGA-3’

(172 base pairs; GenBank Accession Number AF041421); HIF-1a

forward: 5’-TTACAGCAGCCAGATGATCG-3’; HIF-1a reverse: 5’-

TGGTCA-GCTGTGGTAATCCA-3’ (178 base pairs; GenBank Acces-

sion Number AY273790); and glyceraldehyde 3-phosphate

dehydrogenase (GAPDH) forward: 5’-TCACCAGGGCTGCTTT-

TAAC-3’; GAPDH reverse: 5’-GCTGAGATGATGACCCTTT-3’ (317

base pairs; GenBank Accession Number L23961). Amplification

was carried out for 35 cycles (948C for 1 minute, 568C for 1

minute, and 728C for 1 minute) for each in a 50-mL reaction

solution containing 1mL of each cDNA, 0.5mM of each pair of

primers, 0.2mM of each dNTP, 1� PCR buffer, 1.5mMMgCl2, and

1 U of taq DNA polymerase (Invitrogen). Standards were

constructed by cloning each PCR product into a 3.9-kb pCR 2.1-

TOPO with a TOPO TA cloning kit (Invitrogen) and then purified

with AIAGEN Plasmid Minikit (Qiagen). Real-time RT-PCR was

carried out for each in a 30-mL reaction solution containing 10mL

NICOTINE DECOUPLES ANGIOGENESIS AND OSTEOGENESIS

of each cDNA, 15mL of 2� Power SYBR Green PCR Master Mix

(Applied Biosystems, Foster City, CA), and 0.5mM of each pair of

primers. The standard curve was created by 10-fold dilutions of

the standard samples. The quantification of mRNA expression

was analyzed using Real Time PCR System software (Applied

Biosystems). Absolute quantification was performed by compar-

ing the target threshold cycles directly with the absolute

standard curve for each amplification. The copy numbers of

BMP2, VEGF, and HIF1a genes were normalized with the copy

numbers of GAPDH.

Statistical analysis

The mRNA expression values between the two groups were

compared by two-sample t test (Version 11.0 of Statistical

Package of Social Sciences software, SPSS, Inc., Chicago, IL). A

statistical result of less than .05 was considered significant.

The t test assumes that the data are sampled from populations

that follow Gaussian distributions and have equal standard

deviations. To compare the values of mRNA expression, five

animals are necessary in each subgroup to pass the assumption

tests. The sample size for the radiographic and immunohisto-

chemical analysis was estimated based on the results from our

previous studies,(5) and three animals in each subgroup were

considered adequate for the present experiment.

Results

Clinical examination

All rabbits completed the experimental process uneventfully.

The animals showed mild weight loss after the operation and

started to regain the weight within 2 weeks. None of the animals

experienced any postoperative complications, and the distrac-

tors remained stable until the time of sacrifice.

Plain radiography

The distraction regenerate was detected by the low radiodensity

between the host bone segments. On day 5 of active distraction,

the distraction gap in both the control and nicotine groups was

radiolucent without obvious signs of new bone formation

(Fig. 2A, B). On day 11 of active distraction, radiopaque streaks

extending from the bony edges with nonunion in the center

were noted. The radiodensity of the regenerate in the nicotine

group was lower than that in the control group (Fig. 2C, D). On

week 1 of consolidation, partial union was noted in the center of

the distraction regenerate in all animals belonging to the control

group (Fig. 2E). Nonunion in the center was noted in 2 of the

3 animals in the nicotine group (Fig. 2F).

mCT

The bone formation in the distraction regenerate was quantified

by mCT analysis. A gradual increase in bone volume fraction from

active distraction to consolidation was noted in both the control

and nicotine groups. When the two groups were compared, the

difference in bone volume fraction was not significant on day 5

of active distraction. However, the bone volume fraction in the

nicotine group was significantly less than that in the control

Journal of Bone and Mineral Research 1307

Fig. 2. Lateral radiographic view of the rabbit hemimandibles (distrac-

tion regenerates shown by arrows). (A) Control group on day 5. (B)

Nicotine group on day 5. (C) Control group on day 11. (D) Nicotine

group on day 11. (E) Control group on day 18. ( F) Nicotine group on

day 18.

Fig. 3. Histologic sections of the distraction regenerate in rabbit mand-

ibles (H&E stain). (A) Control group on day 5. (B) Nicotine group on day 5.

(C) Control group on day 11. (D) Nicotine group on day 11. (E) Control

group ont day 18. ( F) Nicotine group on day 18. H¼hemorrhage;

F¼ fibrous tissue; T¼ trabeculae.

group on day 11 of active distraction andweek 1 of consolidation

(Table 1).

Histology

On day 5 of active distraction, the distracted gap was bridged by

fibrous tissue, and hemorrhage was seen in the central area of

the distraction regenerate in both the control and nicotine

groups (Fig. 3A, B).

On day 11 of active distraction, the distracted gap in the

control group was mainly filled with thin longitudinal bony

trabeculae aligned in the direction of the distraction vector from

both sides of the bony margins. Fibrous tissues were observed in

the central area of the distraction regenerate (Fig. 3C). In the

nicotine group, the distraction zone was bridged mostly by

fibrous tissue, and hemorrhage was seen in the central area of

the distraction regenerate (Fig. 3D).

Table 1. BV/TV (mean� SD, %) in the rabbit mandibular dis-

traction regenerates (n¼ 3).

Group Day 5 Day 11 Day 18

Control 1.36� 0.443 11.96� 1.383 19.95� 1.318

Nicotine 0.93� 0.181 6.34� 1.875 17.24� 0.910

P value 0.1923 0.0140� 0.0425�

�p< .05 is considered statistically significant.


At week 1 of consolidation, the distraction regenerate in the

control group was composed of primary trabeculae and loose

fibrovascular stroma. Small fibrous discontinuities were seen in

the central area (Fig. 3E). In the nicotine group, new bone was

formed at the edges of the distracted gap, and the central area of

the distraction regenerate was occupied with fibrous tissues

(Fig. 3F).

Neovessel density

Col IV expression was observed in the cytoplasm of the vascular

endothelium. In the distraction regenerate, the signals in the

areas adjacent to the host bone were more intense than those in

the central area. Figure 4 presents the Col IV–staining sections in

the distal middle areas of distraction regenerate. On day 5 of

active distraction, the signals occurred in the capillary-like cell

clusters (Fig. 4A, B). On day 11 of active distraction, cannular

vessels were labeled in the control group (Fig. 4C). In the nicotine

group, most of the labeled cell clusters became capillary loops

(Fig. 4D). At week 1 of consolidation, the control group showed

that primary trabeculae were obvious, and cannular vessels

distributed among the trabeculae (Fig. 4E). In the nicotine group,

tiny dense vessels were noted among the slender immature

trabeculae (Fig. 4F). Compared with Col IV, the expression of

CD31 in the endothelia of vessels was weaker or even absent.

The signals of CD31 also were observed in osteoclasts (Fig. 5). The

low intensity of CD31 staining in vessels may be related to the

MA ET AL.

Fig. 4. Col IV expression in the rabbit mandibular distraction regener-

ates. Blood vessels are visualized by 3,30-Diaminobenzidine (DAB) (brown

coloration). (A) Control group on day 5. (B) Nicotine group on day 5. (C)

Control group ont day 11. (D) Nicotine group on day 11. (E) Control group

on day 18. ( F) Nicotine group on day 18.

Table 2. Neovessel Density (mean� SD, vessles/mm2) in the

Rabbit Mandibular Distraction Regenerates (n¼ 3)

Group Day 5 Day 11 Day 18

Control 11� 2.5 9� 2.0 17� 2.1

Nicotine 22� 2.1 19� 2.1 25� 3.1

p Value .0100� .0102� .0269�

�p< .05 is considered statistically significant.

experimental model, as well as to the time point of observation

in the present study.

To quantify the new vessels, the density of the blood vessels

stained by Col IV was evaluated and represented by NVD.

The nicotine group showed a significantly higher NVD than

the control group during active distraction and at week 1 of

consolidation (Table 2).

Fig. 5. CD31 expression in the rabbit mandibular distraction regenerates

on day 18. Panel B is a section in panel A at higher magnification. The

expression is weaker or even absent in the endothelia of vessels. The

signals are also observed in osteoclasts (arrows).


Expression of BMP-2, VEGF, and HIF-1a

Positive signals of BMP-2 were detectable in hemorrhage,

fibroblasts, osteoblasts, and fibrous matrix. VEGF was widely

expressed in hemorrhage, fibroblasts, osteoblasts, osteocytes,

and fibrous matrix and bone matrix of trabeculae. Intense HIF-1a

expression was noted in hemorrhage and osteoblasts lining

the newly formed trabeculae, and very weak signals also were

detected in fibroblasts and some immature osteocytes. The

nicotine group showed much weaker BMP-2 signals in osteo-

blasts, whereas HIF-1a signals in osteoblsts were more intense.

The stronger expression of VEGF in fibroblasts, osteoblasts, and

osteocytes was detected in the nicotine group (Fig. 6).

Fig. 6. The expression of BMP-2 (A, B), VEGF (C, D), and HIF-1a (E, F) in the

rabbit mandibular distraction regenerates at week 1 of consolidation.

(A, C, E) Control group. (B, D, F) Nicotine group. Bars¼ 10mm. Compared

with the control group, the nicotine group shows that BMP-2 expression

in osteoblasts is much weaker, whereas VEGF signals in fibroblasts,

osteoblasts, and osteocytes and HIF-1a signals in osteoblsts are more

intense.


Fig. 7. The quantification of mRNA expression of BMP2 (A), VEGF (B), and

HIF1a (C) in the rabbit mandibular distraction regenerates. �p< .01;��p< .001; ��p< .0001.

mRNA expression of BMP2, VEGF, and HIF1a was detected in

the distraction regenerates. Quantified by real-time RT-PCR, their

expression levels increased gradually from active distraction to

week 1 of consolidation. When themRNA levels between the two

groups were compared, BMP2 expression decreased, whereas

expression of VEGF and HIF1a increased in the nicotine group.

Significant differences in the expression of BMP2 (day 5 of

distraction: p¼ .0003; week 1 of consolidation: p< .0001) and

VEGF (day 5 of distraction: p¼ .0007; week 1 of consolidation:

p¼ .0002) were detected on day 5 of active distraction and at

week 1 of consolidation. HIF1a expression between the two

groups showed a significant difference at week 1 of consolida-

tion (week 1 of consolidation: p¼ .0015). The gene expression for

BMP2 on day 11 of active distraction (p¼ .7293), VEGF on day 11

of active distraction (p¼ .0536), and HIF1a on days 5 and 11 of

active distraction (day 5: p¼ .0585; day 11: p¼ .0869) showed no

statistically significant difference. (Fig. 7)

Discussion

Many studies show that angiogenesis and osteogenesis are

tightly coupled during bone formation.(24,35–37) Angiogenesis

plays a pivotal role in skeletal development and bone

repair.(24,35–37) Enhanced angiogeneis led to the increased bone

coverage and mineral density in bone defect reconstruc-

tions,(25,38,39) whereas the administration of antiangiogenic

agents inhibited bone healing.(24,38,40–44) Interestingly, this study

found an uncoupling of neovessel formation and bone formation

in the nicotine-induced distraction osteogenesis model. Nico-

tine-stimulated angiogenesis should be able to facilitate bone

formation. However, an impairment of bone healing was noted

in this study.

These results revealed a significantly enhanced expression

of HIF-1a and VEGF associated with consistently increased

neovessel density in the nicotine group. In our previous study

using the same experimental model, we found that nicotine

exposure reduced blood perfusion, resulting in ischemia and

lower oxygen level.(5) Tissue hypoxia is the major stimulus for

initiating the angiogenic cascade.(26–28) Nicotine has been found

to stimulate the accumulation of HIF-1a,(45) which is a central

regulator of hypoxia adaptation and activates downstream

hypoxia-responsive genes such as VEGF.(23–28) However, the

increased vessel formation did not lead to an increased blood

supply. Besides carrying oxygen and nutrients to bone tissue,

blood flow play an active role in bone formation and remodeling

by mediating the interactions among osteoblasts, osteocytes,

osteoclasts, and vascular cells at a variety of levels.(46) The

uncoupled vessel density and blood perfusion implied a complex

mechanism of nicotine in controlling angiogenic activity and

blood perfusion. The reduced blood flow indicates that nicotine

may produce vasoconstriction during bone regeneration.

Nicotine was reported to induce vascular endothelial dysfunc-

tion.(47–49) It has a direct effect on small blood vessels in

producing vasoconstriction and systemic venoconstriction,(50–54)

but this effect in bone healing has not been reported by others.

In our bone-healing model, the direct effects of nicotine on

blood vessels may be responsible for the reduced blood flow.


Our results suggest that hypoxia and ischemia owing to nicotine

exposure could stimulate HIF-1a expression, leading to an

increased expression of VEGF. This, in turn, stimulates angiogen-

esis. However, the enhanced vessel formation is incapable of

compensating for the adverse effect of the reduced blood flow

possibly caused by nicotine-induced vasoconstriction.

BMPs are the most important osteogenic growth factors.(15–17)

BMP-2 can reliably induce both ectopic and orthotopic bone

formation at the site of administration.(55–61) The expression of

endogenous BMPs is regarded as one of the indices to evaluate

the biologic environment in distraction regenerate.(5,29) The

effect of nicotine on BMPs has not been fully studied. Our

previous immuhistochemical study demonstrated that nicotine

inhibited BMP expression in osteoblasts. In this study, the

MA ET AL.

inhibitory effect of nicotine exposure on BMP2mRNA expression

was detected in the whole block of distraction regenerates,

which further confirmed that nicotine depressed osteogenic

activity in bone regeneration.

Taking together, two reasons may be responsible for the

impaired bone healing in the present experimental model. First,

nicotine decreases blood perfusion by its direct effects on blood

vessels in producing vasoconstriction and systemic venocon-

striction, even though it increases angiogenesis. Second, nicotine

directly inhibits the osteogenic activity (Fig. 8).

It is known that VEGF and BMP act synergistically during bone

healing.(19,62,63) The synergistic interaction between VEGF and

BMP depends on the ratios of the two factors.(14,19,63) Excessive

VEGF may lead to impairment in bone formation, possibly by

promoting mesenchymal stem cell differentiation toward an

endothelial lineage,(64) consequently reducing the availability of

mesenchymal stem cells (MSCs) for osteogenic differentiation.(65)

Alternatively, excessive VEGF may increase recruitment of

osteoclasts into the bone-regeneration sites and lead to an

excessive bone resorption.(65) The disruption of the optimal ratio

between VEGF and BMP caused by nicotine also might

contribute to the compromised bone healing. In addition, the

inflammatory response to bone fracture or distraction plays an

important role in initiating the repair cascade. It activates

downstream factors such as cytokines and growth factors that

recruit osteoprogenitor and mesenchymal cells to the injury

site.(64,66,67) Nicotine is an anti-inflammatory agent.(68–71) The

suppression of inflammation by nicotine may have an adverse

effect on bone healing. However, the conclusion cannot be

drawn on these speculations before finding hard evidence.

Distraction osteogenesis relies on the application of controlled

mechanical force to promote bone induction and formation

between two osteotomy fronts. It has become a widely accepted

surgical approach in the treatment of congenial and acquired

bone deformities.(9,72,73) In this study, the significant differences

in mRNA expression levels between the nicotine and control

groups were noted on day 5 of distraction and at week 1 after

distraction, except for day 11 of active distraction. During

Fig. 8. Schematic showing the effect of nicotine on bone regeneration.

Nicotine inhibits BMP expression and associated osteogenesis. At the

same time, it causes vasoconstriction, which leads to hypoxia and

ischemia. The induced HIF-1a stimulates VEGF expression and associated

angiogenesis. However, this stimulatory effect cannot compensate for

the adverse effect of nicotine on bone healing.


distraction osteogenesis, the mechanical strain triggers and

sustains molecular signaling. The expression of BMP-2, VEGF, and

HIF-1a can be induced gradually during the active distrac-

tion.(24,74–78) The effect of mechanical strain on the molecular

signaling accumulates gradually and eventually may cover the

effect of nicotine. Thus distraction osteogenesis could be the

preferred choice among the various bone-reconstruction

methods available to treat patients who have compromised

healing ability, such as smokers and those taking nicotine

medication.

In summary, nicotine exposure decouples angiogenesis and

osteogenesis in this experimental model of mandibular distrac-

tion osteogenesis. Nicotine enhances blood vessel density and

stimulates the associated HIF-1a and VEGF expressions but

impairs bone formation and inhibits the associated BMP

expression. The uncoupling of angiogenesis and osteogenesis

may be explained by the complex effects of nicotine on blood

vessels and osteogenic activity during bone healing.

Disclosures

The first two authors contributed equally to this research. All the

authors state that have no conflicts of interest.

Acknowledgments

This study was supported by the Small Project Funding Pro-

gramme (Reference code HKU200507176099) from the Univer-

sity of Hong Kong. We appreciate the valuable advice given by

Professor J Glowacki from the Harvard School of Dental Medi-

cine. We also appreciate the technical assistance provided by the

Laboratory Animal Unit of the Li Ka Shing Faculty of Medicine

and the Centralized Research Laboratories of the Faculty of

Dentistry.

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uncoupled angiogenesis and osteogenesis in nicotine-compromised bone healing

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