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Bone quality of mandibles reconstructed with particulate cellular bone and marrow, and platelet-rich plasma
Journal of Cranio-Maxillo-Facial Surgery 39 (2011) 628e632
Contents lists available at ScienceDirect
Journal of Cranio-Maxillo-Facial Surgery journal homepage: www.jcmfs.com
Bone quality of mandibles reconstructed with particulate cellular bone and marrow, and platelet-rich plasma Akira Matsuo a, *, Hiroshige Chiba a, Hidetoshi Takahashi a, Jun Toyoda a, On Hasegawa a, Satoru Hojo b a b
Department of Oral and Maxillofacial Surgery (Head: Chief Professor Daichi Chikazu, DDS, PhD), Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Department of Maxillofacial Rehabilitation, Kanagawa Dental College (Vicarious Chief: Sadao Sato) 82 Inaoka-cho, Yokosuka-si, Kanagawa 238-8580, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 22 February 2010 Accepted 3 January 2011
The purpose of this study was to evaluate the bone quality of mandibles reconstructed with particulate cellular bone and marrow (PCBM) and platelet-rich plasma (PRP). We compared the bone mineral density (BMD) and microstructure of PCBM and PRP-reconstructed bone and normal bone in patients. Bone biopsies were taken of 11 samples of PCBM and PRP reconstructed bone and 16 samples of normal bone using a trephine bur. BMD and microstructures were assessed using microcomputed tomography. The compact bone resulting from the PCBM and PRP reconstruction was extremely thin. Analysis of the microstructures, showed statistically significant differences only in trabecular bone number and trabecular bone spaces between PCBM and PRP-reconstructed bones and normal bones. In the case of BMD, no statistical differences were found between the two groups. Lamellar structures and osteocytes were observed histologically in the trabecular bone in both groups. In conclusion, the BMD and microstructures of the cancellous bone in the PCBM and PRP-reconstructed mandibles resembled those in the normal mandibles. Ó 2011 European Association for Cranio-Maxillo-Facial Surgery.
Keywords: Bone microstructure Bone mineral density Mandibular reconstruction Particulate cellular bone and marrow Platelet-rich plasma
1. Introduction There are many methods available to the surgeon for mandibular reconstruction, but no consensus on a definitive method has been reached (Goh et al., 2008). Particulate cellular bone and marrow (PCBM) in a tray has many advantages, for example it carries a significantly lower morbidity than a vascularized free bone graft (Carlson and Marx, 1996). Grafts supported by trays can be contoured to allow functional oral rehabilitation. (Carlson and Marx, 1996). Although clinically adequate amounts of bone can be obtained using PCBM in mandibular reconstruction, its radiodensity is often low when compared with that of surrounding normal bone 1 year after surgery (Marx and Garg, 1998; Iwamoto et al., 2009). If the bone quality is poor in PCBM-reconstructed bone, it is likely to fail due to the lack of bone strength. Recently, it has been noted that applying platelet-rich plasma (PRP) to PCBM reconstruction accelerates both bone formation and wound healing (Tayapongsak et al., 1994; Marx et al., 1998). Recent research in metabolic bone diseases has shown that bone mineral density (BMD) and microstructures are the main factors contributing to bone quality (Seemann, 2002). Microcomputed tomography
* Corresponding author. Tel.: þ81 3 3342 6111x5731; fax: þ81 3 3342 172. E-mail address: [email protected] (A. Matsuo).
(micro-CT) is one of the best methods to evaluate these factors simultaneously in ultra high resolution (Muller et al., 1996; Ito et al., 2005). To the best of our knowledge, there has been no clinical study evaluating bone microstructures and BMD simultaneously in mandibles reconstructed using PCBM and PRP. To evaluate the bone quality in the PCBM and PRP-reconstructed mandibles, we compared the BMD and microstructures of the cancellous bone between the PCBM and PRP-reconstructed and normal mandibular bone using micro-CT.
2. Materials and methods 2.1. Subjects 11 samples were taken from 5 patients using a standardized bone biopsy technique with a 2.8 mm inner diameter trephine bur. The patients had been treated with mandibular reconstruction using both PCBM and PRP in our department between 2003 and 2008 (PCBM group). The eligibility criteria were a defect over 30 mm in size (51.9 mm on average). The 5 patients consisted of 4 men and 1 woman with an average age of 48.4 years (range, 29e61). The primary diseases were 4 benign odontogenic tumours and 1 malignant tumour. The resection methods consisted of 4 marginal resections and 1 segmental resection. As controls, 16
1010-5182/$ e see front matter Ó 2011 European Association for Cranio-Maxillo-Facial Surgery. doi:10.1016/j.jcms.2011.01.003
A. Matsuo et al. / Journal of Cranio-Maxillo-Facial Surgery 39 (2011) 628e632
specimens of normal mandibular bone were obtained from 10 patients consisting of 5 men and 5 women whose average age was 53.1 years (range, 32e78) (normal bone group). Two control patients were from the PCBM group. None of the patients had any severe metabolic bone disease. All reconstructions were carried out by a single experienced maxillofacial surgeon (A.M.). All the patients were fully informed about the procedure and gave informed written consent. The research protocol was approved by the ethics committee of Tokyo Medical University. 2.2. Surgery In all patients, PCBM reconstruction was performed according to our clinical protocol (Iwamoto et al., 2009). In brief, prior to the surgical procedure, 40e60 ml of venous blood was collected to produce PRP and autologous thrombin. They were separated using the Smart Prep System (Harvest Technologies Corp., Norwell, MA, USA). The centrifuge was programmed for an initial spin at 2500 rpm for 10 min, followed by a 1-min-interval, and an additional spin at 2300 rpm for 3 min. The surgical procedure was as follows. First, PCBM was harvested from the bilateral posterior iliac crest by an open approach. In 7 patients, PCBM was obtained from a unilateral posterior or anterior iliac crest. Then, a mesh tray was fitted to the shape of the defect after mandibulectomy. The tray was fixed rigidly with screws or a reconstruction plate only for the cortical climb. The harvested PCBM, isolated PRP and autologous thrombin were mixed in a Petri dish forming a platelet gel which was then transferred to the mandibular defect (Fig. 1a and b). Adequate bone formation was seen in all cases on postsurgical macroscopic and panoramic X-ray investigation (Fig. 1c and d). 2.3. Bone sampling and micro-CT scanning Standardized bone biopsies were performed at the time of dental implant insertion format between 8 and 12 months (average, 9.8 months) after the reconstruction using a trephine bur
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(inner diameter, 2.8 mm) (Fig. 2a). Specimens were fixed in 20% buffered formalin for 24 h and then preserved in phosphonate buffered saline. We scanned the individual samples in one piece using a microCT system (Elescan, Nittetsu Elex Co. Ltd., Kitakyushu, Japan). An Xray tube with a microfocus (spot size, 6 8 mm) is used and a maximum resolution of 4 mm (in pixel size) is attainable. Scanning was conducted at 35 kVp and 100 mA. Consecutive tomographic slices with a slice thickness of 20 mm were acquired. The digital data were reconstructed to obtain CT images with a pixel size of 17.91 mm in a 512 512 matrix. 3D image reconstruction for morphological observations and bone microstructural measurements were performed using computer software (TRI/3D Bon, Ratoc Co. Ltd., Tokyo, Japan). A cylindrical ROI of 2 mm height in cancellous bone was selected for microstructural measurement (Fig. 2b and c). Bone volume fractions (BV/TV [%]) were calculated from bone volume (BV) and total tissue volume (TV), then trabecular thickness (Tb.Th [mm]) was determined (Hildebrand and Rüegsegger, 1997). The trabecular number (Tb.N [1/mm]) and trabecular spacing (Tb.Spac [mm]) were estimated based on the plate model (Parfitt et al., 1987). Both the trabecular bone pattern factor (TBPf [1/mm]) (Hahn et al., 1992) and the structure model index (SMI) (Jinnai et al., 2002) were used to evaluate the shape of the trabecular surface as concave or convex. Node-strut (Nd.Nd/TV [1/mm3]) (Mellish et al., 1991) was also evaluated in terms of trabecular connectivity. 2.4. BMD measurements We measured BMD in all the PCBM cases and 5 specimens (5 cases) in the normal bone group. To obtain volumetric BMD data, we scanned the specimens simultaneously with micro-CT using a bone mineral reference phantom containing calibration objects with equivalent densities of 200, 300, 400, 500, 600, 700 and 800 mg/cm3 calcium hydroxyapatite. Reconstructed stacked 3D volume data of the samples with a reference phantom were used
Fig. 1. Clinical findings in a case of PCBM reconstruction. A 38-year-old woman with ameloblastoma (Case 1). (a, b) Intraoperative view and panorama X-ray findings immediately after PCBM reconstruction. Although clinically sufficient amounts of bone formation were obtained by PCBM reconstruction (c), bone radiopacity was low compared with that of surrounding normal bone 6 months after the operation (d: arrow).
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Fig. 2. Specimen sampling and sample preparation. Arrow: Core bone biopsy was performed using a trephine bur. *Regions of interest (ROIs) were set in the cancellous bone.
for the determination of volumetric BMD using computer software (TRI/3D Bon-BMD-PNTM2, Ratoc Co. Ltd.) (Fig. 2b and d). 2.5. Histology After micro-CT scanning, specimens were decalcified with 10% EDTA and embedded in paraffin. Histological sections were sliced and stained with hematoxylin and eosin (Fig. 2e). 2.6. Statistical analysis All data were presented as means SD. We compared the variables of each parameter between the PCBM group and the normal bone group. The ManneWhitney U-test was used for statistical evaluation. The variance of each parameter was also evaluated using the F-test. All analyses were performed using statistical software (SPSS version 11.5 J; SAS Institute; Cary, NC, USA). P values <0.05 were considered to indicate a statistically significant difference. 3. Results 3.1. Morphological observations using micro-CT The compact bone in the PCBM group was extremely thin. The trabecular structures of the cancellous bone in both groups were similar (Fig. 3). 3.2. Bone microstructures and BMD measurement of the cancellous bone Only Tb.N (P < 0.05) and Tb.Spac (P < 0.01) were significantly different between the PCBM group and the normal bone group, but
Fig. 3. 2D and 3D morphological observations of the PCBM-reconstructed and normal mandibles using micro-CT. (a) Normal mandible. (b) PCBM-reconstructed mandible
no significant differences were found for the other parameters. The standard deviations of the normal bone group were larger in all parameters than those of the PCBM group, and variances were significantly larger in almost all parameters (P < 0.01), except Tb.N and TBPf. The average BMD of the PCBM group was 1248.27 124.63 mg/ cm3 and that of the normal bone group was 139.76 165.46 mg/ cm3. There were no statistically significant differences or variances between the two groups (Table 1).
A. Matsuo et al. / Journal of Cranio-Maxillo-Facial Surgery 39 (2011) 628e632
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Table 1 Evaluation of bone microstructures and bone mineral density between the PCBM and PRP-reconstructed mandible and normal mandible. Bone microstructures
BMD
(N)
Parameters
BV/TV
Tb.Th
Tb.N
Tb.Spac
PCBM þ PRP
11
Average SD
29.24 8.26
105.9 16.7
2.75 0.64
377.31 85.22
Normal
16
Average SD
40.85 19.74
180.9 65.67
2.16 0.64
P value P value
n.s. <0.01
n.s. <0.01
<0.05 n.s.
U-test* F-test
TBPf
SMI
Nd.Nd/TV
(N)
4.95 3.33
2.14 0.92
32.44 9.77
11
1240.09 165.4
492.97 363.42
2.73 4.44
1.73 1.97
33.46 24.75
5
1324.17 220.25
<0.01 <0.01
n.s. n.s.
n.s. <0.01
n.s. <0.01
n.s. n.s.
Bone volume fraction (BV/TV [%]), trabecular thickness (Tb.Th [mm]), trabecular number (Tb.N [1/mm]), trabecular spacing (Tb.Spac [mm]), trabecular bone pattern factor (TBPf [1/mm]), structure model index (SMI), node strut (Nd.Nd/TV [1/mm3]) and bone mineral density (BMD [mg/cm3]). * ManneWhitney U-test.
Fig. 4. Histological observations of HE-stained mandibular tissue. (a) Normal mandible. (b) PCBM-reconstructed mandible. Arrow: osteocyte. Triangle: lining cell
3.3. Histology Lamellar structures and osteocytes were mainly observed on the trabecular bone in the normal bone group, and similar structures were also seen in the titanium tray group (Fig. 4). 4. Discussion In this study, we evaluated the quality of bone obtained using PCBM and PRP in mandibular reconstruction. Reconstructed bone requires sufficient strength to withstand functional forces, especially in continuity defects, because a large occlusal force is loaded onto the mandible (Tie et al., 2006). In cases of PCBM reconstruction, fracture after reconstruction has been reported (Aytak et al., 2005). Satisfactory bone formation has been observed in all previous clinical studies of PCBM reconstruction with PRP (Tayapongsak et al., 1994; Marx et al., 1998; Merkx et al., 2004; Simon et al., 2004; Iwamoto et al., 2009), but recent animal studies have indicated contradictory results, Choi et al. (2004) reported inhibition whereas Fennis et al. (2002) reported promotion. BMD and bone microstructure are the main factors determining bone strength (Seemann, 2002), and several morphometrical studies have been performed to evaluate bone microstructures in normal bone (Acocella et al., 2010), and grafted or reconstructed jaw bone (Leimbruckner et al., 1995; Marx et al., 1998; SchultzeMosagau et al., 2001; Torres-Lagares et al., 2010). These studies used 2D histological sections. It is difficult to precisely evaluate the structure of cancellous bone and BMD simultaneously using conventional methods because a 20 mm resolution is necessary to assess the trabecular structures (Muller et al., 1996). Histological
examination is the most common method for evaluating trabecular structures, but it is difficult to evaluate BMD using this method (Muller et al., 1996). Micro-CT has greatly improved the study of bone metabolic disease because it can evaluate 3D bone microstructures without destruction of the specimens, and additional histological and additional biological evaluation can be performed (Odgaard, 1997; Link et al., 1998; Kazama et al., 2009). This method can also simultaneously assess BMD using a bone mineral reference phantom (Ito et al., 2005). Considering bone microstructures, previous histomorphometrical studies of grafted and reconstructed jaw bone have used metrical parameters (i.e., Tb.Th or Tb.N) based on the parallel plate model (Leimbruckner et al., 1995; Marx et al., 1998; SchultzeMosagau et al., 2001). However, SMI or TBPf, which represents the nonmetric features of trabecular structures, would be more useful than metric parameters (Ito et al., 2005). The node-strut has also been recognized as one of the factors affecting bone strength (Kazama et al., 2009). In the present study, bone microstructure evaluation using micro-CT showed no significant differences in most structural parameters, except Tb.N and Tb.Spac, between the normal bone group and the PCBM group. The standard deviations of many parameters were significantly higher in the normal bone group than in the PCBM group. These results indicate that the bone microstructures of the cancellous bone in the PCBM group are similar in quality to, but are more uniform than, those of the cancellous bone in the normal bone group. No statistically significant differences in BMD were found between the PCBM group and the normal bone group. In several morphological studies cortical thickness in the PCBM-reconstructed mandible was thinner than that in the normal mandible (Marx and Garg, 1998; Iwamoto et al., 2009), and the findings 3D
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A. Matsuo et al. / Journal of Cranio-Maxillo-Facial Surgery 39 (2011) 628e632
morphological findings in this study support these previous findings. Our results suggest that the low radiopacity on X-ray findings or low Hounsfield unit values on the conventional CT in the PCBMreconstructed bone were caused not by the low mineral density of the cancellous bone but by the reduced cortical thickness. Both bone microstructural parameters and BMD are indicators of bone fragility (Seemann, 2002). We therefore conclude that good quality cancellous bone in PCBM and PRP-reconstructed mandibles contributed to good bone strength. Vital trabecular bone with lamellar structures was demonstrated histologically. Regarding bone formation, both bone conduction and induction were reported to be more active in the PCBM graft than in the block bone graft (Goldberg and Akhavan, 2005). In the present study, the higher Tb.N between 8 and 12 months after the PCBM reconstruction indicated the progression of bone remodelling. These results suggest that rapid bone remodelling was progressing actively in the PCBM-reconstructed bone, and a sufficient quantity of bone mineral content and microstructures were obtained by 12 months after reconstruction in the cancellous bone. This method can be applied to every reconstruction method (e.g., vascularized free bone graft). In the near future, a recently developed high-resolution multi-detector CT (Ito et al., 2005) can be used for the evaluation of consequent changes of bone quality in a patient noninvasively. The present study has several limitations. Firstly, sampling of the control was not controlled and the number of cases was small. However the present results showed that the bone quality of PCBM and PRP-reconstructed bone was uniform regardless of location. Secondly, although Marx et al. (1998) reported higher bone volume in PCBM-reconstructed bone with PRP than that without PRP, we did not evaluate the effect of PRP in the present study because we used PRP for all PCBM reconstruction cases according to our clinical protocol (Iwamoto et al., 2009). It is important to note that the purpose of the present study was not to compare the bone quality of the PCBM-reconstructed bone with or without PRP, but with the normal mandible. Finally, only the cancellous bone was evaluated in the present study. We believe that the structure of the PCBMreconstructed bone is mainly affected by the cancellous bone because the cortical bone is very thin morphologically. In future studies, we will investigate the quality of the cortical bone. 5. Conclusions The BMD and microstructures of the cancellous bone in mandibles reconstructed with PCBM and PRP were similar to those of the cancellous bone in normal mandibles. Acknowledgements The authors are indebted to Assistant Professor Edward F. Barroga and Professor J. Patrick Barron of the Department of International Medical Communication of Tokyo Medical University for reviewing this manuscript. References Acocella A, Bertolai R, Colafranceschi M, Sacco R: Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts for alveolar ridge augmentation before implant placement. J Cranio-Maxillofac Surg 38: 222e230, 2010
Aytak C, Ozbec S, Kahveci R, Ozgenel Y, Akin S, Ozcan M: Titanium mesh fracture in mandibular reconstruction. J Craniofac Surg 16: 1120e1122, 2005 Carlson ER, Marx RE: Mandibular reconstruction using cancellous cellular bone grafts. J Oral Maxillofac Surg 54: 889e897, 1996 Choi BH, Huh JY, Suh JJ, Lee SH: Effect of platelet-rich plasma on bone regeneration in autogenous bone graft. Int J Oral Maxillofac Surg 33: 56e59, 2004 Fennis JP, Stoelinga PJ, Jansen JA: Mandibular reconstruction: a clinical and radiographic animal study on the use of autogenous scaffolds and platelet-rich plasma. Int J Oral Maxillofac Surg 31: 281e286, 2002 Goh BT, Lee S, Tiedman H, Stoelinga PJW: Mandibular reconstruction in adults: a review. Int J Oral Maxillofac Surg 37: 597e605, 2008 Goldberg VM, Akhavan S: Biology of bone graft. In: Lieberman JR, Friedlaender GE (eds), Bone regeneration and repair. Biology and clinical applications. Totowa, New Jersey: Humana Press, 57e66, 2005 Hahn M, Vogel M, Pompesius-Kempa M, Delling G: Trabecular bone pattern factor e a new parameter for simple quantification of bone microstructure. Bone 13: 327e330, 1992 Hildebrand T, Rüegsegger P: A new method for the model-independent assessment of thickness in three-dimensional images. J Microsc 185: 67e75, 1997 Ito M, Ikeda K, Nishiguchi M, Nishiguchi M, Shido H, Uetani M, et al: Multi-detector row CT imaging of vertebral microstructure for evaluation of fracture risk. J Bone Miner Res 20: 1828e1836, 2005 Iwamoto I, Matsuo A, Kato N, Takeuchi S, Takahashi H, Hojo S, et al: Computed tomographic evaluation of bone quality of the mandible reconstructed by particular cellular bone and marrow combined with platelet-rich plasma. Oral Sci Int 6: 63e72, 2009 Jinnai H, Watashiba H, Kajihara T, Nishkawa Y, Takahashi M, Ito M: Surface curvatures of trabecular bone microarchitecture. Bone 30: 191e194, 2002 Kazama JJ, Koda R, Yamamoto S, Narita I, Gejyo F, Tokumoto A: Comparison of quantitative cancellous bone connectivity analyses at two- and three-dimensional levels in dialysis patients. Calcif Tissue Int 84: 38e44, 2009 Leimbruckner BD, Dielert E, Wolf K: Histomorphometric examinations of free revascularized iliac crest bone after transplantation for mandibular reconstruction. Microsurg 16: 377e382, 1995 Link TM, Majumdar S, Lin J, Augat P, Gould R, Newitt D, et al: Assessment of trabecular structure using high-resolution CT images and texture analysis. J Comput Assist Tomogr 22: 15e24, 1998 Marx RE, Garg AK: Bone structure, metabolism, and physiology: its impact on dental implantology. Implant Dentistry 7: 267e276, 1998 Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR: Platelet-rich plasma e growth factor enhancement for bone graft. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85: 638e646, 1998 Mellish RW, Ferguson-pell MW, Cochran GV, Lindsay R, Dempster DW: A new manual method for assessing two-dimensional cancellous bone structure: between iliac crest and lumbar vertebrae. J Bone Mineral Res 6: 689e696, 1991 Merkx MAW, Fennis JPM, Verhagen CM, Stoelinga PJW: Reconstruction of the mandible using preshaped 2.3 mm titanium plates, autogenous particulate cortico-cancellous bone grafts and platelet rich plasma: a report on eight patients. Int Oral Maxillofac Surg 33: 733e739, 2004 Muller R, Hahn M, Vogel M, Delling G, Ruegsegger P: Morphometric analysis of noninvasively assessed bone biopsies: comparison of high-resolution computed tomography and histologic sections. Bone 18: 215e220, 1996 Odgaard A: Three-dimensional methods for quantification of cancellous bone architecture. Bone 20: 315e328, 1997 Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al: Bone histomorphometry: standardization of nomenclature, symbols, and units. J Bone Miner Res 2: 595e610, 1987 Schultze-Mosagau S, Keweloh M, Wiltfang J, Kessler P, Neukam FW: Histomorphometric and densitometric changes in bone volume and structure after bone grafting in the extremely atrophic maxilla. Brit J Oral Maxillofac Surg 39: 439e447, 2001 Seemann E: Pathogenesis of bone fragility in women and men. Lancet 359: 1841e1850, 2002 Simon ENM, Merkx MAW, Shubi FM, Kalyanyama BM, Stoelinga: Reconstruction of the mandible after ablative surgery for the treatment of aggressive, benign odontogenic tumours in Tanzania: a preliminary study. Int J Oral Maxillofac Surg 33: 421e426, 2004 Tayapongsak P, O’Brian DA, Monteiro CB, Arceo-Diaz LY: Autologous fibrin adhesive in mandibular reconstruction with particulate cancellous bone and marrow. J Oral Maxillofac Surg 52: 161e165, 1994 Tie Y, Wang DM, Ji T, Wang CT, Zhang CP: Three-dimensional finite-element analysis investigating the biomechanical effects of human mandibular reconstruction with autogenous bone grafts. J Cranio-Maxillofac Surg 34: 290e298, 2006 Torres-Lagares D, Tulasne JF, Pouget C, Llorens A: Structure and remodelling of the human parietal bone: an age and gender histomorphometric study. J CranioMaxillofac Surg 38: 325e330, 2010
Contents lists available at ScienceDirect
Journal of Cranio-Maxillo-Facial Surgery journal homepage: www.jcmfs.com
Bone quality of mandibles reconstructed with particulate cellular bone and marrow, and platelet-rich plasma Akira Matsuo a, *, Hiroshige Chiba a, Hidetoshi Takahashi a, Jun Toyoda a, On Hasegawa a, Satoru Hojo b a b
Department of Oral and Maxillofacial Surgery (Head: Chief Professor Daichi Chikazu, DDS, PhD), Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Department of Maxillofacial Rehabilitation, Kanagawa Dental College (Vicarious Chief: Sadao Sato) 82 Inaoka-cho, Yokosuka-si, Kanagawa 238-8580, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 22 February 2010 Accepted 3 January 2011
The purpose of this study was to evaluate the bone quality of mandibles reconstructed with particulate cellular bone and marrow (PCBM) and platelet-rich plasma (PRP). We compared the bone mineral density (BMD) and microstructure of PCBM and PRP-reconstructed bone and normal bone in patients. Bone biopsies were taken of 11 samples of PCBM and PRP reconstructed bone and 16 samples of normal bone using a trephine bur. BMD and microstructures were assessed using microcomputed tomography. The compact bone resulting from the PCBM and PRP reconstruction was extremely thin. Analysis of the microstructures, showed statistically significant differences only in trabecular bone number and trabecular bone spaces between PCBM and PRP-reconstructed bones and normal bones. In the case of BMD, no statistical differences were found between the two groups. Lamellar structures and osteocytes were observed histologically in the trabecular bone in both groups. In conclusion, the BMD and microstructures of the cancellous bone in the PCBM and PRP-reconstructed mandibles resembled those in the normal mandibles. Ó 2011 European Association for Cranio-Maxillo-Facial Surgery.
Keywords: Bone microstructure Bone mineral density Mandibular reconstruction Particulate cellular bone and marrow Platelet-rich plasma
1. Introduction There are many methods available to the surgeon for mandibular reconstruction, but no consensus on a definitive method has been reached (Goh et al., 2008). Particulate cellular bone and marrow (PCBM) in a tray has many advantages, for example it carries a significantly lower morbidity than a vascularized free bone graft (Carlson and Marx, 1996). Grafts supported by trays can be contoured to allow functional oral rehabilitation. (Carlson and Marx, 1996). Although clinically adequate amounts of bone can be obtained using PCBM in mandibular reconstruction, its radiodensity is often low when compared with that of surrounding normal bone 1 year after surgery (Marx and Garg, 1998; Iwamoto et al., 2009). If the bone quality is poor in PCBM-reconstructed bone, it is likely to fail due to the lack of bone strength. Recently, it has been noted that applying platelet-rich plasma (PRP) to PCBM reconstruction accelerates both bone formation and wound healing (Tayapongsak et al., 1994; Marx et al., 1998). Recent research in metabolic bone diseases has shown that bone mineral density (BMD) and microstructures are the main factors contributing to bone quality (Seemann, 2002). Microcomputed tomography
* Corresponding author. Tel.: þ81 3 3342 6111x5731; fax: þ81 3 3342 172. E-mail address: [email protected] (A. Matsuo).
(micro-CT) is one of the best methods to evaluate these factors simultaneously in ultra high resolution (Muller et al., 1996; Ito et al., 2005). To the best of our knowledge, there has been no clinical study evaluating bone microstructures and BMD simultaneously in mandibles reconstructed using PCBM and PRP. To evaluate the bone quality in the PCBM and PRP-reconstructed mandibles, we compared the BMD and microstructures of the cancellous bone between the PCBM and PRP-reconstructed and normal mandibular bone using micro-CT.
2. Materials and methods 2.1. Subjects 11 samples were taken from 5 patients using a standardized bone biopsy technique with a 2.8 mm inner diameter trephine bur. The patients had been treated with mandibular reconstruction using both PCBM and PRP in our department between 2003 and 2008 (PCBM group). The eligibility criteria were a defect over 30 mm in size (51.9 mm on average). The 5 patients consisted of 4 men and 1 woman with an average age of 48.4 years (range, 29e61). The primary diseases were 4 benign odontogenic tumours and 1 malignant tumour. The resection methods consisted of 4 marginal resections and 1 segmental resection. As controls, 16
1010-5182/$ e see front matter Ó 2011 European Association for Cranio-Maxillo-Facial Surgery. doi:10.1016/j.jcms.2011.01.003
A. Matsuo et al. / Journal of Cranio-Maxillo-Facial Surgery 39 (2011) 628e632
specimens of normal mandibular bone were obtained from 10 patients consisting of 5 men and 5 women whose average age was 53.1 years (range, 32e78) (normal bone group). Two control patients were from the PCBM group. None of the patients had any severe metabolic bone disease. All reconstructions were carried out by a single experienced maxillofacial surgeon (A.M.). All the patients were fully informed about the procedure and gave informed written consent. The research protocol was approved by the ethics committee of Tokyo Medical University. 2.2. Surgery In all patients, PCBM reconstruction was performed according to our clinical protocol (Iwamoto et al., 2009). In brief, prior to the surgical procedure, 40e60 ml of venous blood was collected to produce PRP and autologous thrombin. They were separated using the Smart Prep System (Harvest Technologies Corp., Norwell, MA, USA). The centrifuge was programmed for an initial spin at 2500 rpm for 10 min, followed by a 1-min-interval, and an additional spin at 2300 rpm for 3 min. The surgical procedure was as follows. First, PCBM was harvested from the bilateral posterior iliac crest by an open approach. In 7 patients, PCBM was obtained from a unilateral posterior or anterior iliac crest. Then, a mesh tray was fitted to the shape of the defect after mandibulectomy. The tray was fixed rigidly with screws or a reconstruction plate only for the cortical climb. The harvested PCBM, isolated PRP and autologous thrombin were mixed in a Petri dish forming a platelet gel which was then transferred to the mandibular defect (Fig. 1a and b). Adequate bone formation was seen in all cases on postsurgical macroscopic and panoramic X-ray investigation (Fig. 1c and d). 2.3. Bone sampling and micro-CT scanning Standardized bone biopsies were performed at the time of dental implant insertion format between 8 and 12 months (average, 9.8 months) after the reconstruction using a trephine bur
629
(inner diameter, 2.8 mm) (Fig. 2a). Specimens were fixed in 20% buffered formalin for 24 h and then preserved in phosphonate buffered saline. We scanned the individual samples in one piece using a microCT system (Elescan, Nittetsu Elex Co. Ltd., Kitakyushu, Japan). An Xray tube with a microfocus (spot size, 6 8 mm) is used and a maximum resolution of 4 mm (in pixel size) is attainable. Scanning was conducted at 35 kVp and 100 mA. Consecutive tomographic slices with a slice thickness of 20 mm were acquired. The digital data were reconstructed to obtain CT images with a pixel size of 17.91 mm in a 512 512 matrix. 3D image reconstruction for morphological observations and bone microstructural measurements were performed using computer software (TRI/3D Bon, Ratoc Co. Ltd., Tokyo, Japan). A cylindrical ROI of 2 mm height in cancellous bone was selected for microstructural measurement (Fig. 2b and c). Bone volume fractions (BV/TV [%]) were calculated from bone volume (BV) and total tissue volume (TV), then trabecular thickness (Tb.Th [mm]) was determined (Hildebrand and Rüegsegger, 1997). The trabecular number (Tb.N [1/mm]) and trabecular spacing (Tb.Spac [mm]) were estimated based on the plate model (Parfitt et al., 1987). Both the trabecular bone pattern factor (TBPf [1/mm]) (Hahn et al., 1992) and the structure model index (SMI) (Jinnai et al., 2002) were used to evaluate the shape of the trabecular surface as concave or convex. Node-strut (Nd.Nd/TV [1/mm3]) (Mellish et al., 1991) was also evaluated in terms of trabecular connectivity. 2.4. BMD measurements We measured BMD in all the PCBM cases and 5 specimens (5 cases) in the normal bone group. To obtain volumetric BMD data, we scanned the specimens simultaneously with micro-CT using a bone mineral reference phantom containing calibration objects with equivalent densities of 200, 300, 400, 500, 600, 700 and 800 mg/cm3 calcium hydroxyapatite. Reconstructed stacked 3D volume data of the samples with a reference phantom were used
Fig. 1. Clinical findings in a case of PCBM reconstruction. A 38-year-old woman with ameloblastoma (Case 1). (a, b) Intraoperative view and panorama X-ray findings immediately after PCBM reconstruction. Although clinically sufficient amounts of bone formation were obtained by PCBM reconstruction (c), bone radiopacity was low compared with that of surrounding normal bone 6 months after the operation (d: arrow).
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Fig. 2. Specimen sampling and sample preparation. Arrow: Core bone biopsy was performed using a trephine bur. *Regions of interest (ROIs) were set in the cancellous bone.
for the determination of volumetric BMD using computer software (TRI/3D Bon-BMD-PNTM2, Ratoc Co. Ltd.) (Fig. 2b and d). 2.5. Histology After micro-CT scanning, specimens were decalcified with 10% EDTA and embedded in paraffin. Histological sections were sliced and stained with hematoxylin and eosin (Fig. 2e). 2.6. Statistical analysis All data were presented as means SD. We compared the variables of each parameter between the PCBM group and the normal bone group. The ManneWhitney U-test was used for statistical evaluation. The variance of each parameter was also evaluated using the F-test. All analyses were performed using statistical software (SPSS version 11.5 J; SAS Institute; Cary, NC, USA). P values <0.05 were considered to indicate a statistically significant difference. 3. Results 3.1. Morphological observations using micro-CT The compact bone in the PCBM group was extremely thin. The trabecular structures of the cancellous bone in both groups were similar (Fig. 3). 3.2. Bone microstructures and BMD measurement of the cancellous bone Only Tb.N (P < 0.05) and Tb.Spac (P < 0.01) were significantly different between the PCBM group and the normal bone group, but
Fig. 3. 2D and 3D morphological observations of the PCBM-reconstructed and normal mandibles using micro-CT. (a) Normal mandible. (b) PCBM-reconstructed mandible
no significant differences were found for the other parameters. The standard deviations of the normal bone group were larger in all parameters than those of the PCBM group, and variances were significantly larger in almost all parameters (P < 0.01), except Tb.N and TBPf. The average BMD of the PCBM group was 1248.27 124.63 mg/ cm3 and that of the normal bone group was 139.76 165.46 mg/ cm3. There were no statistically significant differences or variances between the two groups (Table 1).
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Table 1 Evaluation of bone microstructures and bone mineral density between the PCBM and PRP-reconstructed mandible and normal mandible. Bone microstructures
BMD
(N)
Parameters
BV/TV
Tb.Th
Tb.N
Tb.Spac
PCBM þ PRP
11
Average SD
29.24 8.26
105.9 16.7
2.75 0.64
377.31 85.22
Normal
16
Average SD
40.85 19.74
180.9 65.67
2.16 0.64
P value P value
n.s. <0.01
n.s. <0.01
<0.05 n.s.
U-test* F-test
TBPf
SMI
Nd.Nd/TV
(N)
4.95 3.33
2.14 0.92
32.44 9.77
11
1240.09 165.4
492.97 363.42
2.73 4.44
1.73 1.97
33.46 24.75
5
1324.17 220.25
<0.01 <0.01
n.s. n.s.
n.s. <0.01
n.s. <0.01
n.s. n.s.
Bone volume fraction (BV/TV [%]), trabecular thickness (Tb.Th [mm]), trabecular number (Tb.N [1/mm]), trabecular spacing (Tb.Spac [mm]), trabecular bone pattern factor (TBPf [1/mm]), structure model index (SMI), node strut (Nd.Nd/TV [1/mm3]) and bone mineral density (BMD [mg/cm3]). * ManneWhitney U-test.
Fig. 4. Histological observations of HE-stained mandibular tissue. (a) Normal mandible. (b) PCBM-reconstructed mandible. Arrow: osteocyte. Triangle: lining cell
3.3. Histology Lamellar structures and osteocytes were mainly observed on the trabecular bone in the normal bone group, and similar structures were also seen in the titanium tray group (Fig. 4). 4. Discussion In this study, we evaluated the quality of bone obtained using PCBM and PRP in mandibular reconstruction. Reconstructed bone requires sufficient strength to withstand functional forces, especially in continuity defects, because a large occlusal force is loaded onto the mandible (Tie et al., 2006). In cases of PCBM reconstruction, fracture after reconstruction has been reported (Aytak et al., 2005). Satisfactory bone formation has been observed in all previous clinical studies of PCBM reconstruction with PRP (Tayapongsak et al., 1994; Marx et al., 1998; Merkx et al., 2004; Simon et al., 2004; Iwamoto et al., 2009), but recent animal studies have indicated contradictory results, Choi et al. (2004) reported inhibition whereas Fennis et al. (2002) reported promotion. BMD and bone microstructure are the main factors determining bone strength (Seemann, 2002), and several morphometrical studies have been performed to evaluate bone microstructures in normal bone (Acocella et al., 2010), and grafted or reconstructed jaw bone (Leimbruckner et al., 1995; Marx et al., 1998; SchultzeMosagau et al., 2001; Torres-Lagares et al., 2010). These studies used 2D histological sections. It is difficult to precisely evaluate the structure of cancellous bone and BMD simultaneously using conventional methods because a 20 mm resolution is necessary to assess the trabecular structures (Muller et al., 1996). Histological
examination is the most common method for evaluating trabecular structures, but it is difficult to evaluate BMD using this method (Muller et al., 1996). Micro-CT has greatly improved the study of bone metabolic disease because it can evaluate 3D bone microstructures without destruction of the specimens, and additional histological and additional biological evaluation can be performed (Odgaard, 1997; Link et al., 1998; Kazama et al., 2009). This method can also simultaneously assess BMD using a bone mineral reference phantom (Ito et al., 2005). Considering bone microstructures, previous histomorphometrical studies of grafted and reconstructed jaw bone have used metrical parameters (i.e., Tb.Th or Tb.N) based on the parallel plate model (Leimbruckner et al., 1995; Marx et al., 1998; SchultzeMosagau et al., 2001). However, SMI or TBPf, which represents the nonmetric features of trabecular structures, would be more useful than metric parameters (Ito et al., 2005). The node-strut has also been recognized as one of the factors affecting bone strength (Kazama et al., 2009). In the present study, bone microstructure evaluation using micro-CT showed no significant differences in most structural parameters, except Tb.N and Tb.Spac, between the normal bone group and the PCBM group. The standard deviations of many parameters were significantly higher in the normal bone group than in the PCBM group. These results indicate that the bone microstructures of the cancellous bone in the PCBM group are similar in quality to, but are more uniform than, those of the cancellous bone in the normal bone group. No statistically significant differences in BMD were found between the PCBM group and the normal bone group. In several morphological studies cortical thickness in the PCBM-reconstructed mandible was thinner than that in the normal mandible (Marx and Garg, 1998; Iwamoto et al., 2009), and the findings 3D
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morphological findings in this study support these previous findings. Our results suggest that the low radiopacity on X-ray findings or low Hounsfield unit values on the conventional CT in the PCBMreconstructed bone were caused not by the low mineral density of the cancellous bone but by the reduced cortical thickness. Both bone microstructural parameters and BMD are indicators of bone fragility (Seemann, 2002). We therefore conclude that good quality cancellous bone in PCBM and PRP-reconstructed mandibles contributed to good bone strength. Vital trabecular bone with lamellar structures was demonstrated histologically. Regarding bone formation, both bone conduction and induction were reported to be more active in the PCBM graft than in the block bone graft (Goldberg and Akhavan, 2005). In the present study, the higher Tb.N between 8 and 12 months after the PCBM reconstruction indicated the progression of bone remodelling. These results suggest that rapid bone remodelling was progressing actively in the PCBM-reconstructed bone, and a sufficient quantity of bone mineral content and microstructures were obtained by 12 months after reconstruction in the cancellous bone. This method can be applied to every reconstruction method (e.g., vascularized free bone graft). In the near future, a recently developed high-resolution multi-detector CT (Ito et al., 2005) can be used for the evaluation of consequent changes of bone quality in a patient noninvasively. The present study has several limitations. Firstly, sampling of the control was not controlled and the number of cases was small. However the present results showed that the bone quality of PCBM and PRP-reconstructed bone was uniform regardless of location. Secondly, although Marx et al. (1998) reported higher bone volume in PCBM-reconstructed bone with PRP than that without PRP, we did not evaluate the effect of PRP in the present study because we used PRP for all PCBM reconstruction cases according to our clinical protocol (Iwamoto et al., 2009). It is important to note that the purpose of the present study was not to compare the bone quality of the PCBM-reconstructed bone with or without PRP, but with the normal mandible. Finally, only the cancellous bone was evaluated in the present study. We believe that the structure of the PCBMreconstructed bone is mainly affected by the cancellous bone because the cortical bone is very thin morphologically. In future studies, we will investigate the quality of the cortical bone. 5. Conclusions The BMD and microstructures of the cancellous bone in mandibles reconstructed with PCBM and PRP were similar to those of the cancellous bone in normal mandibles. Acknowledgements The authors are indebted to Assistant Professor Edward F. Barroga and Professor J. Patrick Barron of the Department of International Medical Communication of Tokyo Medical University for reviewing this manuscript. References Acocella A, Bertolai R, Colafranceschi M, Sacco R: Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts for alveolar ridge augmentation before implant placement. J Cranio-Maxillofac Surg 38: 222e230, 2010
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