International Orthopaedics (SICOT) DOI 10.1007/s00264-014-2321-2

ORIGINAL PAPER

Efficacy of two different demineralised bone matrix grafts to promote bone healing in a critical-size-defect: a radiological, histological and histomorphometric study in rat femurs Mirja Fassbender & Susann Minkwitz & Mario Thiele & Britt Wildemann

Received: 22 January 2014 / Accepted: 7 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Purpose The aim of the study was to compare two different demineralised bone matrices used clinically regarding their ability to induce bone healing in a critical-size-defect rat model. Methods We stabilised 4 mm femur defects with a custommade plate and filled them either with demineralised bone matrix (DBM) or DBX (DBX Putty®). Bone morphogenetic protein 2 (BMP-2)-loaded collagen and an empty defect served as controls. The outcome was followed after 21 and 42 days by radiology (Faxitron; microCT) and histology. Results Defect healing did not occur in any animal from the empty control, DBM or DBX group. Residuals of the implanted material were still found after six weeks, but only limited callus formation was visible. In contrast, the BMP-2 control demonstrated enhanced formation of callus tissue and undisturbed healing. After 21 days, 11 out of 16 and after 42 days, 7 out of 8 BMP-2-treated animals showed complete defect bridging by cancellous bone tissue.

M. Fassbender : S. Minkwitz : M. Thiele : B. Wildemann (*) Julius Wolff Institute, Center for Musculoskeletal Surgery, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany e-mail: [email protected] M. Fassbender e-mail: [email protected] S. Minkwitz e-mail: [email protected] M. Thiele e-mail: [email protected] M. Fassbender : S. Minkwitz : B. Wildemann Berlin-Brandenburg Center for Regenerative Therapies, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany

Conclusions Demineralised bone grafts were not capable of defect reconstruction; only BMP-2 was able to provide sufficient stimulus to induce uneventful bridging under the specific experimental conditions. Keywords Bone grafts . Critical-size-defect . BMP-2 . MicroCT . Histology

Introduction The treatment of large bone defects is still a major clinical challenge in trauma surgery [1]. The reconstruction of critically sized defects requires the use of bone transplants or substitutes. Only autologous bone transplantation meets osteogenic, osteoinductive and osteoconductive criteria, and it is considered the gold standard for reconstruction of extensive long-bone defects [2, 3]. Harvesting grafts from the iliac crest can provide a sufficient quantity of cortical or cancellous bone, but it bears the risk of intra- and peri-operative complications, such as pain, hypersensitivity, instability, and infections in up to 30 % of patients [4–6] Therefore, alternative materials are needed, and allogenic bone transplants or biomaterials, such as demineralised bone matrix, are available in a huge variety of customised and commercial types [2, 7]. However, the osteoinductive effect of demineralised bone matrix is controversial, and there is little information about when and where to use different graft and carrier materials [8]. Bone grafts have shown unpredictable biologic activity and variable clinical responses [3–9] depending on donor-specific characteristics and postprocessing methods [10–14]. The goal of this study was to evaluate the ability of two clinically available demineralised bone matrices to promote bone repair in an experimental critical-size-defect model. Outcomes were followed by conventional radiography, micro computed tomography (microCT) and histology and were compared

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with empty defects and bone morphogenetic protein-2 (BMP2)-treated defects. BMP-2 was chosen as positive control due to its well-documented osteoinductive potential and clinical approval [15].

Materials and methods If not stated otherwise, all suppliers of materials or technical equipment used in this study were located in Germany. Grafting samples Graft materials tested were: 1. DBM: human bone matrix, demineralised and peracetic acid-ethanol sterilised (PES) [16], (Tissue Bank of the Charité-Universitätsmedizin, Berlin) 2. DBX: DBX Putty® (Synthes Inc., West Chester, PA, USA), human DBM + sodium hyaluronate carrier 3. BMP-2 control: lyophilised BMP-2 (Osteogenetics GmbH, Würzburg; 1 mg/ml diluted in 5 mM aqueous hydrochloric acid) Approximately 60±10 mg of DBM- or DBX matrix were moulded into the defect. For BMP-2 control, a collagencarrier matrix (Lyostypt®, B. Braun, Melsungen) was cut into 5×30-mm strips, and 5 μl of the BMP-2 stock solution were added to 25 μl of 5 mM aqueous hydrochloric acid (HCl) and dropped on the fleece matrix (resulting in 5 μg BMP-2 per matrix). The collagen stripe was rolled up lengthwise for insertion. An empty defect served as negative control. BMP-2 quantification BMP-2 quantification was performed as described previously [12]. Briefly, proteins were extracted with the guanidine HCl/ ethylenediaminetetraacetic acid (EDTA) method. Approximately 20–30 mg of each sample was placed in 1.5ml Eppendorf tubes, with 1.9 ml of 4 M guanidine HCl, 50 mM EDTA in 50 ml Tris-pH 7.4 plus 5 mM benzamidine–HCl, 1 mM phenylmethylsulfonyl and 0.1 mM aminocaproic acid. The samples were dialysed against aqua dest. at 4 °C for 24 hours with four changes. After extraction, the samples were centrifuged at 14,000× g for five minutes, and the supernatant was stored at −80 °C. BMP-2 concentration in the samples was quantified using human-specific enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Wiesbaden). Operative procedure and experimental design Animal experiments were approved by the local legal representatives. Anesthesia was performed with ketamine

hydrochlorid (80 mg/kg) and xylazine (12 mg/kg). Adult female Sprague-Dawley rats (340±15 g; Charles River Inc., Sulzfeld) were used. The femoral bone was prepared and bicortical drilling performed (surgical motor ImplantMED SI-923; straight surgical hand piece S11, W&H, Laufen). A 4-mm osteotomy gap was cut (reciprocating surgical saw hand piece SR 8 and saw blades, 20 mm in length, W&H) in the femoral midshaft. A three-component plate (customised by Steingross Feinmechanik, Berlin, according to the design of the Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA [17]) was used for bone fixation. The plate was composed of two stainlesssteel anchor plates affixed directly to the bone via two titanium screws (1.3-mm self-tapping screws, Plus Drive, Synthes, Umkirch) and one polymer bridging plate secured to the steel plates by two stainless-steel screws. Defects were filled either with DBM-, DBX or BMP-2-loaded collagen. The muscles were reattached and the skin closed. Gentamicin ointment was applied. All animals received analgesic medication for the first three days after surgery. They were checked daily, and signs of pain (no weight bearing, lack of grooming, cowering position) were documented. In case of an implant failure, animals were sacrificed. Originally, 16 animals (eight per time point) were intended in all experimental groups and eight for time point 42 days in the empty control. Due to complications, only eight animals in the DBM group and six in the empty control were operated. Radiography and microCT At day 21 and 42, radiographic follow-up was performed under general anesthesia (ketamine hydrochlorid 10 mg/ animal and medetomidine 0.15 mg/animal). Standard radiographs for controlling the plate position were obtained at 30 kV and ten seconds exposure time (Faxitron Bioptics LLC, Tuscon, AZ, USA). MicroCT scanning was done with Viva40 microCT (Scanco medical AG, Brüttisellen, Switzerland) at 55 kV and 145 μA at a voxel size of 10.5 μm. The total volume of interest comprised 3.1 mm in the proximal and distal direction from the midline of the osteotomy gap. A global threshold of 50 % of the mineral density of the intact cortex, equivalent to 398.2 mg HA/ccm, was used to distinguish mineralised from unmineralised tissue. Histology and histomorphometry Animals were sacrificed either at day 21 and 42. Femora were embedded in polymethylmethacrylate (PMMA, Technovit 9100 neu, Hereaus Kluzer, Wehrheim), and 6-μm-thick sections were cut and stained by Safranin orange/van Kossa. The region of interest was defined at 3.5 mm proximally and distally to the centre of the gap, and tissue composition was quantified using semiautomated software (KS400 3.0

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software; Carl Zeiss MicroImaging GmbH, Eching). Further tissue sections were stained with Movat pentachrome. In the figures, the femur is always shown oriented in the same position. Statistical analysis For statistical analysis, the Mann–Whitney U test and Bonferroni-Holm correction were used (PASW Statistics 18.0; SPSS, IBM, New York, NY< USA). A p value≤0.05 was taken as a significant difference. Data are presented in box plots showing median and 25th and 75th percentile, and minimum and maximum whiskers.

Results BMP-2 quantification Quantification resulted in 0.25 ng±0.09 and 0.66 ng±0.32 [mean ± standard deviation (SD)] BMP-2/mg DBM and DBX, respectively. Experimental groups In total ten, animals had to be excluded from the study. One animal had a fast-growing mammary tumour, one animal died due to a heart failure while sedated for radiographic analysis and another animal was sacrificed due to a postoperative haematoma and hindlimb ischemia and necrosis. Further, radiographic control revealed fixation failure, such as plate breakage and screw loosening, in seven animals (DBM group: n=1; DBX group: n=4; empty control n=2). Those animals were euthanised as soon as the complications were detected. During necropsy, fixation failure was accompanied by purulent fluid accumulation in the DBX group in one animal and in the empty control in both animals. Contrary to these findings, there were no complications in the BMP-2 control. Conventional X-ray and microCT Defect healing did not occur in any animal from the DBM and DBX groups or from the empty control up to six weeks postimplantation; bone ends appeared in the X-rays as either cut sharply or rounded off (Fig. 1). The 3D microCT rendering revealed irregular-shaped or fragmented radio-opaque tissue distributed throughout the defect (Fig. 2a). Apart from one exception in the DBM group, defect sites receiving DBM or DBX showed no evident bone ingrowth. Completely different findings were made in animals treated with BMP-2. A distinct callus formation was visible; after 21 days, 11 of 16 animals, and after 42 days seven out of eight animals, showed a complete bridging of the gap. Animals included in the 42-

day group revealed an increase in callus density, a smoothing of the callus surface and an initial reconstitution of the diaphyseal bone morphology. In the empty control, no radiodense tissue was found within the defect. Bone volume and bone mineral content was highest in the BMP-2 control at both time points (Fig. 2b). Histology and histomorphometry At day 21, no histology was made for the DBM and empty control groups. The DBX group revealed sharp-edged cortical ends. The connective tissue within the defect was mixed with DBX material containing concentric osteon-like structures, with partial remaining mineralised areas. Four animals in the BMP-2 control had a bridged gap with complete mineralisation of surrounding collagen carrier material. The other four showed mineralised callus invasion from the cortical ends towards the centre of the gap (not shown). At day 42 cortical ends had predominately sharp edges in the DBM group, and mineralised DBM remnants were still visible (Figs. 3 and 4). Mineralised callus tissue was found at the cortical ends and the periosteal cortical sides. In two animals, callus tissue formed a thin cap-like structure along the proximal cortical ends (Fig. 3). The Movat-pentachromestained sections revealed that cortex-associated callus tissue was lined with osteoblasts. Some of the Haversian canals within the DBM residuals were newly vascularised, but the implanted material was surrounded predominantly by connective tissue, and bone formation was not observed (Fig. 4). In the DBX group, very limited callus formation occurred (Figs. 3 and 4). In the Movat-pentachrome-stained sections, DBX residuals were clearly visible and appeared as intensive red islets within the gap, but those areas were free of cells (Fig. 4). Except for one animal, in the BMP-2 control, all showed bridging of the defect by woven bone (Fig. 3) and partial reconstitution of the medullary canal. Movat pentachrome staining showed that mineralised callus areas within the gap were lined by osteoblasts and that gaps between the mineralised tissue were filled with bone marrow cells (Fig. 4). The empty defect of the control was mainly filled with prolapsed muscle tissue. In one animal, mineralised callus tissue was found at the proximal endosteal and at the distal periosteal cortical region (Fig. 3). Staining did not indicate cartilage formation within the defect site in any treatment group. Mineralised area within the gap was highest in BMP-2 at days 21 and 42 (Fig. 5).

Discussion The results of this study showed only a poor osteoinductive outcome and no clear superiority for any of the two graft preparations tested. BMP-2 was detected by ELISA in both

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Fig. 1 Radiographs of animals in the different treatment groups (upper row: day 21; lower row: day 42). Scale bar represent 10 mm

preparations. Radio-opaque tissue was found within the defect of animals treated with DBM and DBX, but the amount of this material decreased distinctly over time, indicating a biodegradation of the originally implanted grafts rather than an

anabolic bone activity. An appositional callus formation was induced, but no material was able to provide sufficient stimulus to achieve defect bridging under the experimental conditions. Other studies comparing different bone graft

Fig. 2 a Micro computed tomography (microCT) 3D reconstructions of femoral bone of the different treatment groups. Scale bar represent 1 mm. b MicroCT evaluation of bone volume (mm³) and bone mineral content (mg) at days 21 and 42. Lines with asterisk significant differences between groups and time points

International Orthopaedics (SICOT) Fig. 3 Histological slices (Safranin orange/van Kossa staining) of femoral defects in different treatment groups at day 42. i implant remnants, mc mineralised callus tissue, mt muscle tissue

preparations in experimental models showed inconsistent findings. Quite satisfactory healing results were reported when using demineralised bone cylinders in femoral [18] or demineralised bone powder in radius [19] defects. It can be speculated that the grafts of human origin used in this study achieved less notable success; however, Hansen et al. [20] reported at week six a mineralised fraction of only 16 % after filling cranial-osteotomy defects with DBM obtained from rats of the same breeding strain. However, a much higher stimulating effect of the DBX material was assumed in our study reported here for several reasons: 1. Tissue processing of the graft might have influenced the material properties and growth-factor activity. The DBM was sterilised with peracetic acid, whereas the DBX

material was not sterilised (according to the US Musculoskeletal Transplant Foundation). 2. Growth-factor quantification revealed an approximately 2.5 times higher BMP-2 content per milligram for DBX compared with DBM material. The quantification method detects active but also inactive growth factors, and it therefore could be assumed that the measured quantity provides no information regarding bioactivity of this factor. However, both bone graft materials were tested for their in vitro osteoinductive potential before starting the animal experiments, and osteogenic differentiation of C2C12 cells was significantly enhanced after six days of cell culture with DBX [14]. 3. Demineralised and granulated cortical bone of DBX was augmented with sodium hyaluronate. This substance is a

Fig. 4 Detailed sections (Movat pentachrome staining) of the tissue found within the gap at day 42. bm bone marrow, con connective tissue, i implant remnants, mc mineralised callus tissue, * osteoblastic lining, # Haversian canal of the newly vascularised demineralised bone matrix (DBM)

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bone segments coated with a recombinant human (rh)BMP-2retaining paste into a femoral bone defect and reported encased allograft segments and a biomechanical strength of 80 % compared with intact bone at the same BMP-2 dosage 12 weeks postsurgery.

Conclusion Adequate BMP-2 dosage promotes healing even in defects with an unfavourable prognosis. Results of this study emphasise the need for the addition of strong, osteoinductive stimulus in combination with defect-filling materials to implanted scaffolds or bone substitutes in order to promote bone formation. When sufficient stimulus was lacking, complications were observed. These complications were probably not caused by implanted materials but, rather, were a consequence of instability due to lack of bridging. Fig. 5 Histomorphometric evaluation of mineralised tissue area within the gap (mm²). Lines with asterisk significant differences between groups and time points

naturally occurring polysaccharide reported to have a positive effect on bone formation and vascularisation when used as a cofactor to bovine spongiosa [20] or DBM [21]. Complications occurred in both graft groups as well as in the untreated empty control. The fixation device was introduced by Guldberg et al. [17] and was successfully used in a variety of studies on treating defects in rat femur with growth-factor-supplemented voidfilling materials [22–25]. In contrast to the study we report here, no complications occurred in animals treated with substance-free scaffolds or in empty controls. Animals used in those studies were 13 weeks of age, and although there were no concrete data on body weight, it might be assumed that they had a much lower body weight (∼250 g according to data given by the breeding company). Probably, the fixation device did not provide sufficient mechanical strength for weightbearing adult rats weighing ∼340 g. Unstable hardware might further propagate an inflammatory response and might therefore explain purulent fluid accumulation found in one animal in the DBX group and two in the empty control. Under the specific experimental conditions, only the collagen sponge containing BMP-2 was able to provide sufficient stimulus to promote defect healing; it also achieved uneventful bridging of the bone defect as early as after three weeks postsurgery. Collagen is commonly used clinically and might contribute to mineral deposition, vascular ingrowth, and growth-factor binding [26], but itself does not promote healing in a criticalsize rat-femur defect [25]. In contrast to other experimental studies with a similar design [27, 28], a much lower dosage was applied and achieved satisfying healing results, although no biomechanical testing was performed to evaluate loading capacity. Yasuda et al. [29] placed frozen, stocked, rat-femur

Acknowledgments The authors gratefully thank Prof. Robert E. Guldberg (Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA) and his former co-worker Joel D. Boerckel, PhD, for providing detailed information on the segmental defect model and technical drawings of the fixation device. Sincere thanks to Anke Kadow-Romacker for her help analysing microCT data and to Bettina Willie, PhD, for correcting grammar and spelling. This study was supported by the BMBF (BCRT, FKZ 1315848A). Conflict of interest None.

References 1. Fayaz HC et al (2011) The role of stem cells in fracture healing and nonunion. Int Orthop 35:1587–1597 2. Calori GM, Mazza E, Colombo M, Ripamonti C (2011) The use of bone-graft substitutes in large bone defects: any specific needs? Injury 42(Suppl 2):S56–S63 3. Rogers GF, Greene AK (2012) Autogenous bone graft: basic science and clinical implications. J Craniofacial Surg 23:323–327 4. Boone DW (2003) Complications of iliac crest graft and bone grafting alternatives in foot and ankle surgery. Foot Ankle Clin 8: 1–14 5. Sasso RC, LeHuec JC, Shaffrey C, Spine Interbody Research G (2005) Iliac crest bone graft donor site pain after anterior lumbar interbody fusion: a prospective patient satisfaction outcome assessment. J Spinal Disord Tech 18(Suppl):S77–S81 6. Silber JS et al (2003) Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion. Spine (Phila Pa 1976) 28:134–139 7. Gruskin E, Doll BA, Futrell FW, Schmitz JP, Hollinger JO (2012) Demineralized bone matrix in bone repair: history and use. Adv Drug Deliv Rev 64:1063–1077 8. De Long WG Jr et al (2007) Bone grafts and bone graft substitutes in orthopaedic trauma surgery. A critical analysis. J Bone Joint Surg Am 89:649–658 9. Dinopoulos HT, Giannoudis PV (2006) Safety and efficacy of use of demineralised bone matrix in orthopaedic and trauma surgery. Expert Opin Drug Saf 5:847–866 10. Blum B, Moseley J, Miller L, Richelsoph K, Haggard W (2004) Measurement of bone morphogenetic proteins and other growth factors in demineralized bone matrix. Orthopedics 27:s161–s165

International Orthopaedics (SICOT) 11. Li H, Pujic Z, Xiao Y, Bartold PM (2000) Identification of bone morphogenetic proteins 2 and 4 in commercial demineralized freezedried bone allograft preparations: pilot study. Clin Implant Dent Relat Res 2:110–117 12. Wildemann B, Kadow-Romacker A, Haas NP, Schmidmaier G (2007) Quantification of various growth factors in different demineralized bone matrix preparations. J Biomed Mater Res A 81: 437–442 13. Wildemann B, Kadow-Romacker A, Pruss A, Haas NP, Schmidmaier G (2007) Quantification of growth factors in allogenic bone grafts extracted with three different methods. Cell Tissue Bank 8:107–114 14. Bormann N, Pruss A, Schmidmaier G, Wildemann B (2010) In vitro testing of the osteoinductive potential of different bony allograft preparations. Arch Orthop Trauma Surg 130:143–149 15. McKay WF, Peckham SM, Badura JM (2007) A comprehensive clinical review of recombinant human bone morphogenetic protein2 (INFUSE Bone Graft). Int Orthop 31:729–734 16. Pruss A et al (2001) Validation of the sterilization procedure of allogeneic avital bone transplants using peracetic acid-ethanol. Biologicals 29:59–66 17. Guldberg RE et al (2004) Functional integration of tissue-engineered bone constructs. J Musculoskelet Neuronal Interact 4:399–400 18. Einhorn TA, Lane JM, Burstein AH, Kopman CR, Vigorita VJ (1984) The healing of segmental bone defects induced by demineralized bone matrix. A radiographic and biomechanical study. J Bone Joint Surg Am 66:274–279 19. Gepstein R, Weiss RE, Hallel T (1987) Bridging large defects in bone by demineralized bone matrix in the form of a powder. A

20.

21.

22. 23. 24.

25.

26. 27. 28.

29.

radiographic, histological, and radioisotope-uptake study in rats. J Bone Joint Surg Am 69:984–992 Hansen A et al (2001) Demineralized bone matrix-stimulated bone regeneration in rats enhanced by an angiogenic dipeptide derivate. Cell Tissue Bank 2:69–75 Raines AL et al (2011) Hyaluronic acid stimulates neovascularization during the regeneration of bone marrow after ablation. J Biomed Mater Res A 96:575–583 Boerckel JD et al (2011) Effects of protein dose and delivery system on BMP-mediated bone regeneration. Biomaterials 32:5241–5251 Boerckel JD et al (2012) Effects of in vivo mechanical loading on large bone defect regeneration. J Orthop Res 30:1067–1075 Johnson MR, Boerckel JD, Dupont KM, Guldberg RE (2011) Functional restoration of critically sized segmental defects with bone morphogenetic protein-2 and heparin treatment. Clin Orthop Relat Res 469:3111–3117 Wojtowicz AM et al (2010) Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair. Biomaterials 31:2574–2582 Cornell CN (1999) Osteoconductive materials and their role as substitutes for autogenous bone grafts. Orthop Clin N Am 30:591–598 Schwarz C et al (2013) Mechanical load modulates the stimulatory effect of BMP2 in a rat nonunion model. Tissue Eng Part A 19:247–254 Schutzenberger S et al (2012) The optimal carrier for BMP-2: a comparison of collagen versus fibrin matrix. Arch Orthop Trauma Surg 132:1363–1370 Yasuda H et al (2012) Repair of critical long bone defects using frozen bone allografts coated with an rhBMP-2-retaining paste. J Orthop Sci 17:299–307

Efficacy of two different demineralised bone matrix grafts to promote bone healing in a critical-size-defect: a radiological, histological and histomorphometric study in rat femurs.

The aim of the study was to compare two different demineralised bone matrices used clinically regarding their ability to induce bone healing in a crit...
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