Evaluation of the clinical effectiveness of bioactive glass (S53P4) in the treatment of non-unions of the tibia and femur: study protocol of a randomized controlled non-inferiority trial

Evaluation of the clinical effectiveness of bioactive glass (S53P4) in the treatment of... Background: Treatment of non-union remains challenging and often necessitates augmentation of the resulting defect with an autologous bone graft (ABG). ABG is limited in quantity and its harvesting incurs an additional surgical intervention leaving the risk for associated complications and morbidities. Therefore, artificial bone graft substitutes that might replace autologous bone are needed. S53P4-type bioactive glass (BaG) is a promising material which might be used as bone graft substitute due to its osteostimulative, conductive and antimicrobial properties. In this study, we plan to examine the clinical effectiveness of BaG as a bone graft substitute in Masquelet therapy in comparison with present standard Masquelet therapy using an ABG with tricalciumphosphate to fill the bone defect. Methods/design: This randomized controlled, clinical non-inferiority trial will be carried out at the Department of Orthopedics and Traumatology at Heidelberg University. Patients who suffer from tibial or femoral non-unions with a segmental bone defect of 2–5 cm and who are receiving Masquelet treatment will be included in the study. The resulting bone defect will either be filled with autologous bone and tricalciumphosphate (control group, N = 25) or BaG (S53P4) (study group, N = 25). Subsequent to operative therapy, all patients will receive the same standardized follow-up procedures. The primary endpoint of the study is union achieved 1year after surgery. Discussion: The results from the current study will help evaluate the clinical effectiveness of this promising biomaterial in non-union therapy. In addition, this randomized trial will help to identify potential benefits and limitations regarding the use of BaG in Masquelet therapy. Data from the study will increase the knowledge about BaG as a bone graft substitute as well as identify patients possibly benefiting from Masquelet therapy using BaG and those who are more likely to fail, thereby improving the quality of non-union treatment. Trial registration: German Clinical Trials Register (DRKS), ID: DRKS00013882. Registered on 22 January 2018. Keywords: Non-union, Bioactive glass, Masquelet therapy, Bone regeneration, S53P4, Pseudarthrosis, Bone grafting * Correspondence: patrick.haubruck@med.uni-heidelberg.de HTRG – Heidelberg Trauma Research Group, Centre for Orthopaedics, Trauma Surgery and Spinal Cord Injury, Trauma and Reconstructive Surgery, Heidelberg University Hospital, Schlierbacher Landstrasse 200a, 69118 Heidelberg, Germany © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tanner et al. Trials (2018) 19:299 Page 2 of 9 Background 26]. Radiological union can be assumed when bridging Fracture healing is a complex physiological process of three out of four cortices is apparent. However, evalu- dependent on the intricate interaction of numerous part- ation of osseous consolidation of non-unions relying ners [1]. Delayed or failed fracture healing can lead to merely on x-rays can be misleading. In a recent study by enormous limitations in the quality of life due to pain, Akiho et al. [25] the authors compared conventional reduced mobility, and considerably longer duration of x-rays and computed tomography (CT) scans of pubic disease. In addition, non-union of a fracture can also bone non-unions. Their data showed that CT scans were lead to debilitating economic and social circumstances able to identify a larger number of delayed unions. Thus, [2]. Despite recent research advances and modern treat- even when osseous consolidation is presumed on x-ray, ment options, up to 30% of long bone fractures develop where there are numerous layers superimposed upon non-unions [1, 3, 4]. Especially the treatment of atrophic one another in both planes, persistent non-unions can and infected non-unions, as well as large defect sizes, re- only be detected reliably via CT scans [25]. However, mains a challenge in trauma and orthopedic surgery. utilization of CT scans is limited due to their higher ra- Treatment of segmental defects can be divided into two diation exposure. Therefore, a combination of both general categories: (1). bone transport and (2). bone fill- methods is beneficial to assess radiological outcome of ing [5, 6]. In defects larger than 4–6 cm in length the non-union treatment. Promising new diagnostic modal- “gold standard” remains bone transport [6]; whereas, in ities contributing to a timely identification of successful segmental defects of between 2- to 5-cm bone filling has non-union treatment have been introduced in recent shown good clinical and radiological outcomes [5, 6]. years. In particular, analysis of serum cytokine expres- Therefore, the induced membrane technique, also sion pattern was established as a valid method in the known as the Masquelet technique, was established [7– evaluation of the biological processes occurring during 11]. It is a two-staged procedure; during the first step a bone regeneration [27, 28]. In addition, dynamic vascularized membrane, containing growth factors and contrast-enhanced magnetic resonance imaging supporting the proliferation of human bone marrow (DCE-MRI) perfusion analysis after non-union treatment stromal cells, is induced via a foreign-body reaction [7]. was able to successfully predict the outcome of The second step involves surgically augmenting the non-union therapy [29]. Furthermore, the combination membranous tube via an ABG, due to its osteostimula- of DCE-MRI and contrast enhanced ultrasound (CEUS) tive and osteoconductive properties as well as its osteo- was able to distinguish between infected and aseptic genic potential [8, 10]. The most frequently accessed non-unions pre-operatively [30]. Hence, a combination donor site remains the iliac crest. However, complica- of standardized and innovative diagnostic modalities tions, such as donor-site morbidity, pain and quantita- contributes to a precise and timely identification of suc- tive limitations, are well-documented [12–15]. In recent cessful non-union treatment and, furthermore, helps years, the reamer/irrigator/aspirator (RIA) system has identify patients at risk for infected non-unions. shown numerous advantages in harvesting autologous Regardless of the source, harvesting of autologous bone from the medullary canal of long bones [16]. bone necessitates an additional surgical intervention Reaming debris became a reliable alternative as a source with a potential risk for associated complications and for autologous bone and the RIA system has shown de- morbidities. Also, in some patients, either harvesting of creased morbidity at the harvest site and none of the autologous bone via RIA is anatomically impossible or complications of the iliac crest site [17–21]. From a cel- they may have already have had both iliac crests de- lular aspect, mesenchymal stem cells harvested by the pleted, or both may apply. Hence, alternative methods, RIA system show significantly superior osteogenic differ- such as allogenic bone, demineralized bone matrix and entiation and higher sensitivity towards stimulation with biomaterials designed as artificial bone graft substitutes, differentiation factors compared to mesenchymal stem have been extensively studied, but found to be lacking in cells isolated from iliac crest bone marrow [22, 23]. comparison to an ABG [31, 32]. In order to replace the Evaluation of the outcome of non-union therapy re- ABG, the substituting biomaterial must be bioactive (the mains challenging. A standardized approach was estab- effect of the materials on cells that activate specific re- lished by recent studies [11, 24] containing both sponses), degradable, osteoconductive and osteostimula- radiological and clinical parameters. Clinical outcome tive [33, 34]. BaG (S53P4) is such a material and is can be determined via assessment of mechanical stabil- currently used as bone graft substitute and in the treat- ity, pain associated with weight-bearing and the subject- ment of osteomyelitis [33]. BaG has osteostimulative ive health of patients [24]. Standard of care in the properties; the release of calcium ions leads to formation radiological assessment of bone healing and consolida- of hydroxyapatite. It is also osteoconductive, serving as a tion of non-union subsequent to treatment remains peri- scaffold for bone formation in vivo [33]. In addition, odical conventional x-rays of the affected bone [11, 24– BaG has been shown to have antimicrobial properties Tanner et al. Trials (2018) 19:299 Page 3 of 9 due to its release of ions from its surface resulting in an consent and patients receiving an amputation because of increasing osmotic pressure and pH leading to a micro- persistent infection or extended soft tissue defects will environment unsuitable for microbial growth [35]. be excluded from the study. Therefore, S53P4-type BaG (53% SiO ,4%P O , 23% 2 2 5 Na O and 20% CaO in wt%) is a promising material to Setting employ as bone graft substitute in context with The study is carried out at the Department of Orthope- non-union therapy. dics and Traumatology at Heidelberg University (level 1 The current study is a randomized controlled trial trauma center). Surgical treatment of non-union is (RCT) regarding the non-inferiority of the effectiveness established and a standardized follow-up setting is devel- and safety of the use of BaG (S53P4) as bone graft sub- opment to monitor response to the treatment and clin- stitute in Masquelet therapy for treating ical consolidation [4, 11, 24]. large-sized-defect non-unions of the tibia and femur in comparison to the standard therapy. The study protocol Randomization for the RCT is described in the present manuscript. Due to the sample size a block randomization procedure with randomly chosen block sizes is used to assign par- Methods/design ticipants to each group (1:1 ratio), resulting in one inter- Objectives vention and one control group. This method helps in The primary objective of this study is to evaluate the maintaining the balance of treatment assignment while non-inferiority of the clinical effectiveness of BaG as a reducing the potential for selection bias [36]. bone graft substitute in Masquelet therapy when com- Randomization is performed by an employee not in- pared to present standard Masquelet therapy using an volved in treatment, assessment or data collection re- ABG in combination with ceramic bone substitutes, garding the present study using opaque, sealed such as tricalciumphosphates, to fill the bone defect. envelopes. Secondary objectives include subjective patient quality of life directly post-operative as well as during the time of Surgical treatment recovery, documentation of perfusion of the bone graft After information and randomization, all patients receive using CEUS as well as DCE-MRI [26, 29]. Furthermore, contrast-enhanced ultrasound sonography (CEUS) patient data (such as smoking status, drug abuse, profes- pre-operatively to evaluate local perfusion. Hereafter, sion, time necessary to return to work and pre-existing they are scheduled for Masquelet therapy of the condition) will be assessed and evaluated. Therefore, the non-union. Masquelet therapy is based on the principles results from the current study will facilitate the evalu- of the “diamond concept” [37] and is a two-step proced- ation of BaG as a bone graft substitute regarding object- ure (step I and step II). During the first step, radical de- ive parameters associated with consolidation as well as bridement of the non-union, non-viable bone and subjective parameters associated with the patients’ qual- surrounding tissue is performed [38]. The resulting seg- ity of life. Furthermore, patients who are at risk for un- mental bone defect is subsequently filled with poly- successful treatment with one or the other approach methylmethacrylate (PMMA), which induces a might be identified. Hence, results from the study will foreign-body reaction, resulting in a vascularized Mas- contribute to thoroughly assess whether BaG is a suit- quelet membrane [7]. In addition, multiple bone and soft able bone graft substitute in non-union therapy using tissue samples are harvested for microbiological examin- the Masquelet method. ation. The first step is repeated until all samples are ster- ile. Once sterile, the spacer is left in situ for 6 weeks to Study design guarantee a fully grown Masquelet membrane [7, 10]. In This is a registered, prospective, single-center, two-arm, a second step the spacer is removed while leaving the parallel-group, randomized controlled non-inferiority membrane unimpaired and the resulting bone defect trial (DRKS00013882). filled with either autologous bone and tricalcium phos- phate (control group) or BaG (S53P4) (study group). Inclusion and exclusion criteria Due to the decreased morbidity at the harvest site and Patients older than 18 years who suffer from tibial or significantly superior osteogenic differentiation and femoral non-unions with a segmental bone defect of 2– higher sensitivity towards stimulation with differenti- 5 cm and who are receiving Masquelet treatment will be ation factors harvesting of autologous bone graft will be included into the study after giving informed consent. performed with the RIA system [39]. However, if the Patients who do not agree to participate in the study, quantity of the reaming material threatens to be insuffi- who are not applicable for harvesting autologous bone cient, additional harvesting of the iliac crest might be using the RIA system, who are not able to give informed necessary to achieve sufficient filling of the osseous Tanner et al. Trials (2018) 19:299 Page 4 of 9 defect. De novo osteosynthesis utilizing plates, nails or Discharge from the hospital will be realized as soon as external fixators is performed depending on the bio- soft tissue conditions, patient mobility and pain level mechanical stability during the first or second step of allow it. Afterwards, patients will receive physiotherapy the Masquelet therapy. The eligibility of the utilized on a regular basis of at least twice a week. Clinical and method of osteosynthesis will be based on anatomical radiological follow-up visits at our hospital are planned premises as well as morphology of the non-union and at 6 weeks, 3, 6, 9, 12 and 24 months post-operatively, will be carefully evaluated pre-operatively. A flow chart following the standardized procedure for non-union pa- of the surgical treatment is depicted in Fig. 1. tients treated in our hospital (see Table 1)[27]. By using Post-operatively, all patients regardless of the method of a questionnaire pre-operatively and post-operatively fixation will be treated with partial weight-bearing of after 3, 12 and 24 months, patients can give information 20 kg for 6 weeks; afterwards patients will gradually in- on pain, mobility of limbs and life quality (SF-12) during crease weight-bearing with approximately 10 kg per the course of treatment. General patient data, such as week until full weight-bearing is achieved. profession, Body Mass Index (BMI), risk factors, medica- tion, pre-existing conditions, previous surgeries and ac- Follow-up cident data will be obtained pre-operatively. Subsequent Subsequent to the operative therapy, all patients will re- to step II, the outcome of therapy will be evaluated ceive the same follow-up procedures. Follow-up at our based on clinical and radiological examination. Potential institution is standardized and all procedures and diag- for vascularization will be evaluated pre-operatively and nostics are based solely on medical indications. Initial at 12 weeks subsequent to step II via CEUS using the radiological and clinical evaluation of the surgical treat- established protocol [26]. After 12 weeks, patients also ment will be performed on day 2 after surgery. receive DCE-MRI to assess vitality of the graft as a Fig. 1 Flow chart of the Masquelet therapy and treatment pattern of included patients Tanner et al. Trials (2018) 19:299 Page 5 of 9 Table 1 Structure of clinical and radiological follow-up Pre-op 2 D post-op 6 W post-op 3 M post-op 6 M post-op 9 M post-op 12 M post-op 24 M post-op Clinical examination x x x x x x x x X-ray x x x x x x x x DCE-MRI x CT x CEUS x x Questionnaire (SF-12) x x x x Laboratory work x x x x x x x x Abbreviations: pre-op pre-operative, post-op post-operative, D days, W weeks, M months, DCE-MRI dynamic contrast-enhanced MRI, CT computer tomography, CEUS contrast-enhanced ultrasound, SF-12 12-item Short Form health survey previous study has shown that DCE-MRI perfusion ana- evaluated based on x-rays in two planes (defined as cor- lysis after non-union surgery predicts successful out- tical bridging of at least three out of four cortices) and come [29]. possible differences regarding socioeconomic factors After12months, aCTofthe affected bonewillbeper- (time necessary to return to work, time of recovery) are formed to further evaluate osseous consolidation. Patients assessed and compared between groups. in both study groups will be declared responder/non-re- sponder due to radiological signs of consolidation and clin- Criteria that lead to termination of study ical signs of mechanical stability and full weight-bearing. Data of included patient will be continuously monitored Furthermore, blood samples will be obtained during the regarding outcome and unexpected risk for participating course of treatment and analyzed regarding parameters of patients. If initial data indicates either an inferior out- infection, growth factors and cytokines associated with come of patients included into the study group, or an in- angiogenesis. Previous studies have shown that analysis of creased risk for patients of the study group that is serological cytokine expression pattern is a valid tool in potentially harmful for patients, the study will be termi- evaluation of the potential for angiogenesis and effective- nated. Furthermore, if patients want to withdraw their ness for additional non-union therapy [27, 28]. After 12 consent to the study, they will be excluded from the and 24 months, results of the groups will be statistically an- current study. Withdrawal from the study will not im- alyzed and compared. Duration of patient enrollment will pact the quality of the medical treatment of patients. be 2 years. Data will be stored and monitored using pseu- donyms. Only PH and MCT have access to the full names of the participants. Follow-up will be 2 years with data ana- Statistical analysis lysis after 1 year and 2 years. The duration of the study is 4 Statistical calculation will be conducted with R version 3.4.3 years (Table 1). [41], figures will be created using the package “ggplot2” [42]. Receiver operator characteristics analysis will be per- Primary outcome measure formed via the “pROC” package [43]. Correlation analyses The primary endpoint of the study is union achieved 1 will be performed between all variables. Non-parametric year after surgery by evaluation of x-rays in two planes tests (Mann-Whitney U test for independent variables, Wil- (defined as cortical bridging of at least three out of four coxon signed-rank test for dependent variables) will be uti- cortices) and CT scan [40]. The radiographic datasets lized to investigate location shifts between groups. will be blinded and evaluated by a group of experienced Differences between categorical variables will be examined orthopedic surgeons. via the chi-square test. The Kruskal-Wallis test will be used to assess differences in more than two independent sam- Secondary outcome measure ples. To evaluate the predictive power of variables regard- Secondary endpoints include subjective evaluation of the ing the criterion “consolidation” adjusting for potentially quality of life (assessed by the 12-item Short Form clinically relevant covariates logistic regression models will health survey (SF-12) questionnaire) and pain (Visual be set up and constructed via backwards selection. Analo- Analog Scale (VAS) of affected patients. In addition, per- gous to our previous studies [44–46], predictive perform- fusion of the graft is evaluated using CEUS and ance will be assessed through estimation of the models DCE-MRI and compared between groups. Expression AUC (area under the curve) of the corresponding ROC patterns of inflammatory and angiogenic cytokines are curve and AIC (Akaike information criterion). Continuous evaluated during the course of the study and compared variables will be expressed as absolute mean concentrations between groups regarding possible differences. Addition- ± SD (standard deviation) and the level of significance (α)is ally, union achieved 2 years after surgery will be setat5%. Tanner et al. Trials (2018) 19:299 Page 6 of 9 Sample size determination Discussion Currently, there are no comparable studies available in the This study aims to investigate into the non-inferiority of academic literature. In order to determine the necessary the clinical effectiveness of BaG (S53P4) as a bone graft sample size data from a previous study was utilized (Au- substitute in Masquelet therapy compared to the stand- thor: Haubruck P, Tanner M, Vlachopoulos W, Hagels- ard Masquelet therapy using autograft. kamp S, Miska M, Ober J, Fischer C, Schmidmaier G. BaG has been established in previous case series and in Title: Comparison of the clinical effectiveness of Bone vivo animal studies, but not yet in RCTs as a promising bio- Morphogenic Protein (BMP) -2 and -7 in the adjunct material due to both its osteostimulative, osteoconductive treatment of lower limb non-unions: a matched pair ana- (serving as a scaffold for bone formation in vivo) and anti- lysis. Submitted 2018). In this study, a similar patient col- microbial properties [33]. In particular, after implantation a lective suffering from non-unions of the same anatomical surface reaction occurs, resulting in formation of a calcium region being treated with Masquelet therapy were evalu- phosphate layer [33, 47]. Release of various ions increases ated regarding their osseous consolidation. Based on our local pH and osmotic pressure, then a silica gel layer is previous study we performed the sample size calculation formed on the surface of the biomaterial and amorphous for the binary-outcome non-inferiority trial in R [41]using calcium phosphate precipitates on this layer [33]. There- the package “‘SampleSize4ClinicalTrials” by Hongchao after, crystallization to natural hydroxyapatite occurs, which Qi. Additionally, assuming an alpha level of .05 and a starts the activation of osteoblasts and initiates the forma- power of .90 as well as an equal number of subjects tion of new bone [33, 48]. During this process, new bone is in the experimental and control groups we estimated constituted, the BaG absorbed and the antibacterial micro- that 50 patients in total (25 patients each group) to environment maintained due to a persistently increased pH be required. Intervention group sizes will match this [33]. Hence, BaG as an artificial bone substitute might con- determined sample size. tribute to successful non-union treatment by both Fig. 2 Study process schedule (according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines) Tanner et al. Trials (2018) 19:299 Page 7 of 9 osteostimulative bone regeneration and prevention of an in- PMMA: Polymethylmethacrylate; RCT: Randomized controlled trial; RIA: Reamer/irrigator/aspirator fection via its antimicrobial properties. Furthermore, im- plantation of BaG prevents the surgical intervention Acknowledgements necessary for harvesting of the ABG and, therefore, might We acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding programme Open Access Publishing, by the Baden- contribute to a lower complication rate and lower comor- Württemberg Ministry of Science, Research and Arts and by the Ruprechts- bidities associated with Masquelet therapy. A potential limi- Karls-Universität Heidelberg. tation of the planned study lies in the utilization of different Funding methods of osteosynthesis that might influence the out- The current trial is an investigator-initiated trial funded by BonAlive Biomate- come of non-union treatment. However, a study by Vallier rials Ltd. (Biolinja 12, 20750 Turku, Finland). The funders play no role in the et al. compared the results between plate fixation and intra- study design, surgical treatment, collection, management, analysis and inter- pretation of data or the final report and its publication, nor do they have ul- medullary nail fixation of tibial shaft fractures. The authors timate authority over any of these actions. concluded that rates of union, infection and secondary pro- cedures were similar [49]. In addition, all included patients Availability of data and materials will employ the same post-operative weight-bearing pattern. Access to all data sets and statistical code will be granted individually upon request. Therefore, we believe that the influence of different methods of osteosynthesis on the findings of the planned Authors’ contributions study to be minimal. The results from the current study Study conception and design: PH, RH, CF, FW, GS, MCT, MM, TF, SG. Acquisition of data: PH, RH, CF, FW, GS, MCT, MM, TF. Data monitoring and will help evaluate the clinical effectiveness of this promising statistical analysis: PH, RH, GS, CF, MCT, SG. Analysis and interpretation of biomaterial in non-union therapy. Our hypothesis is that data: MCT, TF, CF, PH, RH, MM, SG. Drafting of manuscript: PH, MCT, RH, FW. S53P4-type BaG will have the same rate of consolidation as Critical revision: PH, RH, CF, FW, GS, MCT, MM, TF, SG. All authors read and approved the final version of this manuscript. Authorship eligibility autologous bone when used in the second step of the Mas- guidelines according to the ICMJE were followed. The use of professional quelet therapy. Furthermore, we assume that the rate of writers is not intended. perioperative infection in patients treated with S53P4-BaG will be reducedcomparedto the controlgroup andthat pa- Ethics approval and consent to participate Ethical approval was obtained by the Ethics Committee of the University tients of the study group will have fewer post-operative of Heidelberg Medical Faculty prior to the beginning of the study complications and morbidities. The results of the study (Ethikkommission I der Medizinischen Fakultät Heidelberg, S-472/2017). should, therefore, help investigate the potential benefits and This trial was registered with the German Clinical Trials Register (DRKS) in Freiburg, a primary registry within the WHO Registry Network, Germany, on limitations regarding the use of S53P4-BaG in Masquelet 22 January 2018 with the trial registration number DRKS00013882. The therapy. Data from the study will increase the knowledge CONSORT extension for non-pharmacological interventions and the Standard about S53P4-BaG as a bone graft substitute as well as iden- Protocol Items: Recommendations for Interventional Trials (SPIRIT) Checklist for the implementation of study protocols were followed (Fig. 2 and Additional file 1) tify patients who might benefit from Masquelet therapy [50, 51]. Written informed consent will be obtained from each patient. In the event using this type of BaG and those who are more likely to fail. that a patient’s physical or mental health becomes jeopardized because of Ultimately, the current study might contribute to an im- participation in the present study, the patient will be dismissed immediately and excluded from the study. All protocol modifications will be registered with the provement in the quality of non-union treatment. DRKS, published in the final paper and communicated to the participants. Competing interests Trial status The authors declare that they have no competing interests. The RCT recruitment and surgical treatment are planned from April 2018 until April 2020. Follow-up will be con- Publisher’sNote ducted over 24 months for each included patient. Data Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. analysis and evaluation will be performed after 12 months and 24 months. 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Int J Surg. 2011;9:672–7. https://doi.org/10.1016/j.ijsu.2011.09.004. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Trials Springer Journals

Evaluation of the clinical effectiveness of bioactive glass (S53P4) in the treatment of non-unions of the tibia and femur: study protocol of a randomized controlled non-inferiority trial

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Medicine & Public Health; Medicine/Public Health, general; Biomedicine, general; Statistics for Life Sciences, Medicine, Health Sciences
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Abstract

Background: Treatment of non-union remains challenging and often necessitates augmentation of the resulting defect with an autologous bone graft (ABG). ABG is limited in quantity and its harvesting incurs an additional surgical intervention leaving the risk for associated complications and morbidities. Therefore, artificial bone graft substitutes that might replace autologous bone are needed. S53P4-type bioactive glass (BaG) is a promising material which might be used as bone graft substitute due to its osteostimulative, conductive and antimicrobial properties. In this study, we plan to examine the clinical effectiveness of BaG as a bone graft substitute in Masquelet therapy in comparison with present standard Masquelet therapy using an ABG with tricalciumphosphate to fill the bone defect. Methods/design: This randomized controlled, clinical non-inferiority trial will be carried out at the Department of Orthopedics and Traumatology at Heidelberg University. Patients who suffer from tibial or femoral non-unions with a segmental bone defect of 2–5 cm and who are receiving Masquelet treatment will be included in the study. The resulting bone defect will either be filled with autologous bone and tricalciumphosphate (control group, N = 25) or BaG (S53P4) (study group, N = 25). Subsequent to operative therapy, all patients will receive the same standardized follow-up procedures. The primary endpoint of the study is union achieved 1year after surgery. Discussion: The results from the current study will help evaluate the clinical effectiveness of this promising biomaterial in non-union therapy. In addition, this randomized trial will help to identify potential benefits and limitations regarding the use of BaG in Masquelet therapy. Data from the study will increase the knowledge about BaG as a bone graft substitute as well as identify patients possibly benefiting from Masquelet therapy using BaG and those who are more likely to fail, thereby improving the quality of non-union treatment. Trial registration: German Clinical Trials Register (DRKS), ID: DRKS00013882. Registered on 22 January 2018. Keywords: Non-union, Bioactive glass, Masquelet therapy, Bone regeneration, S53P4, Pseudarthrosis, Bone grafting * Correspondence: patrick.haubruck@med.uni-heidelberg.de HTRG – Heidelberg Trauma Research Group, Centre for Orthopaedics, Trauma Surgery and Spinal Cord Injury, Trauma and Reconstructive Surgery, Heidelberg University Hospital, Schlierbacher Landstrasse 200a, 69118 Heidelberg, Germany © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tanner et al. Trials (2018) 19:299 Page 2 of 9 Background 26]. Radiological union can be assumed when bridging Fracture healing is a complex physiological process of three out of four cortices is apparent. However, evalu- dependent on the intricate interaction of numerous part- ation of osseous consolidation of non-unions relying ners [1]. Delayed or failed fracture healing can lead to merely on x-rays can be misleading. In a recent study by enormous limitations in the quality of life due to pain, Akiho et al. [25] the authors compared conventional reduced mobility, and considerably longer duration of x-rays and computed tomography (CT) scans of pubic disease. In addition, non-union of a fracture can also bone non-unions. Their data showed that CT scans were lead to debilitating economic and social circumstances able to identify a larger number of delayed unions. Thus, [2]. Despite recent research advances and modern treat- even when osseous consolidation is presumed on x-ray, ment options, up to 30% of long bone fractures develop where there are numerous layers superimposed upon non-unions [1, 3, 4]. Especially the treatment of atrophic one another in both planes, persistent non-unions can and infected non-unions, as well as large defect sizes, re- only be detected reliably via CT scans [25]. However, mains a challenge in trauma and orthopedic surgery. utilization of CT scans is limited due to their higher ra- Treatment of segmental defects can be divided into two diation exposure. Therefore, a combination of both general categories: (1). bone transport and (2). bone fill- methods is beneficial to assess radiological outcome of ing [5, 6]. In defects larger than 4–6 cm in length the non-union treatment. Promising new diagnostic modal- “gold standard” remains bone transport [6]; whereas, in ities contributing to a timely identification of successful segmental defects of between 2- to 5-cm bone filling has non-union treatment have been introduced in recent shown good clinical and radiological outcomes [5, 6]. years. In particular, analysis of serum cytokine expres- Therefore, the induced membrane technique, also sion pattern was established as a valid method in the known as the Masquelet technique, was established [7– evaluation of the biological processes occurring during 11]. It is a two-staged procedure; during the first step a bone regeneration [27, 28]. In addition, dynamic vascularized membrane, containing growth factors and contrast-enhanced magnetic resonance imaging supporting the proliferation of human bone marrow (DCE-MRI) perfusion analysis after non-union treatment stromal cells, is induced via a foreign-body reaction [7]. was able to successfully predict the outcome of The second step involves surgically augmenting the non-union therapy [29]. Furthermore, the combination membranous tube via an ABG, due to its osteostimula- of DCE-MRI and contrast enhanced ultrasound (CEUS) tive and osteoconductive properties as well as its osteo- was able to distinguish between infected and aseptic genic potential [8, 10]. The most frequently accessed non-unions pre-operatively [30]. Hence, a combination donor site remains the iliac crest. However, complica- of standardized and innovative diagnostic modalities tions, such as donor-site morbidity, pain and quantita- contributes to a precise and timely identification of suc- tive limitations, are well-documented [12–15]. In recent cessful non-union treatment and, furthermore, helps years, the reamer/irrigator/aspirator (RIA) system has identify patients at risk for infected non-unions. shown numerous advantages in harvesting autologous Regardless of the source, harvesting of autologous bone from the medullary canal of long bones [16]. bone necessitates an additional surgical intervention Reaming debris became a reliable alternative as a source with a potential risk for associated complications and for autologous bone and the RIA system has shown de- morbidities. Also, in some patients, either harvesting of creased morbidity at the harvest site and none of the autologous bone via RIA is anatomically impossible or complications of the iliac crest site [17–21]. From a cel- they may have already have had both iliac crests de- lular aspect, mesenchymal stem cells harvested by the pleted, or both may apply. Hence, alternative methods, RIA system show significantly superior osteogenic differ- such as allogenic bone, demineralized bone matrix and entiation and higher sensitivity towards stimulation with biomaterials designed as artificial bone graft substitutes, differentiation factors compared to mesenchymal stem have been extensively studied, but found to be lacking in cells isolated from iliac crest bone marrow [22, 23]. comparison to an ABG [31, 32]. In order to replace the Evaluation of the outcome of non-union therapy re- ABG, the substituting biomaterial must be bioactive (the mains challenging. A standardized approach was estab- effect of the materials on cells that activate specific re- lished by recent studies [11, 24] containing both sponses), degradable, osteoconductive and osteostimula- radiological and clinical parameters. Clinical outcome tive [33, 34]. BaG (S53P4) is such a material and is can be determined via assessment of mechanical stabil- currently used as bone graft substitute and in the treat- ity, pain associated with weight-bearing and the subject- ment of osteomyelitis [33]. BaG has osteostimulative ive health of patients [24]. Standard of care in the properties; the release of calcium ions leads to formation radiological assessment of bone healing and consolida- of hydroxyapatite. It is also osteoconductive, serving as a tion of non-union subsequent to treatment remains peri- scaffold for bone formation in vivo [33]. In addition, odical conventional x-rays of the affected bone [11, 24– BaG has been shown to have antimicrobial properties Tanner et al. Trials (2018) 19:299 Page 3 of 9 due to its release of ions from its surface resulting in an consent and patients receiving an amputation because of increasing osmotic pressure and pH leading to a micro- persistent infection or extended soft tissue defects will environment unsuitable for microbial growth [35]. be excluded from the study. Therefore, S53P4-type BaG (53% SiO ,4%P O , 23% 2 2 5 Na O and 20% CaO in wt%) is a promising material to Setting employ as bone graft substitute in context with The study is carried out at the Department of Orthope- non-union therapy. dics and Traumatology at Heidelberg University (level 1 The current study is a randomized controlled trial trauma center). Surgical treatment of non-union is (RCT) regarding the non-inferiority of the effectiveness established and a standardized follow-up setting is devel- and safety of the use of BaG (S53P4) as bone graft sub- opment to monitor response to the treatment and clin- stitute in Masquelet therapy for treating ical consolidation [4, 11, 24]. large-sized-defect non-unions of the tibia and femur in comparison to the standard therapy. The study protocol Randomization for the RCT is described in the present manuscript. Due to the sample size a block randomization procedure with randomly chosen block sizes is used to assign par- Methods/design ticipants to each group (1:1 ratio), resulting in one inter- Objectives vention and one control group. This method helps in The primary objective of this study is to evaluate the maintaining the balance of treatment assignment while non-inferiority of the clinical effectiveness of BaG as a reducing the potential for selection bias [36]. bone graft substitute in Masquelet therapy when com- Randomization is performed by an employee not in- pared to present standard Masquelet therapy using an volved in treatment, assessment or data collection re- ABG in combination with ceramic bone substitutes, garding the present study using opaque, sealed such as tricalciumphosphates, to fill the bone defect. envelopes. Secondary objectives include subjective patient quality of life directly post-operative as well as during the time of Surgical treatment recovery, documentation of perfusion of the bone graft After information and randomization, all patients receive using CEUS as well as DCE-MRI [26, 29]. Furthermore, contrast-enhanced ultrasound sonography (CEUS) patient data (such as smoking status, drug abuse, profes- pre-operatively to evaluate local perfusion. Hereafter, sion, time necessary to return to work and pre-existing they are scheduled for Masquelet therapy of the condition) will be assessed and evaluated. Therefore, the non-union. Masquelet therapy is based on the principles results from the current study will facilitate the evalu- of the “diamond concept” [37] and is a two-step proced- ation of BaG as a bone graft substitute regarding object- ure (step I and step II). During the first step, radical de- ive parameters associated with consolidation as well as bridement of the non-union, non-viable bone and subjective parameters associated with the patients’ qual- surrounding tissue is performed [38]. The resulting seg- ity of life. Furthermore, patients who are at risk for un- mental bone defect is subsequently filled with poly- successful treatment with one or the other approach methylmethacrylate (PMMA), which induces a might be identified. Hence, results from the study will foreign-body reaction, resulting in a vascularized Mas- contribute to thoroughly assess whether BaG is a suit- quelet membrane [7]. In addition, multiple bone and soft able bone graft substitute in non-union therapy using tissue samples are harvested for microbiological examin- the Masquelet method. ation. The first step is repeated until all samples are ster- ile. Once sterile, the spacer is left in situ for 6 weeks to Study design guarantee a fully grown Masquelet membrane [7, 10]. In This is a registered, prospective, single-center, two-arm, a second step the spacer is removed while leaving the parallel-group, randomized controlled non-inferiority membrane unimpaired and the resulting bone defect trial (DRKS00013882). filled with either autologous bone and tricalcium phos- phate (control group) or BaG (S53P4) (study group). Inclusion and exclusion criteria Due to the decreased morbidity at the harvest site and Patients older than 18 years who suffer from tibial or significantly superior osteogenic differentiation and femoral non-unions with a segmental bone defect of 2– higher sensitivity towards stimulation with differenti- 5 cm and who are receiving Masquelet treatment will be ation factors harvesting of autologous bone graft will be included into the study after giving informed consent. performed with the RIA system [39]. However, if the Patients who do not agree to participate in the study, quantity of the reaming material threatens to be insuffi- who are not applicable for harvesting autologous bone cient, additional harvesting of the iliac crest might be using the RIA system, who are not able to give informed necessary to achieve sufficient filling of the osseous Tanner et al. Trials (2018) 19:299 Page 4 of 9 defect. De novo osteosynthesis utilizing plates, nails or Discharge from the hospital will be realized as soon as external fixators is performed depending on the bio- soft tissue conditions, patient mobility and pain level mechanical stability during the first or second step of allow it. Afterwards, patients will receive physiotherapy the Masquelet therapy. The eligibility of the utilized on a regular basis of at least twice a week. Clinical and method of osteosynthesis will be based on anatomical radiological follow-up visits at our hospital are planned premises as well as morphology of the non-union and at 6 weeks, 3, 6, 9, 12 and 24 months post-operatively, will be carefully evaluated pre-operatively. A flow chart following the standardized procedure for non-union pa- of the surgical treatment is depicted in Fig. 1. tients treated in our hospital (see Table 1)[27]. By using Post-operatively, all patients regardless of the method of a questionnaire pre-operatively and post-operatively fixation will be treated with partial weight-bearing of after 3, 12 and 24 months, patients can give information 20 kg for 6 weeks; afterwards patients will gradually in- on pain, mobility of limbs and life quality (SF-12) during crease weight-bearing with approximately 10 kg per the course of treatment. General patient data, such as week until full weight-bearing is achieved. profession, Body Mass Index (BMI), risk factors, medica- tion, pre-existing conditions, previous surgeries and ac- Follow-up cident data will be obtained pre-operatively. Subsequent Subsequent to the operative therapy, all patients will re- to step II, the outcome of therapy will be evaluated ceive the same follow-up procedures. Follow-up at our based on clinical and radiological examination. Potential institution is standardized and all procedures and diag- for vascularization will be evaluated pre-operatively and nostics are based solely on medical indications. Initial at 12 weeks subsequent to step II via CEUS using the radiological and clinical evaluation of the surgical treat- established protocol [26]. After 12 weeks, patients also ment will be performed on day 2 after surgery. receive DCE-MRI to assess vitality of the graft as a Fig. 1 Flow chart of the Masquelet therapy and treatment pattern of included patients Tanner et al. Trials (2018) 19:299 Page 5 of 9 Table 1 Structure of clinical and radiological follow-up Pre-op 2 D post-op 6 W post-op 3 M post-op 6 M post-op 9 M post-op 12 M post-op 24 M post-op Clinical examination x x x x x x x x X-ray x x x x x x x x DCE-MRI x CT x CEUS x x Questionnaire (SF-12) x x x x Laboratory work x x x x x x x x Abbreviations: pre-op pre-operative, post-op post-operative, D days, W weeks, M months, DCE-MRI dynamic contrast-enhanced MRI, CT computer tomography, CEUS contrast-enhanced ultrasound, SF-12 12-item Short Form health survey previous study has shown that DCE-MRI perfusion ana- evaluated based on x-rays in two planes (defined as cor- lysis after non-union surgery predicts successful out- tical bridging of at least three out of four cortices) and come [29]. possible differences regarding socioeconomic factors After12months, aCTofthe affected bonewillbeper- (time necessary to return to work, time of recovery) are formed to further evaluate osseous consolidation. Patients assessed and compared between groups. in both study groups will be declared responder/non-re- sponder due to radiological signs of consolidation and clin- Criteria that lead to termination of study ical signs of mechanical stability and full weight-bearing. Data of included patient will be continuously monitored Furthermore, blood samples will be obtained during the regarding outcome and unexpected risk for participating course of treatment and analyzed regarding parameters of patients. If initial data indicates either an inferior out- infection, growth factors and cytokines associated with come of patients included into the study group, or an in- angiogenesis. Previous studies have shown that analysis of creased risk for patients of the study group that is serological cytokine expression pattern is a valid tool in potentially harmful for patients, the study will be termi- evaluation of the potential for angiogenesis and effective- nated. Furthermore, if patients want to withdraw their ness for additional non-union therapy [27, 28]. After 12 consent to the study, they will be excluded from the and 24 months, results of the groups will be statistically an- current study. Withdrawal from the study will not im- alyzed and compared. Duration of patient enrollment will pact the quality of the medical treatment of patients. be 2 years. Data will be stored and monitored using pseu- donyms. Only PH and MCT have access to the full names of the participants. Follow-up will be 2 years with data ana- Statistical analysis lysis after 1 year and 2 years. The duration of the study is 4 Statistical calculation will be conducted with R version 3.4.3 years (Table 1). [41], figures will be created using the package “ggplot2” [42]. Receiver operator characteristics analysis will be per- Primary outcome measure formed via the “pROC” package [43]. Correlation analyses The primary endpoint of the study is union achieved 1 will be performed between all variables. Non-parametric year after surgery by evaluation of x-rays in two planes tests (Mann-Whitney U test for independent variables, Wil- (defined as cortical bridging of at least three out of four coxon signed-rank test for dependent variables) will be uti- cortices) and CT scan [40]. The radiographic datasets lized to investigate location shifts between groups. will be blinded and evaluated by a group of experienced Differences between categorical variables will be examined orthopedic surgeons. via the chi-square test. The Kruskal-Wallis test will be used to assess differences in more than two independent sam- Secondary outcome measure ples. To evaluate the predictive power of variables regard- Secondary endpoints include subjective evaluation of the ing the criterion “consolidation” adjusting for potentially quality of life (assessed by the 12-item Short Form clinically relevant covariates logistic regression models will health survey (SF-12) questionnaire) and pain (Visual be set up and constructed via backwards selection. Analo- Analog Scale (VAS) of affected patients. In addition, per- gous to our previous studies [44–46], predictive perform- fusion of the graft is evaluated using CEUS and ance will be assessed through estimation of the models DCE-MRI and compared between groups. Expression AUC (area under the curve) of the corresponding ROC patterns of inflammatory and angiogenic cytokines are curve and AIC (Akaike information criterion). Continuous evaluated during the course of the study and compared variables will be expressed as absolute mean concentrations between groups regarding possible differences. Addition- ± SD (standard deviation) and the level of significance (α)is ally, union achieved 2 years after surgery will be setat5%. Tanner et al. Trials (2018) 19:299 Page 6 of 9 Sample size determination Discussion Currently, there are no comparable studies available in the This study aims to investigate into the non-inferiority of academic literature. In order to determine the necessary the clinical effectiveness of BaG (S53P4) as a bone graft sample size data from a previous study was utilized (Au- substitute in Masquelet therapy compared to the stand- thor: Haubruck P, Tanner M, Vlachopoulos W, Hagels- ard Masquelet therapy using autograft. kamp S, Miska M, Ober J, Fischer C, Schmidmaier G. BaG has been established in previous case series and in Title: Comparison of the clinical effectiveness of Bone vivo animal studies, but not yet in RCTs as a promising bio- Morphogenic Protein (BMP) -2 and -7 in the adjunct material due to both its osteostimulative, osteoconductive treatment of lower limb non-unions: a matched pair ana- (serving as a scaffold for bone formation in vivo) and anti- lysis. Submitted 2018). In this study, a similar patient col- microbial properties [33]. In particular, after implantation a lective suffering from non-unions of the same anatomical surface reaction occurs, resulting in formation of a calcium region being treated with Masquelet therapy were evalu- phosphate layer [33, 47]. Release of various ions increases ated regarding their osseous consolidation. Based on our local pH and osmotic pressure, then a silica gel layer is previous study we performed the sample size calculation formed on the surface of the biomaterial and amorphous for the binary-outcome non-inferiority trial in R [41]using calcium phosphate precipitates on this layer [33]. There- the package “‘SampleSize4ClinicalTrials” by Hongchao after, crystallization to natural hydroxyapatite occurs, which Qi. Additionally, assuming an alpha level of .05 and a starts the activation of osteoblasts and initiates the forma- power of .90 as well as an equal number of subjects tion of new bone [33, 48]. During this process, new bone is in the experimental and control groups we estimated constituted, the BaG absorbed and the antibacterial micro- that 50 patients in total (25 patients each group) to environment maintained due to a persistently increased pH be required. Intervention group sizes will match this [33]. Hence, BaG as an artificial bone substitute might con- determined sample size. tribute to successful non-union treatment by both Fig. 2 Study process schedule (according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines) Tanner et al. Trials (2018) 19:299 Page 7 of 9 osteostimulative bone regeneration and prevention of an in- PMMA: Polymethylmethacrylate; RCT: Randomized controlled trial; RIA: Reamer/irrigator/aspirator fection via its antimicrobial properties. Furthermore, im- plantation of BaG prevents the surgical intervention Acknowledgements necessary for harvesting of the ABG and, therefore, might We acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding programme Open Access Publishing, by the Baden- contribute to a lower complication rate and lower comor- Württemberg Ministry of Science, Research and Arts and by the Ruprechts- bidities associated with Masquelet therapy. A potential limi- Karls-Universität Heidelberg. tation of the planned study lies in the utilization of different Funding methods of osteosynthesis that might influence the out- The current trial is an investigator-initiated trial funded by BonAlive Biomate- come of non-union treatment. However, a study by Vallier rials Ltd. (Biolinja 12, 20750 Turku, Finland). The funders play no role in the et al. compared the results between plate fixation and intra- study design, surgical treatment, collection, management, analysis and inter- pretation of data or the final report and its publication, nor do they have ul- medullary nail fixation of tibial shaft fractures. The authors timate authority over any of these actions. concluded that rates of union, infection and secondary pro- cedures were similar [49]. In addition, all included patients Availability of data and materials will employ the same post-operative weight-bearing pattern. Access to all data sets and statistical code will be granted individually upon request. Therefore, we believe that the influence of different methods of osteosynthesis on the findings of the planned Authors’ contributions study to be minimal. The results from the current study Study conception and design: PH, RH, CF, FW, GS, MCT, MM, TF, SG. Acquisition of data: PH, RH, CF, FW, GS, MCT, MM, TF. Data monitoring and will help evaluate the clinical effectiveness of this promising statistical analysis: PH, RH, GS, CF, MCT, SG. Analysis and interpretation of biomaterial in non-union therapy. Our hypothesis is that data: MCT, TF, CF, PH, RH, MM, SG. Drafting of manuscript: PH, MCT, RH, FW. S53P4-type BaG will have the same rate of consolidation as Critical revision: PH, RH, CF, FW, GS, MCT, MM, TF, SG. All authors read and approved the final version of this manuscript. Authorship eligibility autologous bone when used in the second step of the Mas- guidelines according to the ICMJE were followed. The use of professional quelet therapy. Furthermore, we assume that the rate of writers is not intended. perioperative infection in patients treated with S53P4-BaG will be reducedcomparedto the controlgroup andthat pa- Ethics approval and consent to participate Ethical approval was obtained by the Ethics Committee of the University tients of the study group will have fewer post-operative of Heidelberg Medical Faculty prior to the beginning of the study complications and morbidities. The results of the study (Ethikkommission I der Medizinischen Fakultät Heidelberg, S-472/2017). should, therefore, help investigate the potential benefits and This trial was registered with the German Clinical Trials Register (DRKS) in Freiburg, a primary registry within the WHO Registry Network, Germany, on limitations regarding the use of S53P4-BaG in Masquelet 22 January 2018 with the trial registration number DRKS00013882. The therapy. Data from the study will increase the knowledge CONSORT extension for non-pharmacological interventions and the Standard about S53P4-BaG as a bone graft substitute as well as iden- Protocol Items: Recommendations for Interventional Trials (SPIRIT) Checklist for the implementation of study protocols were followed (Fig. 2 and Additional file 1) tify patients who might benefit from Masquelet therapy [50, 51]. Written informed consent will be obtained from each patient. In the event using this type of BaG and those who are more likely to fail. that a patient’s physical or mental health becomes jeopardized because of Ultimately, the current study might contribute to an im- participation in the present study, the patient will be dismissed immediately and excluded from the study. All protocol modifications will be registered with the provement in the quality of non-union treatment. DRKS, published in the final paper and communicated to the participants. Competing interests Trial status The authors declare that they have no competing interests. The RCT recruitment and surgical treatment are planned from April 2018 until April 2020. Follow-up will be con- Publisher’sNote ducted over 24 months for each included patient. Data Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. analysis and evaluation will be performed after 12 months and 24 months. 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