Regenerate Augmentation with Bone Marrow Concentrate After Traumatic

Orthopedic Reviews 2012; volume 4:e14

Regenerate augmentation with rates. The therapeutic potential of facilitating callus maturation lies in both reducing the Correspondence: Jan Gessmann, Department of bone marrow concentrate after healing index in more basic cases and increas- General and Trauma Surgery, BG University traumatic bone loss ing the success rates in more difficult cases. Hospital Bergmannsheil, Bürkle-de-la-Camp- Currently, augmentation with percutaneously Platz 1, 44789 Bochum, Germany. Jan Gessmann,1 Manfred Köller,2 applied adjuvants to reduce consolidation time Tel. +49.234.302.3425. E-mail: [email protected] Holger Godry,1 has been designated as one of the major goals Thomas Armin Schildhauer,1 for future research in distraction osteogene- Key words: distraction osteogenesis, bone mar- Dominik Seybold1 sis.1 There is already clinical evidence that the row, posttraumatic bone defect, mesenchymal application of cells can accelerate healing of stem cell. 1Department of General and Trauma the regenerate: Kitoh et al.2 were able to Surgery and 2Department of Surgical demonstrate that the percutaneous transplan- Received for publication: 4 February 2012. Research, BG University Hospital tation of culture-expanded bone marrow cells Accepted for publication: 4 March 2012. Bergmannsheil, Bochum, Germany and platelet-rich plasma accelerates bone This work is licensed under a Creative Commons regeneration during distraction osteogenesis Attribution NonCommercial 3.0 License (CC BY- and thereby reduce overall complication rates.2 NC 3.0). However, the directives for the expansion, dif- Abstract ferentiation and re-transplantation of bone ©Copyright J. Gessmann et al., 2012 marrow cells in Europe follow certain, very Licensee PAGEPress, Italy Distraction osteogenesis after post-traumat- strict requirements.3 There are also potential Orthopedic Reviews 2012; 4:e14 doi:10.4081/or.2012.e14 ic segmental bone loss of the tibia is a complex risks, such as contamination or depletion of and time-consuming procedure that is often proliferative capacity, as well as additional complicated due to prolonged consolidation or costs, including personnel expenses, culture complete insufficiency of the regenerate. The instruments, chemicals, and contamination (mean 44.4) years with posttraumatic bone aim of this feasibility study was to investigate tests.2,4 Furthermore, a one-step procedure defectsonly of the tibia were admitted to our clinic the potential of bone marrow aspiration con- with on-table preparation for immediate trans- (Table 1). All patients were treated with differ- centrate (BMAC) for percutaneous regenerate plantation may prevent complications related ent operative procedures before attending our augmentation to accelerate bony consolidation to a reduced quality of the transplanted cells, clinic, with a mean of 4.8 prior operative proce- of the regenerate. Eight patients (age 22-64) such as pre-aging, reduced viability, or dedif- dures. Patients who reported prior treatment with an average posttraumatic bone defect of ferentiation (all of which are associateduse with after open trauma and posttraumatic infection 82.4 mm and concomitant risk factors (nico- in vitro cultivation).5,6 were treated with debridement, bone resection tine abuse, soft-tissue defects, obesity and/or In the treatment of aseptic non-unions, per- and soft-tissue coverage by local and free flaps circulatory disorders) were treated with a mod- cutaneous injection of autologous bone mar- and temporary stabilisation with a monolateral ified Ilizarov external frame using an row aspirate has been shown to be clinically fixator (Figure 1). The sizes of the bone intramedullary cable transportation system. At effective.7,8 Hernigou et al. were able to demon- defects were between 44 and 126 mm (mean the end of the distraction phase, each patient strate that efficacy seems to be related to the 82.4 mm). A free flap transfer was necessary in was treated with a percutaneously injection of number of progenitor cells in the graft.9 two patients (Patients 1 (latissimus dorsi flap) autologous BMAC into the centre of the regen- Therefore, the authors concentrated the bone and 5 (anterolateral thigh (ALT) and latissimus erate. The concentration factor was analysed marrow aspirate for increasing the number of dorsi flap) and local flap coverage in five using flow cytometry. The mean follow up after progenitor cells before intraosseous reinjec- patients (Patients 2-4, 7, 8). After soft-tissue frame removal was 10 (4-15) months. With a tion at the site of non-union.9 Recently, there healing, the monolateral external fixation was mean healing index (HI) of 36.9 d/cm, bony have been reports with promising results on replaced by an Ilizarov ring fixator. Segmental consolidation of the regenerate was achieved transplantation of bone marrow concentrate bone transport was performed using an in all eight cases. The mean concentration fac- for poor-healing bone sites.10-13 The advantage intramedullary cable transport system (Figure tor of the bone marrow aspirate was 4.6 (SD of concentrated bone marrow aspirate relies 2). For distraction osteogenesis, percutaneous 1.23). No further operations concerning the on the possibility of on-table preparation and osteotomy of the proximal tibia (Gigli saw) regenerate were needed and no adverseNon-commercial effects reinjection in one operation without the need was performed in the seven cases of antero- were observed with the BMAC procedure. This to take the cells from the operation room into grade transport; percutaneous osteotomy of procedure can be used for augmentation of the a laboratory. The positive results of this new the distal tibia was performed in the case of regenerate in cases of segmental bone trans- technique together with the on-table prepara- retrograde transport. The bone segment transport. Further studies with a larger number of tion were the motivation to test the feasibility port was started after a delay of 7 days. The dis- patients and control groups are needed to eval- for distraction osteogenesis. traction rate was 1 mm/day in the anterograde uate a possible higher success rate and accel- transport and 0.5 mm in the one patient with erating effects on regenerate healing. retrograde transport. Six patients were heavy smokers before bone transport, and four of Materials and Methods them continued to smoke throughout the entire treatment phase (Patients 1, 4, 5, 6). Introduction This prospective study was approved by the Patients were taught to work the transport ethics committee of the Ruhr Universität clickers by themselves during the first days Distraction osteogenesis for segmental Bochum, Germany (ethical approval number after the operation and were seen every two bone defect reconstruction is an established 3594-09). All patients gave their written con- weeks for X-ray control of distraction progress. procedure. However, the long duration of the sent to participate in this study. At the time of the docking of the transported time in external fixation is often cause of con- Between June 2009 and August 2010, two segment, the patients were admitted to the hos- siderable morbidity and high complication female and six male patients aged 22-64 pital. The cable system and transport clickers

[page 62] [Orthopedic Reviews 2012; 4:e14] Article were removed, and the transport-segment was Patients were regularly seen every two weeks fixed to the frame with K-wires and/or half pins for frame and soft-tissue control and for retight- Results in the operating room. Local debridement of the ening of the Ilizarov screws. X-rays in two docking zone and autologous bone grafting planes were taken every four weeks to evaluate Bony consolidation of the regenerate was from the iliac crest were performed in each the healing of the docking zone and the regen- achieved in all patients. The mean healing patient. Before harvesting the autologous bone erate. Frames were removed when bony healing index was 36.9 d/cm (range 27.9 to 48.6) while from the iliac crest, a total of 60 mL bone mar- with at least three cortices was present in both the mean consolidation index was 23.1 d/cm row aspirate was obtained by Jamshidi vacuum the regenerate and the docking zone. Peri- and (range 14.8 to 32.7; Table 1). The mean dis- aspiration from the same iliac crest. Thereby, postoperative complications were divided to traction phase lasted for 107 (60-156) days. only a few millilitres were aspirated at once frame-associated complication, BMAC trans- The longer distraction phase in relation to the before the aspiration needle was placed in dif- plantation-associated complications and com- mean defect size was caused by different fac- ferent areas of the iliac crest to ensure a cell- rich aspirate. The bone marrow aspiration con- plications not related to the procedures. The tors. Retrograde transport (Patient 2) was per- centrate (BMAC) was produced via density gra- healing index (=time in frame (in days)/length formed with 0.5 mm/d, which doubled the dis- dient centrifugation using the Smartprep2TM of regenerate (in cm)) and the consolidation traction phase in this patient. Furthermore, centrifuge (Harvest Technologies, Plymouth, index (=time in frame after distraction phase the longer defect sizes (Patients 3-8) required MA, USA), in accordance with the manufactur- [in days)/length of the regenerate (in cm)] an exchange of the distraction clickers with re- er’s directions in the operation room. After 3after BMAC transplantation were calculated. tensioning of the cable. During that procedure, centrifugation, the plasma was removed, and cells were resuspended, leaving a total volume of 8 mL. Two mL of autologous thrombin were Table 1. Mean bone defect size, soft tissue procedure, healing index and consolidation added to the cell suspension before transplan- index. tation into the regenerate. The Jamshidi needle was used as a syringe and was placed under Patient Age Sex Bone Additional soft tissue HI CI in Follow up X-ray control percutaneously in the centre of defect procedures in d/cm d/cm in in mm months the regenerate, and the BMAC-thrombin-sus- only pension was slowly injected. Both aspirate and 1 51 m 60 Latissimus dorsi free flap 42.7 32.7 14 concentrate (2 mL were saved of each sample 2 44 m 44 Local flap 48.6 25.4 10 for in vitro analyses) were analysed using flow 3 50 f 85 Local flap 28.5 16.9 13 cytometry (FACS) regarding the containing use cells. The concentration factor was calculated 4 42 m 97 Local flap 43.7 31.3 15 by the cell count-quotient BMAC/aspirate after 5 42 m 96 Anterior lateral thigh free flap, the centrifugation procedure. Latissimus dorsi free flap 40.1 23.9 13 6 40 m 81 None 35.2 22.2 9 7 22 m 70 Local flap 27.9 14.8 5 8 64 f 126 Local flap 28.7 17.5 4

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Figure 1. X-rays (anterior (A) and lateral view (B)) of Patient 7 at the day of admis- Figure 2. X-rays (A, B) of Patient 7 during the distraction phase (anterograde bone seg- sion with central bone defect after an open mental transport) and clinical picture (C) of the Ilizarov external frame with the applied fracture of the tibia (Gustilo type III B). cable system and transport clickers.

[Orthopedic Reviews 2012; 4:e14] [page 63] Article the bone segment slipped back several mm in 4.4)25 and Jäger et al. (mean factor of 5.2).10 the reverse-direction of transport. During the Discussion Very recently, other authors have demonstrat- distraction phase, patient 4 suffered from a ed beneficial effects on bone healing in distal femoral fracture due to an accidental fall Several experimental studies demonstrated patients with bone cysts, osteo-necrosis and on the same leg. This fracture was fixed with a that transplantation of progenitor cells into a non-unions with the transplantation of percutaneous locking-plate system (NCB, bone-healing site improves bone forma- BMAC.5,10,11,13,26 To the knowledge of the Zimmer, USA). In Patient 5, the previously tion.4,14,15 This suggests that in many situations authors, the procedure has not been described transplanted ALT-flap showed partial necrosis, tissue and bone repair may be limited by the for distraction osteogenesis. which exposed the bone ends in the docking presence and number of local progenitor cells.4 Three essentials constituents have been zone. Before the docking procedure, coverage There are different approaches to increase the assumed besides biomechanical stability and with a latissimus dorsi flap was necessary, and population of these cells, but there is prelimi- vascularization in accelerating new bone for- the BMAC procedure was performed after soft- nary evidence that local application of these mation (referred to as the diamond concept): tissue healing to reduce the risk of infection. cells may be of benefit:16,17 either immediate growth factors, osteoprogenitor cells and Local pin infections were seen in six implantation of harvested cells from bone mar- extracellular matrix/natural scaffold.27 The patients (Patient 2, 4, 5-8) and were treated row aspiration or transplantation of cells after BMAC system provides the first two con- with local skin incision and oral antibiotics. No culture expansion and differentiation. The lat- stituents. The third - the natural scaffold - is pin or wire removal was necessary. With regard ter approach is already in clinical use for carti- generated during the distraction process: to major complications, Patient 2 displayed lage repair18-20 but has also been proven clini- During the distraction phase a fibrovascular non-union of the docking site. The patient cally effective in distraction osteogenesis.2,21-23 lattice is generated between the two distracted refused any further surgery and was treated Connolly et al.7 and Garg et al.8 suggested a bone ends, which consist of longitudinally ori- with a leg brace. However, the regenerate beneficial effect of the percutaneous trans- ented fibrous connective tissue with a protec- showed sound bony healing. plantation of autologous bone marrow aspirate tive covering membrane.28,29 The BMAC was At the mean follow-up of 10.4 months after in cases of non-union in long bones. Hernigou injected at the end of the distraction phase removal of the frame, all patients except patient et al. demonstrated that by centrifugation of into this membranaceous tube that is assumed 2 were able to walk at full weight-bearing with- bone marrow aspirate, the mononuclear cells to function as a natural scaffold for the cell out the help of crutches. Figures 3 and 4 show can be concentrated, resulting in further suspension.only It has been shown that a so-called the radiological results of Patient 7 and 8. No enhancements of osteogenesis.9 The effective- growth zone forms in the centre of the length- adverse effects of cell injection into the regener- ness of the BMAC procedure in harvesting ening segment, in which no ossification ate were seen. The in vitro analysis of aspirate mononuclear cells, including mesenchymal occurs until the consolidation phase and BMAC showed that the percentage of cells stem cells, has been demonstrated in differentusebegins. 28,29 In a patient treated previously to was not changed signifantly by the centrifuga- studies.10,24,25 Jäger et al. showed that relevant the present study we could visualize the diffu- tion process (percentage of cells before/after amounts of potent mesenchymal stem cells, sion of non-concentrated iliac crest blood centrifugation: granulocytes 75.9/76.6; lympho- which differentiate into osteoblasts in vitro, mixed with a contrast medium in the centre of cytes 17.7/16.9; monocytes 3.1/3.6; CD34+ 1.1/1.3; can be harvested with the BMAC system.10 The the regenerate (Figure 5). Furthermore, the CD45-/CD90+ 0.76/0.93). The average concen- mean concentration factor of 4.6 that we found addition of autologous thrombin transferred tration factor of the BMAC compared to the in the present study lies between the results the fluid cell suspension into a viscous, gel- aspirate was 4.6 (SD 1.23). presented by Hermann et al. (mean factor of like constitution, and may lead to a local adher-

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Figure 3. X-rays of Patient 7 four months after frame removal with bony consolidation both of the regenerate and the distal Figure 4. X-rays of Patient 8 with a pathologic fracture due to a chronic osteomyelitis at the day docking site. of admission (A, B) during transportation (C) and five months after frame removal (C, D, E).

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ciency of the callus is of great need in clinical concentrate reduces autologous bone practice. The safety demands for an augmenta- grafting in osseous defects. J Orthop Res tion procedure must be very strict; the tech- 2010;29:173-80. nique needs to be of minimal risk and minimal 11. Garnavos C, Mouzopoulos G, Morakis E. invasiveness, without compromising the Fixed intramedullary nailing and percuta- regenerated callus and surrounding soft tis- neous autologous concentrated bone-mar- sues. Based on the recent literature and these row grafting can promote bone healing in preliminary results, this seems to apply for the humeral-shaft fractures with delayed BMAC procedure. We did not detect any union. Injury 2010;41:563-7. adverse effects on the side of harvesting or at 12. Murawski CD, Kennedy JG. Percutaneous the regenerate. Further studies with a larger internal fixation of proximal fifth number of patients and a control group are metatarsal jones fractures (zones II and needed to evaluate the effects on accelerated III) with Charlotte Carolina screw and regenerate healing. bone marrow aspirate concentrate: an out- Figure 5. Intraoperatively taken X-ray pic- come study in athletes. Am J Sports Med ture showing the diffusion of iliac crest 2011;39:1295-301. blood aspirate mixed with contrast medium 13. Wang BL, Sun W, Shi ZC, et al. Treatment in the centre of the regenerate in a patient References of nontraumatic osteonecrosis of the treated with segmental bone transport. femoral head with the implantation of core 1. Watson JT, Kuldjanov, D. Bone Defects. In: decompression and concentrated autolo- Rozbruch SR, Ilizarov, S., editor. Limb gous bone marrow containing mononu- ence of the injected cells as described similar- Lengthening and Reconstruction Surgery. clear cells. 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Percutaneous gous chondrocyte implantation compared appear rather short in comparison to the above autogenous bone marrow grafting in 20 with microfracture for chondral lesions of named studies concerning posttraumatic bone cases of ununited fracture. Acta Orthop the knee: prospective nonrandomized defects but must be interpreted with care due Scand 1993;64:671-2. study at 5 years. Am J Sports Med 2009;37: to limitations of the present feasibility study. 9. Hernigou P, Poignard A, Beaujean F, 33-41. Rouard H. Percutaneous autologous bone- 21. Kitoh H, Kitakoji T, Tsuchiya H, et al. marrow grafting for nonunions. Influence Transplantation of marrow-derived mes- of the number and concentration of pro- enchymal stem cells and platelet-rich plas- Conclusions genitor cells. J Bone Joint Surg Am ma during distraction osteogenesis--a pre- 2005;87:1430-7. liminary result of three cases. Bone 2004; Accelerating the consolidation phase during 10. 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