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Three-Dimensionally Printed Hyperelastic Bone Scaffolds Accelerate Bone Regeneration in Critical-Size Calvarial Bone Defects

Huang, Yu-Hui, M.S.; Jakus, Adam E., Ph.D.; Jordan, Sumanas W., M.D., Ph.D.; Dumanian, Zari, B.S.; Parker, Kelly, B.S.; Zhao, Linping, Ph.D.; Patel, Pravin K., M.D.; Shah, Ramille N., Ph.D.

Plastic and Reconstructive Surgery: May 2019 - Volume 143 - Issue 5 - p 1397–1407
doi: 10.1097/PRS.0000000000005530
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Background: Autologous bone grafts remain the gold standard for craniofacial reconstruction despite limitations of donor-site availability and morbidity. A myriad of commercial bone substitutes and allografts are available, yet no product has gained widespread use because of inferior clinical outcomes. The ideal bone substitute is both osteoconductive and osteoinductive. Craniofacial reconstruction often involves irregular three-dimensional defects, which may benefit from malleable or customizable substrates. “Hyperelastic Bone” is a three-dimensionally printed synthetic scaffold, composed of 90% by weight hydroxyapatite and 10% by weight poly(lactic-co-glycolic acid), with inherent bioactivity and porosity to allow for tissue integration. This study examines the capacity of Hyperelastic Bone for bone regeneration in a critical-size calvarial defect.

Methods: Eight-millimeter calvarial defects in adult male Sprague-Dawley rats were treated with three-dimensionally printed Hyperelastic Bone, three-dimensionally printed Fluffy–poly(lactic-co-glycolic acid) without hydroxyapatite, autologous bone (positive control), or left untreated (negative control). Animals were euthanized at 8 or 12 weeks postoperatively and specimens were analyzed for new bone formation by cone beam computed tomography, micro–computed tomography, and histology.

Results: The mineralized bone volume–to–total tissue volume fractions for the Hyperelastic Bone cohort at 8 and 12 weeks were 74.2 percent and 64.5 percent of positive control bone volume/total tissue, respectively (p = 0.04). Fluffy–poly(lactic-co-glycolic acid) demonstrated little bone formation, similar to the negative control. Histologic analysis of Hyperelastic Bone scaffolds revealed fibrous tissue at 8 weeks, and new bone formation surrounding the scaffold struts by 12 weeks.

Conclusion: Findings from our study suggest that Hyperelastic Bone grafts are effective for bone regeneration, with significant potential for clinical translation.

Chicago and Evanston, Ill.

From Shriners Hospitals for Children-Chicago; The Craniofacial Center, Department of Surgery, Division of Plastic and Reconstructive Surgery, University of Illinois Health; and the Department of Materials Science and Engineering, the Simpson Querrey Institute for BioNanotechnology, the Department of Surgery, Division of Plastic and Reconstructive Surgery, the Department of Biomedical Engineering, and the Division of Organ Transplantation, Department of Surgery, Northwestern University.

Received for publication March 19, 2018; accepted October 9, 2018.

Disclosure:Patents pertaining to this work have been filed and are pending: (1) room-temperature synthesis and three-dimensional printing of bioactive elastic bone for tissue engineering applications (inventors: A.E.J. and R.N.S.), (2) ink compositions for three-dimensional printing and methods of forming objects using the ink compositions (inventors: A.E.J. and R.N.S.), and additive manufacturing of porous materials through creation and three-dimensional printing of salt containing liquid feedstock materials followed by multi-step leaching. The other authors declare that they have no competing interests. A.E.J. and R.N.S. are co-founders of and shareholders in Dimension Inx, LLC, which develops and manufactures new advanced manufacturing compatible materials and devices for medical and nonmedical applications. As of August of 2017, A.E.J. is currently full-time chief technology officer of Dimension Inx, and R.N.S. serves part time as chief science officer of Dimension Inx. A.E.J. and R.N.S. are inventors on patents that are licensed to Dimension Inx. Dimension Inx did not influence the conduct, description, or interpretation of the findings in this article.

A “Hot Topic Video” by Editor-in-Chief Rod J. Rohrich, M.D., accompanies this article. Go to PRSJournal.com and click on “Plastic Surgery Hot Topics” in the “Digital Media” tab to watch.

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Ramille N. Shah, Ph.D., Department of Bioengineering, University of Illinois at Chicago, 851 South Morgan Street, 2nd floor, Chicago, Ill. 60607-7043, ramille@uic.edu

Autologous bone grafts, harvested from the iliac crest, cranium, ribs, tibia, and other areas, are considered to be the standard of care because of their osteoconductive, osteoinductive, and osteogenic nature.1,2 However, donor-site morbidity, including pain, infection, bleeding, and injury to surrounding structures, and the limited supply of available autologous tissue motivate the need for efficacious, off-the-shelf bone substitutes.3–5 For select applications, processed allografts and alloplastic materials, including various preformed implants, cements, putties, powders, and ceramics, circumvent supply constraints and provide an alternative for bony reconstruction; however, they do so at the added risk of infection, extrusion, and resorption.2,6

Regardless of materials used, craniomaxillofacial defects, whether they are congenital, traumatic, or oncologic, are often geometrically complex. Patient-specific implants for structural reconstruction, such as cranioplasty, have become readily available, although these are not intended to regenerate bone, nor do they grow with the patient.7 For other applications, cancellous bone, demineralized bone matrix, synthetic bone chips, or formable bone putties are packed into the defect site and rely on compression from the surrounding soft tissues to remain in place.8–10 Chips, powders, granules, and putties are nonporous or have limited connected porosity. This mitigates effective surface-guided cell migration and vascularization, leading to encapsulation rather than tissue integration, and can increase the risk of infection.11

Three-dimensional printing offers a platform for creating patient-matched, defect-fitting constructs, with defined internal architecture. The capability to fabricate complex, three-dimensional objects without the need for expensive tooling makes three-dimensional printing technologies ideal for creating customized items for individualized medicine. Although many advances are being made within the broad realm of biomedical three-dimensional printing, a major deficiency is the lack of three-dimensionally printable materials that are not only biocompatible and bioactive but also can be manufactured on relevant scales and rates with ease of handling and implantation during surgery while retaining high biofunctionality.12–14 “Hyperelastic Bone” is an osteoregenerative scaffold fabricated by room-temperature extrusion of 90 percent by weight (solids content) hydroxyapatite and 10 percent by weight poly(lactic-co-glycolic acid) liquid ink into self-supporting three-dimensional forms without a need for curing, sintering, or other chemical or physical stabilization.15–17 The three-dimensionally printed scaffold is compressible and elastic and highly absorbent, and has been shown to induce osteogenic differentiation of bone marrow–derived human mesenchymal stem cells in vitro without added osteoinducing stimuli.15 In vivo, Hyperelastic Bone did not elicit a negative immune response, became vascularized and integrated with surrounding tissues, and supported new bone growth in a rat posterolateral spinal fusion model by 8 weeks and in a nonhuman primate calvarial defect case study by 4 weeks.15 Hyperelastic Bone scaffolds have also been demonstrated to be effective carriers for transduced human adipose-derived stem cells to promote bone repair.17 In the current study, we compared the osteoregenerative capacity of Hyperelastic Bone to the clinical standard, autologous bone, in rat critical-size calvarial defects.

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MATERIALS AND METHODS

Material and Three-Dimensionally Printed Scaffold Preparation

Hyperelastic Bone was synthesized and characterized as described previously using good medical practices–grade hydroxyapatite (Merz North America, Raleigh, N.C.) and poly(lactic-co-glycolic acid) (Evonik Cyro, Parsippany, N.J.).15 (See Video, Supplemental Digital Content 1, which shows a micro–computed tomographic scan of the Hyperelastic Bone scaffold, http://links.lww.com/PRS/D412.) Fluffy–poly(lactic-co-glycolic acid) was fabricated from copper sulfate (Alfa Aesar, Ward Hill, Mass.)/poly(lactic-co-glycolic acid) (Evonik Cyro) inks that had been three-dimensionally printed and salt-leached. Fluffy–poly(lactic-co-glycolic acid)18 served as a three-dimensionally printed, hydroxyapatite-free control, that retained a degree of porosity and form similar to the three-dimensionally printed Hyperelastic Bone scaffolds. All samples were three-dimensionally printed using a three-dimensional BioPlotter Manufacturing Series (envisionTEC GmbH, Gladbeck, Germany); 5 × 5-cm sheets of each material, five layers thick (120 μm per layer, for a total of 0.6 mm) and with a progressive 120-degree pore pattern, were produced using a 200-μm nozzle and 250-μm spacing between adjacent parallel fibers. Eight-millimeter-diameter scaffolds for implantation were punched from the 5 × 5-cm sheets using a biopsy punch and washed and sterilized according to previously described protocols15 (Fig. 1).

Fig. 1

Fig. 1

Video

Video

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Surgical Procedure

Adult male Sprague-Dawley rats weighing approximately 500 g each were obtained from Charles River Laboratories International, Inc. (Wilmington, Mass.). All animal surgical procedures were approved by and performed according to the guidelines established by the University of Illinois at Chicago Animal Care and Use Committee. Rats were maintained with general anesthesia (2% isoflurane/100% oxygen) during the procedure. Under routine sterile conditions, the calvaria between the coronal and lambdoid sutures was exposed through a 1.5-cm sagittal incision. A handheld drill fitted with a trephine was used at low speed with sterile normal saline irrigation to create a full-thickness 8-mm diameter calvarial defect.19 The defects were randomly allocated into the following study groups: (1) empty defect (negative control) (n = 7); (2) defect implanted with autologous calvarial bone (positive control) (n = 6); (3) defect implanted with Fluffy–poly(lactic-co-glycolic acid) scaffold (n = 6); and (4) defect implanted with Hyperelastic Bone scaffold (n = 10) (Fig. 2). Periosteum and skin were closed using a running absorbable suture, and Buprenorphine SR LAB (0.1 mg/kg) (SR Veterinary Technologies, Windsor, Colo.) was administered subcutaneously for analgesia. Animals were housed two per cage with ad libitum access to water and food. Rats were euthanized at 8 (n = 16) and 12 weeks (n = 13) postoperatively. The skull samples containing defect sites were retrieved and fixed in 10% neutral buffered formalin for analysis.

Fig. 2

Fig. 2

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Cone Beam Computed Tomographic Imaging

Skull samples were analyzed using cone beam computed tomography (iCAT scanner, Next Generation 17-19; Imaging Sciences International, Hatfield, Pa.) with an exposition time of 26.9 seconds, 100 kVp, and 5 mA. The window size used was 16 × 4-cm height with 0.125-mm voxel size.

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Micro–Computed Tomographic Imaging

The regions of interest were cut from the calvarial bone, placed in 70% ethanol, and scanned with a micro–computed tomography device (micro-CT 40; SCANCO Medical AG, Brüttisellen, Switzerland) at a source voltage of 55 kVp and beam current of 145 μA, with a voxel size of 10 μm and an integration time of 300 msec.

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Image Analyses

Cone beam computed tomographic and micro–computed tomographic images were imported in Digital Imaging and Communications in Medicine format and analyzed with Mimics Medical 19.0 (Materialise, Leuven, Belgium). The region of interest was centered in the machined defect with an 8-mm-diameter circular region and matched the thickness of the defect margin. The bone density was profiled and segmented for each individual rat to minimize the selection of hydroxyapatite. Using the manufacturer’s analysis module, bone volume per total volume values were calculated.

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Histology and Scanning Electron Microscopic Analyses

After completing micro–computed tomography, the explants were dissected into halves for histologic analyses and imaged using scanning electron microscopy. The halves of the explants for histologic analyses were decalcified using a Surgipath Decalcifier II procedure. Sectioning (5 μm thick) of paraffin-embedded blocks in the central portion of the wound, slide preparation in the sagittal plane, and hematoxylin and eosin staining were performed by the University of Illinois Veterinary Diagnostic Laboratory staff under the direction of a certified histotechnologist. To evaluate in vivo bone regeneration after 8 and 12 weeks; three sections, representing the central area of each defect, including intact native bone margins surrounding the reconstructed defects, were used to assess new bone formation and bridging of the created defect under light microscopy. The other halves of explanted scaffolds were prepared for and imaged using scanning electron microscopy. Briefly, sample halves were fixed for 1 hour in an aqueous solution of 4% glutaraldehyde and 3% sucrose, transferred to 70% ethanol, and stored at 4°C ready for preparation. Sample halves were then dehydrated successively in 80, 90, and 100% ethanol and critical point dried (Samdri critical point dryer; Tousimis, Rockville, Md.), and mounted on scanning electron microscopy stubs coated with carbon tape. Before scanning electron microscopic imaging, the samples were coated with approximately 12-nm of osmium metal using osmium plasma. Samples were imaged using a LEO Gemini 1525 (Carl Zeiss, Oberkochen, Germany) scanning electron microscope.

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Statistical Analyses

Numerical data are represented as box-and-whisker plots depicting medians, quartiles, and ranges. All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, Calif.). Unless stated otherwise, the following statistics were used: treatment groups were normalized to the median of the autologous graft group serving as positive controls and compared using Kruskal-Wallis with Dunnett post hoc analysis comparing all treatment groups (α = 0.05).

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RESULTS

Bone Volume Quantification

Cone beam computed tomographic and micro–computed tomographic three-dimensional reconstructions are displayed in Figure 3, above. The amount of bone tissue regenerated was quantified by the mineralized bone volume as a fraction of the total tissue volume of interest. Bone volume per total volume fraction for Hyperelastic Bone, Fluffy–poly(lactic-co-glycolic acid), and negative control were normalized against the bone volume per total volume fraction for the autologous graft (positive control) group. Cone beam computed tomographic and micro–computed tomographic scans revealed increased quantities of mineralized bone matrix in the calvarial bone defects treated with Hyperelastic Bone scaffolds, compared to empty defect and poly(lactic-co-glycolic acid) scaffold groups (Fig. 3, below). Before normalization to the median bone volume per total volume fraction of the autologous graft group, the median bone volume per total volume fraction of the Hyperelastic Bone graft cohort increased from 55.7 percent to 57.0 percent at 8 and 12 weeks for cone beam computed tomography and from 36.1 percent to 37.1 percent on micro–computed tomography. After normalization to the median bone volume per total volume fraction of the autologous graft group, the median bone volumes for the Hyperelastic Bone cohort were 95.6 and 82.0 percent of positive control bone volume per total volume fraction at 8 and 12 weeks on cone beam computed tomography, respectively (p = 0.03). For micro–computed tomography, the median bone volumes for the Hyperelastic Bone cohort were 74.2 and 64.5 percent of positive control bone volume per total volume fraction at 8 and 12 weeks, respectively (p = 0.04). Fluffy–poly(lactic-co-glycolic acid) had median bone volume per total volume fractions of 12.1 and 20.0 percent of positive control bone volume per total volume fraction at 8 and 12 weeks, respectively, on cone beam computed tomography (p = 0.03), and median bone volume per total volume fractions of 16.6 and 22.5 percent of positive control bone volume per total volume fractions at 8 and 12 weeks, respectively, on micro–computed tomography (p = 0.04). Negative control had median bone volume per total volume fractions of 10.3 and 13.8 percent of positive control bone volume per total volume fractions at 8 and 12 weeks, respectively, on cone beam computed tomography (p = 0.03), and median bone volume per total volume fractions of 14.5 and 19.5 percent of positive control bone volume per total volume fractions at 8 and 12 weeks, respectively, on micro–computed tomography (p = 0.04). The bone volume per total volume fractions were 7.81-fold and 5.75-fold higher in defects treated with Hyperelastic Bone scaffolds at 8 and 12 weeks postoperatively compared with the negative control group, respectively. The distribution of bone volume per total volume fraction of defects treated with Hyperelastic Bone was significantly higher compared with negative controls at 8 weeks according to cone beam computed tomography (p = 0.02, Kruskal-Wallis test) and micro–computed tomography (p = 0.04, Kruskal-Wallis test) and 12 weeks postoperatively according to cone beam computed tomography (p = 0.04, Kruskal-Wallis test) and micro–computed tomography (p = 0.04, Kruskal-Wallis test) (Fig. 3, fifth row and below).

Fig. 3

Fig. 3

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Histologic Analyses

Histologic analyses further validated cone beam computed tomographic and micro–computed tomographic results. The defect edges were identified and new bone was stained in eosin. Fibrous tissue was evident in the empty defect, but bone tissue was minimal. Similarly, poly(lactic-co-glycolic acid) scaffolds contained mostly fibrous tissue, with only small amounts of new bone formation at the defect margins. For the Hyperelastic Bone scaffolds, bridging of the defect by mineralized bone tissue was observed (Fig. 4). The defect sites of the Hyperelastic Bone scaffold rats show fibrous tissue and membranous cellular components within the scaffold at 8 weeks and new bone formation at the defect margins at 12 weeks. New bone formation was observed surrounding the struts of the scaffold by 12 weeks.

Fig. 4

Fig. 4

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Scanning Electron Microscopic Analyses

Scanning electron microscopic imaging of 12-week explanted scaffold tissues revealed that the tissue formed intimate, cellularized contact with the material within and throughout the volumes of the Hyperelastic Bone and Fluffy–poly(lactic-co-glycolic acid) scaffolds. Tissue surrounding Hyperelastic Bone more closely resembled healthy extracellular matrix with defined collagenous extracellular matrix, whereas in the Fluffy–poly(lactic-co-glycolic acid) scaffolds, tissue appears to directly infiltrate porous material struts, making it difficult to distinguish porous poly(lactic-co-glycolic acid) from tissue and cells (Fig. 5).

Fig. 5

Fig. 5

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DISCUSSION

There is an ongoing clinical need for osteogenic biomaterials that are not only highly efficacious but also easy to surgically implement, cost-effective, and capable of being manufactured on scales that can address this widespread need. With Hyperelastic Bone, we have previously demonstrated that well-established, safe, clinically used material of pharmaceutical grade poly(lactic-co-glycolic acid) and hydroxyapatite can be processed to create new composite material with unique mechanical, physical, and biological properties, and that this new composite material can be synthesized and manufactured at scales and speeds that are clinically relevant, using synthetic materials that are less expensive than cadaver-derived allografts (demineralized bone matrix). The capacity for this material to be fabricated into the required shape, size, and internal architecture is an advantage of the three-dimensional printing process, making it promising for patient-matched implants. Furthermore, the mechanical and physical properties of the resulting three-dimensionally printed Hyperelastic Bone material allows for it to be intraoperatively manipulated, making Hyperelastic Bone a surgically malleable material, in contrast to ceramics or polymer-ceramic composites.

Through in vitro studies in previous work, we have demonstrated that Hyperelastic Bone can stimulate a significant osteogenic response in adult human mesenchymal stem cells without any addition of osteoinducing factors.15 Further promising findings were evident when mice received subcutaneous Hyperelastic Bone implants, demonstrating biocompatibility, and improved tissue growth and structure over the commonly implemented hot-melt polymer-calcium phosphate composite materials. Similar beneficial observations are reported in a rat posterolateral spinal fusion model, where Hyperelastic Bone is equally efficacious at promoting bone growth as allograft-derived demineralized bone matrix.15 Finally, a case study of Hyperelastic Bone’s implantation in a calvarial bone defect in a rhesus macaque demonstrated that Hyperelastic Bone can be quickly produced on a relevant scale and fashioned intraoperatively to press-fit into the defect site.15 In a short period of 4 weeks, the Hyperelastic Bone implant promoted rapid tissue integration within the defect space with signs of mineralization. The current work demonstrates that by using the Hyperelastic Bone scaffold composed of poly(lactic-co-glycolic acid) and hydroxyapatite microparticles within the scaffold, the osteconductivity was increased to accelerate bone regeneration in critical-sized calvarial defects. When normalized to the clinical standard of autologous grafts, cone beam computed tomography and micro–computed tomographic analysis of bone formation 8 and 12 weeks after implantation showed a significantly greater volume of new bone formation with the Hyperelastic Bone scaffolds when compared to the empty defect controls and poly(lactic-co-glycolic acid) hydroxyapatite-free scaffolds. In fact, Hyperelastic Bone scaffold was 73.8 percent as effective as the clinical standard of autologous grafts on micro–computed tomography at 8 weeks and 64.5 percent at 12 weeks after implantation. We hypothesize that the reported results normalized to the median bone volume per total volume fractions of the autologous graft group decreased because of the gradual degradation of Hyperelastic Bone scaffolds. The predetermined three-dimensionally printed scaffold architecture with high porosity permitted surrounding tissue to integrate with the Hyperelastic Bone scaffold. The chosen progressive 120-degree pore pattern ensured homogeneous, lateral mechanical properties along the implant perimeter loading directions, which was also previously demonstrated in a rat spinal fusion model.15 After 8 and 12 weeks after implantation of the Hyperelastic Bone scaffolds, soft tissue was found covering and connected with the extracranial surface of the Hyperelastic Bone scaffold with an interior that had become integrated with the surrounding tissue. We hypothesize that with more time, newly formed bone tissue would continue to infiltrate and degrade the Hyperelastic Bone scaffold to recapitulate the form of the defect. The results of our study advance previous findings on the safety and efficacy of Hyperelastic Bone through the use of a critical-size calvarial bone defect model, normalization to the clinical gold standard of autologous graft, and evaluation at a longer time point of 12 weeks.

Although the results presented herein are promising, there are limitations to the study. Despite achieving significant differences in defect filling among all four groups on Kruskal-Wallis analysis, Dunn multiple comparisons achieved significance between the Hyperelastic Bone scaffold and empty defect groups only at 8 weeks after implantation and not at 12 weeks after implantation because of the small sample size and resulting low statistical power. Future studies should include larger sample sizes and obtain in vivo serial computed tomographic images at multiple time points such as 48 hours and 2, 4, 6, 8, and 12 weeks postoperatively to evaluate bone regeneration within the same animal. Another confounding variable is that the Hyperelastic Bone scaffold consists of hydroxyapatite microparticles that display similar density as bone on cone beam computed tomography and micro–computed tomography, leading to potential overestimation of new bone volume in the Hyperelastic Bone scaffold group; however, the bone density was profiled and segmented for individual rats to minimize the selection of hydroxyapatite. For future consideration, a Hyperelastic Bone variant can be made with bioresorbable tricalcium phosphate rather than hydroxyapatite, or a combination of hydroxyapatite and tricalcium phosphate, to tailor the degradation and remodeling rate of the Hyperelastic Bone scaffold that may further enhance bone formation.

In this study, the safety and efficacy of Hyperelastic Bone in bone regeneration is demonstrated for the first time in a rat calvarial bone model. We conclude that three-dimensionally printed Hyperelastic Bone is capable of inducing bone formation in vivo in a critical-size calvarial defect without additional osteostimulating factors, such as growth factors or cells. This is supported by cone beam computed tomographic and micro–computed tomographic evidence of defect filling and histologic confirmation of bone regeneration. Hyperelastic Bone implant promoted rapid tissue integration within the defect space, with signs of mineralization. In addition, it is clear that Hyperelastic Bone surpassed Fluffy–poly(lactic-co-glycolic acid) in bone regeneration capacity. As previously highlighted (see earlier), Hyperelastic Bone has significant potential to be translated to craniofacial reconstructive surgery, where the need for cost-effective bone replacement grafts is enormous. Our study underscores the promising translational potential of this novel strategy for tissue-engineering applications, particularly bone regeneration. Future studies will explore additional material ink compositions and scaffold architectural designs to further optimize and enhance tissue regeneration.

The value of Hyperelastic Bone’s technical and medical advantages is further enhanced through its capacity to be rapidly manufactured into any size and shape by means of simple, room-temperature extrusion-based three-dimensional printing of instantly drying three-dimensional ink. These qualities mark Hyperelastic Bone as a promising new synthetic bone graft biomaterial with substantial translational potential, to be confirmed in comprehensive, indication-specific, large-animal model studies to evaluate Hyperelastic Bone’s efficacy for specific craniofacial indications.

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ACKNOWLEDGMENTS

This work was funded by support from Shriners Hospitals for Children grant 85300-CHI-16. Adam E. Jakus, Ph.D., was supported in part by the Hartwell Foundation. The authors are grateful to Ilham Putra, M.D., for assistance in animal surgery; Xin Li, M.D., Ph.D., for data collection and analysis of the project; Google Gift (Ramille N. Shah, Ph.D.); the Hartwell Foundation (Adam E. Jakus, Ph.D.); and for scaffolds produced by Adam E. Jakus, Ph.D., and Ramille N. Shah, Ph.D., TEAM Lab at the Simpson Querrey Institute for BioNanotechnology, which was funded by the U.S. Army Research Office, the U.S. Army Medical Research and Materiel Command, and Northwestern University. The Northwestern University Center for Advanced Microscopy was supported by National Cancer Institute Cancer Center Support Grant P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. The Electron Probe Instrumentation Center facility (NUANCE Center, Northwestern University) was supported by National Science Foundation grants DMR-1121262 and EEC-0118025|003. This study was also supported by the Office of Naval Research MURI Program (N00014-11-1-0690).

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