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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, osteoinduc-
tive, and osteogenic nature.1,2 However, donor-site
morbidity, including pain, infection, bleeding, and
injury to surrounding structures, and the limited sup-
ply of available autologous tissue motivate the need
Copyright © 2019 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0000000000005530
Yu-Hui Huang, M.S.
Adam E. Jakus, Ph.D.
Sumanas W. Jordan, M.D.,
Ph.D.
Zari Dumanian, B.S.
Kelly Parker, B.S.
Linping Zhao, Ph.D.
Pravin K. Patel, M.D.
Ramille N. Shah, Ph.D.
Chicago and Evanston, Ill.
Background: Autologous bone grafts remain the gold standard for craniofacial
reconstruction despite limitations of donor-site availability and morbidity. A myr-
iad 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 reconstruc-
tion often involves irregular three-dimensional defects, which may benefit from
malleable or customizable substrates. “Hyperelastic Bone” is a three-dimension-
ally 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-dimen-
sionally printed Fluffy–poly(lactic-co-glycolic acid) without hydroxyapatite, au-
tologous 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–comput-
ed 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 re-
vealed 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. (Plast. Reconstr. Surg. 143: 1397, 2019.)
From Shriners Hospitals for Children-Chicago; The Cranio-
facial 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 Reconstruc-
tive 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.
Three-Dimensionally Printed Hyperelastic Bone
Scaffolds Accelerate Bone Regeneration in
Critical-Size Calvarial Bone Defects
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.
SUPPLEMENTAL DIGITAL CONTENT IS AVAIL-
ABLE IN THE TEXT.
Supplemental digital content is available for
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text; simply type the URL address into any Web
browser to access this content. Clickable links
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PRSJournal.com).
EXPERIMENTAL
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
1398
Plastic and Reconstructive Surgery • May 2019
for efficacious, off-the-shelf bone substitutes.3–5 For
select applications, processed allografts and alloplas-
tic materials, including various preformed implants,
cements, putties, powders, and ceramics, circum-
vent 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, craniomaxil-
lofacial 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 lim-
ited connected porosity. This mitigates effective
surface-guided cell migration and vascularization,
leading to encapsulation rather than tissue integra-
tion, and can increase the risk of infection.11
Three-dimensional printing offers a platform
for creating patient-matched, defect-fitting con-
structs, 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 individual-
ized 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 scaf-
fold fabricated by room-temperature extrusion of
90 percent by weight (solids content) hydroxyapa-
tite and 10 percent by weight poly(lactic-co-glycolic
acid) liquid ink into self-supporting three-dimen-
sional forms without a need for curing, sintering,
or other chemical or physical stabilization.15–17 The
three-dimensionally printed scaffold is compress-
ible 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 sup-
ported 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 dem-
onstrated 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.
MATERIALS AND METHODS
Material and Three-Dimensionally Printed
Scaffold Preparation
Hyperelastic Bone was synthesized and charac-
terized as described previously using good medi-
cal 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 Hyper-
elastic Bone scaffold, http://links.lww.com/PRS/
D412.) Fluffy–poly(lactic-co-glycolic acid) was fab-
ricated 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,
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 (in-
ventors: 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 materi-
als 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 manufac-
tures new advanced manufacturing compatible ma-
terials and devices for medical and nonmedical ap-
plications. 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. Dimen-
sion Inx did not influence the conduct, description,
or interpretation of the findings in this article.
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Volume 143, Number 5 • Hyperelastic Bone
1399
hydroxyapatite-free control, that retained a degree
of porosity and form similar to the three-dimen-
sionally 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 pro-
gressive 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 pro-
tocols15 (Fig. 1).
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% isoflu-
rane/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 Hyperelas-
tic 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 Vet-
erinary Technologies, Windsor, Colo.) was admin-
istered 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.
Cone Beam Computed Tomographic Imaging
Skull samples were analyzed using cone beam
computed tomography (iCAT scanner, Next Gen-
eration 17-19; Imaging Sciences International,
Hatfield, Pa.) with an exposition time of 26.9 sec-
onds, 100 kVp, and 5 mA. The window size used
was 16 × 4-cm height with 0.125-mm voxel size.
Micro–Computed Tomographic Imaging
The regions of interest were cut from the cal-
varial 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.
Image Analyses
Cone beam computed tomographic and micro–
computed tomographic images were imported in
Digital Imaging and Communications in Medi-
cine format and analyzed with Mimics Medi-
cal 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 analy-
sis module, bone volume per total volume values
were calculated.
Histology and Scanning Electron Microscopic
Analyses
After completing micro–computed tomogra-
phy, 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
Video. Supplemental Digital Content 1 shows a micro–com-
puted tomographic scan of the Hyperelastic Bone scaold,
http://links.lww.com/PRS/D412.
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
1400
Plastic and Reconstructive Surgery • May 2019
central portion of the wound, slide preparation
in the sagittal plane, and hematoxylin and eosin
staining were performed by the University of Illi-
nois 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 cen-
tral area of each defect, including intact native
Fig. 1. (Above) Photographs of Hyperelastic Bone and copper sulfate (CuSO4) three-dimensional (3D)
paints and resulting three-dimensionally printed objects before and after being biopsy punched
to produce 8-mm-diameter scaolds. Note that copper sulfate scaolds undergo additional wash-
ing to yield Fluy–poly(lactic-co-glycolic acid) scaolds. (Below, left) Micro–computed tomographic
reconstruction (top-down and cross-sectional views) of an 8-mm-diameter Hyperelastic Bone scaf-
fold. (Below, right) Detail of micro–computed tomographic cross-sectional view highlighting relevant
dimensions and features.
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Volume 143, Number 5 • Hyperelastic Bone
1401
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 scaf-
folds were prepared for and imaged using scan-
ning 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 prep-
aration. Sample halves were then dehydrated suc-
cessively in 80, 90, and 100% ethanol and critical
point dried (Samdri critical point dryer; Tousi-
mis, Rockville, Md.), and mounted on scanning
electron microscopy stubs coated with carbon
tape. Before scanning electron microscopic imag-
ing, 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.
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 con-
trols and compared using Kruskal-Wallis with
Dunnett post hoc analysis comparing all treat-
ment groups (α = 0.05).
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 quan-
tified 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) scaf-
fold groups (Fig. 3, below). Before normalization
to the median bone volume per total volume frac-
tion 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
Fig. 2. (Left) Intraoperative photograph and (right) detail showing scaold place-
ment in a rat calvarial defect.
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1402
Plastic and Reconstructive Surgery • May 2019
Fig. 3. Cone beam computed tomographic (CBCT) and micro–computed tomographic (μCT) analyses of calvarial
defect healing at 8 and 12 weeks after implantation. (Above) Representative cone beam computed tomographic
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Volume 143, Number 5 • Hyperelastic Bone
1403
bone volume per total volume fraction of the
autologous graft group, the median bone vol-
umes 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 vol-
ume 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 tomogra-
phy (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 frac-
tions 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 tomogra-
phy (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 distribu-
tion of bone volume per total volume fraction of
defects treated with Hyperelastic Bone was signifi-
cantly higher compared with negative controls at 8
weeks according to cone beam computed tomog-
raphy (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, Krus-
kal-Wallis test) and micro–computed tomography
(p = 0.04, Kruskal-Wallis test) (Fig. 3, fifth row and
below).
Histologic Analyses
Histologic analyses further validated cone
beam computed tomographic and micro–com-
puted 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 for-
mation at the defect margins. For the Hyperelastic
Bone scaffolds, bridging of the defect by mineral-
ized bone tissue was observed (Fig. 4). The defect
sites of the Hyperelastic Bone scaffold rats show
fibrous tissue and membranous cellular compo-
nents 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.
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 extracellu-
lar 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 distin-
guish porous poly(lactic-co-glycolic acid) from tis-
sue and cells (Fig. 5).
DISCUSSION
There is an ongoing clinical need for osteo-
genic biomaterials that are not only highly effi-
cacious but also easy to surgically implement,
cost-effective, and capable of being manufac-
tured on scales that can address this widespread
need. With Hyperelastic Bone, we have previ-
ously demonstrated that well-established, safe,
clinically used material of pharmaceutical grade
Fig. 3. (Continued). and micro–computed tomographic scans
showing empty defects (negative control), autologous graft
(positive control), Fluy–poly(lactic-co-glycolic acid) (F-PLGA)
scaold, and Hyperelastic Bone (HB) scaold at 8 and 12 weeks
after implantation. (Below) Amount of mineralized tissue was
quantied by bone volume to total tissue volume of interest
and normalized to positive control group. Cone beam com-
puted tomographic and micro–computed tomographic analy-
ses revealed increased mineralized tissue around the defect
edges in the Hyperelastic Bone scaold group compared to the
negative control and Fluy–poly(lactic-co-glycolic acid) scaold
groups. The bone volume–to–total tissue volume of interest
values for the Hyperelastic Bone cohort are 95.6 and 82.0 per-
cent of positive control values at 8 and 12 weeks, respectively
(p=0.03), on cone beam computed tomography (below, left),
and 74.2 and 64.5 percent of positive control bone volume–to–
total issue volume of interest values at 8 and 12 weeks, respec-
tively (p=0.04), on micro–computed tomography (below, right).
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
1404
Plastic and Reconstructive Surgery • May 2019
Fig. 4. Histologic evaluation of the calvarial defect repair in an empty defect (negative control),
autologous bone (positive control), Fluy–poly(lactic-co-glycolic acid) scaold, and Hyperelastic
Bone scaold at 8 and 12 weeks postoperatively. (Above) The defect sites of the empty defect rats
show incomplete healing, as the untreated defects remained unlled at 8 and 12 weeks. (Second
row) The defect sites of the positive controls show interdigitating bone processes at defect mar-
gins with viable bone at 8 and 12 weeks. (Third row) The defect sites of the Fluy–poly(lactic-co-
glycolic acid) scaold group show defects primarily bridged with brous tissue and membranous
cellular components within the scaold at 8 and 12 weeks. (Below) The defect sites of the Hyper-
elastic Bone scaold group show brous tissue and membranous cellular components within the
scaold at 8 weeks and new bone formation at the defect margins at 12 weeks (scale bar=500
µm; arrow, defect margin; Ft, brous tissue; Mc, membranous cellular component; Nb, new bone).
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Volume 143, Number 5 • Hyperelastic Bone
1405
poly(lactic-co-glycolic acid) and hydroxyapatite
can be processed to create new composite mate-
rial with unique mechanical, physical, and bio-
logical 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 fab-
ricated into the required shape, size, and internal
architecture is an advantage of the three-dimen-
sional printing process, making it promising for
patient-matched implants. Furthermore, the
mechanical and physical properties of the result-
ing three-dimensionally printed Hyperelastic
Bone material allows for it to be intraoperatively
manipulated, making Hyperelastic Bone a surgi-
cally 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 addi-
tion of osteoinducing factors.15 Further promising
findings were evident when mice received subcuta-
neous Hyperelastic Bone implants, demonstrating
biocompatibility, and improved tissue growth and
structure over the commonly implemented hot-
melt polymer-calcium phosphate composite mate-
rials. Similar beneficial observations are reported
in a rat posterolateral spinal fusion model, where
Hyperelastic Bone is equally efficacious at pro-
moting bone growth as allograft-derived demin-
eralized 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 pro-
moted rapid tissue integration within the defect
space with signs of mineralization. The current
Fig. 5. Scanning electron micrographs of cross-sections of single (above, left) Hyperelastic Bone and (above, right)
Fluy–poly(lactic-co-glycolic acid) bers, comprising the larger implanted scaolds, surrounded by tissues from
12-week explants. (Below) Higher magnication views of the same images, highlighting tissue material interac-
tions. Scale bar=50 µm.
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
1406
Plastic and Reconstructive Surgery • May 2019
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 com-
puted tomography and micro–computed tomo-
graphic analysis of bone formation 8 and 12 weeks
after implantation showed a significantly greater
volume of new bone formation with the Hyper-
elastic Bone scaffolds when compared to the
empty defect controls and poly(lactic-co-glycolic
acid) hydroxyapatite-free scaffolds. In fact, Hyper-
elastic 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 hypoth-
esize 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-dimension-
ally printed scaffold architecture with high poros-
ity permitted surrounding tissue to integrate with
the Hyperelastic Bone scaffold. The chosen pro-
gressive 120-degree pore pattern ensured homo-
geneous, 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 implan-
tation of the Hyperelastic Bone scaffolds, soft tis-
sue was found covering and connected with the
extracranial surface of the Hyperelastic Bone scaf-
fold 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 Hyperelas-
tic Bone scaffold to recapitulate the form of the
defect. The results of our study advance previous
findings on the safety and efficacy of Hyperelas-
tic 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 scaf-
fold and empty defect groups only at 8 weeks after
implantation and not at 12 weeks after implanta-
tion because of the small sample size and resulting
low statistical power. Future studies should include
larger sample sizes and obtain in vivo serial com-
puted tomographic images at multiple time points
such as 48 hours and 2, 4, 6, 8, and 12 weeks post-
operatively 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 tomog-
raphy 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 indi-
vidual rats to minimize the selection of hydroxy-
apatite. For future consideration, a Hyperelastic
Bone variant can be made with bioresorbable tri-
calcium phosphate rather than hydroxyapatite, or
a combination of hydroxyapatite and tricalcium
phosphate, to tailor the degradation and remodel-
ing rate of the Hyperelastic Bone scaffold that may
further enhance bone formation.
In this study, the safety and efficacy of Hyper-
elastic 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 Hyper-
elastic 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-engineer-
ing 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-temper-
ature 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 sub-
stantial translational potential, to be confirmed in
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Volume 143, Number 5 • Hyperelastic Bone
1407
comprehensive, indication-specific, large-animal
model studies to evaluate Hyperelastic Bone’s effi-
cacy for specific craniofacial indications.
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
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 scaf-
folds 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 Medi-
cal Research and Materiel Command, and Northwest-
ern 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 facil-
ity (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 Pro-
gram (N00014-11-1-0690).
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