Percutaneous autologous concentrated bone marrow grafting in the treatment for nonunion

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1 DOI /s ORIGINAL ARTICLE Percutaneous autologous concentrated bone marrow grafting in the treatment for nonunion Hisashi Sugaya Hajime Mishima Katsuya Aoto Meihua Li Yukiyo Shimizu Tomokazu Yoshioka Shinsuke Sakai Hiroshi Akaogi Naoyuki Ochiai Masashi Yamazaki Received: 14 September 2013 / Accepted: 10 November 2013 Ó Springer-Verlag France 2013 Abstract The purpose of this study was to evaluate the clinical and radiographic treatment effects of percutaneous autologous concentrated bone marrow grafting in nonunion cases and to evaluate the effectiveness of this grafting procedure. We enrolled 17 cases those had atrophic changes due to continuous nonunion for over 9 months after injury and had undergone low-intensity pulsed ultrasound treatment for more than 3 months. The site of nonunion was the femur in 10 cases, the tibia in 5 cases, the humerus in 1 case, and the ulna in 1 case. They underwent percutaneous autologous concentrated bone marrow grafting and continued low-intensity pulsed ultrasound stimulation treatment after grafting. Patients were evaluated using the visual analogue scale for pain at immediately before the procedure, 3, 6, and 12 months after grafting. Plain radiographs of the affected site were taken and evaluated about the healing of the nonunion site at each clinical evaluation. As quantitative assessment, CT scans were undertaken before the procedure and 6 months after grafting. The visual analogue scale pain score was reduced consistently after grafting in all patients. About the healing at the nonunion site, 11 and 13 cases of bone union were observed at 6 and 12 months after grafting. The mean volume of callus formation based on CT images was 4,147 (262 27,392) mm 3 total between grafting and 6 months. Percutaneous autologous concentrated bone marrow H. Sugaya H. Mishima (&) K. Aoto M. Li Y. Shimizu T. Yoshioka S. Sakai H. Akaogi N. Ochiai M. Yamazaki Department of Orthopaedic Surgery, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki , Japan hmishima@md.tsukuba.ac.jp grafting is an effective procedure for the treatment of patients with nonunion. Keywords Nonunion Bone marrow Concentration Percutaneous grafting Minimally invasive technique Introduction Nonunion, which is failure of normal healing of a bone, remains challenging for patients as well as healthcare professionals, with nonunion following long bone fracture having an incidence of 5 10 % [1]. Nonunion causes continued chronic pain and significant psychological, financial, and physical burden [2, 3]. The standard treatment for nonunion is autologous bone grafting that is performed to accelerate osteogenesis by stimulating the local microenvironment at the nonunion site. However, this treatment is relatively invasive, requires bone harvesting and freshening of the fracture site. Also, it postoperatively may cause pain, neurovascular injury, or infection at the donor site. Other treatment options for nonunion include multiple drilling, allografting, bone graft substitutes, bone marrow grafting, low-intensity pulsed ultrasound stimulation (LIPUS), shockwave stimulation, and growth factor treatment [4]. Our group developed autologous concentrated bone marrow grafting for osteonecrosis of femoral head from 2003 and reported the safeness and effectiveness [5]. So we applied this technique for the treatment for nonunion from The purpose of this study was to evaluate the clinical and radiographic treatment effects of percutaneous autologous concentrated bone marrow grafting in nonunion cases and to evaluate the effectiveness of this grafting procedure.

2 Table 1 Patient demographics: The characteristics of the 17 cases are shown Case Age Sex Fracture site Initial injury Initial treatment Time since injury (months) 1 37 F Femur (shaft) Closed Plate fixation M Femur (shaft) Closed Intramedullary nailing M Femur (distal) Closed Intramedullary nailing M Femur (shaft) Closed Intramedullary nailing M Femur (shaft) Open Plate fixation M Femur (distal) Closed External Fixation M Femur (distal) Closed Plate fixation M Femur (shaft) Open Plate fixation F Femur (proximal) Closed Intramedullary nailing M Femur (proximal) Closed Intramedullary nailing F Tibia (shaft) Closed Intramedullary nailing M Tibia (shaft) Closed Intramedullary nailing F Tibia (distal) Closed Plate fixation M Tibia (distal) Open Plate fixation M Tibia (distal) Closed Plate fixation F Humerus (shaft) Open Plate fixation M Ulna (proximal) Open Plate fixation 63 The mean time from initial injury to bone marrow grafting was 25 months Materials and methods Patients From 2006 to 2012, we performed a total of 22 percutaneous autologous concentrated bone marrow grafting procedures in patients with nonunion. Of these cases, 17 were included in the present study. Five cases in less than 12 months after grafting were excluded. Since 1 patient had nonunion at 2 locations, the study included 17 cases in 16 patients. The characteristics of the 17 cases are shown in Table 1. There were 11 men and 5 women in the present study with a mean age of 40.7 (22 80) years. As a treatment for nonunion, LIPUS was administered for C3 months in all cases. Over 9 months were passed from injury to grafting, and no visible progressive signs of healing at the fracture site were continued for three consecutive months in all cases, which fulfilled the definition of nonunion as per the Food and Drug Administration. There were no fixation problems in any case, and all the cases were of atrophic nonunion. All procedures were approved by the Institutional Ethical Review Committee of the University of Tsukuba. Bone marrow aspiration and concentration Bone marrow aspiration and concentration were performed according to a previously published method [5]. Firstly, bone marrow was aspirated from both anterior iliac crests under general anaesthesia using a bone marrow harvest needle (Medical Device Technologies, Inc., Gainesville, FL, USA). The contents of bone marrow aspirates were collected into the bone marrow collection kit (Baxter, Deerfield, IL, USA) and then transferred to a quadruple blood bag (Terumo, Tokyo, Japan) for the concentration. The bone marrow aspirates were processed by a two-step centrifugation method (KUBOTA 9800, Kubota, Japan) at room temperature. First centrifugation of the blood bag was performed at 1,200g for 10 min, following which the erythrocytes were transferred into the satellite bag. Second centrifugation was performed at 3,870g for 7 min, following which the plasma and anticoagulants were transferred into the satellite bag, and the bone marrow concentrates containing buffy coat were extracted. This technique reduced the typical 300 ml of bone marrow aspirate to bone marrow concentrates of approximately ml. Percutaneous grafting Under fluoroscopic control, 2.4-mm Kirshner wire was inserted for drilling through the nonunion and the normal peripheral bones. After the drilling repeated several times, the tip of a bone marrow harvest needle, which was identical to that used for bone marrow aspiration, was placed in the nonunion gap. After this, the bone marrow concentrate was slowly injected for a minute and gradually withdrawn (Fig. 1). The fibrous tissue of the nonunion site was not removed or disturbed.

3 Fig. 1 Percutaneous grafting: the bone marrow concentrate was slowly injected for a minute through the bone marrow harvest needle Management after bone marrow grafting For patients with lower extremity nonunion, walking was permitted from the day following the grafting procedure, depending on the pain level. After bone marrow grafting, LIPUS was performed every day for 20 min. Plain radiographs were obtained every 4 weeks until 3 months after grafting and every 3 months thereafter. Computed tomography (CT) was also performed before grafting and 6 months after grafting to quantitatively evaluate callus development. When bone union was not achieved 6 months after the grafting procedure, CT was performed again 12 months after grafting. Level of pain and radiographic evaluation The level of pain was evaluated using the visual analogue scale (VAS). The healing at the nonunion site was evaluated using plain anteroposterior and lateral radiographs. The healing at the nonunion site, which meant bone union, was determined whether the loss of the fracture margin or development of a bone-to-bone callus bridge was observed in at least 3 of the following sites: the anterior, posterior, medial, or lateral cortical bone [6]. Three orthopaedic surgeons independently judged the healing at the nonunion site, and the final diagnosis was based on majority opinion. The CT images of the entire long bone, including that of 1-mm-thick sections at the nonunion site, were obtained and saved in DICOM format. After visually confirming that the CT images were taken at the same levels at all times, the region 10 mm proximal and 10 mm distal to the nonunion site was considered the range of measurement. Materialise Mimics 16.0 software (version 16.0; Materialise, Leuven, Belgium) was used to measure the volume of the callus formation. Since this software defines the bony element as the fixed threshold interval (226 1,613 grey levels), only the bony element within the measurement range of the obtained image data was extracted. Thus, the extracted region was reconstructed three dimensionally, and the volume of the bony elements was measured at each time point. The volume of the bony elements before grafting and at 6 months after grafting was measured, and the volume increase in the bony elements was defined as the volume of callus formation (Fig. 2). Haematological analysis and fibroblastic colonyforming unit (CFU-F) assay Haematological analysis and CFU-F assay were performed according to a previously published method [7]. The numbers of nucleated cells were determined with an automated haematology analyser (K-4500; Sysmex, Kobe, Japan). The recovery rates of the nucleated cells after concentration were calculated as follows: recovery rate (%) = (total number of nucleated cells in samples after concentration)/(total number of nucleated cells in samples before concentration) To determine the presence and proportion of progenitor cells, CFU-F assays were performed with bone marrow aspirates and concentrates from all the individuals. Phosphate-buffered saline was used to wash 100 ll of the samples twice, and these were suspended in 3 ml of growth medium. The growth medium consisted of Dulbecco s modified Eagle s medium (Sigma) supplemented with 10 % foetal bovine serum (Gibco) and antibiotics (antibiotic antimicotic solution; Gibco BRL). Samples were seeded onto 60-cm 2 dishes and cultured at 37 C ina humidified atmosphere of 5 % CO 2. The medium was replaced 2 days after plating, and nonadherent cells were

4 Fig. 2 The volume of callus formation: the reconstruction images of a 45-year-old patient who had sustained a closed fracture of femoral shaft were shown (left before grafting, right at 6 months after grafting). By the reconstruction of the extracted area on slices of the bony element before grafting and at 6 months after grafting, respectively, the volume of the bony elements was computed automatically. The volume of callus formation was calculated as the volume increase in the bony elements removed. Thereafter, it was replaced twice a week. After 2 weeks, the medium was removed and the dishes were stained with 0.5 % crystal violet (Sigma) in methanol for 5 min. The cells were washed twice with distilled water, and the number of CFU-Fs was counted. Colonies \2 mm in diameter and those that were only faintly stained were ignored. The prevalence of the progenitor cells was calculated as the number of CFU-Fs per 10 6 nucleated cells. Statistical analysis The concentrations of nucleated cells before and after the concentration procedure were analysed using the paired t test. VAS results before grafting and the 3-, 6-, and 12-month evaluation after grafting were analysed using the one-way analysis of variance with repeated measurement. Post hoc analyses were performed using the Bonferroni comparisons test. The probability level accepted for statistical significance was P \ All statistical analyses were performed with IBM SPSS Statistics 19.0 (IBM Corporation, Armonk, USA). Results Visual analogue scale In the VAS pain evaluation, pain alleviation was consistently observed after grafting in all the cases. The mean VAS score was 31 ± 24 mm before grafting, and after grafting, it was 12 ± 15 mm at 3 months, 8 ± 10 mm at 6 months, and 6 ± 8 mm at 12 months (Fig. 3). Differences in VAS score over the time after grafting were statistically significant (P \ 0.001) by the one-way analysis of variance with repeated measurement. With post hoc Bonferroni correction, statistically significant differences Fig. 3 VAS pain evaluation: pain alleviation was consistently observed in all the cases after grafting. Statistically significant differences were observed as below. Before grafting versus 3 months after grafting, P = Before grafting versus 6 months after grafting, P = Before grafting versus 12 months after grafting, P \ were observed between VAS scale before grafting and 3 (P = 0.001), 6 (P = 0.001), and 12 months (P \ 0.001) after grafting. Plain radiographs There were no cases of bone union 3 months after grafting; however, eleven cases of bone union were observed at 6 months, respectively. At 12 months, radiographic evidence of bone union was observed (Fig. 4) in thirteen of the seventeen cases. Computed tomography The mean volume of callus formation 6 months after grafting was 4,147 (262 27,392) mm 3 (Fig. 5). When this result was combined with those of bone union on plain radiographs, the mean volume of callus formation was

5 Fig. 4 Radiographic evaluation of bone union: plain radiographs of a 22-year-old patient who had sustained a closed fracture of femoral shaft. The radiographs were made at the time of initial treatment (a); at the time of bone marrow grafting (b); at 1 month after grafting (c); at 3 months after grafting (d); and at 6 months after grafting (e) was 5.5 ± 1.6, and the number of CFU-F per 10 6 nucleated cells was 2.17 ± Discussion Fig. 5 The mean volume of callus formation 6 months after grafting was 4,147 (262 27,392) mm 3. Based on plain radiograph, black bars indicate bone union and grey bars indicate nonunion. The mean volume of callus formation was 5,982 mm 3 in the 11 cases of bone union and 783 mm 3 in the 6 cases of nonunion 5,982 (1,999 27,392) mm 3 in the 11 cases of bone union and 783 (262 1,455) mm 3 in the 6 cases of nonunion. In the 6 cases of nonunion, the mean volume of callus formation 12 months after grafting was 2,154 (379 5,006) mm 3. The volume of callus formation increased in 2 cases, which were judged to be bone union cases according to plain radiographs. There was almost no increase in the volume of callus formation in the other 4 cases, which were also judged to be nonunion cases according to plain radiographs. Haematological analysis and fibroblastic colonyforming unit assay The mean number of nucleated cells included in the bone marrow-derived blood was 0.86 ± cells/ml before concentration and 4.82 ± cells/ml after concentration (P \ 0.001). The mean concentration rate The methodology for bone grafting used in our study has not been reported previously. In the literature, excluding case reports, 12 studies have reported on autologous bone marrow grafting for nonunion, as shown in Table 2 [8 19], which includes cases of single grafting of bone marrow performed without measuring the concentration or cases in which 2 graftings were performed at several week intervals. Despite this, the bone union rate in those reports and in the present study was similar (57 94 vs. 76 % (13/17), respectively). The previous studies included cases that only involved the lower extremities; therefore, for better comparison, we extracted 15 cases involving the lower extremity in the present study and found a bone union rate of 87 % (13/15), which was relatively favourable. One of the common symptoms for nonunion is chronic pain. Although most of our cases were troubled by chronic pain in the site of nonunion, the symptoms remitted or disappeared in the cases of bone union. We think chronic pain was healed by getting the complete stability at the nonunion gap, and the relief of pain is one of the indicators as the healing of nonunion. With an aim of grafting as many osteogenic stem cells as possible, we grafted concentrated aspirates. In the literature, 2 studies have reported percutaneous grafting of autologous bone marrow concentrates for nonunion cases [10, 14] in which in one study, the plasma component was eliminated after a single step centrifugation, and in the other study, a cell separator was applied to extract the buffy

6 Table 2 The reports of autologous bone marrow grafting for nonunion: 12 studies have reported on autologous bone marrow grafting for nonunion First author Year Number of nonunion cases Type Union (N) Union (%) Healey Femur Connolly Tibia Garg Tibia Humerus 3 Ulna 2 Sim Tibia Femur 1 Humerus 1 Ulna 1 Matsuda Femur Siwach Tibia Femur 8 Humerus 12 Forearm 10 Wang Tibia Goel Tibia Hernigou Tibia Bhargava Tibia Femur 2 Ulna 1 Singh Ulna Femur 3 Humerus 2 Metacarpal 1 Braly Tibia Our study Femur Tibia 5 Humerus 1 Ulna 1 coat for concentrating the aspirate. However, our study involved a two-step centrifugation method. Thus, although the methodologies of these reports were different from that of this study, the tibial bone union rates were 80 % (8/10) and 88 % (53/60) of these studies, respectively, and that in this study it was 87 % (13/15) for the lower extremities and 83 % (5/6) for the tibia, indicating similar results. As the quantitative assessment of bone union, one study has reported a mean volume of callus formation of 3,100 (800 5,300) mm 3 at 4 months after grafting based on CT images [14], whereas that in this study at 6 months after grafting was 4,147 (262 27,392) mm 3. Although both studies differed in terms of methodology of grafting, time of evaluation, and imaging conditions, the volume of callus formation was similar between both studies. Among previous studies that have reported percutaneous grafting for upper extremity nonunion, the study by Siwach et al. [18] only reported the bone union rate of both upper and lower extremities, whereas reports by Garg et al. [12] and Singh et al. [19] found a rate of 80 % (4/5) and 75 % (6/8), respectively. In this study, there were only 2 cases of upper extremity involvement, and bone union was not obtained in either case. This result can probably be attributed to the number of cases in the present study; thus, we would like to make a reassessment after accumulating a sufficient number of cases. In the present study, atrophic nonunion was observed in all cases. Although no fixation complications were observed at the nonunion site, the osteogenic response had likely halted. In our procedure, multiple drilling was performed to accelerate the inflow of bone marrow cells or growth factors from the normal peripheral bones. Also, we expected that the bone marrow cells or growth factors derived from the grafted bone marrow concentrates would intensify the osteogenic effect at the nonunion site and aid bone formation. We believe that the supply of these cells or growth factors would be limited with multiple drilling alone and that bone formation was accelerated by grafting autologous concentrated bone marrow. In addition, we believe that continuous LIPUS after grafting further accelerated osteogenesis and angiogenesis. LIPUS has been reported to improve local blood flow [20], accelerate cytokine production inducing angiogenesis [21, 22], accelerate oxygen and nutrient transportation to living cells [23], accelerate differentiation from mesenchymal stem cells to osteoblasts [24, 25], inhibit the differentiation and production of osteoclasts [26, 27], and accelerate endochondral ossification [28, 29]. Although LIPUS was provided[3 months prior to grafting in all the cases in this study, the effect of LIPUS before grafting was likely limited, as the bone union effect had probably halted. We believe that there was an abundance of bone marrow cells and growth factors as osteogenic materials, and the bone union resumed after grafting; thus, LIPUS acted effectively and contributed to bone formation. Multiple drilling was expected to accelerate the inflow of bone marrow cells or growth factors from the normal peripheral bones. LIPUS was expected to intensify the osteogenic effect at the nonunion site for the bone marrow cells or growth factors derived from the grafted bone marrow concentrates. We think it is difficult for nonunion to heal with multiple drilling only or LIPUS only. In regard to multiple drilling, the diameter of the drill we used is 2.4 mm for the prevention of the refracture, and there is only a small effect of osteogenesis. In regard to LIPUS, all of our cases had undergone LIPUS for over 3 months before our treatment, but none of those were healed. There are no reports of healing with LIPUS only, multiple drilling

7 only, and the combination of LIPUS and multiple drilling for the treatment for atrophic nonunion. The supply of the bone marrow cells or growth factors derived from multiple drilling is limited, and the osteogenic effect of LIPUS, which affect the bone marrow cells or growth factors, can be less expected. So we think it is difficult for atrophic nonunion to heal with the combination therapy of multiple drilling and LIPUS. Percutaneous autologous concentrated bone marrow grafting is a less invasive surgery than autologous bone grafting, that is the conventional surgery for nonunions. According to the study by Ahlmann et al. [30], the rate of the complication of autologous bone grafting is 15 % (10/ 88). In our study, there were no cases of the complication at the site of the bone marrow aspiration and no damages to the iliac crest bone regarding the conservation of bone stock. We encountered no local or systemic complications. The theoretical criticism of our procedure is that there is a risk of infection or carcinogenesis. However, in our study, there were no cases of infection at the site of the bone marrow grafting. Also, with the inclusion of other studies on autologous bone marrow grafting for nonunion, there were no reports of carcinogenesis after the grafting. So this concentrate bone marrow grafting is much safer than the cell therapy with cell expansion in ex vivo. The limitation of our study is that the sample size is small and the study design is retrospective. To evaluate the efficacy of our procedure for various sites of nonunion, the prospective study like randomised clinical trial about our procedure and other treatments can be needed. Also, this procedure cannot be used in cases where there is already existing angular deformity, shortening, or unstable fixation at the nonunion site. In these cases, the direct access to the nonunion site is needed. The percutaneous autologous concentrated bone marrow grafting performed in this study was a relatively minimally invasive and safe procedure. Since this procedure does not require special equipment such as a cell separator, it seems economical and feasible for one-stage treatment. Since the treatment results are equivalent to those of other reports, we believe that percutaneous autologous concentrated bone marrow grafting is a useful treatment method for nonunion. Conclusion After grafting, all the patients showed pain alleviation, and bone union was observed in most cases, radiographically. Percutaneous autologous concentrated bone marrow grafting is an effective procedure for the treatment of patients with nonunion. Conflict of interest of interest. References The authors declare that they have no conflicts 1. Pountos I, Georgouli T, Kontakis G, Giannoudis PV (2010) Efficacy of minimally invasive techniques for enhancement of fracture healing: evidence today. 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