ArticlePDF Available

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


Abstract and Figures

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.
Content may be subject to copyright.
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited. 1397
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.,
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 and click on “Plastic Surgery
Hot Topics” in the “Digital Media” tab to watch.
Supplemental digital content is available for
this article. Direct URL citations appear in the
text; simply type the URL address into any Web
browser to access this content. Clickable links
to the material are provided in the HTML text
of this article on the Journal’s website (www.
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
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.
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,
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
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
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 scaold,
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
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 scaolds. Note that copper sulfate scaolds undergo additional wash-
ing to yield Fluy–poly(lactic-co-glycolic acid) scaolds. (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
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).
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 scaold place-
ment in a rat calvarial defect.
Copyright © 2019 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
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
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
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).
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), Fluy–poly(lactic-co-glycolic acid) (F-PLGA)
scaold, and Hyperelastic Bone (HB) scaold at 8 and 12 weeks
after implantation. (Below) Amount of mineralized tissue was
quantied 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 scaold group compared to the
negative control and Fluy–poly(lactic-co-glycolic acid) scaold
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.
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), Fluy–poly(lactic-co-glycolic acid) scaold, and Hyperelastic
Bone scaold at 8 and 12 weeks postoperatively. (Above) The defect sites of the empty defect rats
show incomplete healing, as the untreated defects remained unlled 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 Fluy–poly(lactic-co-
glycolic acid) scaold group show defects primarily bridged with brous tissue and membranous
cellular components within the scaold at 8 and 12 weeks. (Below) The defect sites of the Hyper-
elastic Bone scaold group show brous tissue and membranous cellular components within the
scaold 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
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)
Fluy–poly(lactic-co-glycolic acid) bers, comprising the larger implanted scaolds, surrounded by tissues from
12-week explants. (Below) Higher magnication 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.
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
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
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).
1. Akbay E, Aydogan F. Reconstruction of isolated mandibular
bone defects with non-vascularized corticocancellous bone
autograft and graft viability. Auris Nasus Larynx 2014;41:56–62.
2. Chung EJ, Sugimoto MJ, Ameer GA. The role of hydroxyapa-
tite in citric acid-based nanocomposites: Surface character-
istics, degradation, and osteogenicity in vitro. Acta Biomater.
3. Misch CM. Autogenous bone: Is it still the gold standard?
Implant Dent. 2010;19:361.
4. Banwart JC, Asher MA, Hassanein RS. Iliac crest bone graft
harvest donor site morbidity: A statistical evaluation. Spine
(Phila Pa 1976) 1995;20:1055–1060.
5. Mangano FG, Zecca PA, van Noort R, et al. Custom-made
biphasic calcium-phosphate scaffold for augmentation of
an atrophic mandibular anterior ridge. Case Rep Dent. 2015;
6. Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. Biodegradable
and bioactive porous polymer/inorganic composite scaffolds
for bone tissue engineering. Biomaterials 2006;27:3413–3431.
7. Lethaus B, Bloebaum M, Koper D, Poort-Ter Laak M,
Kessler P. Interval cranioplasty with patient-specific implants
and autogenous bone grafts: Success and cost analysis. J
Craniomaxillofac Surg. 2014;42:1948–1951.
8. Amini AR, Laurencin CT, Nukavarapu SP. Bone tissue engi-
neering: Recent advances and challenges. Crit Rev Biomed
Eng. 2012;40:363–408.
9. Delloye C, Cornu O, Druez V, Barbier O. Bone allografts:
What they can offer and what they cannot. J Bone Joint Surg
Br. 2007;89:574–579.
10. Arcos D, Izquierdo-Barba I, Vallet-Regí M. Promising trends
of bioceramics in the biomaterials field. J Mater Sci Mater
Med. 2009;20:447–455.
11. Brooks BD, Sinclair KD, Grainger DW, Brooks AE. A resorb-
able antibiotic-eluting polymer composite bone void filler
for perioperative infection prevention in a rabbit radial
defect model. PLoS One 2015;10:e0118696.
12. Jakus AE, Rutz AL, Shah RN. Advancing the field of 3D bio-
material printing. Biomed Mater. 2016;11:014102.
13. Jakus AE, Secor EB, Rutz AL, Jordan SW, Hersam MC, Shah
RN. Three-dimensional printing of high-content graphene
scaffolds for electronic and biomedical applications. ACS
Nano 2015;9:4636–4648.
14. Rutz AL, Hyland KE, Jakus AE, Burghardt WR, Shah RN. A
multimaterial bioink method for 3D printing tunable, cell-
compatible hydrogels. Adv Mater. 2015;27:1607–1614.
15. Jakus AE, Rutz AL, Jordan SW, et al. Hyperelastic “bone”:
A highly versatile, growth factor-free, osteoregenerative,
scalable, and surgically friendly biomaterial. Sci Transl Med.
16. Jakus AE, Shah RN. Multi and mixed 3D-printing of gra-
phene-hydroxyapatite hybrid materials for complex tissue
engineering. J Biomed Mater Res A 2017;105:274–283.
17. Alluri R, Jakus A, Bougioukli S, et al. 3D printed hyperelastic
“bone” scaffolds and regional gene therapy: A novel approach
to bone healing. J Biomed Mater Res A 2018;106:1104–1110.
18. Jakus AE, Geisendorfer NR, Lewis PL, Shah RN. 3D-printing
porosity: A new approach to creating elevated porosity mate-
rials and structures. Acta Biomater. 2018;72:94–109.
19. Spicer PP, Kretlow JD, Young S, Jansen JA, Kasper FK, Mikos
AG. Evaluation of bone regeneration using the rat critical
size calvarial defect. Nat Protoc. 2012;7:1918–1929.
... In most cases, a trephine 119-152 is used to create cylindrical defects in the calvaria and the mandible. Other bone cutting devices for cylindrical defects include a circular knife, 153 a biopsy punch, 154 a drill 126,[155][156][157][158][159] and rongeurs. 160 The most used cutting device for creating segmental defects in the mandibular is the reciprocating bone saw, which is mainly used for large animals. ...
... (3 out of 66 studies)126,142,166 is a positive control, such as an autologous bone graft, used as a comparator to assess ultimate efficacy of the tested material. This creates a knowledge gap in the current literature: how does the bonemimetic material perform in a preclinical model against the SOC? ...
Full-text available
Bone tissue engineering is a rapidly developing field with potential for the regeneration of craniomaxillofacial (CMF) bones, with 3D printing being a suitable fabrication tool for patient‐specific implants. The CMF region includes a variety of different bones with distinct functions. The clinical implementation of tissue engineering concepts is currently poor, likely due to multiple reasons including the complexity of the CMF anatomy and biology, and the limited relevance of the currently used preclinical models. The ‘recapitulation of a human disease’ is a core requisite of preclinical animal models, but this aspect is often neglected, with a vast majority of studies failing to identify the specific clinical indication they are targeting and/or the rationale for choosing one animal model over another. Currently, there are no suitable guidelines that propose the most appropriate animal model to address a specific CMF pathology and no standards are established to test the efficacy of biomaterials or tissue engineered constructs in the CMF field. This review reports the current clinical scenario of CMF reconstruction, then discusses the numerous limitations of currently used preclinical animal models employed for validating 3D‐printed tissue engineered constructs and the need to reduce animal work that does not address a specific clinical question. We will highlight critical research aspects to consider, to pave a clinically driven path for the development of new tissue engineered materials for CMF reconstruction. Reconstruction of bone defects of the CMF region is a major and difficult surgical intervention. 3D printing is an ideal biofabrication tool to create patient‐specific tissue engineered bone scaffolds. An appropriate pre‐clinical animal model should be based on the targeted clinical indication. Implementing standardised guidelines for preclinical studies can improve translation.
... Organ transplant difficulties including extended waiting times for a donor or immunological rejection of the transplanted organ may be avoided by harvesting stem cells from transplant recipients and printing them into a replacement organ. Recent experiments have shown that 3D tissue bioprinting can produce organ-level structures including bone, cornea, cartilage, heart, and skin" [12][13][14][15][16][17][18][19]. ...
Three-dimensional (3D) printing is one of the most well-liked new innovative and promising manufacturing techniques, which has demonstrated tremendous potential for the creation of biostructures in tissue engineering, particularly for bones, orthopaedic tissues, and related organs. 3D printing for the medical industry was considered a lofty pipe dream. Time and money, though, made it a reality. Today's 3D printing technology has a significant possibility to assist pharmaceutical and medical corporations in developing more specialised pharmaceuticals, enabling the quick creation of medical implants, and transforming how doctors and surgeons approach surgical planning. In today's practise of precision medicine and for individualised therapies, patient-specific anatomical models that are 3D printed are becoming increasingly helpful tools. In contrast to the conventional use of 3D printing to create cell-free scaffolds, 3D bioprinting requires various technical methods, such as biomimicry, autonomous self-assembly, and mini-tissue building blocks, to create 3D structures with mechanical and biological properties suitable for the deposition of living cells and the restoration of tissue and organ function. Cells, bioinks, and bioprinters are all necessary components of the bioprinting process, and each one of them has biological, technological, ethical, and cost- and clinically-effectiveness-related issues. As a result, there are several difficulties in integrating 3D bioprinting into widespread clinical practise. Currently, there are multiple applications for 3D bioprinting such as in surgery, cardiovascular system, musculoskeletal and even in drug screening. All of which will be discussed in this review.
... Their limited success in clinical applications, despite the promising in vitro and in vivo characteristics, is considered the result of the lack of proper osteoconductive cues in the bioink, biomaterial-related infections, and insufficient engraftment [227]. Continuous research in this area, provided the hyperelastic bone osteoregenerative bioink, composed of hydroxyapatite and PLGA, which allows printing of nanoparticle-functionalized bone scaffold systems with enhanced bacteriostatic properties and a complex, porous, and customized structure [228,229]. ...
Full-text available
3D-printing application in dentistry not only enables the manufacture of patient-specific devices and tissue constructs, but also allows mass customization, as well as digital workflow, with predictable lower cost and rapid turnaround times. 4D printing also shows a good impact in dentistry, as it can produce dynamic and adaptable materials, which have proven effective in the oral environment, under its continuously changing thermal and humidity conditions. It is expected to further boost the research into producing a whole tooth, capable to harmoniously integrate with the surrounding periodontium, which represents the ultimate goal of tissue engineering in dentistry. Because of their high versatility associated with the wide variety of available materials, additive manufacturing in dentistry predominantly targets the production of polymeric constructs. The aim of this narrative review is to catch a glimpse of the current state-of-the-art of additive manufacturing in dentistry, and the future perspectives of this modern technology, focusing on the specific polymeric materials.
... Different materials were directly combined via interlocking interfaces in earlier studies to form biphasic scaffolds (Figure 2a). 39 More recently, composite scaffolds with uniform compositions, such as scaffolds combining BG and HA, 40 BG and TCP, 41,42 polypropylene (PP) and TCP, 43 HA and PLGA or PCL (hyperelastic bone), 44 and DCB particles and PCL (Figure 2b), 45 have been 3D printed and many have been applied to critical-sized craniofacial defect treatment in vivo. PCL/HA mixtures with varying compositional and structural gradients have also been fabricated (Figure 2c). ...
Full-text available
Three-dimensional (3D) printing of scaffolds for tissue engineering applications has grown substantially in the past two decades. Unlike conventional autografts and allografts, 3D-printed scaffolds can satisfy the growing need for personalized bony reconstruction following massive craniofacial bone loss. Employing layer-by-layer manufacturing techniques, it is possible to produce patient-specific structures to rebuild complicated geometries for esthetic purposes and restore mechanical and respiratory functions. Here, we summarize the trends and current state-of-the-art studies in 3D-printing technologies for craniofacial bone reconstruction. We describe the design and development of the craniofacial scaffolds, including material choices, scaffold fabrication workflows, and the mechanical, structural, and biological considerations impacting scaffold application and function. Finally, we summarize the remaining hurdles and opportunities for growth to transition to the widespread clinical adoption of this technology. Graphical abstract
... In the same line of evidence, the addition of perforations of 300-400 µm in diameter in the scaffold material was shown to promote bone formation in comparison to other-sized perforations (106-600 µm) [27][28][29]. Recent three-dimensional (3D) printing technology has allowed precise control in designing complex scaffold structures featuring perforations of various sizes, with a high degree of porosity, and pore interconnectivity [30,31]. However, a limitation of this technology exists since there is limited compatibility with regard to fitting the scaffold material into a 3D printer and using direct injection to form the scaffold [32]. ...
Full-text available
The receptor activator of NF-κB ligand (RANKL)-binding peptide, OP3-4, is known to stimulate bone morphogenetic protein (BMP)-2-induced bone formation, but peptides tend to aggregate and lose their bioactivity. Cholesterol-bearing pullulan (CHP) nanogel scaffold has been shown to prevent aggregation of peptides and to allow their sustained release and activity; however, the appropriate design of CHP nanogels to conduct local bone formation needs to be developed. In the present study, we investigated the osteoconductive capacity of a newly synthesized CHP nanogel, CHPA using OP3-4 and BMP-2. We also clarified the difference between perforated and nonperforated CHPA impregnated with the two signaling molecules. Thirty-six, five-week-old male BALB/c mice were used for the calvarial defect model. The mice were euthanized at 6 weeks postoperatively. A higher cortical bone mineral content and bone formation rate were observed in the perforated scaffold in comparison to the nonperforated scaffold, especially in the OP3-4/BMP-2 combination group. The degradation rate of scaffold material in the perforated OP3-4/BMP-2 combination group was lower than that in the nonperforated group. These data suggest that perforated CHPA nanogel could lead to local bone formation induced by OP3-4 and BMP-2 and clarified the appropriate degradation rate for inducing local bone formation when CHPA nanogels are designed to be perforated.
... Autogenous bone grafts are the gold standard treatment used for this purpose due to their osteoconductive, osteoinductive, and osteogenic properties. However, their clinical use has been confined due to limited sources of autogenous bone tissue, bleeding, donor site morbidity, and prolonged grafting time [6][7][8]. Allogenic bone graft is an alternative treatment that commonly associated with the risk of immunological rejection, the transmission of infectious diseases, and limited incorporation into host bone [8,9]. Consequently, bone tissue engineering has attracted significant attention as a potential alternative solution to overcome these limitations and fulfill the clinical need [5,8,9]. ...
Full-text available
Background Repair of large-sized bone defects is a challengeable obstacle in orthopedics and evoked the demand for the development of biomaterials that could induce bone repair in such defects. Recently, UiO-66 has emerged as an attractive metal–organic framework (MOF) nanostructure that is incorporated in biomedical applications due to its biocompatibility, porosity, and stability. In addition, its osteogenic properties have earned a great interest as a promising field of research. Thus, the UiO-66 was prepared in this study and assessed for its potential to stimulate and support osteogenesis in vitro and in vivo in a rabbit femoral condyle defect model. The nanomaterial was fabricated and characterized using x-ray diffraction (XRD) and transmission electron microscopy (TEM). Afterward, in vitro cytotoxicity and hemolysis assays were performed to investigate UiO-66 biocompatibility. Furthermore, the material in vitro capability to upregulate osteoblast marker genes was assessed using qPCR. Next, the in vivo new bone formation potential of the UiO-66 nanomaterial was evaluated after induction of bone defects in rabbit femoral condyles. These defects were left empty or filled with UiO-66 nanomaterial and monitored at weeks 4, 8, and 12 after bone defect induction using x-ray, computed tomography (CT), histological examinations, and qPCR analysis of osteocalcin (OC) and osteopontin (OP) expressions. Results The designed UiO-66 nanomaterial showed excellent cytocompatibility and hemocompatibility and stimulated the in vitro osteoblast functions. The in vivo osteogenesis was enhanced in the UiO-66 treated group compared to the control group, whereas evidence of healing of the treated bone defects was observed grossly and histologically. Interestingly, UiO-66 implanted defects displayed a significant osteoid tissue and collagen deposition compared to control defects. Moreover, the UiO-66 nanomaterial demonstrated the potential to upregulate OC and OP in vivo. Conclusions The UiO-66 nanomaterial implantation possesses a stimulatory impact on the healing process of critical-sized bone defects indicating that UiO-66 is a promising biomaterial for application in bone tissue engineering.
... The natural meniscus is composed of a heterogeneous group of connective tissue cells [9,10]. 3D printing technology using biodegradable polymers can be used for the regeneration of various tissues or organs [11][12][13][14]. ...
Full-text available
Meniscus is a wedge-shaped fibrocartilaginous tissue, playing important roles in maintaining joint stability and function. Meniscus injuries are difficult to heal and frequently progress into structural breakdown, which then leads to osteoarthritis. Regeneration of heterogeneous tissue engineering meniscus (TEM) continues to be a scientific and translational challenge. The morphology, tissue architecture, mechanical strength, and functional applications of the cultivated TEMs have not been able to meet clinical needs, which may due to the negligent attention on the importance of microenvironment in vitro and in vivo. Herein, we combined the 3D (three-dimensional)-printed gradient porous scaffolds, spatiotemporal partition release of growth factors, and anti-inflammatory and anti-oxidant microenvironment regulation of Ac2-26 peptide to prepare a versatile meniscus composite scaffold with heterogeneous bionic structures, excellent biomechanical properties and anti-inflammatory and anti-oxidant effects. By observing the results of cell activity and differentiation, and biomechanics under anti-inflammatory and anti-oxidant microenvironments in vitro, we explored the effects of anti-inflammatory and anti-oxidant microenvironments on construction of regional and functional heterogeneous TEM via the growth process regulation, with a view to cultivating a high-quality of TEM from bench to bedside.
This review focuses on the advancements in additive manufacturing techniques that are utilized for fabricating bioceramic scaffolds and their characterizations leading to bone tissue regeneration. Bioscaffolds are made by mimicking the human bone structure, material composition, and properties. Calcium phosphate apatite materials are the most commonly used scaffold materials as they closely resemble live bone in their inorganic composition. The functionally graded scaffolds are fabricated by utilizing the right choice of the 3D printing method and material combinations to achieve the requirement of the bioscaffold. To tailor the physical, mechanical, and biological properties of the scaffold, certain materials are reinforced, doped, or coated to incorporate the functionality. The biomechanical loading conditions that involve flexion, torsion, and tension exerted on the implanted scaffold are discussed. The finite element analysis (FEA) technique is used to investigate the mechanical property of the scaffold before fabrication. This helps in reducing the actual number of samples used for testing. The FEA simulated results and the experimental result are compared. This review also highlights some of the challenges associated while processing the scaffold such as shrinkage, mechanical instability, cytotoxicity, and printability. In the end, the new materials that are evolved for tissue engineering applications are compiled and discussed.
There has been a lack of research for developing functional polymer composites for biomedical implants. Even though metals are widely used as implant materials, there is a need for developing polymer composites as implant materials because of the stress shielding effect that causes a lack of compatibility of metals with the human body. This review aims to bring out the latest developments in polymer composite materials for body implants and to emphasize the significance of polymer composites as a viable alternative to conventional materials used in the biomedical industry for ease of life. This review article explores the developments in functional polymer composites for biomedical applications and provides distinct divisions for their applications based on the part of the body where they are implanted. Each application has been covered in some detail. The various applications covered are bone transplants and bone regeneration, cardiovascular implants (stents), dental implants and restorative materials, neurological and spinal implants, and tendon and ligament replacement.
The field of bone tissue engineering seeks to mimic the bone extracellular matrix composition, balancing the organic and inorganic components. In this regard, additive manufacturing (AM) of high content calcium phosphate (CaP)-polymer composites holds great promise towards the design of bioactive scaffolds. Yet, the biological performance of such scaffolds is still poorly characterized. In this study, melt extrusion AM (ME-AM) was used to fabricate poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT)-nanohydroxyapatite (nHA) scaffolds with up to 45 wt% nHA, which presented significantly enhanced compressive mechanical properties, to evaluate their in vitro osteogenic potential as a function of nHA content. While osteogenic gene upregulation and matrix mineralization were observed on all scaffold types when cultured in osteogenic media, human mesenchymal stromal cells did not present an explicitly clear osteogenic phenotype, within the evaluated timeframe, in basic media cultures (i.e. without osteogenic factors). Yet, due to the adsorption of calcium and inorganic phosphate ions from cell culture media and simulated body fluid, the formation of a CaP layer was observed on PEOT/PBT-nHA 45 wt% scaffolds, which is hypothesized to account for their bone forming ability in the long term in vitro, and osteoconductivity in vivo.
Full-text available
Statement of significance: Porosity plays an essential role in the performance and function of biomaterials, tissue engineering, and clinical medicine. For the same material chemistry, the level of porosity can dictate if it is cell, tissue, or organ friendly; with low porosity materials being far less favorable than high porosity materials. Despite its importance, it has been difficult to create three-dimensionally printed structures that are comprised of materials that have extremely high levels of internal porosity yet are surgically friendly (able to handle and utilize during surgical operations). In this work, we extend a new materials-centric approach to 3D-printing, 3D-Painting, to 3D-printing structures made almost entirely out of water-soluble salt. The structures are then washed in a specific way that not only extracts the salt but causes the structures to increase in size. With the salt removed, the resulting medical polymer structures are almost entirely porous and contain very little solid material, but the maintain their 3D-printed form and are highly compatible with adult human stem cells, are mechanically robust enough to use in surgical manipulations, and can be filled with and act as carriers for biologically active liquids and gels. We can also extend this process to three-dimensionally printing other porous materials, such as graphene, metals, and even ceramics.
Full-text available
Despite substantial attention given to the development of osteoregenerative biomaterials, severe deficiencies remain in current products. These limitations include an inability to adequately, rapidly, and reproducibly regenerate new bone; high costs and limited manufacturing capacity; and lack of surgical ease of handling. To address these shortcomings, we generated a new, synthetic osteoregenerative biomaterial, hyperelastic "bone" (HB). HB, which is composed of 90 weight % (wt %) hydroxyapatite and 10 wt % polycaprolactone or poly(lacticco- glycolic acid), could be rapidly three-dimensionally (3D) printed (up to 275 cm3/hour) from room temperature extruded liquid inks. The resulting 3D-printed HB exhibited elastic mechanical properties (32 to 67% strain to failure, ~4 to 11 MPa elastic modulus), was highly absorbent (50% material porosity), supported cell viability and proliferation, and induced osteogenic differentiation of bone marrow-derived human mesenchymal stem cells cultured in vitro over 4 weeks without any osteo-inducing factors in the medium. We evaluated HB in vivo in a mouse subcutaneous implant model for material biocompatibility (7 and 35 days), in a rat posterolateral spinal fusion model for new bone formation (8 weeks), and in a large, non-human primate calvarial defect case study (4 weeks). HB did not elicit a negative immune response, became vascularized, quickly integrated with surrounding tissues, and rapidly ossified and supported new bone growth without the need for added biological factors.
Full-text available
With the emergence of 3D-printing (3DP) as a vital tool in tissue engineering and medicine, there is an ever growing need to develop new biomaterials that can be 3D-printed and also emulate the compositional, structural, and functional complexities of human tissues and organs. In this work, we probe the 3D-printable biomaterials spectrum by combining two recently established functional 3D-printable particle-laden biomaterial inks: one that contains hydroxyapatite microspheres (Hyperelastic Bone, HB) and another that contains graphene nanoflakes (3D-Graphene, 3DG). We demonstrate that not only can these distinct, osteogenic and neurogenic inks be co-3D-printed to create complex, multi-material constructs, but that composite inks of HB and 3DG can also be synthesized. Specifically, the printability, microstructural, mechanical, electrical, and biological properties of a hybrid material comprised of 1:1 HA:graphene by volume is investigated. The resulting HB-3DG hybrid exhibits mixed characteristics of the two distinct systems, while maintaining 3D-printability, electrical conductivity, and flexibility. In vitro assessment of HB-3DG using mesenchymal stem cells demonstrates the hybrid material supports cell viability and proliferation, as well as significantly upregulates both osteogenic and neurogenic gene expression over 14 days. This work ultimately demonstrates a significant step forward towards being able to 3D-print graded, multi-compositional, and multi-functional constructs from hybrid inks for complex composite tissue engineering. This article is protected by copyright. All rights reserved.
Full-text available
3D biomaterial printing has emerged as a potentially revolutionary technology, promising to transform both research and medical therapeutics. Although there has been recent progress in the field, on-demand fabrication of functional and transplantable tissues and organs is still a distant reality. To advance to this point, there are two major technical challenges that must be overcome. The first is expanding upon the limited variety of available 3D printable biomaterials (biomaterial inks), which currently do not adequately represent the physical, chemical, and biological complexity and diversity of tissues and organs within the human body. Newly developed biomaterial inks and the resulting 3D printed constructs must meet numerous interdependent requirements, including those that lead to optimal printing, structural, and biological outcomes. The second challenge is developing and implementing comprehensive biomaterial ink and printed structure characterization combined with in vitro and in vivo tissue- and organ-specific evaluation. This perspective outlines considerations for addressing these technical hurdles that, once overcome, will facilitate rapid advancement of 3D biomaterial printing as an indispensable tool for both investigating complex tissue and organ morphogenesis and for developing functional devices for a variety of diagnostic and regenerative medicine applications.
Full-text available
This report documents the clinical, radiographic, and histologic outcome of a custom-made computer-aided-design/computer-aided-manufactured (CAD/CAM) scaffold used for the alveolar ridge augmentation of a severely atrophic anterior mandible. Computed tomographic (CT) images of an atrophic anterior mandible were acquired and modified into a 3-dimensional (3D) reconstruction model; this was transferred to a CAD program, where a custom-made scaffold was designed. CAM software generated a set of tool-paths for the manufacture of the scaffold on a computer-numerical-control milling machine into the exact shape of the 3D design. A custom-made scaffold was milled from a synthetic micromacroporous biphasic calcium phosphate (BCP) block. The scaffold closely matched the shape of the defect: this helped to reduce the time for the surgery and contributed to good healing. One year later, newly formed and well-integrated bone was clinically available, and two implants (AnyRidge, MegaGen, Gyeongbuk, South Korea) were placed. The histologic samples retrieved from the implant sites revealed compact mature bone undergoing remodelling, marrow spaces, and newly formed trabecular bone surrounded by residual BCP particles. This study demonstrates that custom-made scaffolds can be fabricated by combining CT scans and CAD/CAM techniques. Further studies on a larger sample of patients are needed to confirm these results.
Full-text available
The exceptional properties of graphene enable applications in electronics, optoelectronics, energy storage, and structural composites. Here we demonstrate a 3D printable graphene (3DG) composite consisting of majority graphene and minority polylactide-co-glycolide, a biocompatible elastomer, 3D printed from a liquid ink. This ink can be utilized under ambient conditions via extrusion-based 3D printing to create graphene structures with features as small as 100 µm comprised of as few as two layers (<300 µm thick object) or many hundreds of layers (>10 cm thick object). The resulting 3DG material is mechanically robust and flexible while retaining electrical conductivities greater than 800 S/m, an order of magnitude increase over previously reported 3D printed carbon materials. In vitro experiments in simple growth medium, in the absence of neurogenic stimuli, reveal that 3DG supports human mesenchymal stem cell (hMSC) adhesion, viability, proliferation, and neurogenic differentiation with significant upregulation of glial and neuronal genes. This coincides with hMSCs adopting highly elongated morphologies with features similar to axons and presynaptic terminals. In vivo experiments indicate that 3DG has promising biocompatibility over the course of at least 30 days. Surgical tests using a human cadaver nerve model also illustrate that 3DG has exceptional handling characteristics and can be intraoperatively manipulated and applied to fine surgical procedures. With this unique set of properties, combined with ease of fabrication, 3DG could be applied towards the design and fabrication of a wide range of functional electronic, biological, and bioelectronic medical and non-medical devices.
Full-text available
Nearly 1.3 million total joint replacement procedures are performed in the United States annually, with numbers projected to rise exponentially in the coming decades. Although finite infection rates for these procedures remain consistently low, device-related infections represent a significant cause of implant failure, requiring secondary or revision procedures. Revision procedures manifest several-fold higher infection recurrence rates. Importantly, many revision surgeries, infected or not, require bone void fillers to support the host bone and provide a sufficient tissue bed for new hardware placement. Antibiotic-eluting bone void fillers (ABVF), providing both osteoconductive and antimicrobial properties, represent one approach for reducing rates of orthopedic device-related infections. Using a solvent-free, molten-cast process, a polymer-controlled antibiotic-eluting calcium carbonate hydroxyapatite (HAP) ceramic composite BVF (ABVF) was fabricated, characterized, and evaluated in vivo using a bacterial challenge in a rabbit radial defect window model. ABVF loaded with tobramycin eliminated the infectious burden in rabbits challenged with a clinically relevant strain of Staphylococcus aureus (inoculum as high as 107 CFU). Histological, microbiological, and radiographic methods were used to detail the effects of ABVF on microbial challenge to host bone after 8 weeks in vivo. In contrast to the HAP/BVF controls, which provided no antibiotic protection and required euthanasia 3 weeks post-operatively, tobramycin-releasing ABVF animals showed no signs of infection (clinical, microbiological, or radiographic) when euthanized at the 8-week study endpoint. ABVF sites did exhibit fibrous encapsulation around the implant at 8 weeks. Local antibiotic release from ABVF to orthopedic sites requiring bone void fillers eliminated the periprosthetic bacterial challenge in this 8-week in vivo study, confirming previous in vitro results.
Introduction: The purpose of this study was to evaluate the viability of human adipose-derived stem cells (ADSCs) transduced with a lentiviral (LV) vector to overexpress bone morphogenetic protein-2 (BMP-2) loaded onto a novel 3D printed scaffold. Methods: Human ADSCs were transduced with a LV vector carrying the cDNA for BMP-2. The transduced cells were loaded onto a 3D printed Hyperelastic "Bone" (HB) scaffold. In vitro BMP-2 production was assessed using ELISA analysis. The ability of ADSCs loaded on the HB scaffold to induce in vivo bone formation in a hind limb muscle pouch model was assessed in the following groups: ADSCs transduced with LV-BMP-2, LV-GFP, ADSCs alone, and empty HB scaffolds. Bone formation was assessed using radiographs, histology and histomorphometry. Results: Transduced ADSCs BMP-2 production on the HB scaffold at 24 hours was similar on 3D printed HB scaffolds versus control wells with transduced cells alone, and continued to increase after 1 and 2 weeks of culture. Bone formation was noted in LV-BMP-2 animals on plain radiographs at 2 and 4 weeks after implantation; no bone formation was noted in the other groups. Histology demonstrated that the LV-BMP-2 group was the only group that formed woven bone and mean the bone area/tissue area was significantly greater when compared to the other groups. Conclusions: 3D printed HB scaffolds are effective carriers for transduced ADSCs to promote bone repair. The combination of gene therapy and tissue engineered scaffolds is a promising multidisciplinary approach to bone repair with significant clinical potential. This article is protected by copyright. All rights reserved.
A multimaterial bioink method using polyethylene glycol crosslinking is presented for expanding the biomaterial palette required for 3D bioprinting more mimetic and customizable tissue and organ constructs. Lightly crosslinked, soft hydrogels are produced from precursor solutions of various materials and 3D printed. Rheological and biological characterizations are presented, and the promise of this new bioink synthesis strategy is discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Unlabelled: Different options exist for the reconstruction of craniectomy defects following interval cranioplasty. The standard procedure is still based on the re-implantation of autogenous bone specimen which can be stored in the abdominal wall or be cryopreserved. Alternatively patient-specific implants (PSIs) can be used. We conducted a retrospective study based on 50 consecutive patients with skull bone defects of 100 cm(2) or more being operated on by the same team of surgeons. Thirty-three patients agreed to take part in the study. Seventeen patients who underwent reconstruction with PSIs (titanium and polyether ether ketone, PEEK) (follow-up, 43 months [range, 3-93]) were compared with 16 control subjects who had autogenous bone grafts re-implanted (follow-up, 32 months [range, 5-92]). Criteria analyzed were the success and complication rates, operation time, duration of hospitalization and the treatment costs. Complication rate and the rate of reoperation were significantly lower, and the hospital stay was shorter in the PSI group. The treatment costs for reconstruction with autogenous bone were considerably lower than skull bone reconstruction based on PSIs (average costs: 10849.91 €/patient versus 15532.08 €/patient with PSI). Due to biological reasons some of the autogenous bone implants fail due to infection and resorption and the patients have to undergo another operation with implantation of a PSI in a secondary attempt. For those patients the highest overall treatment costs must be calculated (average costs: 26086.06 €/patient with secondary stage PSI versus 15532.08 €/patient with primary stage PSI). Conclusion: High success rates and reliability of PSIs may change the treatment strategy in patients undergoing interval cranioplasty.