The Spine Journal 15 (2015) 1402–1408
Basic Science
Effect of serum nicotine level on posterior spinal fusion in an in vivo rabbit model Scott D. Daffner, MDa,*, Stacey Waugh, MSa, Timothy L. Norman, PhD, PEb, Nilay Mukherjee, PhDc, John C. France, MDa a
Department of Orthopaedics, West Virginia University, Morgantown, PO Box 9196, WV 26506-9196, USA b Department of Engineering, Cedarville University, 51 North Main St, Cedarville, OH, USA c Covidien, 15 Hampshire Street, Mansfield, MA 02048, USA Received 23 May 2014; revised 30 January 2015; accepted 18 February 2015
Abstract
BACKGROUND CONTEXT: Cigarette smoking has a deleterious effect on spinal fusion. Although some studies have implied that nicotine is primarily responsible for poor fusion outcomes, other studies suggest that nicotine may actually stimulate bone growth. Hence, there may be a dosedependent effect of nicotine on posterior spinal fusion outcomes. PURPOSE: The purpose of this study was to determine if such a relationship could be shown in an in vivo rabbit model. STUDY DESIGN/SETTING: This is a prospective in vivo animal study. METHODS: Twenty-four adult male New Zealand white rabbits were randomly divided into four groups. All groups received a single-level posterolateral, intertransverse process fusion at L5–L6 with autologous iliac crest bone. One group served as controls and only underwent the spine fusion surgery. Three groups received 5.25-, 10.5-, and 21-mg nicotine patches, respectively, for 5 weeks. Serum nicotine levels were recorded for each group. All animals were euthanized 5 weeks postoperatively, and spinal fusions were evaluated radiographically, by manual palpation, and biomechanically. Statistical analysis evaluated the dose response effect of outcomes variables and nicotine dosage. This study was supported by a portion of a $100,000 grant from the Orthopaedic Research and Education Foundation. Author financial disclosures were completed in accordance with the journal’s guidelines; there were no conflicts of interests disclosed that would have led to bias in this work. RESULTS: The average serum levels of nicotine from the different patches were 7.861.9 ng/mL for the 5.25-mg patch group; 99.7617.7 ng/mL for the 10.5-mg patch group; and 149.1624.6 ng/ mL for the 21-mg patch group. The doses positively correlated with serum concentrations of nicotine (correlation coefficient50.8410, p!.001). The 5.25-mg group provided the best fusion rate, trabeculation, and stiffness. On the basis of the palpation tests, the fusion rates were control (50%), 5.25 mg (80%), 10.5 mg (50%), and 21 mg (42.8%). Radiographic assessment of trabeculation and bone incorporation and biomechanical analysis of bending stiffness ratio were also greatest in the 5.25-mg group. Radiographic evaluation showed a significant (p5.0446) quadratic effect of nicotine dose on spinal fusion. CONCLUSIONS: The effects of nicotine on spinal fusion are complex, may be dose dependent, and may not always be detrimental. The uniformly negative effects of smoking reported in patients
FDA device/drug status: Not applicable. Author disclosures: SDD: Grant: OREF (No. 01-053, E, Paid directly to institution); Stock Ownership: Pfizer (Unknown, !1%), Amgen (Unknown (!1%)); Speaking and/or Teaching Arrangements: DePuy-Synthes (AO) Nursing Education (B); Grants: CSRS (F, Paid directly to institution), AO North America (F, Paid directly to institution); Fellowship Support: AO Spine (E, Paid directly to institution). SW: Grant: OREF (No. 01-053, E, Paid directly to institution). TLN: Grant: OREF (E, Paid directly to institution). NM: Grant: OREF (No. 01-053, http://dx.doi.org/10.1016/j.spinee.2015.02.041 1529-9430/Ó 2015 Elsevier Inc. All rights reserved.
E, Paid directly to institution). JCF: Grant: OREF (No. 01-053, E, Paid directly to institution). The disclosure key can be found on the Table of Contents and at www. TheSpineJournalOnline.com. * Corresponding author. Department of Orthopaedics, West Virginia University, PO Box 9196, Morgantown, WV 26506-9196, USA. Tel.: (304) 293-2779; fax: (304) 293-7042. E-mail address: [email protected] (S.D. Daffner)
S.D. Daffner et al. / The Spine Journal 15 (2015) 1402–1408
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undergoing spinal fusion may possibly be attributed to the other components of cigarette smoke. Ó 2015 Elsevier Inc. All rights reserved. Keywords:
Nicotine; Bone healing; Spinal fusion; Smoking; Pseudarthrosis; Lumbar fusion
Introduction Numerous human and animal studies have shown that cigarette smoking is detrimental to bone health and impairs bone healing [1–5]. Nicotine has been implicated as the agent in cigarette smoke that is responsible for the ill effects of smoking on bone health. Animal studies have reported decreased bone healing and lower rates of spinal fusion in rats and rabbits exposed to nicotine [6–8]. Notably, Wing et al. [9] found that, while chronic exposure to nicotine decreased spinal fusion rates in a rabbit model, quitting improved fusion rates. The mechanism by which nicotine affects bone health and healing has yet to be fully elucidated. Some authors suggest that it may be related to vascular changes (vasoconstriction and/or decreased vascularization) induced by nicotine exposure [10–12]. Heeschen et al. [13], however, found nicotine stimulated angiogenesis in three different animal models not involving bone, and Clouse et al. reported that coronary artery bypass grafts in a canine model were unaffected by nicotine [14]. Our group has previously reported that, in a study evaluating the effects of direct current (DC) electrical stimulation on spinal fusion in rabbits exposed to nicotine, both the nicotine control group (fusionþnicotine administration) and the DC stimulation group (fusionþnicotineþDC stimulator) had increased fusion rates compared to the negative control group (fusion alone) [15]. That study, however, did not examine a dose-response relationship. In a follow-up study, we demonstrated a dose-dependent increase in osteoblastic activity with nicotine exposure in an in vitro cell culture model [16]. We therefore hypothesized that there would likewise be a dosedependent impact of nicotine exposure on spinal fusion in vivo. The purpose of this study was to further elucidate in an in vivo model the dose-dependent effect of nicotine on spinal fusion.
Materials and methods All procedures used were in accordance with approval from the Institutional Animal Care and Use Committee. Twenty-four, adult (1 year), 4.0-kg, male, New Zealand white rabbits were obtained and randomly divided into four test groups (n56). All groups received a single-level posterolateral, intertransverse process fusion at L5–L6 with autologous iliac crest bone. One group served as controls and only underwent the spine fusion surgery. Three groups received the autologous bone graft fusion and variable doses
of nicotine administered via transdermal patch (5.25, 10.5, and 21 mg, respectively). The surgical procedure was performed as previously described in the literature [7,17]. All surgeries were performed by a single operative team including the senior author and two experienced laboratory technicians. Postoperative radiographs were taken. The animals were observed during recovery, then placed in individual housing after recovery. Nicotine was administered to rabbits by way of transdermal nicotine patches (Habitrol; Novartis Consumer Health, Inc., Parsippany, NJ, USA) applied to the ear. Patches were changed daily for a period of 5 weeks after surgery. Blood was drawn from the central ear vein at 3 and 5 weeks after surgery, and serum nicotine levels were assessed by an independent laboratory (National Medical Services, Willow Grove, PA, USA). All animals were euthanized 5 weeks (35 days) postoperatively. Five weeks has been demonstrated to be adequate time for fusion to occur and peak biomechanical tensile strength to be achieved [17]. The fusion masses and adjacent unfused segment (L4–L5) were carefully harvested from each animal. The L4 and L5 vertebrae provided a relative scale for control. Spinal fusions were evaluated radiographically, by manual palpation, and then biomechanically tested in a manner consistent with protocols from previous studies [7,12,17,18]. Anterior/posterior radiographs were obtained of the harvested vertebral segments. The radiographs were viewed and graded by two independent orthopedic surgeons blinded to the experiment. The radiographs were graded using a scale of 1 (not fused) to 3 (fused). A fused segment was defined as complete fusion with no lucent clefts or radiolucencies. An unfused segment was defined as 100% clefts. A grade of two defined partial fusion, indicated by the presence of partial clefts. This standard technique is used in the clinical setting. Trabeculation, its continuity and uniformity, and bone incorporation were indicators of fusion used in the evaluation. The lumbar spine segments were manually palpated and evaluated for fusion on the basis of motion, similar to the evaluation standard in humans for determining nonunion and solid fusion. Each sample was tested for gross motion by two different surgeons and graded as fused or not fused (samples were not tested to failure). The presence or absence of motion determined the grade when the segments were stressed in flexion and extension. A grade of fused corresponded to a fused segment with no motion. A grade of unfused corresponded to a lack of fusion with unrestricted motion.
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The average serum levels of nicotine from the different patches were 7.861.9 ng/mL for the 5.25-mg patch group; 99.7617.7 ng/mL for the 10.5-mg patch group; and 149.1624.6 ng/mL for the 21-mg patch group. The doses positively correlated with serum concentrations of nicotine
(correlation coefficient50.84, p!.001) showing that the doses of nicotine were correctly delivered to the rabbits. A regression analysis was performed to check for the effect of nicotine dose on all outcome variables. The 10.5- and 21-mg doses produced very similar outcome variables, and the 21-mg group was neglected in the regression analysis. The analysis showed that there was a statistically significant quadratic effect of dose [radiographic score approximately52.70þ0.02(dose)0.02(dose5.25)2; p5 .045] on radiographic evaluation of fusion. A similar trend was noted for the biomechanical outcomes [stiffness ratio approximately53.41þ0.09(dose)0.06(dose5.25)2], but it was not statistically significant (p5.169). The approximate probability of fusion based on the palpation tests was given by the equation: Probability of fusion511/[1þe(lnf)], where lnf50.090.09(dose)0.06(dose5.25)2; this result also did not achieve statistical significance (p5.1645). The differences in means between individual groups were analyzed by analysis of variance. The 5.25-mg group provided the best fusion rate (Fig. 2), trabeculation (Fig. 3), and stiffness (Fig. 4). On the basis of the palpation tests, the fusion rates were control (50%), 5.25 mg (80%), 10.5 mg (50%), and 21 mg (42.8%). Radiographic assessment revealed trabeculation and bone incorporation as control (2.0860.92), 5.25 mg (2.9060.22), 10.5 mg (2.4260.38), and 21 mg (2.3660.85). The bending stiffness ratio was also greater in the 5.25-mg group control (1.7361.09), 5.25 mg (3.8763.07), 10.5 mg (2.6462.16), and 21 mg (2.1661.99). A power analysis revealed a power of 28% to detect differences between the means of the two groups. This finding indicated that there was not enough statistical power to determine a difference between groups should one exist. The analysis also revealed that, to have a power of 80% to detect a difference of 30% fusion rates between the 5.25 mg and control groups, a sample size of 32 animals per group
Fig. 1. Testing fixture showing cantilever deflection test.
Fig. 2. Fusion rates of treatment groups.
Biomechanical testing was conducted using a Materials Testing System (MTS, Minneapolis, MN, USA). To minimize the chance of damaging the fusion mass, the segment below the fusion was always tested first, then the fused segment, and finally the upper segment. The spines were placed in a testing fixture and bent about the fusion masses and adjacent segments in a cantilever deflection test (Fig. 1). A preload of 10 N was applied followed by cyclic preconditioning for five cycles. A monotonic load was then applied at a rate of 0.1 mm/s up to approximately 25% of the estimated failure load being careful not to cause failure or cracking. The failure load was estimated from previous work and from Bozic [19]. Load displacement data were recorded and graphically displayed with the force plotted against displacement. Stiffness was determined from the linear slope of the load displacement curve, and a ratio between the stiffness of the fused and unfused segments was determined. The radiographic, manual palpation, and biomechanical testing data for the nicotine groups were compared. A regression analysis was performed to check the doseresponse effect of all outcome variables against nicotine dose. An analysis of variance was conducted to detect significant differences (p!.05) between the means of each group. A post hoc analysis (LS Means Differences Student t test) was performed for pairwise comparison among groups. The statistical analysis package JMP was used (SAS Institute Inc., Cary, NC, USA).
Results
S.D. Daffner et al. / The Spine Journal 15 (2015) 1402–1408
Fig. 3. Trabeculation score of treatment groups.
would be required. This large sample size was considered to be outside the scope of the current research efforts.
Discussion Our study demonstrated a statistically significant effect of nicotine dose on radiographic spinal fusion outcomes in vivo. The administration of nicotine via a transdermal patch also provided an enhanced fusion rate in the intertransverse process fusion model in rabbits as assessed by palpation and bending stiffness. The enhancement occurred for the 5.25-mg patch (30% greater fusion rates than controls, p5.16) which provided an average serum level concentration of 7.8 ng/mL. This concentration is at the lower end of the range of humans who are smokers (10–70 ng/mL) [12,20]. Interestingly, rabbits that received the 10.5- and 21-mg patches, which delivered significantly higher doses (average serum
Fig. 4. Stiffness ratio of treatment groups.
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nicotine concentration 99.7 ng/mL and 149.1 ng/mL, respectively), did not experience a fusion outcome different than controls. Unfortunately, our study was not designed to determine why such a dose-dependent effect occurred, but merely that one existed. One possible explanation is that the higher dose patches created supraphysiologic serum nicotine levels (approximately double or triple that of active smokers), which at that level could have inhibited bone growth [21]. In addition, although serum nicotine levels significantly correlated with increasing patch dose, the relative increase from the 5.25-mg patch to the 10.5-mg patch (12.8 times higher) is much greater than the relative increase from 10.5 mg to 21 mg (1.5 times higher), despite that the dose was doubled in each instance. Again, although our study design precludes determining an explanation for this outcome, we hypothesize that it may be due to upregulation of hepatic nicotine metabolism and/or renal excretion stimulated by a higher exposure [21]. Although our findings of increased fusion with nicotine are inconsistent with a number of previous in vivo studies [6–9], several other animal studies have shown positive outcomes with nicotine exposure. For example, Gotfredsen et al. [22], using a rabbit osteotomy model, reported that long-term (6 month) exposure to nicotine did not have a detrimental effect on osseointegration of titanium implants. In a rat femur fracture model, Hastrup et al. reported that torsional strength of healing bone increased with nicotine exposure [23]. A previous report by our group examining the effect of electrical stimulation on bone healing found increased lumbar posterolateral fusion rates in rabbits exposed to nicotine compared to controls [15]. Although Ma et al. reported histologic differences, they found no difference between rabbits exposed to low-dose nicotine and the control group of rabbits with regard to radiographic and micro–computed tomography findings or expression of BMP-2 following mandibular distraction osteogenesis [24]. In some in vivo models of posterior spinal fusion, nicotine administration has been shown to decrease fusion rates. Decreased angiogenesis measured by decreased vascular ingrowth into autogenous cancellous bone grafts [8] and inhibition of the expression of cytokines associated with neovascularization [10] have been observed in these models. However, in other models, such as tumor or ischemia models, nicotine is a potent enhancer of angiogenesis [13]. Nicotine seems to positively affect osteoblasts and negatively affect vascularization in bone and increase angiogenesis in other models. The overall relationship between fusion rates and nicotine may depend on many factors including dose, angiogenesis, and osteoblastic activity. Several in vitro studies have suggested a dose-dependent effect of nicotine on osteoblast activity [16,25–27]. These studies support the fact that nicotine can have a positive role in osteoblast function. Interestingly, Rothem et al. [28] reported a dose-dependent effect of nicotine on bone
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metabolism in osteoblastic cells which mimics what we observed in vivo. They found that a low dose of nicotine (equivalent to a light smoker) upregulated expression of osteocalcin, type I collagen, and alkaline phosphatase, whereas a high concentration inhibited expression of these genes [28]. Hence, the increased fusion rates we observed in rabbits exposed to nicotine could possibly be explained by dose-related enhanced osteoblastic activity due to nicotine. One reason why the results of the present study differ from those of the other in vivo studies could relate to the method of nicotine administration [23]. We previously reported that use of a transdermal patch provided a more constant and sustained level of nicotine than a miniosmotic pump (with which we found widely fluctuating levels) [15]. This technique was further validated by Donigan et al. in a rabbit tibial osteotomy model [29]. In vivo model of Riebal et al. showed a variable response to nicotine, which the investigators attributed to some unspecified underlying ‘‘predisposition’’ of the individual rabbits tested, which may be more of a reflection of the variability in serum nicotine levels encountered due to administration via miniosmotic pump [8]. It is possible that the increased fusion rate we saw was partially due to a more consistent serum nicotine level during the duration of the study when compared to previous reports which used miniosmotic pumps. The effects of nicotine alone on bone health are not conclusive (Table) [22,24,29–33]. On the other hand, numerous clinical studies have consistently documented the deleterious impact of smoking on bone healing and spinal fusion [2,34–36]. One must be careful, therefore, to differentiate smoking exposure from nicotine exposure. Cigarette smoke contains over 4,700 chemical substances [37]. In an in vitro study, Guillihorn et al. reported that, although exposure of osteoblast-like cells to nicotine elicited a dose-dependent increase in metabolic activity,
preparations composed of cigarette smoke condensate with equivalent levels of nicotine showed reduced alkaline phosphatase activity and decreased total protein and collagen synthesis [38]. Using a rat femur fracture model, Skott et al. [39] reported the results of exposure to nicotine, tobacco extract, or both. When mechanically testing fracture healing, they found that ultimate torque and torque at yield point in rats receiving tobacco extract alone were decreased 21% and 23%, respectively, compared to the control (saline infusion) group, and 20% and 26%, respectively, compared to the nicotine-only group. The combined group (tobacco extract plus nicotine) demonstrated an 18% torque reduction compared to the nicotine-only group; no difference was found between the tobacco-only group and the combined group [39]. In a recent human clinical study, Lee et al. [40] examined the correlation of urine cotinine levels (a metabolite used as a proxy for smoking exposure) and bone mineral density in 770 male patients. They found that patients with urine cotinine levels more than 10 mg/mL (equivalent to an active smoker or nonsmoker exposed to secondhand smoke) had significantly lower bone mineral density than those patients with levels below 10 mg/ mL [40]. These studies suggest that smoking—and perhaps exposure to the multitude of other toxins in cigarette smoke, rather than just nicotine—may be the prime culprit. If the detrimental effects on bone healing are attributable to cigarette smoke, but not specifically nicotine, it stands to reason that use of a nicotine patch by a patient undergoing spinal fusion may be acceptable and may not in and of itself lead to pseudarthrosis. This supposition also raises the question of the influence of smokeless tobacco on spinal fusion, which has not been widely studied. In vitro evidence suggests that the inhibitory effects of smokeless tobacco extract on bone metabolism are not due to nicotine [41]. In a
Table Variable impact of nicotine on bone health Study
Model
Nicotine delivery
Findings
Akhter (2003) [30]
Adult female rats
No difference in BMC, BMD, or strength
Broulik (1993) [31] Iwaniec (2000) [32]
Adult male mice Growing female rats
Iwaniec (2001) [33]
Adult female rats
High-dose nicotine (9 mg/kg/d) via osmotic pump for 12 wk Nicotine via drinking water for 56 d Nicotine via osmotic pump (3 or 4.5 mg/kg/d) for 2 or 3 mo Nicotine via osmotic pump (4.5 or 6 mg/kg/d) for 3 mo
Gotfredsen (2009) [22]
Female rabbit, femoral and tibial osteotomy with titanium implant placement Rabbit (gender not specified), mandibular distraction osteogenesis Male rabbit, tibial osteotomy
Ma (2011) [24]
Donigan (2012) [29]
Nicotine via osmotic pump (6 mg/kg/min) for 32 wk Time-release subcutaneous nicotine pellets (0.75 g total), for 7 wk Transdermal nicotine patch (10.5 mg) for 21 d
BMC, bone mineral content; BMD, bone mineral density; PTH, parathyroid hormone.
BMC, BMD lower No effect on BMC, BMD, bone strength, Vitamin D, calcitonin, PTH No impact on BMC, BMD, Ca, vitamin D; elevated PTH in high dose; lower calcitonin in both doses No impact on osseointegration or pullout strength of titanium implants No impact on bone morphogenetic protein-2 expression or radiographic findings, but noted delayed healing histologically Higher nonunion rates and decreased mechanical strength of healing fractures
S.D. Daffner et al. / The Spine Journal 15 (2015) 1402–1408
clinical study of patients undergoing tibial osteotomy, W-Dahl and Toksvig-Larsen found that, when compared to smokers and non–tobacco users, patients who used oral snuff had a significantly lower incidence of delayed bone healing, spent the least amount of time in external fixation, and had significantly fewer complications [42]. Although further clinical research is needed, these findings suggest that patients who smoke cigarettes and require spinal fusion procedures may be able to achieve successful fusion if permitted to use an alternate nicotine delivery system (eg, patch, electronic cigarette) which allows them to avoid smoking. There are several limitations to this study. Multiple sources of nicotine are available on the market. We cannot neglect the possibility that the differences in outcome relative to other studies could be due to differences in the nicotine that was used. In addition, there is always a possibility that the differences noticed could be attributed to chance, as our study design would have required a prohibitively large number of animals to provide a statistical power of 80%. In addition, we did not perform any evaluation of interobserver or intraobserver reliability with regard to manual palpation testing, which is an inherently subjective means of evaluating fusion. We did, however, include more objective radiographic evaluation and biomechanical testing and noted a similar trend in all three outcomes measures. Finally, the findings of an animal model may not directly translate into a human clinical setting, although as previously discussed, both in vivo and in vitro studies have shown the deleterious effects of smoking and tobacco exposure on bone healing, similar to findings in human clinical reports. This study was supported by a portion of a $100,000 grant from the Orthopedic Research and Education Foundation. Author financial disclosures were completed in accordance with the journal’s guidelines; there were no conflicts of interests disclosed that would have led to bias in this work. In conclusion, the results of this study suggest that the effects of nicotine on posterior spinal fusion are complex, dose dependent, and may not always be detrimental. Lowdose nicotine exposure may enhance spinal fusion, but high dose appears to be inhibitory. Our results should be interpreted cautiously, however, as the study is underpowered. Nonetheless, to our knowledge, this is the first study to suggest that low-dose nicotine exposure may improve spinal fusion in a rabbit model. Clearly, more research needs to be performed before the effects of nicotine on healing bone can be determined. The uniformly negative effects of smoking on spinal fusion in patients may be attributable to other components of cigarette smoke. Future studies should focus on better elucidating the reasons behind the dose-dependent impact of nicotine exposure on a molecular and cell level, and the relative impacts of nicotine compared to cigarette smoke exposure on spinal fusion with a goal of ultimately translating the findings to human clinical applicability.
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Basic Science
Effect of serum nicotine level on posterior spinal fusion in an in vivo rabbit model Scott D. Daffner, MDa,*, Stacey Waugh, MSa, Timothy L. Norman, PhD, PEb, Nilay Mukherjee, PhDc, John C. France, MDa a
Department of Orthopaedics, West Virginia University, Morgantown, PO Box 9196, WV 26506-9196, USA b Department of Engineering, Cedarville University, 51 North Main St, Cedarville, OH, USA c Covidien, 15 Hampshire Street, Mansfield, MA 02048, USA Received 23 May 2014; revised 30 January 2015; accepted 18 February 2015
Abstract
BACKGROUND CONTEXT: Cigarette smoking has a deleterious effect on spinal fusion. Although some studies have implied that nicotine is primarily responsible for poor fusion outcomes, other studies suggest that nicotine may actually stimulate bone growth. Hence, there may be a dosedependent effect of nicotine on posterior spinal fusion outcomes. PURPOSE: The purpose of this study was to determine if such a relationship could be shown in an in vivo rabbit model. STUDY DESIGN/SETTING: This is a prospective in vivo animal study. METHODS: Twenty-four adult male New Zealand white rabbits were randomly divided into four groups. All groups received a single-level posterolateral, intertransverse process fusion at L5–L6 with autologous iliac crest bone. One group served as controls and only underwent the spine fusion surgery. Three groups received 5.25-, 10.5-, and 21-mg nicotine patches, respectively, for 5 weeks. Serum nicotine levels were recorded for each group. All animals were euthanized 5 weeks postoperatively, and spinal fusions were evaluated radiographically, by manual palpation, and biomechanically. Statistical analysis evaluated the dose response effect of outcomes variables and nicotine dosage. This study was supported by a portion of a $100,000 grant from the Orthopaedic Research and Education Foundation. Author financial disclosures were completed in accordance with the journal’s guidelines; there were no conflicts of interests disclosed that would have led to bias in this work. RESULTS: The average serum levels of nicotine from the different patches were 7.861.9 ng/mL for the 5.25-mg patch group; 99.7617.7 ng/mL for the 10.5-mg patch group; and 149.1624.6 ng/ mL for the 21-mg patch group. The doses positively correlated with serum concentrations of nicotine (correlation coefficient50.8410, p!.001). The 5.25-mg group provided the best fusion rate, trabeculation, and stiffness. On the basis of the palpation tests, the fusion rates were control (50%), 5.25 mg (80%), 10.5 mg (50%), and 21 mg (42.8%). Radiographic assessment of trabeculation and bone incorporation and biomechanical analysis of bending stiffness ratio were also greatest in the 5.25-mg group. Radiographic evaluation showed a significant (p5.0446) quadratic effect of nicotine dose on spinal fusion. CONCLUSIONS: The effects of nicotine on spinal fusion are complex, may be dose dependent, and may not always be detrimental. The uniformly negative effects of smoking reported in patients
FDA device/drug status: Not applicable. Author disclosures: SDD: Grant: OREF (No. 01-053, E, Paid directly to institution); Stock Ownership: Pfizer (Unknown, !1%), Amgen (Unknown (!1%)); Speaking and/or Teaching Arrangements: DePuy-Synthes (AO) Nursing Education (B); Grants: CSRS (F, Paid directly to institution), AO North America (F, Paid directly to institution); Fellowship Support: AO Spine (E, Paid directly to institution). SW: Grant: OREF (No. 01-053, E, Paid directly to institution). TLN: Grant: OREF (E, Paid directly to institution). NM: Grant: OREF (No. 01-053, http://dx.doi.org/10.1016/j.spinee.2015.02.041 1529-9430/Ó 2015 Elsevier Inc. All rights reserved.
E, Paid directly to institution). JCF: Grant: OREF (No. 01-053, E, Paid directly to institution). The disclosure key can be found on the Table of Contents and at www. TheSpineJournalOnline.com. * Corresponding author. Department of Orthopaedics, West Virginia University, PO Box 9196, Morgantown, WV 26506-9196, USA. Tel.: (304) 293-2779; fax: (304) 293-7042. E-mail address: [email protected] (S.D. Daffner)
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undergoing spinal fusion may possibly be attributed to the other components of cigarette smoke. Ó 2015 Elsevier Inc. All rights reserved. Keywords:
Nicotine; Bone healing; Spinal fusion; Smoking; Pseudarthrosis; Lumbar fusion
Introduction Numerous human and animal studies have shown that cigarette smoking is detrimental to bone health and impairs bone healing [1–5]. Nicotine has been implicated as the agent in cigarette smoke that is responsible for the ill effects of smoking on bone health. Animal studies have reported decreased bone healing and lower rates of spinal fusion in rats and rabbits exposed to nicotine [6–8]. Notably, Wing et al. [9] found that, while chronic exposure to nicotine decreased spinal fusion rates in a rabbit model, quitting improved fusion rates. The mechanism by which nicotine affects bone health and healing has yet to be fully elucidated. Some authors suggest that it may be related to vascular changes (vasoconstriction and/or decreased vascularization) induced by nicotine exposure [10–12]. Heeschen et al. [13], however, found nicotine stimulated angiogenesis in three different animal models not involving bone, and Clouse et al. reported that coronary artery bypass grafts in a canine model were unaffected by nicotine [14]. Our group has previously reported that, in a study evaluating the effects of direct current (DC) electrical stimulation on spinal fusion in rabbits exposed to nicotine, both the nicotine control group (fusionþnicotine administration) and the DC stimulation group (fusionþnicotineþDC stimulator) had increased fusion rates compared to the negative control group (fusion alone) [15]. That study, however, did not examine a dose-response relationship. In a follow-up study, we demonstrated a dose-dependent increase in osteoblastic activity with nicotine exposure in an in vitro cell culture model [16]. We therefore hypothesized that there would likewise be a dosedependent impact of nicotine exposure on spinal fusion in vivo. The purpose of this study was to further elucidate in an in vivo model the dose-dependent effect of nicotine on spinal fusion.
Materials and methods All procedures used were in accordance with approval from the Institutional Animal Care and Use Committee. Twenty-four, adult (1 year), 4.0-kg, male, New Zealand white rabbits were obtained and randomly divided into four test groups (n56). All groups received a single-level posterolateral, intertransverse process fusion at L5–L6 with autologous iliac crest bone. One group served as controls and only underwent the spine fusion surgery. Three groups received the autologous bone graft fusion and variable doses
of nicotine administered via transdermal patch (5.25, 10.5, and 21 mg, respectively). The surgical procedure was performed as previously described in the literature [7,17]. All surgeries were performed by a single operative team including the senior author and two experienced laboratory technicians. Postoperative radiographs were taken. The animals were observed during recovery, then placed in individual housing after recovery. Nicotine was administered to rabbits by way of transdermal nicotine patches (Habitrol; Novartis Consumer Health, Inc., Parsippany, NJ, USA) applied to the ear. Patches were changed daily for a period of 5 weeks after surgery. Blood was drawn from the central ear vein at 3 and 5 weeks after surgery, and serum nicotine levels were assessed by an independent laboratory (National Medical Services, Willow Grove, PA, USA). All animals were euthanized 5 weeks (35 days) postoperatively. Five weeks has been demonstrated to be adequate time for fusion to occur and peak biomechanical tensile strength to be achieved [17]. The fusion masses and adjacent unfused segment (L4–L5) were carefully harvested from each animal. The L4 and L5 vertebrae provided a relative scale for control. Spinal fusions were evaluated radiographically, by manual palpation, and then biomechanically tested in a manner consistent with protocols from previous studies [7,12,17,18]. Anterior/posterior radiographs were obtained of the harvested vertebral segments. The radiographs were viewed and graded by two independent orthopedic surgeons blinded to the experiment. The radiographs were graded using a scale of 1 (not fused) to 3 (fused). A fused segment was defined as complete fusion with no lucent clefts or radiolucencies. An unfused segment was defined as 100% clefts. A grade of two defined partial fusion, indicated by the presence of partial clefts. This standard technique is used in the clinical setting. Trabeculation, its continuity and uniformity, and bone incorporation were indicators of fusion used in the evaluation. The lumbar spine segments were manually palpated and evaluated for fusion on the basis of motion, similar to the evaluation standard in humans for determining nonunion and solid fusion. Each sample was tested for gross motion by two different surgeons and graded as fused or not fused (samples were not tested to failure). The presence or absence of motion determined the grade when the segments were stressed in flexion and extension. A grade of fused corresponded to a fused segment with no motion. A grade of unfused corresponded to a lack of fusion with unrestricted motion.
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The average serum levels of nicotine from the different patches were 7.861.9 ng/mL for the 5.25-mg patch group; 99.7617.7 ng/mL for the 10.5-mg patch group; and 149.1624.6 ng/mL for the 21-mg patch group. The doses positively correlated with serum concentrations of nicotine
(correlation coefficient50.84, p!.001) showing that the doses of nicotine were correctly delivered to the rabbits. A regression analysis was performed to check for the effect of nicotine dose on all outcome variables. The 10.5- and 21-mg doses produced very similar outcome variables, and the 21-mg group was neglected in the regression analysis. The analysis showed that there was a statistically significant quadratic effect of dose [radiographic score approximately52.70þ0.02(dose)0.02(dose5.25)2; p5 .045] on radiographic evaluation of fusion. A similar trend was noted for the biomechanical outcomes [stiffness ratio approximately53.41þ0.09(dose)0.06(dose5.25)2], but it was not statistically significant (p5.169). The approximate probability of fusion based on the palpation tests was given by the equation: Probability of fusion511/[1þe(lnf)], where lnf50.090.09(dose)0.06(dose5.25)2; this result also did not achieve statistical significance (p5.1645). The differences in means between individual groups were analyzed by analysis of variance. The 5.25-mg group provided the best fusion rate (Fig. 2), trabeculation (Fig. 3), and stiffness (Fig. 4). On the basis of the palpation tests, the fusion rates were control (50%), 5.25 mg (80%), 10.5 mg (50%), and 21 mg (42.8%). Radiographic assessment revealed trabeculation and bone incorporation as control (2.0860.92), 5.25 mg (2.9060.22), 10.5 mg (2.4260.38), and 21 mg (2.3660.85). The bending stiffness ratio was also greater in the 5.25-mg group control (1.7361.09), 5.25 mg (3.8763.07), 10.5 mg (2.6462.16), and 21 mg (2.1661.99). A power analysis revealed a power of 28% to detect differences between the means of the two groups. This finding indicated that there was not enough statistical power to determine a difference between groups should one exist. The analysis also revealed that, to have a power of 80% to detect a difference of 30% fusion rates between the 5.25 mg and control groups, a sample size of 32 animals per group
Fig. 1. Testing fixture showing cantilever deflection test.
Fig. 2. Fusion rates of treatment groups.
Biomechanical testing was conducted using a Materials Testing System (MTS, Minneapolis, MN, USA). To minimize the chance of damaging the fusion mass, the segment below the fusion was always tested first, then the fused segment, and finally the upper segment. The spines were placed in a testing fixture and bent about the fusion masses and adjacent segments in a cantilever deflection test (Fig. 1). A preload of 10 N was applied followed by cyclic preconditioning for five cycles. A monotonic load was then applied at a rate of 0.1 mm/s up to approximately 25% of the estimated failure load being careful not to cause failure or cracking. The failure load was estimated from previous work and from Bozic [19]. Load displacement data were recorded and graphically displayed with the force plotted against displacement. Stiffness was determined from the linear slope of the load displacement curve, and a ratio between the stiffness of the fused and unfused segments was determined. The radiographic, manual palpation, and biomechanical testing data for the nicotine groups were compared. A regression analysis was performed to check the doseresponse effect of all outcome variables against nicotine dose. An analysis of variance was conducted to detect significant differences (p!.05) between the means of each group. A post hoc analysis (LS Means Differences Student t test) was performed for pairwise comparison among groups. The statistical analysis package JMP was used (SAS Institute Inc., Cary, NC, USA).
Results
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Fig. 3. Trabeculation score of treatment groups.
would be required. This large sample size was considered to be outside the scope of the current research efforts.
Discussion Our study demonstrated a statistically significant effect of nicotine dose on radiographic spinal fusion outcomes in vivo. The administration of nicotine via a transdermal patch also provided an enhanced fusion rate in the intertransverse process fusion model in rabbits as assessed by palpation and bending stiffness. The enhancement occurred for the 5.25-mg patch (30% greater fusion rates than controls, p5.16) which provided an average serum level concentration of 7.8 ng/mL. This concentration is at the lower end of the range of humans who are smokers (10–70 ng/mL) [12,20]. Interestingly, rabbits that received the 10.5- and 21-mg patches, which delivered significantly higher doses (average serum
Fig. 4. Stiffness ratio of treatment groups.
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nicotine concentration 99.7 ng/mL and 149.1 ng/mL, respectively), did not experience a fusion outcome different than controls. Unfortunately, our study was not designed to determine why such a dose-dependent effect occurred, but merely that one existed. One possible explanation is that the higher dose patches created supraphysiologic serum nicotine levels (approximately double or triple that of active smokers), which at that level could have inhibited bone growth [21]. In addition, although serum nicotine levels significantly correlated with increasing patch dose, the relative increase from the 5.25-mg patch to the 10.5-mg patch (12.8 times higher) is much greater than the relative increase from 10.5 mg to 21 mg (1.5 times higher), despite that the dose was doubled in each instance. Again, although our study design precludes determining an explanation for this outcome, we hypothesize that it may be due to upregulation of hepatic nicotine metabolism and/or renal excretion stimulated by a higher exposure [21]. Although our findings of increased fusion with nicotine are inconsistent with a number of previous in vivo studies [6–9], several other animal studies have shown positive outcomes with nicotine exposure. For example, Gotfredsen et al. [22], using a rabbit osteotomy model, reported that long-term (6 month) exposure to nicotine did not have a detrimental effect on osseointegration of titanium implants. In a rat femur fracture model, Hastrup et al. reported that torsional strength of healing bone increased with nicotine exposure [23]. A previous report by our group examining the effect of electrical stimulation on bone healing found increased lumbar posterolateral fusion rates in rabbits exposed to nicotine compared to controls [15]. Although Ma et al. reported histologic differences, they found no difference between rabbits exposed to low-dose nicotine and the control group of rabbits with regard to radiographic and micro–computed tomography findings or expression of BMP-2 following mandibular distraction osteogenesis [24]. In some in vivo models of posterior spinal fusion, nicotine administration has been shown to decrease fusion rates. Decreased angiogenesis measured by decreased vascular ingrowth into autogenous cancellous bone grafts [8] and inhibition of the expression of cytokines associated with neovascularization [10] have been observed in these models. However, in other models, such as tumor or ischemia models, nicotine is a potent enhancer of angiogenesis [13]. Nicotine seems to positively affect osteoblasts and negatively affect vascularization in bone and increase angiogenesis in other models. The overall relationship between fusion rates and nicotine may depend on many factors including dose, angiogenesis, and osteoblastic activity. Several in vitro studies have suggested a dose-dependent effect of nicotine on osteoblast activity [16,25–27]. These studies support the fact that nicotine can have a positive role in osteoblast function. Interestingly, Rothem et al. [28] reported a dose-dependent effect of nicotine on bone
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metabolism in osteoblastic cells which mimics what we observed in vivo. They found that a low dose of nicotine (equivalent to a light smoker) upregulated expression of osteocalcin, type I collagen, and alkaline phosphatase, whereas a high concentration inhibited expression of these genes [28]. Hence, the increased fusion rates we observed in rabbits exposed to nicotine could possibly be explained by dose-related enhanced osteoblastic activity due to nicotine. One reason why the results of the present study differ from those of the other in vivo studies could relate to the method of nicotine administration [23]. We previously reported that use of a transdermal patch provided a more constant and sustained level of nicotine than a miniosmotic pump (with which we found widely fluctuating levels) [15]. This technique was further validated by Donigan et al. in a rabbit tibial osteotomy model [29]. In vivo model of Riebal et al. showed a variable response to nicotine, which the investigators attributed to some unspecified underlying ‘‘predisposition’’ of the individual rabbits tested, which may be more of a reflection of the variability in serum nicotine levels encountered due to administration via miniosmotic pump [8]. It is possible that the increased fusion rate we saw was partially due to a more consistent serum nicotine level during the duration of the study when compared to previous reports which used miniosmotic pumps. The effects of nicotine alone on bone health are not conclusive (Table) [22,24,29–33]. On the other hand, numerous clinical studies have consistently documented the deleterious impact of smoking on bone healing and spinal fusion [2,34–36]. One must be careful, therefore, to differentiate smoking exposure from nicotine exposure. Cigarette smoke contains over 4,700 chemical substances [37]. In an in vitro study, Guillihorn et al. reported that, although exposure of osteoblast-like cells to nicotine elicited a dose-dependent increase in metabolic activity,
preparations composed of cigarette smoke condensate with equivalent levels of nicotine showed reduced alkaline phosphatase activity and decreased total protein and collagen synthesis [38]. Using a rat femur fracture model, Skott et al. [39] reported the results of exposure to nicotine, tobacco extract, or both. When mechanically testing fracture healing, they found that ultimate torque and torque at yield point in rats receiving tobacco extract alone were decreased 21% and 23%, respectively, compared to the control (saline infusion) group, and 20% and 26%, respectively, compared to the nicotine-only group. The combined group (tobacco extract plus nicotine) demonstrated an 18% torque reduction compared to the nicotine-only group; no difference was found between the tobacco-only group and the combined group [39]. In a recent human clinical study, Lee et al. [40] examined the correlation of urine cotinine levels (a metabolite used as a proxy for smoking exposure) and bone mineral density in 770 male patients. They found that patients with urine cotinine levels more than 10 mg/mL (equivalent to an active smoker or nonsmoker exposed to secondhand smoke) had significantly lower bone mineral density than those patients with levels below 10 mg/ mL [40]. These studies suggest that smoking—and perhaps exposure to the multitude of other toxins in cigarette smoke, rather than just nicotine—may be the prime culprit. If the detrimental effects on bone healing are attributable to cigarette smoke, but not specifically nicotine, it stands to reason that use of a nicotine patch by a patient undergoing spinal fusion may be acceptable and may not in and of itself lead to pseudarthrosis. This supposition also raises the question of the influence of smokeless tobacco on spinal fusion, which has not been widely studied. In vitro evidence suggests that the inhibitory effects of smokeless tobacco extract on bone metabolism are not due to nicotine [41]. In a
Table Variable impact of nicotine on bone health Study
Model
Nicotine delivery
Findings
Akhter (2003) [30]
Adult female rats
No difference in BMC, BMD, or strength
Broulik (1993) [31] Iwaniec (2000) [32]
Adult male mice Growing female rats
Iwaniec (2001) [33]
Adult female rats
High-dose nicotine (9 mg/kg/d) via osmotic pump for 12 wk Nicotine via drinking water for 56 d Nicotine via osmotic pump (3 or 4.5 mg/kg/d) for 2 or 3 mo Nicotine via osmotic pump (4.5 or 6 mg/kg/d) for 3 mo
Gotfredsen (2009) [22]
Female rabbit, femoral and tibial osteotomy with titanium implant placement Rabbit (gender not specified), mandibular distraction osteogenesis Male rabbit, tibial osteotomy
Ma (2011) [24]
Donigan (2012) [29]
Nicotine via osmotic pump (6 mg/kg/min) for 32 wk Time-release subcutaneous nicotine pellets (0.75 g total), for 7 wk Transdermal nicotine patch (10.5 mg) for 21 d
BMC, bone mineral content; BMD, bone mineral density; PTH, parathyroid hormone.
BMC, BMD lower No effect on BMC, BMD, bone strength, Vitamin D, calcitonin, PTH No impact on BMC, BMD, Ca, vitamin D; elevated PTH in high dose; lower calcitonin in both doses No impact on osseointegration or pullout strength of titanium implants No impact on bone morphogenetic protein-2 expression or radiographic findings, but noted delayed healing histologically Higher nonunion rates and decreased mechanical strength of healing fractures
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clinical study of patients undergoing tibial osteotomy, W-Dahl and Toksvig-Larsen found that, when compared to smokers and non–tobacco users, patients who used oral snuff had a significantly lower incidence of delayed bone healing, spent the least amount of time in external fixation, and had significantly fewer complications [42]. Although further clinical research is needed, these findings suggest that patients who smoke cigarettes and require spinal fusion procedures may be able to achieve successful fusion if permitted to use an alternate nicotine delivery system (eg, patch, electronic cigarette) which allows them to avoid smoking. There are several limitations to this study. Multiple sources of nicotine are available on the market. We cannot neglect the possibility that the differences in outcome relative to other studies could be due to differences in the nicotine that was used. In addition, there is always a possibility that the differences noticed could be attributed to chance, as our study design would have required a prohibitively large number of animals to provide a statistical power of 80%. In addition, we did not perform any evaluation of interobserver or intraobserver reliability with regard to manual palpation testing, which is an inherently subjective means of evaluating fusion. We did, however, include more objective radiographic evaluation and biomechanical testing and noted a similar trend in all three outcomes measures. Finally, the findings of an animal model may not directly translate into a human clinical setting, although as previously discussed, both in vivo and in vitro studies have shown the deleterious effects of smoking and tobacco exposure on bone healing, similar to findings in human clinical reports. This study was supported by a portion of a $100,000 grant from the Orthopedic Research and Education Foundation. Author financial disclosures were completed in accordance with the journal’s guidelines; there were no conflicts of interests disclosed that would have led to bias in this work. In conclusion, the results of this study suggest that the effects of nicotine on posterior spinal fusion are complex, dose dependent, and may not always be detrimental. Lowdose nicotine exposure may enhance spinal fusion, but high dose appears to be inhibitory. Our results should be interpreted cautiously, however, as the study is underpowered. Nonetheless, to our knowledge, this is the first study to suggest that low-dose nicotine exposure may improve spinal fusion in a rabbit model. Clearly, more research needs to be performed before the effects of nicotine on healing bone can be determined. The uniformly negative effects of smoking on spinal fusion in patients may be attributable to other components of cigarette smoke. Future studies should focus on better elucidating the reasons behind the dose-dependent impact of nicotine exposure on a molecular and cell level, and the relative impacts of nicotine compared to cigarette smoke exposure on spinal fusion with a goal of ultimately translating the findings to human clinical applicability.
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