Background: Smoking has been suggested to increase the rate of perioperative complications including soft-tissue complications, to decrease the rate of fracture union, and to prolong healing time. The purpose of our study was to systematically evaluate and analyze the literature regarding the relationship between smoking and healing following operative treatment of long-bone fractures.

Methods: We searched the MEDLINE, Embase, and Cochrane databases by pairing the search terms “smoking,” “tobacco,” and “nicotine” with the terms “fracture,” “nonunion,” delayed union,” and “healing.” Articles and citations were evaluated for relevance. Inclusion and exclusion criteria were established to maintain data quality for analysis. Relevant information was independently extracted and compared to ensure agreement. The methodological quality of the studies was determined. A random-effects model was used. The adjusted odds ratios (ORs) and frequency-weighted means for the primary and secondary outcome measures were calculated.

Results: Our initial search identified 7110 articles. Of the 237 articles that underwent further evaluation of the abstract, nineteen (seven prospective and twelve retrospective cohort studies) were included. The adjusted OR of nonunion in the smoking group compared with the nonsmoking group was 2.32 (95% confidence interval [CI], 1.76 to 3.06; p < 0.001). An increased nonunion rate was observed in smokers with a tibial fracture (OR, 2.16; 95% CI, 1.55 to 3.01; p < 0.001) and those with an open fracture (OR, 1.95; 95% CI, 1.3 to 2.9; p < 0.001). For all fractures, the mean healing time was longer for smokers (30.2 weeks; 95% CI, 22.7 to 37.7 weeks) than for nonsmokers (24.1 weeks; 95% CI, 17.3 to 30.9 weeks) (p = 0.18). Trends toward more superficial and deep infections of postoperative or traumatic wounds in smokers were noted; however, the differences in superficial and deep infection rates were not significant (p = 0.13 and p = 0.33, respectively).

Conclusions: Smoking significantly increased the risk of nonunion of fractures overall, tibial fractures, and open fractures. Nonsignificant trends toward increased time to union in all fractures and toward increased postoperative rates of superficial and deep infections were noted in smokers compared with nonsmokers.

Level of Evidence: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Cigarette smoking increases complications following fracture: a systematic review

Non-union continues to be the most devastating complication after fracture fixation. Its treatment can be prolonged and often unpredictable. The burden to the patient, surgeon and health care system can be immense. Strategies to prevent it and or identify early its development are desirable in order to improve the clinical course of the affected patients and their outcomes. We undertook a systematic review of the literature in order to identify the most common and important risk factors based on the hierarchy of level of evidence. Accordingly, a stratification scale was formed which highlighted 10 risk factors including; an open method of fracture reduction, open fracture, presence of post-surgical fracture gap, smoking, infection, wedge or comminuted types of fracture, high degree of initial fracture displacement, lack of adequate mechanical stability provided by the implant used, fracture location in the poor zone of vascularity of the affected bone, and the presence of the fracture in the tibia. Clinicians should take in to account these findings when managing patients with long bone fractures, particularly the femur and tibia in order to minimise the risk of non-union.

Risk factors for long bone fracture non-union: a stratification approach based on the level of the existing scientific evidence

Objectives To review the systemic impact of smoking on bone healing as evidenced
within the orthopaedic literature. Methods A protocol was established and studies were sourced from five
electronic databases. Screening, data abstraction and quality assessment
was conducted by two review authors. Prospective and retrospective
clinical studies were included. The primary outcome measures were
based on clinical and/or radiological indicators of bone healing.
This review specifically focused on non-spinal orthopaedic studies. Results Nine tibia studies and eight other orthopaedic studies were considered
for systematic review. Of these 17 studies, 13 concluded that smoking
negatively influenced bone healing. Conclusions Smoking has a negative effect on bone healing, in terms of delayed
union, nonunion and more complications.

The effect of smoking on bone healing: A systematic review

After studying this article, the participant should be able to: 1. Describe the basic physiologic events in normal wound healing. 2. Understand the differences in healing among skin, bone, cartilage, and tendon. 3. Identify factors that may compromise or delay wound healing. 4. Describe methods for optimal closure of a wound.
Understanding the physiology and pathophysiology of normal wound healing and potential impediments to its end will allow the plastic surgeon to maximize postoperative outcomes and, in some instances, avoid unnecessary surgical interventions. Continuous advancements in our understanding of this process require frequent reviews of available data to permit reliable, evidence-based recommendations for clinical application. This is the first of a two-part article summarizing the science and clinical recommendations necessary for successful wound healing.

Wound Healing: Part I. Basic Science

Objectives:This study on patients with operatively treated ankle fractures aimed to investigate the impact of smoking on postoperative complications and especially deep wound infections.Design:Cohort study with prospective follow-up.Setting:University-associated teaching hospital with advanced traum

The Impact of Smoking on Complications After Operatively Treated Ankle Fractures—A Follow-Up Study of 906 Patients

Tobacco smoking is the single most avoidable cause of premature death worldwide. In fracture healing, it has been found to be a contributory factor to delayed union, and smokers are significantly disadvantaged, as healing times are often prolonged. The orthopaedic surgeon is likely to be knowledgeable about the detrimental effects of smoking on healing bones, as the problem has been known for some time. Smoking adversely affects bone mineral density, lumbar disc degeneration, the incidences of hip fractures and the dynamics of bone and wound healing. Clinical trials and demographic studies have been more widespread than biochemical analyses, and have reported poor prognosis for fracture patients who smoke. Scientific research has elucidated some of the negative impacts of tobacco use and investigations involving several animal models in cellular and humoral analyses have shown damage caused by various toxicological processes. Cessation of the habit perioperatively, therefore, is routinely advised to improve outcomes for patients. The current review describes some of the consequences of tobacco smoking in fracture healing.

The effects of smoking on fracture healing

Tobacco smoking has a detrimental impact on fracture healing, and has been implicated
in the non-union and delayed union of bone. Whilst previous studies have concentrated
on the clinical manifestations of smoking, little work has been undertaken biochemically.
Aim: To study the effects of smoking at the cellular and molecular level in vitro.
Methods: Cell-culture assay: Fracture haematomas were collected from anaesthetised
patients (n=16; 5 smokers vs. 11 non-smokers) that had sustained a tibial fracture. The
semisolid material was explanted into tissue culture fl asks and allowed to clot. Complete
culture media was introduced into the fl asks, which were placed in an incubator (37°C;
humidifi ed CO2). Cultured cells were characterised via immunofl uorescence and
immunophenotyping using known mesenchymal stem cell antibody markers (CD29,
CD44, and CD166); CD34 was applied as a negative control. A fl ow cytometer was used
to count cell populations at the end of each passage. ELISA assays: Serum and cells
obtained from the fracture haematomas were subjected to an ELISA to compare the
amount of VEGF-A (serum) and IL-6 (cells) between smokers and non-smokers.
Results: Cell counting showed a reduction in the rate of proliferation of cells in smokers
over 3 passages (~-200%). The VEGF-A (serum) and IL-6 (cells) ELISA revealed a
reduction of these acute phase proteins in patients who were smokers (VEGF-A ~-10%;
IL-6 ~-15%).
Conclusion: Fracture haematoma mesenchymal stem cells proliferate at a slower rate
in vitro in smokers than non-smokers. The amounts of VEGF-A and IL-6 are reduced in
smokers’ serum and cell cytoplasm respectively.

Inhibition of tibial fracture healing in smokers: cellular and molecular aspects

Introduction: Clinical and demographic studies have shown that tobacco smoking is a major contributor to non- and delayed-union in fracture healing. The cellular and molecular basis for this is poorly understood, and few studies in human fractures have been undertaken.Aims: To analyse the in vitro biological effects of tobacco smoking at the cellular level within the human fracture microenvironment, with specific regard to mesenchymal stem cell (MSC) proliferation and to ascertain whether the application of bone morphogenetic factor-2 (BMP-2) could be used therapeutically to improve fracture healing.Methods: Fracture haematomas (n=10) were collected from anaesthetised, non-smoking patients who had sustained a tibial fracture, and who were undergoing surgical operative fixation. The semi-solid material was dissected and explanted into tissue culture flasks. Complete culture media was introduced, and cultures were incubated at 37oC in a humidified 5% CO2 environment. Cigarette smoke extract (CSE) was produced and infused into the cell cultures to establish an in vitro smoking environment. A control group (n=10) was set-up and left  ntreated by CSE. Harvested, spindle-shaped adherent cells were characterised by immunocytochemistry. Cell populations were counted via flow cytometry to assess and compare proliferation rates between CSE-treated and untreated cell cultures. BMP-2 concentrations (10 and 100 ng/mL (an additional dose of 500 ng/mL in CSE- reated cells)) were infused into cell cultures to enhance in vitro cellular viability, which was analysed by means of the MTT assay.Results: There was a significant reduction in the rate of proliferation of osteoblastic MSCs in CSE-treated cells after 5 days of culture (p < 0.05). At a dose of 100 ng/mL, BMP-2 augmented cellular growth and improved cellular viability in cultures not treated with CSE (p < 0.0001). No significant improvement was seen in CSE-treated cell cultures.Summary: The effect of smoking on bone fracture healing appears to contribute to the inhibition of osteoblast proliferation, which may not be reversible with the therapeutic use of exogenous BMP-2. Moreover, the improvement seen in non-smokers does strengthen the case for smokers to cease using tobacco in the perioperative setting in order that such treatments are rendered more effective.

In Vitro effect of Bone Morphogenetic Protein-2 (BMP-2) and Cigarette Smoke Extract (CSE) on Osteoblastic Mesenchymal Stem Cells: Beneficial Biological Effects of BMP-2 Negated By CSE

Introduction: Tobacco smoking has been shown to have a detrimental impact on fracture healing and is often implicated in the non- and delayed-union of bone. Whilst numerous studies have concentrated on the demographic and clinical manifestations of fracture healing and smoking, very little analyses have been undertaken at the biochemical level. Aims: This research project will assess the impact of smoking on the cellular and molecular mechanisms of bone healing by analysing human tibial fracture haematomas using a variety of laboratory techniques. Materials and methods: Cell culture: Fracture haematomas (~2 mL) were collected from anaesthetised patients (n=48), who had sustained a fracture of the tibia. The semisolid material was explanted into 25 cm2 non-coated tissue culture flasks and allowed to clot. Complete culture media was prepared and pipetted into the vessels, which were then placed in an incubator (37˚C; humidified 5% CO2). After characterisation via immunocytochemistry (using antigen-specific markers to CD29, CD44, CD73, CD105, CD166 and CD34), isolated cells were counted using flow cytometry and proliferation rates were compared between non-smokers and smokers. Cigarette smoke extract (CSE) was manufactured based on the methods of Bernhard et al. (2004), in order to synthesise an in vitro smoking environment. Cells harvested from non-smokers (n=10) were cultured and infused with the CSE (2.5%) and proliferation and cell recovery rates were compared between the treated and untreated groups. Cellular nitric oxide levels and necrosis rates (using propidium iodide for the latter) were also compared in a similar way. An MTT assay was conducted to assess response to various concentrations of BMP-2 (10, 100 and 500 ng mL-1) infusion in cell
xvi
cultures. Molecular assays: Serum was extracted from the haematomas and resolved on 16% acrylamide gels before staining with either Coomassie blue or silver nitrate. The resolved gels were photographed using a Kodak gel imaging system and unknown bands were identified using MALDI-TOF. Intracellular TGF- and IL-6 were probed using antibodies, which were then analysed via flow cytometry in order to confirm the presence of these acute-phase proteins. In additional experiments, the ELISA was undertaken to quantify and compare the amounts of VEGF and IL-6 in haematoma serum and lysed fracture cells respectively. Biochemical testing on serum for b-ALP, albumin and total protein amounts in fracture serum was carried out, comparing smoker (n=10) and non-smoker (n=10) levels. Results: Cell culture: Spindle-shaped, fibroblast-like cells were visible in the primary culture, which were expanded through at least 10 further passages. The immunostaining of cells suggested a mesenchymal stem cell (MSC) lineage (CD29+, CD44+, CD73+, CD105+, CD166+, CD34-). Absolute cell counting using flow cytometry revealed marked proliferation of cells after 3 passages of culture. There was a reduction in the rate of proliferation of MSCs in smokers over 2 passages compared with non-smokers (-20%). A 5-day proliferation study in the CSE-treated vs. untreated cells showed a reduction in the rate of doubling in the treated group (-40% p<0.05). Cells showed a recovery response after CSE was withdrawn from culture, but was not significant. Cell necrosis analysis of CSE-treated vs. untreated cells showed that the CSE-treated cultures had a higher rate of necrosis than the untreated cells (CSE-treated 14.37% vs. untreated 7.98% p<0.05). Nitric oxide levels were lower in the CSE-treated cells (CSE-treated 3.0 M vs. untreated 3.6 M; p<0.05). MTT assay; BMP-2 infusions all improved cell viability compared to the non-infused cells,
xvii
with a BMP-2 concentration of 10 ng mL-1 increasing cell viability the most, though not significantly (+23%; p>0.05). Molecular assay: After staining, SDS-PAGE gels showed numerous bands of serum proteins and the haematoma serum lanes displayed unknown proteins which were later found to be haptoglobin (~20 kDa) and haemoglobin (~14 kDa) chains via mass spectrometry (MALDI-TOF). Cells were strongly positive (>96%) for the intracellular protein markers TGF- and IL-6. The VEGF-A (serum) and IL-6 (cells) preliminary data from the ELISA revealed a reduction of these acute-phase proteins in patients who were smokers (VEGF-A -10%; IL-6 -15%). In the biochemical assays, albumin was reduced in smokers’ serum (-13%, p 0.05 n.s.) and total protein lowered (-12%; p<0.01). Conclusions: The haematoma cultures produced colonies of adherent fibroblast-like cells that were of a MSC lineage. With the exception of a residual haemolysis-derived haptoglobin band, SDS-PAGE did not appear to show an over-expression of acute phase proteins, although it was useful for the characterisation of the fracture milieu, in that it was suggestive of a consistently haemolysed haematoma. The effect of smoking on bone fracture healing, therefore, appears to contribute to the inhibition of MSC proliferation, angiogenesis and the acute phase stress response. Cigarette smoke was also shown to cause excessive necrosis and reduce the amount of NO in those MSC cultures treated with CSE, which may indicate reduced vasodilatation to the fracture site. Bone alkaline phosphatase (b-ALP) was raised in the fracture serum of smokers, suggesting abnormally-high bone turnover, whilst the reduction of total protein and serum albumin likely indicates a lowered capacity for acute-phase protein synthesis and calcium transport in these patients. Exogenous BMP may be indicated for rectification of malunion in smokers.

Andrew Sloan

Papers

The effects of smoking on fracture healing

Inhibition of tibial fracture healing in smokers: cellular and molecular aspects

In Vitro effect of Bone Morphogenetic Protein-2 (BMP-2) and Cigarette Smoke Extract (CSE) on Osteoblastic Mesenchymal Stem Cells: Beneficial Biological Effects of BMP-2 Negated By CSE

Inhibition of the fracture healing process in smokers: deregulation of cellular and molecular milieu in tibial fracture micro-environment