CRANIOMAXILLOFACIAL DEFORMITIES/COSMETIC SURGERY
Bone Tissue Engineering by Way of Allograft Revitalization: Mechanistic and Mechanical Investigations Using a Porcine Model Christopher M. Runyan, MD, PhD,* Samantha T. Ali, BS,y Wendy Chen, MD,z Bennet W. Calder, MD,x Aaron E. Rumburg, BS,k David A. Billmire, MD,{ and Jesse A. Taylor, MD# Purpose:
‘‘Allograft revitalization’’ is a process in which cadaveric bone is used to generate wellvascularized living bone. We had previously found that porcine allograft hemimandibles filled with autologous adipose-derived stem cells (ASCs) and recombinant human bone morphogenetic protein-2–soaked absorbable collagen sponge (rhBMP-2/ACS) were completely replaced by vascularized bone, provided the construct had been incubated within a periosteal envelope. The present study sought to deepen our understanding of allograft revitalization by investigating the individual contributions of ASCs and rhBMP-2 in the process and the mechanical properties of the revitalized allograft.
Materials and Methods:
Porcine allograft hemimandible constructs were implanted bilaterally into rib periosteal envelopes in 8 pigs. To examine the contributions of ASCs and rhBMP-2, the following groups were assessed: group 1, periosteum alone; group 2, periosteum+ASCs; group 3, periosteum+rhBMP-2/ ACS; and group 4, periosteum+ASCs+rhBMP-2/ACS. After 8 weeks, the allograft constructs were harvested for micro-computed tomography (CT) and histologic analyses and 3-point bending to assess the strength.
Results:
On harvesting, the constructs receiving rhBMP-2/ACS had significantly greater bone shown by micro-CT than those receiving periosteum only (51,463 vs 34,310 mm3; P = .031). The constructs receiving ASCs had increased bone compared to group 1 (periosteum only), although not significantly (P = .087). The combination of rhBMP-2/ACS with ASCs produced bone (50,399 mm3) equivalent to that of the constructs containing rhBMP-2/ACS only. The 3-point bending tests showed no differences between the 4 groups and a nonimplanted allograft or native mandible (P = .586), suggesting the absence of decreased strength of the allograft bone when revitalized.
Conclusions: These data have shown that rhBMP-2/ACS significantly stimulates new bone formation by way of allograft revitalization and that the revitalized allograft has equivalent mechanical strength to native bone. Ó 2014 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 72:1000.e1-1000.e11, 2014
Autologous bone grafting became the standard for mandible reconstruction after reports of successful application in wounded soldiers during World War I and II.1,2
After World War II, oral cancer and odontogenic tumors became the predominant sources of critical mandibular defects. For smaller defects, cancellous bone grafts will
*Resident, Division of Plastic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH.
#Attending Surgeon, Division of Plastic Surgery, Children’s Hospital of Philadelphia, Philadelphia, PA.
yMedical Student, Division of Plastic Surgery, Cincinnati
Address correspondence and reprint requests to Dr Taylor: Perel-
Children’s Hospital Medical Center, Cincinnati, OH
man School of Medicine at the University of Pennsylvania, Division of
zResident, Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA.
Plastic Surgery, Children’s Hospital of Philadelphia, 3501 Civic Center Boulevard, 9th Floor, Philadelphia, PA 19104; e-mail: Taylorj5@
xResident, Department of Surgery, Medical University of South Carolina, Charleston, SC.
Received September 26 2013
kMedical Student, Division of Plastic Surgery, Cincinnati
Accepted January 18 2014 Ó 2014 American Association of Oral and Maxillofacial Surgeons
Children’s Hospital Medical Center, Cincinnati, OH. {Attending Surgeon, Division of Plastic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH.
email.chop.edu
0278-2391/14/00110-4$36.00/0 http://dx.doi.org/10.1016/j.joms.2014.01.017
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often be sufficient. For larger segmental deficiencies, cortical bone grafts, including allogeneic cribs, have frequently been used to provide structural integrity during bone healing. Complex cases, including those with radiated beds, soft tissue deficiencies, and large anterior defects, have been particularly challenging to repair using bone grafting. Marx3 has shown excellent results in these instances using hyperbaric oxygen therapy and soft tissue flaps as an adjuvant to bone graft-based mandible reconstruction. With the advent of microsurgery, vascularized bone flaps have largely supplanted the use of bone grafts for the reconstruction of large mandible defects.4 Comparisons of the 2 approaches have shown that bone grafting results in a much greater failure rate, in particular, with larger defects (>10 cm) and requires more operations to achieve union.5-7 By carrying their own blood supply, vascularized bone flaps will be resistant to radiation-related complications and more predictable than bone grafts for restoring function and form. However, vascularized bone flaps are not without limitations. Fibula flaps have had a 30% complication rate, including wound healing difficulties, hardware exposure, poor shape match, and donor site morbidity, including leg weakness, decreased ankle mobility, gait disturbance, and great toe contracture.8,9 An ideal reconstructive approach would provide a predictable outcome and minimize donor morbidity. Bone tissue engineering (BTE) has the potential to produce an alternative to vascularized bone flaps for repair of critical bony defects.10-13 BTE involves the use of osteogenic growth factors (eg, bone morphogenetic protein [BMP]-2), mesenchymal stem cells, and a scaffold to guide bone growth and provide support. Preclinical, large-animal studies have shown promise for healing critical defects using either bone marrow-derived stem cells (BMSCs)14-16 or BMP-2,17,18 or a combination of the 2.19,20 The results from early clinical reports have been mixed. Herford and Boyne21 reported bony union using a BMP-2–soaked collagen sponge to repair defects in 14 of 14 nonirradiated patients. Clokie and Sandor22 found similar success in 10 of 10 patients using BMP-7 with demineralized bone matrix. However, others have reported high rates of BMP-containing graft failure due to infection and fracture.10,23 One factor limiting the success of BTE-based approaches has been a lack of consensus on the best scaffold to use. Perioperative swelling with the use of BMPs will only worsen pocket collapse because of the lack of a rigid scaffold support.23 However, tenting open a soft tissue pocket with mesh or plates will increase the risk of exposure and infection. Mandible allografts have excellent potential as a scaffold for BTE. Structural allografts will have equivalent strength to native bone and can be selected to perfectly fit a defect, without donor morbidity. Numerous reports have been published of using auto- or allograft mandible as cribs filled with cancel-
lous bone for the repair of large defects; however, these have frequently failed owing to a lack of incorporation.24-28 Similar to others, we believed that were large allografts able to be revascularized, they would be the ideal scaffold for BTE.29-31 We, therefore, conducted an experiment comparing a periosteal envelope and a vascularized muscle flap for revascularizing an allograft hemimandible crib construct in pigs.32 All constructs were filled with recombinant human BMP-2/absorbable collagen sponge (rhBMP-2/ACS) and autologous adiposederived stem cells (ASCs) as a mesenchymal stem cell source. The allografts that had incubated for 2 months within a periosteal envelope had been completely replaced by large amounts of wellvascularized bone. We concluded that large bony allografts could be an effective scaffold for BTE and that vascularized periosteum would be necessary in this process. The purpose of the present study was to more fully understand this ‘ allograft revitalization’’ process, preparatory to its clinical application. We sought, using our described porcine hemimandible allograft model, to determine whether ASCs and rhBMP-2 are necessary for the process, and whether the bone produced by allograft revitalization would be mechanically sound. We hypothesized that both ASCs and rhBMP-2 would contribute to allograft revitalization and that the bone produced would be mechanically equivalent to native bone.
Materials and Methods ALLOGRAFT PREPARATION AND ANIMAL HUSBANDRY
Mandibles were harvested from 6-week-old Yorkshire/Hampshire pigs. The soft tissues were removed, the teeth extracted, and the mandibles bisected at the symphysis. The hemimandibles were then sent for allograft processing, including detergent and alcohol washes,33 sterility validation, packaging, and freeze drying (Veterinary Transplant Services, Kent, WA). The processed hemimandibles were stored at 20 C until implantation. Eight additional female Yorkshire/ Hampshire pigs were used as the allograft recipients. The pigs were housed in a standard 12-hour light/ dark-cycle room, with twice daily swine chow and ad lib access to water. The pigs were fasted for at least 12 hours before all surgical procedures. The Cincinnati Children’s Hospital Medical Center Institutional Animal Care and Use Committee approved and monitored all procedures (approval no. 1D03029). LIPOSUCTION
At 6.5 weeks of age, select pigs (n = 4) were anesthetized with intramuscular ketamine (30 mg/kg) and intramuscular acepromazine (1.1 mg/kg) and
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maintained under general endotracheal anesthesia with isoflurane. Approximately 150 mL of tumescent anesthetic (40 mL of 1% lidocaine with 1:100,000 epinephrine in 1 L of saline) was infused in each of 4 locations: bilateral abdomen and bilateral flanks. A 4F liposuction cannula was used to collect the lipoaspirate. The incisions were closed in a single layer, and intramuscular buprenorphine (0.05 to 0.10 mg/kg) was administered postoperatively for analgesia. ASC ISOLATION, CULTURE, AND HISTOLOGIC EXAMINATION
The lipoaspirate tissue and fluid were processed to obtain ASCs, as previously described.32 The ASCs were cultured in stromal media containing 199 media (HyClone; Thermo Fisher Scientific, Waltham, MA), 10% fetal bovine serum (FBS; USA Scientific, Ocala, FL), 1 antibiotic-antimycotic (no. 15420; Gibco, Life Technologies, Thermo Fisher Scientific). The processed lipoaspirate cells from the individual pigs were plated in separate 75-cm2 flasks, allowed to adhere for 48 hours, and rinsed with fresh media to remove debris and nonadherent cells. The cells were passaged on reaching near confluency (85%) using trypsinization (0.05% trypsin, no. 25300; Gibco) and split 1:2 or 1:3 to 225-cm2 flasks. Culture of the ASCs continued for 3 passages. On the day of implantation, all cells were harvested by trypsinization, washed, and resuspended in 1 mL of stromal media lacking FBS for autologous loading of the allograft constructs. Undifferentiated ASCs were subjected to different culture media to assess their multipotency, as described previously.34-36 The base media used was Dulbecco’s modified Eagle medium+GlutaMAX media (Gibco) containing 10% FBS and 1% antibioticantimycotic. Osteogenic media also contained 10mM b-glycerophosphate (G9422; Sigma-Aldrich, St. Louis, MO) and 250-mM ascorbic acid (A5960; SigmaAldrich). Adipogenic differentiation also contained 10-mM insulin (91077C; Sigma-Aldrich), 1-mM dexamethasone (D4902; Sigma-Aldrich), 0.5-mM isobutylmethylxanthine (I5879; Sigma-Aldrich), and 200-mM indomethacin (D4902; Sigma-Aldrich). The chondrogenic media also contained 6.25-mg/mL insulin, 10 ng/mL transforming growth factor-b1 (T4567; SigmaAldrich), and 50-nM ascorbate-2-phosphate (A8960; Sigma-Aldrich). At their fourth passage, the ASCs were plated at a density of 100,000 cells/well in a 6well tissue culture plate and switched to the above differentiation media. The media were changed twice weekly for a 2-week period before staining. Histologic stains were performed using Alizarin Red (A5533), silver nitrate (209139), Oil-Red O (O0625) and Alcian Blue (A3157) reagents, per the manufacturer’s (SigmaAldrich) recommendations.
BONE TISSUE ENGINEERING BY ALLOGRAFT REVITALIZATION ALLOGRAFT IMPLANTATION AND HARVEST OPERATIONS
At 8 weeks of age, each pig (n = 8) received bilateral hemimandible allograft constructs, implanted within a periosteal envelope made by the extraction of 2 adjacent ribs. The allograft construct assembly was nearly identical to our previously reported technique.32 In brief, absorbable collagen sponges (ACSs; Medtronic, Minneapolis, MN) were soaked in 8 mL of rhBMP-2 solution (0.5 mg/mL, Infuse, Medtronic) for 15 to 45 minutes, cut into 0.5-cm-wide strips, and placed within the hemimandible crib. Autologous ASCs (500 mL/ hemimandible) were then pipetted onto the collagen sponges and covered with additional rhBMP-2–soaked ACSs. The 8 pigs were divided into 4 groups (2 pigs and 4 implants per group) according to the composition of the allograft construct (Table 1). Group 1 received allograft hemimandibles containing only ACS. The allografts in group 2 were filled with rhBMP-2–soaked ACS. In group 3, the allografts were filled with ACS and autologous ASCs. Finally, in group 4, the allografts were filled with both ASCs and rhBMP2-soaked ACS. All the grafts were implanted in a rib periosteal envelope (bilaterally in each pig) and allowed to incubate for 7 weeks. The implantation operations were performed with the pigs in the supine position, with anesthesia as described for liposuction. A 14-cm incision was made on the right thorax parallel to and between ribs 5 and 6, and the ribs were removed subperiosteally for a distance of 14 cm. The interval between the ribs was excluded from the periosteal sheaths, and the 2 sheaths were sewn around the thoracic constructs. The thoracic wall was then closed in layered fashion using interrupted, absorbable suture. The pigs were administered prophylactic intramuscular penicillin G, and intramuscular buprenorphine (0.05 to 0.10 mg/kg) was administered postoperatively for analgesia. After a 7- to 8-week incubation, the pigs were anesthetized using intramuscular ketamine and acepromazine and sacrificed using 85 mg/kg of intravenous Fatal Plus (Vortech Pharmaceuticals, Dearborn, MI). The allograft constructs were harvested with the surrounding soft tissue structures (eg, periosteal sheath with intercostal muscle and native rib sections). The constructs were fixed immediately in formalin for processing and histologic evaluation. ALLOGRAFT CONSTRUCT IMAGING
The harvested constructs were scanned using a Concorde MicroCAT II micro-computed tomography (CT) system (Imtek, Knoxville, TN) in 3 scans and digitally assembled for analysis using Amira, version 5, software (Visage Imaging, San Diego, CA). Calcific tissue densities were calculated by measuring the saturated
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Table 1. PIG WEIGHTS AT SURGERY, LIPOASPIRATE VOLUMES, AND IMPLANTED CELL COUNTS
Weight (kg)
Pig No. 1 2 3 4 5 6 7 8
Age* (days)
ASCs
rhBMP-2
Lipoaspirate Volume (mL)
Cells Implanted (n)
Liposuction
Allograft Implantation
Allograft Harvest
62 62 62 62 61 61 61 61
No No Yes Yes No No Yes Yes
No No No No Yes Yes Yes Yes
— — 85 155 — — 95 125
— — 3.13 107 3.18 107 — — 3.18 107 3.33 107
— — 15.0 15.0 — — 15.4 13.6
17.7 16.4 22.7 25.2 17.0 14.0 12.0 22.0
67.9 68.6 74.5 77.7 51.0 NAy 65.9 49.0
Dashes indicate absence of liposuction operations for these pigs. Abbreviations: ASCs, adipose-derived stem cells; NA, not applicable; rhBMP-2, recombinant human bone morphogenetic protein-2. * Age at implantation of allograft. y Pig 6 was found dead 4 weeks into the experiment, with the autopsy findings negative for cardiopulmonary pathologic features. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
pixels (lower threshold set at 800 for trabecular bone) on the micro-CT scans. The adjoining ribs were digitally cropped from the images before this calculation. The calculated volumes of calcific tissue in the constructs from the different groups were compared using paired Student t tests. HISTOLOGIC EVALUATION
The harvested constructs were fixed in 10% formalin, and the soft tissues were manually removed to facilitate mechanical testing centrally, with strips of soft tissue left intact for imaging of the axial sections. Representative sections from each construct were sent to Flagship Biosciences (Aurora, CO) for processing and embedding in glycol methacrylate (GMA) for nondecalcified histologic evaluation. Standard hematoxylin and eosin (H&E) and trichrome stains were performed on each section. To identify the retained allograft, nondecalcified GMA-embedded sections were stained with 40 ,6diamidino-2-phenylindolesolution (Prolong Gold with DAPI; Molecular Probes, Eugene, OR), which stains the nuclei of living cells. Two or three slides per sample, constituting an entire axial section, were imaged and digitally scanned at 20 magnification using a slide scanner and automated Aperio software (Aperio, Vista, CA). To quantify the amount of retained allograft, the images were divided into 16 equal grids and manually examined for the presence of DAPInegative (dead allograft) bone. The percentage of these 16 grids containing any DAPI-negative bone was calculated for each sample and compared among the groups using the Student t test.
MECHANICAL TESTING
Using a band saw, the 5-cm central portion from each graft was trimmed into 3 blocks, 50 10 10 mm, of nondecalcified bone. Nonimplanted allografts and adult porcine native mandibles were prepared in the same manner. Immediately before testing, the sections were maintained in saline to prevent dehydration. Mechanical testing was accomplished using an Instron 8501 Universal Mechanical Testing System (Instron, Norwood, MA). During the 3-point bending test, the specimen was placed on the lower supports of the bending apparatus. Each specimen was positioned such that the lower supports were exactly 10 mm from each end, with 30 mm of the specimen unsupported between the supports. An initial preload (1 N) was applied to the center of the allograft, at a point 15 mm from each of the lower supports, and a displacement of 15 mm was applied at a rate of 5.0 mm/second until failure. Load-deflection data were collected using Instron software, and failure was determined as the maximum load recorded immediately before an absolute decrease in load. The mean failure strengths of the samples were compared using analysis of variance (SYSTAT 13; Systat Software, Chicago, IL).
Results Harvested porcine ASCs had previously been found to express mesenchymal stem cell markers CD34, CD90, and CD105, but not the hematopoietic marker CD45.32 To assess their multipotency, we subjected them to different mesenchymal differentiation media in vitro and found that they could differentiate into
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FIGURE 1. Adipocyte stem cell (ASC) differentiation in vitro. ASCs exposed to osteogenic differentiation media were subjected to different stains. A, von Kossa staining indicated extracellular matrix calcification in osteogenically differentiated cells (6.3). B, Positive Alizarin Red staining indicating calcium deposits in terminally differentiated osteogenic cells (2). C, Oil-Red O staining in adipogenic-differentiated ASCs showing intracellular lipid accumulation (12). D, Chondrogenically differentiated ASCs stained positive for Alcian Blue, indicating the presence of sulfated proteoglycans in a cartilaginous matrix (4). ASCs not exposed to differentiation media were negative for these stains (data not shown). A Leica M165 FC microscope (Leica Microsystems, Wetzlar, Germany) was used at the stated magnifications for these images. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
all mesenchymal cell lines tested, including chondrocytes, adipocytes, and osteoblasts (Fig 1). The pigs were divided into 4 groups (Table 1) according to the composition of the allograft constructs: group 1, incubated within rib periosteal envelope without the addition of ASCs and rhBMP-2/ACS (hereafter rhBMP-2); group 2, periosteum and autologous ASCs; group 3, periosteum and rhBMP-2; and group 4, periosteum, ASCs, and rhBMP-2. All pigs tolerated liposuction and construct implantation (Fig 2) without difficulty. One pig from group 3 was found dead 4 weeks after allograft placement; however, this death was not related to the grafts or previous surgery. On harvest, the grafts from all groups were found to have fusion with the cut ends of the ribs (Fig 3A). By both gross examination and micro-CT, the grafts from groups 2 and 3 subjectively had fewer surface irregularities and greater
bone production than did group 1. Gross axial sections of the harvested grafts showed a cortical rim of bone surrounding predominately cancellous bone with small, usually central, areas of fibrous tissue (Fig 3B). The H&E sections showed marrow-rich cancellous bone on the axial sections in all groups. To compare the volume of bone generated among the different groups, the calcific tissue volumes were obtained from the micro-CT images. Only the bone between the proximal and distal ends of the original graft was included to exclude the surrounding fused rib. Group 2 (44,499 mm3) and group 3 (51,463 mm3) both had increased bone production compared with group 1 (34,310 mm3; Fig 4). However, this difference was statistically significant for group 3 (P = .031), but not for group 2 (P = .087), suggesting a greater role for rhBMP-2 than for ASCs in the process. The group 4
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FIGURE 2. Allograft construct assembly. The upper 2 images show superior views of the porcine allograft hemimandible with the A, medullary cavity, which was then filled B, with absorbable collagen sponge (ACS, red arrowheads), with or without recombinant human bone morphogenetic protein-2 (rhBMP-2) and autologous adipose-derived stem cells (ASCs). The constructs in groups 2 and 4 received ASCs and those in groups 3 and 4 received rhBMP-2/ACS, as described in the ‘‘Materials and Methods’’ section. C, Resection of 2 adjacent ribs was then performed in young pigs bilaterally, forming periosteal pockets. D, The allograft constructs were placed within the periosteal envelope, with the periosteum and intercostal muscles to be closed over the allograft, completely encasing the graft. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
constructs (50,399 mm3) also had greater bone production than group 1 (P = .032), but essentially equal to that of group 3, suggesting no synergistic effect between rhBMP-2 and ASCs for bone production in this model. On both CT and histologic analysis, some of the samples appeared to have some retained allograft. To quantify this, axial sections from each sample were stained with DAPI, which binds living nuclei. Entire sections were stained, scanned, divided into a grid of 16 images, and manually scored for the presence of dead bone. Nearly 14% of the grids from the group 1 sections contained some retained allograft bone (Fig 5). rhBMP-2–treated grafts had less retained allograft (1.5% for group 3 and 0.86% for group 4); however, these differences were not statistically significant, given the wide distribution for group 1. To assess the mechanical strength of the harvested bone, three 1 1 5-cm cores of bone from each sample were subjected to the 3-point bending test. The
4 experimental groups were compared with native mandible and nonimplanted mandibular allograft (Fig 6). With 3-point bending, all samples fractured at the midpoint, and analysis of variance (P = .578) showed that the failure load of the allograft bone did not differ significantly among the treatment groups and the nonimplanted allograft (Fig 7). Neither the side of the pig nor the individual pig variables were found to have a significant effect (P = .405, side; P = .888, pig) on failure load.
Discussion The present study showed that rhBMP-2 significantly increased the volume of bone produced using the allograft revitalization technique. This was consistent with other reports,20,21 suggesting rhBMP-2 could be an important ingredient when using allograft revitalization for the repair of critical bony defects. We also observed a trend (P = .087) toward increased
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FIGURE 3. A, Gross and micro-computed tomography (CT) findings of harvested grafts. Representative grafts harvested from each group are shown. Top Row, The soft tissue was removed from the gross samples, with the exception of 2 strips that were left for histologic purposes. All samples had fusion with the adjoining 2 ribs. Little heterotopic bone was found in the surrounding soft tissues. Bottom Row, An examination of the micro-CT scans of these samples showed increased bone formation, in particular, within the center of the construct, in the samples receiving recombinant human bone morphogenetic protein-2 (rhBMP-2). Scale bars measure 1 cm. B, Cross-sectional findings of harvested grafts. Top Row, Gross and Middle, micro-CT axial cuts of the harvested constructs showing a cortical rim of bone predominately surrounding cancellous bone. Centrally, all constructs contained areas of fibrous tissue devoid of bone, possibly corresponding with the allograft medullary canal, which was subjectively larger in group 1 than in the other groups. Bottom Row, Hematoxylin-eosin–stained sections of the boxed areas confirmed the presence of marrow-rich cancellous bone in all 4 groups (magnification 4). ASCs, adipose-derived stem cells. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
bone produced with the addition of ASCs. The lack of statistical significance for the addition of ASCs might have resulted from an actual lack of biologic contribution of the ASCs or our small sample size (n = 4 per group). ASCs were selected for this model, because they have osteogenic properties comparable to those of BMSCs and are more readily available than BMSCs,
given the absence of a difference in the numbers of stem cells found in equivalent volumes of bone marrow and lipoaspirate.37,38 Given that the ASCs showed osteogenic capacity in vitro, it is likely that the addition of ASCs might contribute to the process, although our study was underpowered to detect this difference. However, the lack of synergism when both
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FIGURE 4. The volume of calcific tissue of harvested grafts was calculated from the micro-CT scans. Groups 2 to 4 all had increased bone compared with the periosteum-only group (group 1). However, this was only significant for grafts receiving recombinant human bone morphogenetic protein-2 (rhBMP-2; groups 3 and 4). The number of samples (n) per group is depicted just above the x-axis. Error bars represent the standard deviation. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
rhBMP-2 and ASCs were added suggests a limited benefit for ASCs greater than the effect of rhBMP-2 in our model. Therefore, we cannot recommend including or withholding ASCs from allograft constructs when applying this clinically. Future study, including fate mapping of ASCs, would help elucidate their contribution, including whether they provide a direct or indirect paracrine benefit, as suggested.39 Similar to our previous findings, the bone generated in this model was larger than the implanted graft, more closely resembling 2 conjoined ribs rather than a hemimandible. Additionally, the inner face of the graft in contact with the intercostal muscle suture line did not revitalize as well as that directly apposed to the periosteum. This suggests that intimate contact of the graft with vascularized periosteum is helpful for graft desorption and replacement and that periosteal support helps determine the shape of the bone produced. We also observed that all regenerated bone was contained within the confines of the periosteal sleeve, suggesting that the periosteum might act as an important barrier to heterotopic ossification in allograft revitalization. We also found that although the harvested explants were predominately well-vascularized cancellous bone, there were small areas of dead, nonresorbed allograft present. This is in contrast to our previous experiment in which no retained allograft was found. We attributed this change to differences in allograft processing, because no other differences were present in our model. Our initial study used a commercial allograft preparation process that had included treatment with proprietary detergents and sterilants under oscil-
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FIGURE 5. Histomorphometric percentage of nonabsorbed allograft in harvested grafts showing percentages of each sample containing DAPI-negative (dead allograft) bone. A decreased percentage of retained allograft was found in grafts receiving autologous adipose-derived stem cells (ASCs) and/or recombinant human bone morphogenetic protein-2 (rhBMP-2; groups 2 to 4); however, this was not statistically significant, given the wide variability in the periosteum-only group (group 1). Error bars depict standard deviations. Statistical testing used unpaired Student’s t tests for comparison of group 1 to all other groups, individually. The number for each group was the same as in Figure 3. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
lating positive and negative pressure (Biocleanse, RTI Biologics, Alachua, FL). This was not an option for the present study. Instead, the harvested mandibles were prepared by an allograft processing service (Veterinary Transplant Services) using a published protocol that included detergent, alcohol, and water washes in a sonicator.33 We speculated that the former process was more rigorous and that the degree of graft processing might correlate temporally with absorption in vivo. It was also interesting that the graft constructs loaded with autologous ASCs and rhBMP-2 had less retained allograft (albeit not significantly), suggesting more rapid graft absorption as a part of the process of new bone generation in our model. Three-point bending is an established model for mechanical testing of bone.40 This model allowed us to test multiple cores from each sample while preserving nonfractured portions for histologic examination. With most types of preparations, allograft bone will have mechanical strength equivalent to that of native bone.41-43 Our data have confirmed this. Furthermore, we found no differences in strength among the unimplanted allograft, native mandible, and harvested grafts. Thus, the bone produced by way of allograft revitalization had strength comparable to that of native bone, at least in the short term. This might not reflect the long-term fate of revitalized bone; however, we believe the comparable strength will continue as the new bone remodels over time. Furthermore, our model did not examine the effects of loading on the allograft
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FIGURE 6. Mechanical testing experimental design. From each sample, three 1 1 5-cm sections of the graft were cut with a band saw. A, The location of these ‘‘cores’’ was standardized between all the samples, with 2 cores taken from the lateral aspect and 1 from the central aspect (purple boxes). B, The cores were then stressed to fracture on a 3-point bending rig. The number of Newtons of force required for fracture was measured for each individual core, and all the cores for each group were combined for the intergroup analysis of variance statistical analysis. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
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mote allograft revascularization. Perhaps the most beneficial application would be for repair of mandibular or maxillofacial defects using a local periosteal flap,49 because this would obviate the need for free tissue transfer.
References
FIGURE 7. Force required to fracture mandible cores during the 3point mechanical testing depicted in Figure 5. The box plots display the force required to achieve fracture for the cores of bone harvested from each of the 4 groups and, for comparison, from both native porcine mandible and unimplanted porcine allograft mandible. The boxes display the range of the 25th to 75th percentiles of the samples for each group. The horizontal lines within each box indicate the 50th percentile (mean) for each group. The error bars represent the standard deviation. Using analysis of variance, no difference in the force required to achieve fracture was found among the groups (P = .578). Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
constructs. During bone fracture healing, mechanical loads have been shown to improve strength as the bone remodels,44 and we hypothesized that a load applied to allograft would further stimulate the process of revitalization. Importantly, the histologic finding of revitalized bone throughout the construct was in contrast to that of explanted structural allograft alone. Enneking and Mindell,45 Enneking and Campanacci,46 and Wheeler and Enneking47 reported that the late clinical failures in the axial skeleton were due to chronic stress on a nonrevitalized, mineral scaffold whose ability to remodel and regenerate was limited. In summary, a large cadaveric allograft might normally be expected to fail when used for reconstruction; however, when revitalized, it can be expected to function as normal bone, including the ability to heal when fractured and to resist infection. We believe these data can meaningfully guide the clinical application of allograft revitalization. The next step will be preclinical animal modeling to test whether the revitalized allograft is effective in repairing a critical defect and whether it is equivalent to the reference standard, the vascularized autograft. Our data suggest that rhBMP-2 and vascularized periosteum will be key ingredients for the process. If efficacious in animal models, allograft revitalization could be considered for repair of critical defects of the craniofacial and axial skeleton. One can envision its use as a reliable method of prelaminating a bony free flap for future transfer to fill a defect or to repair a critical defect in situ using a free periosteal flap48 to pro-
1. Cole P, Bubb CH: Bone-grafting in ununited fractures of the mandible, with special reference to the pedicle graft. Proc R Soc Med 12:13, 1919 2. Blocker TG, Weiss LR: Use of cancellous bone in the repair of defects around the jaws. Ann Surg 123:622, 1946 3. Marx RE: Current advances in reconstruction of the mandible in head and neck cancer surgery. Semin Surg Oncol 7:47, 1991 4. Hidalgo DA: Fibula free flap mandibular reconstruction. Clin Plast Surg 21:25, 1994 5. Pogrel MA, Podlesh S, Anthony JP, Alexander J: A comparison of vascularized and nonvascularized bone grafts for reconstruction of mandibular continuity defects. J Oral Maxillofac Surg 55:1200, 1997 6. Foster RD, Anthony JP, Sharma A, Pogrel MA: Vascularized bone flaps versus nonvascularized bone grafts for mandibular reconstruction: An outcome analysis of primary bony union and endosseous implant success. Head Neck 21:66, 1999 7. August M, Tompach P, Chang Y, Kaban L: Factors influencing the long-term outcome of mandibular reconstruction. J Oral Maxillofac Surg 58:731, 2000 8. Momoh AO, Yu P, Skoracki RJ, et al: A prospective cohort study of fibula free flap donor-site morbidity in 157 consecutive patients. Plast Reconstr Surg 128:714, 2011 9. Lopez-Arcas JM, Arias J, Del Castillo JL, et al: The fibula osteomyocutaneous flap for mandible reconstruction: A 15-year experience. J Oral Maxillofac Surg 68:2377, 2010 10. Warnke PH, Wiltfang J, Springer I, et al: Man as living bioreactor: Fate of an exogenously prepared customized tissue-engineered mandible. Biomaterials 27:3163, 2006 11. Zuk PA: Tissue engineering craniofacial defects with adult stem cells? Are we ready yet? Pediatr Res 63:478, 2008 12. Torroni A: Engineered bone grafts and bone flaps for maxillofacial defects: State of the art. J Oral Maxillofac Surg 67:1121, 2009 13. Zaky SH, Cancedda R: Engineering craniofacial structures: Facing the challenge. J Dent Res 88:1077, 2009 14. Abukawa H, Shin M, Williams WB, et al: Reconstruction of mandibular defects with autologous tissue-engineered bone. J Oral Maxillofac Surg 62:601, 2004 15. He Y, Zhang ZY, Zhu HG, et al: Experimental study on reconstruction of segmental mandible defects using tissue engineered bone combined bone marrow stromal cells with three-dimensional tricalcium phosphate. J Craniofac Surg 18:800, 2007 16. Yuan J, Cui L, Zhang WJ, et al: Repair of canine mandibular bone defects with bone marrow stromal cells and porous betatricalcium phosphate. Biomaterials 28:1005, 2007 17. Carstens MH, Chin M, Li XJ: In situ osteogenesis: Regeneration of 10-cm mandibular defect in porcine model using recombinant human bone morphogenetic protein-2 (rhBMP-2) and Helistat absorbable collagen sponge. J Craniofac Surg 16:1033, 2005 18. Marukawa E, Asahina I, Oda M, et al: Functional reconstruction of the non-human primate mandible using recombinant human bone morphogenetic protein-2. Int J Oral Maxillofac Surg 31: 287, 2002 19. Seto I, Asahina I, Oda M, Enomoto S: Reconstruction of the primate mandible with a combination graft of recombinant human bone morphogenetic protein-2 and bone marrow. J Oral Maxillofac Surg 59:53, 2001 20. Seto I, Marukawa E, Asahina I: Mandibular reconstruction using a combination graft of rhBMP-2 with bone marrow cells expanded in vitro. Plast Reconstr Surg 117:902, 2006 21. Herford AS, Boyne PJ: Reconstruction of mandibular continuity defects with bone morphogenetic protein-2 (rhBMP-2). J Oral Maxillofac Surg 66:616, 2008
1000.e11 22. Clokie CM, Sandor GK: Reconstruction of 10 major mandibular defects using bioimplants containing BMP-7. J Can Dent Assoc 74:67, 2008 23. Carter TG, Brar PS, Tolas A, Beirne OR: Off-label use of recombinant human bone morphogenetic protein-2 (rhBMP-2) for reconstruction of mandibular bone defects in humans. J Oral Maxillofac Surg 66:1417, 2008 24. Harding RL: Replantation of the mandible in cancer surgery. Plast Reconstr Surg (1946) 19:373, 1957 25. Hamaker RC, Singer MI: Irradiated mandibular autografts update. Arch Otolaryngol Head Neck Surg 112:277, 1986 26. Leipzig B, Cummings CW: The current status of mandibular reconstruction using autogenous frozen mandibular grafts. Head Neck Surg 6:992, 1984 27. Rochanawutanon S, Suddhasthira T, Pairuchvej V, Vajaradul Y: Long term follow-up of reconstruction with allogeneic mandibular bone crib packed with autogenous particulate cancellous bone marrow. Cell Tissue Bank 3:183, 2002 28. Cantu G, Bimbi G, Colombo S, et al: Autologous freeze-treated bone for mandibular reconstruction after malignant tumor resection: A study of 72 patients. Am J Otolaryngol 30:383, 2009 29. Follmar KE, Prichard HL, DeCroos FC, et al: Combined bone allograft and adipose-derived stem cell autograft in a rabbit model. Ann Plast Surg 58:561, 2007 30. Frohlich M, Grayson WL, Wan LQ, et al: Tissue engineered bone grafts: Biological requirements, tissue culture and clinical relevance. Curr Stem Cell Res Ther 3:254, 2008 31. Hoffman MD, Benoit DS: Emerging ideas: Engineering the periosteum: Revitalizing allografts by mimicking autograft healing. Clin Orthop Relat Res 471:721, 2013 32. Runyan CM, Jones DC, Bove KE, et al: Porcine allograft mandible revitalization using autologous adipose-derived stem cells, bone morphogenetic protein-2, and periosteum. Plast Reconstr Surg 125:1372, 2010 33. Beebe KS, Benevenia J, Tuy BE, et al: Effects of a new allograft processing procedure on graft healing in a canine model: A preliminary study. Clin Orthop Relat Res 467:273, 2009 34. Zuk PA, Zhu M, Ashjian P, et al: Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13:4279, 2002 35. Yoshimura K, Shigeura T, Matsumoto D, et al: Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol 208:64, 2006
BONE TISSUE ENGINEERING BY ALLOGRAFT REVITALIZATION 36. Gupta DM, Panetta NJ, Longaker MT: Osteogenic differentiation of human multipotent mesenchymal stromal cells. Methods Mol Biol 698:201, 2011 37. Dragoo JL, Choi JY, Lieberman JR, et al: Bone induction by BMP-2 transduced stem cells derived from human fat. J Orthop Res 21: 622, 2003 38. De Ugarte DA, Morizono K, Elbarbary A, et al: Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 174:101, 2003 39. Zuk PA: The ASC: Critical Participants in Paracrine-Mediated Tissue Health and Function. In Andrades JA (ed): Regenerative Medicine and Tissue Engineering, 2013. Available at: http://www. intechopen.com/books/regenerative-medicine-and-tissueengineering/the-asc-critical-participants-in-paracrine-mediatedtissue-health-and-function. Accessed October 15, 2013. 40. Russell NA, Rives A, Pelletier MH, et al: The effect of sterilization on the mechanical properties of intact rabbit humeri in threepoint bending, four-point bending and torsion. Cell Tissue Bank 14:231, 2013 41. Mroz TE, Lin EL, Summit MC, et al: Biomechanical analysis of allograft bone treated with a novel tissue sterilization process. Spine J 6:34, 2006 42. Mikhael MM, Huddleston PM, Zobitz ME, et al: Mechanical strength of bone allografts subjected to chemical sterilization and other terminal processing methods. J Biomech 41:2816, 2008 43. Balsly CR, Cotter AT, Williams LA, et al: Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts. Cell Tissue Bank 9:289, 2008 44. Ulstrup AK: Biomechanical concepts of fracture healing in weight-bearing long bones. Acta Orthop Belg 74:291, 2008 45. Enneking WF, Mindell ER: Observations on massive retrieved human allografts. J Bone Joint Surg Am 73:1123, 1991 46. Enneking WF, Campanacci DA: Retrieved human allografts: A clinicopathological study. J Bone Joint Surg Am 83-A:971, 2001 47. Wheeler DL, Enneking WF: Allograft bone decreases in strength in vivo over time. Clin Orthop Relat Res 435:36, 2005 48. Maercks RA, Runyan CM, Jones DC, Taylor JA: The vastus intermedius periosteal (VIP) flap: A novel flap for osteoinduction. J Reconstr Microsurg 26:335, 2010 49. Patel PA, Chen W, Wilkening MW, et al: Extended composite temporoparietal fascial flap: Clinical implications for tissue engineering in mandibular reconstruction. J Craniofac Surg 24:273, 2013
Bone Tissue Engineering by Way of Allograft Revitalization: Mechanistic and Mechanical Investigations Using a Porcine Model Christopher M. Runyan, MD, PhD,* Samantha T. Ali, BS,y Wendy Chen, MD,z Bennet W. Calder, MD,x Aaron E. Rumburg, BS,k David A. Billmire, MD,{ and Jesse A. Taylor, MD# Purpose:
‘‘Allograft revitalization’’ is a process in which cadaveric bone is used to generate wellvascularized living bone. We had previously found that porcine allograft hemimandibles filled with autologous adipose-derived stem cells (ASCs) and recombinant human bone morphogenetic protein-2–soaked absorbable collagen sponge (rhBMP-2/ACS) were completely replaced by vascularized bone, provided the construct had been incubated within a periosteal envelope. The present study sought to deepen our understanding of allograft revitalization by investigating the individual contributions of ASCs and rhBMP-2 in the process and the mechanical properties of the revitalized allograft.
Materials and Methods:
Porcine allograft hemimandible constructs were implanted bilaterally into rib periosteal envelopes in 8 pigs. To examine the contributions of ASCs and rhBMP-2, the following groups were assessed: group 1, periosteum alone; group 2, periosteum+ASCs; group 3, periosteum+rhBMP-2/ ACS; and group 4, periosteum+ASCs+rhBMP-2/ACS. After 8 weeks, the allograft constructs were harvested for micro-computed tomography (CT) and histologic analyses and 3-point bending to assess the strength.
Results:
On harvesting, the constructs receiving rhBMP-2/ACS had significantly greater bone shown by micro-CT than those receiving periosteum only (51,463 vs 34,310 mm3; P = .031). The constructs receiving ASCs had increased bone compared to group 1 (periosteum only), although not significantly (P = .087). The combination of rhBMP-2/ACS with ASCs produced bone (50,399 mm3) equivalent to that of the constructs containing rhBMP-2/ACS only. The 3-point bending tests showed no differences between the 4 groups and a nonimplanted allograft or native mandible (P = .586), suggesting the absence of decreased strength of the allograft bone when revitalized.
Conclusions: These data have shown that rhBMP-2/ACS significantly stimulates new bone formation by way of allograft revitalization and that the revitalized allograft has equivalent mechanical strength to native bone. Ó 2014 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 72:1000.e1-1000.e11, 2014
Autologous bone grafting became the standard for mandible reconstruction after reports of successful application in wounded soldiers during World War I and II.1,2
After World War II, oral cancer and odontogenic tumors became the predominant sources of critical mandibular defects. For smaller defects, cancellous bone grafts will
*Resident, Division of Plastic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH.
#Attending Surgeon, Division of Plastic Surgery, Children’s Hospital of Philadelphia, Philadelphia, PA.
yMedical Student, Division of Plastic Surgery, Cincinnati
Address correspondence and reprint requests to Dr Taylor: Perel-
Children’s Hospital Medical Center, Cincinnati, OH
man School of Medicine at the University of Pennsylvania, Division of
zResident, Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA.
Plastic Surgery, Children’s Hospital of Philadelphia, 3501 Civic Center Boulevard, 9th Floor, Philadelphia, PA 19104; e-mail: Taylorj5@
xResident, Department of Surgery, Medical University of South Carolina, Charleston, SC.
Received September 26 2013
kMedical Student, Division of Plastic Surgery, Cincinnati
Accepted January 18 2014 Ó 2014 American Association of Oral and Maxillofacial Surgeons
Children’s Hospital Medical Center, Cincinnati, OH. {Attending Surgeon, Division of Plastic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH.
email.chop.edu
0278-2391/14/00110-4$36.00/0 http://dx.doi.org/10.1016/j.joms.2014.01.017
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often be sufficient. For larger segmental deficiencies, cortical bone grafts, including allogeneic cribs, have frequently been used to provide structural integrity during bone healing. Complex cases, including those with radiated beds, soft tissue deficiencies, and large anterior defects, have been particularly challenging to repair using bone grafting. Marx3 has shown excellent results in these instances using hyperbaric oxygen therapy and soft tissue flaps as an adjuvant to bone graft-based mandible reconstruction. With the advent of microsurgery, vascularized bone flaps have largely supplanted the use of bone grafts for the reconstruction of large mandible defects.4 Comparisons of the 2 approaches have shown that bone grafting results in a much greater failure rate, in particular, with larger defects (>10 cm) and requires more operations to achieve union.5-7 By carrying their own blood supply, vascularized bone flaps will be resistant to radiation-related complications and more predictable than bone grafts for restoring function and form. However, vascularized bone flaps are not without limitations. Fibula flaps have had a 30% complication rate, including wound healing difficulties, hardware exposure, poor shape match, and donor site morbidity, including leg weakness, decreased ankle mobility, gait disturbance, and great toe contracture.8,9 An ideal reconstructive approach would provide a predictable outcome and minimize donor morbidity. Bone tissue engineering (BTE) has the potential to produce an alternative to vascularized bone flaps for repair of critical bony defects.10-13 BTE involves the use of osteogenic growth factors (eg, bone morphogenetic protein [BMP]-2), mesenchymal stem cells, and a scaffold to guide bone growth and provide support. Preclinical, large-animal studies have shown promise for healing critical defects using either bone marrow-derived stem cells (BMSCs)14-16 or BMP-2,17,18 or a combination of the 2.19,20 The results from early clinical reports have been mixed. Herford and Boyne21 reported bony union using a BMP-2–soaked collagen sponge to repair defects in 14 of 14 nonirradiated patients. Clokie and Sandor22 found similar success in 10 of 10 patients using BMP-7 with demineralized bone matrix. However, others have reported high rates of BMP-containing graft failure due to infection and fracture.10,23 One factor limiting the success of BTE-based approaches has been a lack of consensus on the best scaffold to use. Perioperative swelling with the use of BMPs will only worsen pocket collapse because of the lack of a rigid scaffold support.23 However, tenting open a soft tissue pocket with mesh or plates will increase the risk of exposure and infection. Mandible allografts have excellent potential as a scaffold for BTE. Structural allografts will have equivalent strength to native bone and can be selected to perfectly fit a defect, without donor morbidity. Numerous reports have been published of using auto- or allograft mandible as cribs filled with cancel-
lous bone for the repair of large defects; however, these have frequently failed owing to a lack of incorporation.24-28 Similar to others, we believed that were large allografts able to be revascularized, they would be the ideal scaffold for BTE.29-31 We, therefore, conducted an experiment comparing a periosteal envelope and a vascularized muscle flap for revascularizing an allograft hemimandible crib construct in pigs.32 All constructs were filled with recombinant human BMP-2/absorbable collagen sponge (rhBMP-2/ACS) and autologous adiposederived stem cells (ASCs) as a mesenchymal stem cell source. The allografts that had incubated for 2 months within a periosteal envelope had been completely replaced by large amounts of wellvascularized bone. We concluded that large bony allografts could be an effective scaffold for BTE and that vascularized periosteum would be necessary in this process. The purpose of the present study was to more fully understand this ‘ allograft revitalization’’ process, preparatory to its clinical application. We sought, using our described porcine hemimandible allograft model, to determine whether ASCs and rhBMP-2 are necessary for the process, and whether the bone produced by allograft revitalization would be mechanically sound. We hypothesized that both ASCs and rhBMP-2 would contribute to allograft revitalization and that the bone produced would be mechanically equivalent to native bone.
Materials and Methods ALLOGRAFT PREPARATION AND ANIMAL HUSBANDRY
Mandibles were harvested from 6-week-old Yorkshire/Hampshire pigs. The soft tissues were removed, the teeth extracted, and the mandibles bisected at the symphysis. The hemimandibles were then sent for allograft processing, including detergent and alcohol washes,33 sterility validation, packaging, and freeze drying (Veterinary Transplant Services, Kent, WA). The processed hemimandibles were stored at 20 C until implantation. Eight additional female Yorkshire/ Hampshire pigs were used as the allograft recipients. The pigs were housed in a standard 12-hour light/ dark-cycle room, with twice daily swine chow and ad lib access to water. The pigs were fasted for at least 12 hours before all surgical procedures. The Cincinnati Children’s Hospital Medical Center Institutional Animal Care and Use Committee approved and monitored all procedures (approval no. 1D03029). LIPOSUCTION
At 6.5 weeks of age, select pigs (n = 4) were anesthetized with intramuscular ketamine (30 mg/kg) and intramuscular acepromazine (1.1 mg/kg) and
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maintained under general endotracheal anesthesia with isoflurane. Approximately 150 mL of tumescent anesthetic (40 mL of 1% lidocaine with 1:100,000 epinephrine in 1 L of saline) was infused in each of 4 locations: bilateral abdomen and bilateral flanks. A 4F liposuction cannula was used to collect the lipoaspirate. The incisions were closed in a single layer, and intramuscular buprenorphine (0.05 to 0.10 mg/kg) was administered postoperatively for analgesia. ASC ISOLATION, CULTURE, AND HISTOLOGIC EXAMINATION
The lipoaspirate tissue and fluid were processed to obtain ASCs, as previously described.32 The ASCs were cultured in stromal media containing 199 media (HyClone; Thermo Fisher Scientific, Waltham, MA), 10% fetal bovine serum (FBS; USA Scientific, Ocala, FL), 1 antibiotic-antimycotic (no. 15420; Gibco, Life Technologies, Thermo Fisher Scientific). The processed lipoaspirate cells from the individual pigs were plated in separate 75-cm2 flasks, allowed to adhere for 48 hours, and rinsed with fresh media to remove debris and nonadherent cells. The cells were passaged on reaching near confluency (85%) using trypsinization (0.05% trypsin, no. 25300; Gibco) and split 1:2 or 1:3 to 225-cm2 flasks. Culture of the ASCs continued for 3 passages. On the day of implantation, all cells were harvested by trypsinization, washed, and resuspended in 1 mL of stromal media lacking FBS for autologous loading of the allograft constructs. Undifferentiated ASCs were subjected to different culture media to assess their multipotency, as described previously.34-36 The base media used was Dulbecco’s modified Eagle medium+GlutaMAX media (Gibco) containing 10% FBS and 1% antibioticantimycotic. Osteogenic media also contained 10mM b-glycerophosphate (G9422; Sigma-Aldrich, St. Louis, MO) and 250-mM ascorbic acid (A5960; SigmaAldrich). Adipogenic differentiation also contained 10-mM insulin (91077C; Sigma-Aldrich), 1-mM dexamethasone (D4902; Sigma-Aldrich), 0.5-mM isobutylmethylxanthine (I5879; Sigma-Aldrich), and 200-mM indomethacin (D4902; Sigma-Aldrich). The chondrogenic media also contained 6.25-mg/mL insulin, 10 ng/mL transforming growth factor-b1 (T4567; SigmaAldrich), and 50-nM ascorbate-2-phosphate (A8960; Sigma-Aldrich). At their fourth passage, the ASCs were plated at a density of 100,000 cells/well in a 6well tissue culture plate and switched to the above differentiation media. The media were changed twice weekly for a 2-week period before staining. Histologic stains were performed using Alizarin Red (A5533), silver nitrate (209139), Oil-Red O (O0625) and Alcian Blue (A3157) reagents, per the manufacturer’s (SigmaAldrich) recommendations.
BONE TISSUE ENGINEERING BY ALLOGRAFT REVITALIZATION ALLOGRAFT IMPLANTATION AND HARVEST OPERATIONS
At 8 weeks of age, each pig (n = 8) received bilateral hemimandible allograft constructs, implanted within a periosteal envelope made by the extraction of 2 adjacent ribs. The allograft construct assembly was nearly identical to our previously reported technique.32 In brief, absorbable collagen sponges (ACSs; Medtronic, Minneapolis, MN) were soaked in 8 mL of rhBMP-2 solution (0.5 mg/mL, Infuse, Medtronic) for 15 to 45 minutes, cut into 0.5-cm-wide strips, and placed within the hemimandible crib. Autologous ASCs (500 mL/ hemimandible) were then pipetted onto the collagen sponges and covered with additional rhBMP-2–soaked ACSs. The 8 pigs were divided into 4 groups (2 pigs and 4 implants per group) according to the composition of the allograft construct (Table 1). Group 1 received allograft hemimandibles containing only ACS. The allografts in group 2 were filled with rhBMP-2–soaked ACS. In group 3, the allografts were filled with ACS and autologous ASCs. Finally, in group 4, the allografts were filled with both ASCs and rhBMP2-soaked ACS. All the grafts were implanted in a rib periosteal envelope (bilaterally in each pig) and allowed to incubate for 7 weeks. The implantation operations were performed with the pigs in the supine position, with anesthesia as described for liposuction. A 14-cm incision was made on the right thorax parallel to and between ribs 5 and 6, and the ribs were removed subperiosteally for a distance of 14 cm. The interval between the ribs was excluded from the periosteal sheaths, and the 2 sheaths were sewn around the thoracic constructs. The thoracic wall was then closed in layered fashion using interrupted, absorbable suture. The pigs were administered prophylactic intramuscular penicillin G, and intramuscular buprenorphine (0.05 to 0.10 mg/kg) was administered postoperatively for analgesia. After a 7- to 8-week incubation, the pigs were anesthetized using intramuscular ketamine and acepromazine and sacrificed using 85 mg/kg of intravenous Fatal Plus (Vortech Pharmaceuticals, Dearborn, MI). The allograft constructs were harvested with the surrounding soft tissue structures (eg, periosteal sheath with intercostal muscle and native rib sections). The constructs were fixed immediately in formalin for processing and histologic evaluation. ALLOGRAFT CONSTRUCT IMAGING
The harvested constructs were scanned using a Concorde MicroCAT II micro-computed tomography (CT) system (Imtek, Knoxville, TN) in 3 scans and digitally assembled for analysis using Amira, version 5, software (Visage Imaging, San Diego, CA). Calcific tissue densities were calculated by measuring the saturated
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Table 1. PIG WEIGHTS AT SURGERY, LIPOASPIRATE VOLUMES, AND IMPLANTED CELL COUNTS
Weight (kg)
Pig No. 1 2 3 4 5 6 7 8
Age* (days)
ASCs
rhBMP-2
Lipoaspirate Volume (mL)
Cells Implanted (n)
Liposuction
Allograft Implantation
Allograft Harvest
62 62 62 62 61 61 61 61
No No Yes Yes No No Yes Yes
No No No No Yes Yes Yes Yes
— — 85 155 — — 95 125
— — 3.13 107 3.18 107 — — 3.18 107 3.33 107
— — 15.0 15.0 — — 15.4 13.6
17.7 16.4 22.7 25.2 17.0 14.0 12.0 22.0
67.9 68.6 74.5 77.7 51.0 NAy 65.9 49.0
Dashes indicate absence of liposuction operations for these pigs. Abbreviations: ASCs, adipose-derived stem cells; NA, not applicable; rhBMP-2, recombinant human bone morphogenetic protein-2. * Age at implantation of allograft. y Pig 6 was found dead 4 weeks into the experiment, with the autopsy findings negative for cardiopulmonary pathologic features. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
pixels (lower threshold set at 800 for trabecular bone) on the micro-CT scans. The adjoining ribs were digitally cropped from the images before this calculation. The calculated volumes of calcific tissue in the constructs from the different groups were compared using paired Student t tests. HISTOLOGIC EVALUATION
The harvested constructs were fixed in 10% formalin, and the soft tissues were manually removed to facilitate mechanical testing centrally, with strips of soft tissue left intact for imaging of the axial sections. Representative sections from each construct were sent to Flagship Biosciences (Aurora, CO) for processing and embedding in glycol methacrylate (GMA) for nondecalcified histologic evaluation. Standard hematoxylin and eosin (H&E) and trichrome stains were performed on each section. To identify the retained allograft, nondecalcified GMA-embedded sections were stained with 40 ,6diamidino-2-phenylindolesolution (Prolong Gold with DAPI; Molecular Probes, Eugene, OR), which stains the nuclei of living cells. Two or three slides per sample, constituting an entire axial section, were imaged and digitally scanned at 20 magnification using a slide scanner and automated Aperio software (Aperio, Vista, CA). To quantify the amount of retained allograft, the images were divided into 16 equal grids and manually examined for the presence of DAPInegative (dead allograft) bone. The percentage of these 16 grids containing any DAPI-negative bone was calculated for each sample and compared among the groups using the Student t test.
MECHANICAL TESTING
Using a band saw, the 5-cm central portion from each graft was trimmed into 3 blocks, 50 10 10 mm, of nondecalcified bone. Nonimplanted allografts and adult porcine native mandibles were prepared in the same manner. Immediately before testing, the sections were maintained in saline to prevent dehydration. Mechanical testing was accomplished using an Instron 8501 Universal Mechanical Testing System (Instron, Norwood, MA). During the 3-point bending test, the specimen was placed on the lower supports of the bending apparatus. Each specimen was positioned such that the lower supports were exactly 10 mm from each end, with 30 mm of the specimen unsupported between the supports. An initial preload (1 N) was applied to the center of the allograft, at a point 15 mm from each of the lower supports, and a displacement of 15 mm was applied at a rate of 5.0 mm/second until failure. Load-deflection data were collected using Instron software, and failure was determined as the maximum load recorded immediately before an absolute decrease in load. The mean failure strengths of the samples were compared using analysis of variance (SYSTAT 13; Systat Software, Chicago, IL).
Results Harvested porcine ASCs had previously been found to express mesenchymal stem cell markers CD34, CD90, and CD105, but not the hematopoietic marker CD45.32 To assess their multipotency, we subjected them to different mesenchymal differentiation media in vitro and found that they could differentiate into
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FIGURE 1. Adipocyte stem cell (ASC) differentiation in vitro. ASCs exposed to osteogenic differentiation media were subjected to different stains. A, von Kossa staining indicated extracellular matrix calcification in osteogenically differentiated cells (6.3). B, Positive Alizarin Red staining indicating calcium deposits in terminally differentiated osteogenic cells (2). C, Oil-Red O staining in adipogenic-differentiated ASCs showing intracellular lipid accumulation (12). D, Chondrogenically differentiated ASCs stained positive for Alcian Blue, indicating the presence of sulfated proteoglycans in a cartilaginous matrix (4). ASCs not exposed to differentiation media were negative for these stains (data not shown). A Leica M165 FC microscope (Leica Microsystems, Wetzlar, Germany) was used at the stated magnifications for these images. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
all mesenchymal cell lines tested, including chondrocytes, adipocytes, and osteoblasts (Fig 1). The pigs were divided into 4 groups (Table 1) according to the composition of the allograft constructs: group 1, incubated within rib periosteal envelope without the addition of ASCs and rhBMP-2/ACS (hereafter rhBMP-2); group 2, periosteum and autologous ASCs; group 3, periosteum and rhBMP-2; and group 4, periosteum, ASCs, and rhBMP-2. All pigs tolerated liposuction and construct implantation (Fig 2) without difficulty. One pig from group 3 was found dead 4 weeks after allograft placement; however, this death was not related to the grafts or previous surgery. On harvest, the grafts from all groups were found to have fusion with the cut ends of the ribs (Fig 3A). By both gross examination and micro-CT, the grafts from groups 2 and 3 subjectively had fewer surface irregularities and greater
bone production than did group 1. Gross axial sections of the harvested grafts showed a cortical rim of bone surrounding predominately cancellous bone with small, usually central, areas of fibrous tissue (Fig 3B). The H&E sections showed marrow-rich cancellous bone on the axial sections in all groups. To compare the volume of bone generated among the different groups, the calcific tissue volumes were obtained from the micro-CT images. Only the bone between the proximal and distal ends of the original graft was included to exclude the surrounding fused rib. Group 2 (44,499 mm3) and group 3 (51,463 mm3) both had increased bone production compared with group 1 (34,310 mm3; Fig 4). However, this difference was statistically significant for group 3 (P = .031), but not for group 2 (P = .087), suggesting a greater role for rhBMP-2 than for ASCs in the process. The group 4
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FIGURE 2. Allograft construct assembly. The upper 2 images show superior views of the porcine allograft hemimandible with the A, medullary cavity, which was then filled B, with absorbable collagen sponge (ACS, red arrowheads), with or without recombinant human bone morphogenetic protein-2 (rhBMP-2) and autologous adipose-derived stem cells (ASCs). The constructs in groups 2 and 4 received ASCs and those in groups 3 and 4 received rhBMP-2/ACS, as described in the ‘‘Materials and Methods’’ section. C, Resection of 2 adjacent ribs was then performed in young pigs bilaterally, forming periosteal pockets. D, The allograft constructs were placed within the periosteal envelope, with the periosteum and intercostal muscles to be closed over the allograft, completely encasing the graft. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
constructs (50,399 mm3) also had greater bone production than group 1 (P = .032), but essentially equal to that of group 3, suggesting no synergistic effect between rhBMP-2 and ASCs for bone production in this model. On both CT and histologic analysis, some of the samples appeared to have some retained allograft. To quantify this, axial sections from each sample were stained with DAPI, which binds living nuclei. Entire sections were stained, scanned, divided into a grid of 16 images, and manually scored for the presence of dead bone. Nearly 14% of the grids from the group 1 sections contained some retained allograft bone (Fig 5). rhBMP-2–treated grafts had less retained allograft (1.5% for group 3 and 0.86% for group 4); however, these differences were not statistically significant, given the wide distribution for group 1. To assess the mechanical strength of the harvested bone, three 1 1 5-cm cores of bone from each sample were subjected to the 3-point bending test. The
4 experimental groups were compared with native mandible and nonimplanted mandibular allograft (Fig 6). With 3-point bending, all samples fractured at the midpoint, and analysis of variance (P = .578) showed that the failure load of the allograft bone did not differ significantly among the treatment groups and the nonimplanted allograft (Fig 7). Neither the side of the pig nor the individual pig variables were found to have a significant effect (P = .405, side; P = .888, pig) on failure load.
Discussion The present study showed that rhBMP-2 significantly increased the volume of bone produced using the allograft revitalization technique. This was consistent with other reports,20,21 suggesting rhBMP-2 could be an important ingredient when using allograft revitalization for the repair of critical bony defects. We also observed a trend (P = .087) toward increased
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FIGURE 3. A, Gross and micro-computed tomography (CT) findings of harvested grafts. Representative grafts harvested from each group are shown. Top Row, The soft tissue was removed from the gross samples, with the exception of 2 strips that were left for histologic purposes. All samples had fusion with the adjoining 2 ribs. Little heterotopic bone was found in the surrounding soft tissues. Bottom Row, An examination of the micro-CT scans of these samples showed increased bone formation, in particular, within the center of the construct, in the samples receiving recombinant human bone morphogenetic protein-2 (rhBMP-2). Scale bars measure 1 cm. B, Cross-sectional findings of harvested grafts. Top Row, Gross and Middle, micro-CT axial cuts of the harvested constructs showing a cortical rim of bone predominately surrounding cancellous bone. Centrally, all constructs contained areas of fibrous tissue devoid of bone, possibly corresponding with the allograft medullary canal, which was subjectively larger in group 1 than in the other groups. Bottom Row, Hematoxylin-eosin–stained sections of the boxed areas confirmed the presence of marrow-rich cancellous bone in all 4 groups (magnification 4). ASCs, adipose-derived stem cells. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
bone produced with the addition of ASCs. The lack of statistical significance for the addition of ASCs might have resulted from an actual lack of biologic contribution of the ASCs or our small sample size (n = 4 per group). ASCs were selected for this model, because they have osteogenic properties comparable to those of BMSCs and are more readily available than BMSCs,
given the absence of a difference in the numbers of stem cells found in equivalent volumes of bone marrow and lipoaspirate.37,38 Given that the ASCs showed osteogenic capacity in vitro, it is likely that the addition of ASCs might contribute to the process, although our study was underpowered to detect this difference. However, the lack of synergism when both
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FIGURE 4. The volume of calcific tissue of harvested grafts was calculated from the micro-CT scans. Groups 2 to 4 all had increased bone compared with the periosteum-only group (group 1). However, this was only significant for grafts receiving recombinant human bone morphogenetic protein-2 (rhBMP-2; groups 3 and 4). The number of samples (n) per group is depicted just above the x-axis. Error bars represent the standard deviation. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
rhBMP-2 and ASCs were added suggests a limited benefit for ASCs greater than the effect of rhBMP-2 in our model. Therefore, we cannot recommend including or withholding ASCs from allograft constructs when applying this clinically. Future study, including fate mapping of ASCs, would help elucidate their contribution, including whether they provide a direct or indirect paracrine benefit, as suggested.39 Similar to our previous findings, the bone generated in this model was larger than the implanted graft, more closely resembling 2 conjoined ribs rather than a hemimandible. Additionally, the inner face of the graft in contact with the intercostal muscle suture line did not revitalize as well as that directly apposed to the periosteum. This suggests that intimate contact of the graft with vascularized periosteum is helpful for graft desorption and replacement and that periosteal support helps determine the shape of the bone produced. We also observed that all regenerated bone was contained within the confines of the periosteal sleeve, suggesting that the periosteum might act as an important barrier to heterotopic ossification in allograft revitalization. We also found that although the harvested explants were predominately well-vascularized cancellous bone, there were small areas of dead, nonresorbed allograft present. This is in contrast to our previous experiment in which no retained allograft was found. We attributed this change to differences in allograft processing, because no other differences were present in our model. Our initial study used a commercial allograft preparation process that had included treatment with proprietary detergents and sterilants under oscil-
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FIGURE 5. Histomorphometric percentage of nonabsorbed allograft in harvested grafts showing percentages of each sample containing DAPI-negative (dead allograft) bone. A decreased percentage of retained allograft was found in grafts receiving autologous adipose-derived stem cells (ASCs) and/or recombinant human bone morphogenetic protein-2 (rhBMP-2; groups 2 to 4); however, this was not statistically significant, given the wide variability in the periosteum-only group (group 1). Error bars depict standard deviations. Statistical testing used unpaired Student’s t tests for comparison of group 1 to all other groups, individually. The number for each group was the same as in Figure 3. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
lating positive and negative pressure (Biocleanse, RTI Biologics, Alachua, FL). This was not an option for the present study. Instead, the harvested mandibles were prepared by an allograft processing service (Veterinary Transplant Services) using a published protocol that included detergent, alcohol, and water washes in a sonicator.33 We speculated that the former process was more rigorous and that the degree of graft processing might correlate temporally with absorption in vivo. It was also interesting that the graft constructs loaded with autologous ASCs and rhBMP-2 had less retained allograft (albeit not significantly), suggesting more rapid graft absorption as a part of the process of new bone generation in our model. Three-point bending is an established model for mechanical testing of bone.40 This model allowed us to test multiple cores from each sample while preserving nonfractured portions for histologic examination. With most types of preparations, allograft bone will have mechanical strength equivalent to that of native bone.41-43 Our data have confirmed this. Furthermore, we found no differences in strength among the unimplanted allograft, native mandible, and harvested grafts. Thus, the bone produced by way of allograft revitalization had strength comparable to that of native bone, at least in the short term. This might not reflect the long-term fate of revitalized bone; however, we believe the comparable strength will continue as the new bone remodels over time. Furthermore, our model did not examine the effects of loading on the allograft
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FIGURE 6. Mechanical testing experimental design. From each sample, three 1 1 5-cm sections of the graft were cut with a band saw. A, The location of these ‘‘cores’’ was standardized between all the samples, with 2 cores taken from the lateral aspect and 1 from the central aspect (purple boxes). B, The cores were then stressed to fracture on a 3-point bending rig. The number of Newtons of force required for fracture was measured for each individual core, and all the cores for each group were combined for the intergroup analysis of variance statistical analysis. Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
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mote allograft revascularization. Perhaps the most beneficial application would be for repair of mandibular or maxillofacial defects using a local periosteal flap,49 because this would obviate the need for free tissue transfer.
References
FIGURE 7. Force required to fracture mandible cores during the 3point mechanical testing depicted in Figure 5. The box plots display the force required to achieve fracture for the cores of bone harvested from each of the 4 groups and, for comparison, from both native porcine mandible and unimplanted porcine allograft mandible. The boxes display the range of the 25th to 75th percentiles of the samples for each group. The horizontal lines within each box indicate the 50th percentile (mean) for each group. The error bars represent the standard deviation. Using analysis of variance, no difference in the force required to achieve fracture was found among the groups (P = .578). Runyan et al. Bone Tissue Engineering by Allograft Revitalization. J Oral Maxillofac Surg 2014.
constructs. During bone fracture healing, mechanical loads have been shown to improve strength as the bone remodels,44 and we hypothesized that a load applied to allograft would further stimulate the process of revitalization. Importantly, the histologic finding of revitalized bone throughout the construct was in contrast to that of explanted structural allograft alone. Enneking and Mindell,45 Enneking and Campanacci,46 and Wheeler and Enneking47 reported that the late clinical failures in the axial skeleton were due to chronic stress on a nonrevitalized, mineral scaffold whose ability to remodel and regenerate was limited. In summary, a large cadaveric allograft might normally be expected to fail when used for reconstruction; however, when revitalized, it can be expected to function as normal bone, including the ability to heal when fractured and to resist infection. We believe these data can meaningfully guide the clinical application of allograft revitalization. The next step will be preclinical animal modeling to test whether the revitalized allograft is effective in repairing a critical defect and whether it is equivalent to the reference standard, the vascularized autograft. Our data suggest that rhBMP-2 and vascularized periosteum will be key ingredients for the process. If efficacious in animal models, allograft revitalization could be considered for repair of critical defects of the craniofacial and axial skeleton. One can envision its use as a reliable method of prelaminating a bony free flap for future transfer to fill a defect or to repair a critical defect in situ using a free periosteal flap48 to pro-
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