EXPERIMENTAL Cranial Particulate Bone Graft Ossifies Calvarial Defects by Osteogenesis Aladdin H. Hassanein, M.D., M.M.Sc. Praveen R. Arany, Ph.D., M.D.S., M.M.Sc. Rafael A. Couto, B.A. James E. Clune, M.D. Julie Glowacki, Ph.D. Gary F. Rogers, M.D., J.D., M.B.A. John B. Mulliken, M.D. Arin K. Greene, M.D., M.M.Sc. Boston, Mass.
Background: Cranial particulate bone graft heals inlay calvarial defects and can be harvested as early as infancy. The purpose of this study was to test the hypothesis that particulate bone promotes ossification primarily by osteogenesis. Methods: Freshly harvested particulate bone, devitalized particulate bone, and high-speed drilled bone dust from rabbit calvaria were assayed for metabolic activity (resazurin) and viable osteoblasts (alkaline phosphatase). A rabbit cranial defect model was used to test the effect of devitalizing particulate bone on in vivo ossification. A parietal critical-size defect was created and managed in three ways: (1) no implant (n ⫽ 6); (2) particulate bone implant (n ⫽ 6); and (3) devitalized particulate bone implant (n ⫽ 6). Micro– computed tomographic scanning was used to measure ossification 16 weeks later; histology also was studied. Results: Particulate bone contained more viable cells (0.94 percent transmittance per milligram) compared with devitalized particulate bone (0.007 percent) or bone dust (0.21 percent) (p ⫽ 0.01). Particulate bone had greater alkaline phosphatase activity (0.13 U/g) than devitalized particulate bone (0.000) or bone dust (0.06) (p ⫽ 0.01). Critical-size defects treated with particulate bone had more ossification (99.7 percent) compared with devitalized particulate bone implants (42.2 percent) (p ⫽ 0.01); no difference was found between devitalized particulate bone and the control (40.8 percent) (p ⫽ 0.9). Conclusions: Particulate bone graft contains living cells, including osteoblasts, that are required to heal critical-size cranial defects. These data support the hypothesis that particulate bone promotes ossification primarily by osteogenesis. (Plast. Reconstr. Surg. 129: 796e, 2012.)
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econstruction of pediatric calvarial defects is difficult. The use of alloplastic materials generally is not indicated because they do not fully osseointegrate and can become unstable with cranial growth.1 Autologous rib and iliac grafts resorb and cause donor-site morbidity.2– 4 Split cranial bone is preferred for inlay cranioplasty but is difficult to harvest in children younger than 5 years because of the underdeveloped diploe¨.5 One option for overcoming the limited supply of autologous bone in children is to use cranial particulate bone graft. This material may be harvested in infancy, before a diploic space has From the Department of Plastic and Oral Surgery, Children’s Hospital Boston, Harvard Medical School; the Program in Oral and Maxillofacial Pathology, Harvard School of Dental Medicine; and the Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School. Received for publication July 16, 2011; accepted November 21, 2011. Copyright ©2012 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0b013e31824a2bdd
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formed. Using a hand-turned brace and bit, pieces of bone can be obtained from the ectocortical or endocortical surface. We previously have shown that particulate graft repairs nonhealing, criticalsize defects experimentally6 and clinically.7–10 The mechanism by which particulate graft ossifies critical-size defects, however, is unknown. Bone autografts heal primarily by two mechanisms, depending on their architecture: osteogenesis (direct ossification by osteoblasts within the graft) and/or osteoconduction (ingrowth of adjacent bone into the implant).11,12 Cancellous grafts have a large surface area–to–volume ratio, which facilitates revascularization, diffusion of recipientsite nutrients, and osteogenesis. Cortical grafts are resistant to rapid neovascularization; cells on the interior do not survive, and the dense scaffold is repopulated by ingrowth of living tissue.11,12 Al-
Disclosure: The authors have no financial interest to declare in relation to the content of this article.
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Volume 129, Number 5 • Ossification of Calvarial Defects though osteoinduction (differentiation of host mesenchymal cells into osteoblasts) may be involved in autograft healing, demineralization is required for osteoinductive matrix to have activity.13–16 Because the morphology of corticocancellous particulate graft is similar to cancellous bone, we hypothesized that particulate graft contains viable cells that are responsible for healing. The purpose of this study was to determine whether autologous calvarial particulate bone graft ossifies inlay cranioplasty defects primarily by osteogenesis.
MATERIALS AND METHODS Histologic Analysis and Vital Assays of Bone Grafts This study was approved by the Animal Use and Care Committee at Children’s Hospital Boston. Subperiosteal exposure of the frontal and parietal bones of an adult, New Zealand White rabbit was performed, and particulate bone (700 mg) was harvested from the calvaria with a handdriven Hudson brace and 16-mm D’Errico craniotomy bit (Codman & Shurtleff, Inc., Raynham, Mass.).6 Bone dust (350 mg), which does not heal critical-size cranial defects,6 was used as a control; it was procured with an electric pen drive and 4-mm egg burr (Synthes, Inc., West Chester, Pa.). Particulate bone and bone dust were divided into 35-mg aliquots. One-half of the particulate bone was devitalized by two freeze-thaw cycles (–20°C for 18 hours and then room temperature for 6 hours), followed by five deep freeze-thaw cycles (– 80°C for 18 hours and then room temperature for 6 hours).17 Freeze-thawing was chosen as the devitalization method because it does not alter the extracellular matrix or graft morphology.18 Samples from all three graft types were fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin, and 5-m sections were stained with hematoxylin and eosin. Resazurin, an oxidation-reduction indicator of living cells, was used to assess graft viability. Metabolically active cells reduce blue dye to a red fluorescent resorufin (Alamar Blue; Invitrogen, Inc., Carlsbad, Calif.).19,20 One milliliter of resazurin was added to each of five aliquots of particulate bone, devitalized particulate bone, and bone dust. The mixtures were incubated in the dark at 37°C for 2 hours. Absorption was measured using a microplate reader at 570 nm and analyzed with KC4 software (BioTek Instruments, Inc., Winooski, Vt.). These data were normalized to dry weights (per milligram of graft) following 3 days
of lyophilization (FreeZone; Labconco, Inc., Kansas City, Mo.). An assay for alkaline phosphatase was used to measure osteoblastic activity.21,22 One milliliter of lysis buffer (50 mM Tris HCl and 0.1% Triton X-100, pH 9.5) was added to five aliquots of particulate bone, devitalized particulate bone, and bone dust, that were then lysed with sonication. One hundred microliters of 4-methylbelliferyl phosphate (4-MUP Substrate System; Sigma-Aldrich, Inc., St. Louis, Mo.) was added to 20 l of each sample for 25 minutes at 37°C. A standard curve was run for each assay with human placental alkaline phosphatase (Sigma-Aldrich). Fluorescence was measured using a microplate reader with excitation/emission at 365/450 nm and analyzed with KC4 software (BioTek). Fluorescence units were normalized to the amount of protein (in microunits per microgram) in each sample. Protein was determined with the bicinchoninic acid assay reagent according to the manufacturer’s protocol (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific, Inc., Waltham, Mass.).23 Animal Model A rabbit calvarial critical-size defect model was used to test the effect of particulate bone graft devitalization on in vivo ossification.6 Eighteen New Zealand White rabbits aged 16 to 18 months underwent subperiosteal exposure of the frontal and parietal bones through a coronal incision (Fig. 1). A 17 ⫻ 17-mm defect in the parietal bones of 12 animals was made using an osteotome while preserving the underlying dura. Particulate bone graft was harvested from the frontal bones using a hand-driven Hudson brace and 16-mm D’Errico craniotomy bit (Codman & Shurtleff).6 In group I (n ⫽ 6), the particulate bone was discarded and no implant was placed into the defect. In group II (n ⫽ 6), the particulate bone was placed into the parietal defect. In group III (n ⫽ 6), particulate bone first was harvested and devitalized; at a second operation, the parietal defect was created and filled with the devitalized particulate bone. Before closure, fibrin sealant was placed over the criticalsize defect in all three groups (Tisseel; Baxter Healthcare Corporation, Deerfield, Ill.).6 Sixteen weeks postoperatively, rabbits were studied with micro– computed tomography (Siemens MicroCAT II Small Animal Imaging System; Siemens Medical Solutions USA, Inc., Malvern, Pa.). Ossification was determined based on computed tomographic densitometry using Amira software (Visage Imaging, Inc., San Diego, Calif.) and pre-
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Fig. 1. Rabbit inlay cranioplasty model. (Above, left) Exposed frontal and parietal bones. (Above, right) Particulate bone procured from frontal bone with a hand-driven brace and bit. (Below, left) A 17 ⫻ 17-mm parietal critical-size defect and partial-thickness particulate bone donor site. (Below, right) Graft placed into critical-size defect.
sented as the percentage of the critical-size defect that had ossified (1 – residual defect area/original defect area ⫻ 100). The parietal bones were excised, and hematoxylin and eosin staining was performed to evaluate osseous healing of the grafts. Statistical Analysis Power analysis indicated that a sample size of six rabbits in each group provided a 90 percent power to detect a 25 percent difference in ossification (SD, 10 percent) using analysis of variance (nQuery Advisor 7.0; Statistical Solutions, Saugus, Mass.). Data are presented as medians with interquartile ranges (25th to 75th percentile). Differ-
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ences between absorption/fluorescence and ossification of critical-size defects were compared with the Kruskal-Wallis test. Two-tailed values of p ⬍ 0.05 were considered significant. Statistical analysis was performed with GraphPad Prism (GraphPad Software, Inc., La Jolla, Calif.).
RESULTS Histologic Analysis and Vital Assays of Bone Grafts Fresh particulate bone exhibited osteocytefilled lacunae, whereas the lacunae were empty in devitalized particulate bone (Fig. 2). Bone dust showed occasional osteocytes with predominantly
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Fig. 2. Comparison of cranial bone grafts. (Above) Gross images show that nondevitalized and devitalized particulate bone appear similar and are larger than bone dust. (Below) Histologic evaluation illustrates that particulate bone and devitalized particulate bone have equivalent architecture, in contrast to bone dust. Particulate bone contains lacunae that are osteocytefilled; devitalized particulate bone has empty lacunae. Bone dust demonstrates occasional osteocytes with predominantly empty lacunae; dark staining and altered lamellar architecture indicate thermal injury (hematoxylin and eosin; original magnification, ⫻400).
vacant lacunae and areas of darkened staining and altered lamellar structure consistent with thermal injury. Particulate bone had more viable cells by resazurin assay (0.94 percent transmittance per milligram; interquartile range, 0.78 to 1.63 percent) than devitalized particulate bone (0.007 percent; interquartile range, 0.004 to 0.017 percent) or bone dust (0.21 percent; interquartile range, 0.19 to 0.54 percent) (p ⫽ 0.01); bone dust exhibited greater viability than devitalized particulate bone (p ⫽ 0.01) (Table 1). Particulate bone had more alkaline phosphatase activity (0.13 U/ g; interquartile range, 0.11 to 0.23) than devitalized particulate graft (0.000; interquartile range, 0.000 to 0.006) or bone dust (0.06; inter-
quartile range, 0.03 to 0.08) (p ⫽ 0.01); bone dust had elevated alkaline phosphatase activity compared with devitalized particulate bone (p ⫽ 0.01). Repair of Cranial Defects Sixteen weeks after inlay cranioplasty, defects treated with particulate bone were closed, whereas those managed with no implant or with devitalized particulate bone were not healed (Fig. 3). Micro– computed tomography demonstrated that defects filled with particulate bone (group II) had greater ossification (99.7 percent; interquartile range, 98.2 to 100 percent) than those with devitalized particulate bone (group III) (42.2 percent; inter-
Table 1. Comparison of Bone Graft Viability and Osteoblast Activity (n ⴝ 5 per Group)
Viability (resazurin, % transmittance/mg) Osteoblast activity (alkaline phosphatase, U/g)
Particulate Bone (IQR)
Devitalized Particulate Bone (IQR)
Bone Dust (IQR)
p*
0.94 (0.78–1.63) 0.13 (0.11–0.23)
0.007 (0.004–0.017) 0.000 (0.000–0.006)
0.21 (0.19–0.54) 0.06 (0.03–0.08)
0.01 0.01
IQR, interquartile range. *Kruskal-Wallis test.
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Fig. 3. Healing of critical-size defects 16 weeks after inlay cranioplasty. (Above) Ectocortical view shows complete ossification with particulate bone, in contrast to defects filled with devitalized bone, which healed similarly to untreated defects. Boxes represent original 17 ⫻ 17-mm defect. (Below) Coronal micro– computed tomographic images.
quartile range, 33.4 to 46.0 percent) or no implant (group I) (40.8 percent; interquartile range, 23.2 to 45.6 percent) (p ⫽ 0.01) (Fig. 4). Critical-size defects treated with devitalized particulate bone (group III) had healing similar to that of the control (group I) (p ⫽ 0.9). Histologic evaluation of defects filled with particulate bone showed primarily woven bone that was osseointegrated with the adjacent lamellar cranium (Fig. 5). In contrast, defects that had been implanted with devitalized particulate bone contained only a rim of reactive woven bone with fibrous tissue adjacent to the native parietal bone.
DISCUSSION Cranial particulate bone graft has become the cranioplasty technique of choice at our center. In
Fig. 4. Ossification of parietal critical-size defect by micro– computed tomography 16 weeks after inlay cranioplasty. Defects filled with particulate bone exhibited greater ossification than devitalized particulate bone and control (p ⫽ 0.01). No difference was found between devitalized particulate bone and control (p ⫽ 0.9).
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the past, options for cranioplasty in young children were autologous rib/iliac graft, waiting until the patient was old enough to harvest split cranial bone, or placement of alloplastic material. Tissueengineering strategies using recombinant bone morphogenetic proteins or adipose-derived stem cells have been proposed for pediatric cranioplasty but are limited by cost, unclear efficacy, and unknown long-term safety.24 –29 Particulate bone is an ideal material for inlay cranioplasty; it can be obtained from any area of the cranium without neurosurgical assistance or donor-site morbidity. The graft is autologous, does not add material cost, and forms osseointegrated native bone (inducing a diploe¨) as thick as the surrounding calvaria.6 –10 The mechanism by which particulate bone graft heals full-thickness cranial defects is unknown. In this study, we determined that particulate bone graft primarily promotes ossification through osteogenesis by living cells within the graft. The vital assays showed that particulate bone graft contained more viable cells, including osteoblasts, than devitalized particulate bone and bone dust. Implantation of particulate bone graft resulted in fully healed calvarial defects, whereas use of devitalized particulate bone demonstrated ossification similar to defects with no implant. Although repeated freeze-thaw cycles devitalized the particulate bone of living cells as evidenced by the vital assays, the matrix architecture and histologic appearance remained intact. Thus, if osteoconduction was a significant mechanism of healing, animals treated with devitalized particu-
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Fig. 5. Histologic evaluation of grafted cranial defects 16 weeks postoperatively (hematoxylin and eosin; original magnification, ⫻20). (Left) Particulate bone has healed the critical-size defect and osseointegrated with the adjacent lamellar cranium. (Right) Devitalized particulate bone demonstrates reactive woven bone and fibrous tissue adjacent to the native parietal bone.
late bone would have been expected to ossify in a manner similar to those with fresh particulate bone implants. Without viable cells, the size of the devitalized particulate bone graft pieces likely was too small to withstand resorption and thus could not serve as a scaffold for bony ingrowth.6 In addition to osteogenesis, it is possible that other factors may influence particulate bone graft healing. Although particulate bone may contain osteoinductive proteins, the graft is unlikely to cause osteoinduction because it is not demineralized. Demineralization of bone is required for osteoinduction to occur in heterotopic sites.13–16 Recent experimental evidence suggests that circulating progenitor cells may contribute to ossification of calvarial defects.30,31 Bone dust commonly is described as fine pieces of bone generated by a power-driven tool for drilling burr holes or while harvesting split cranial bone.6,32– 42 We chose bone dust as a control for the vital assays because, although bone dust is procured from the same donor site as particulate graft, it does not heal inlay cranioplasty defects.6 The greater viability of particulate bone compared with bone dust may be attributable to thermal injury. During the procurement of bone dust, cells may be subjected to excessive heat from the high-speed drill. Because particulate bone is harvested with a slow, hand-driven brace and bit, osteoblasts are not damaged by heat and thus can survive procurement and transfer to the recipient bed. Bone dust contained more viable cells, including osteoblasts, than devitalized particulate bone, yet bone dust does not heal critical-size defects.6 This finding suggests that there
may be a minimal threshold for the number of viable cells required for bony particles to ossify by osteogenesis. Another possibility is that bone dust may possess adequate osteogenic potential, but the small size of the particles facilitates resorption by macrophages, in contrast to the larger pieces of particulate graft, which are more resistant to phagocytosis.6
CONCLUSIONS This study shows that particulate bone graft contains living cells, including osteoblasts. After devitalization, particulate bone no longer heals critical-size calvarial defects. Thus, the primary mechanism by which particulate bone graft ossifies inlay cranioplasty defects is by osteogenesis from cells within the graft. Arin K. Greene, M.D., M.M.Sc. Department of Plastic and Oral Surgery Children’s Hospital Boston 300 Longwood Avenue Boston, Mass. 02115
[email protected]
ACKNOWLEDGMENT
The authors thank the Kresge Laboratory for Pediatric Imaging Research (Children’s Hospital Boston) for use of the micro– computed tomography and image-processing software. REFERENCES 1. Gosain AK, Chim H, Arneja JS. Application-specific selection of biomaterials for pediatric craniofacial reconstruction: Developing a rational approach to guide clinical use. Plast Reconstr Surg. 2009;123:319–330.
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