Riham Fliefela,b,c, Jan Kühnischd, Michael Ehrenfeldb, Sven Ottob Gene therapy for Bone Defects in Oral and Maxillofacial Surgery: A Systematic Review and Meta-analysis of Animal Studies Riham Fliefela,b,c, Jan Kühnischd, Michael Ehrenfeldb, Sven Ottob a Experimental Surgery and Regenerative Medicine (ExperiMed), Ludwig-Maximilians-University, Munich, Germany. b Department of Oral and Maxillofacial Surgery, Ludwig-Maximilians-University, Munich, Germany. c Department of Oral and Maxillofacial Surgery, Alexandria-University, Alexandria, Egypt. d Department of Conservative Dentistry and Periodontology, Ludwig-Maximilians-University, Munich, Germany. Craniofacial anomalies and bone defects present a challenging problem for maxillofacial surgeons with the goal of restoring facial form, function and occlusion. Conventional therapies are directed towards the use of bone grafts. However, the effectiveness of these techniques is constrained by donor site morbidity and insufficient tissue resources. Recently, DNA-based technology as gene therapy had appeared as a promising method to replace the traditional techniques. Gene therapy application in the craniofacial region is more often directed at regenerating tissues by acting as a delivery system for therapeutic genes promoting healing. INTRODUCTION Meta-analysis Fourteen studies were included in the histological meta-analysis of percentage of area of newly formed bone by gene therapy whereas three studies were included in percentage of volume of newly formed bone. However, four studies were included in the radiographic meta-analysis of the bone formation by calculating the bone volume fraction We have conducted a systematic review summarizing the articles reporting trials of gene therapy worldwide in the field of oral and maxillofacial. OBJECTIVE Medical databases (PubMed, Cochrane library and Web of Knowledge) were searched to 18th December 2015. The data search included the following keywords: ‘‘Gene therapy’’ ‘‘AND’’ ‘‘Maxillofacial surgery’’ ‘‘OR’’ ‘‘Gene therapy’’ ‘‘AND’’ ‘‘Bone tissue engineering’’, ‘‘Genetic Engineering’’ ‘‘AND’’ ‘‘Maxillofacial bone’’, ‘‘Gene therapy’’ ‘‘AND’’ “Distraction Osteogenesis” ‘‘OR’’ ‘‘Gene therapy’’ ‘‘AND’’ “Alveolar bone” ‘‘OR’’ ‘‘Gene therapy’’ ‘‘AND’’ “Periodontal tissue” ‘‘OR’’ ‘‘Gene therapy’’ ‘‘AND’’ “Temporomandibular joint”. Medical subject headings (MeSH terms) without subheading restrictions was used and the heading sequence was ‘‘Gene therapy’’ ‘‘AND’’ ‘‘Dentistry’’. Inclusion criteria: (1) Relevant data on using Gene therapy for treatment of defects, (2) In-vivo or animal studies, (3) Defects surgically induced in the Oral and Maxillofacial region, (4) Any language. Exclusion criteria: (1) Review articles, (2) Letters to the editor, editorials, poster or oral presentations or articles with only abstract, (3) In vitro studies, (4) Studies based on the use of only growth factors ( protein-based) or cell-based therapies, (5) Oral cancer or soft tissue lesions, (6) Calvarial bones defects, (7) Gene therapy in bones other than maxillofacial. The reporting quality of all the included studies was performed based on ARRIVE guidelines. CAMARADES risk of bias tool was applied to assess the internal validity of the included studies. Bone formation was the primary outcome measured by histology or radiograph. The effect size in our review was a continuous outcome variable recorded as mean difference (MD). GRADE approach was used to assess the quality of evidence for each outcome variable included in the systematic review and the strength of recommendation regarding the use of gene therapy to regenerate bone or heal defects in animal studies in the field of Oral and Maxillofacial surgery. A qualitative data analysis was performed with the aim of summarizing the results of the studies included. Meta-analysis as well as forest and funnel plots were conducted using Review Manager [RevMan] Version 5.3. MATERIALS AND METHODS Fig.3: Forest plot of Mean difference (MD) with 95% Confidence Interval (CI) in bone formation by histology and radiograph comparing different subgroups. Panel (A) represents forest plot of percentage area of bone formation by histology. Panel (B) represents forest plot of percentage volume of bone formation by histology. Panel (C) represents forest plot of bone volume fraction (BVF) detected by 3D µCT. the diamond represents the overall effect within each subgroup. Search results The search identified a total of 2081 references from the different databases and hand search of which a total of 57 studies fulfilled the final selection criteria. RESULTS Publication Bias Symmetrical funnel plots were obtained in all the models. The funnel plot of the study standard error by effect size (MD) was symmetric. The funnel plot of standard error versus effect size (MD) was symmetrical indicating the absence of potential publication bias among the meta-analysis of bone formation by histology or radiograph. Fig.1: Flow-chart of the process of literature search and studies included in the review. Fig.4: Funnel plot showing publication bias among the studies. The symmetry of the funnel plot shows there was no evidence of publication bias among the studies. Each symbol on the funnel plot represents an individual study estimate included in the meta-analysis. The y-axis displays the standard error and the x-axis displays the mean difference. SE: Standard Error; MD: mean difference. BMPs were the most commonly used proteins for gene therapy followed by PDGF. Most of the studies were conducted in USA and China. Adenovirus was the universally used vector. Alveolar bone defects with or without dental implant were the prevalent model used. Sprague-Dawley rats were the frequently used animal model in the experimental studies. Risk of bias Overall, all the studies were having low risk of bias in publishing in peer-reviewed journals. Three studies had a low risk of bias in random allocation concealment. Only two studies were reporting sample size calculation The statement of compliance with animal welfare regulations was reported in most of the studies while conflict of interest were missing in most articles. CONCLUSION Challenging approaches had emerged for oral and maxillofacial reconstruction in the last decade due to the complex nature of craniofacial defects. Gene therapy is on the top list of innovative strategies in tissue engineering that developed in the last 10 years. While significant progress has been made towards preclinical studies of gene therapy in the maxillofacial region building the scientific basis of this technique, gene therapy is still in the clinical trials phase in salivary glands and craniofacial defects. REFERENCES 1. Bonadio J and ML Cunningham. (2002). Genetic Approaches to Craniofacial Tissue Repair. Annals of the New York Academy of Sciences 961:48-57. 2. Dai J, AB Rabie, U Hagg and R Xu. (2004). Alternative gene therapy strategies for the repair of craniofacial bone defects. Curr Gene Ther 4:469-85. 3. Kademani D, S Mardini and SL Moran. (2008). Reconstruction of head and neck defects: a systematic approach to treatment. Semin Plast Surg 22:141-55. 4. Nussenbaum B and PH Krebsbach. (2006). The role of gene therapy for craniofacial and dental tissue engineering. Advanced Drug Delivery Reviews 58:577-91. 5. Scheller EL, LG Villa-Diaz and PH Krebsbach. (2012). Gene therapy: implications for craniofacial regeneration. J Craniofac Surg 23:333-7. Fig.2: Risk of bias graph for the studies included in this systematic review. Assessment of risk of bias using modified CAMARADES tool. Risk of bias of all included studies with the percentage of risk of bias for each item of assessment. ACKNOWLEDGEMENT CORRESPONDANCE The authors would like to thank SYRCLE organization for their helpful comments and discussion. Dr.Riham Fliefel Experimental Surgery and Regenerative Medicine Department of Surgery, Ludwig-Maximilians University Nussbaumstraße 20, 80336 München Riham.Fliefel@med.uni-muenchen.de .