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Annals of Biomedical Engineering ( 2016)

DOI: 10.1007/s10439-016-1668-5

Additive Manufacturing of Biomaterials, Tissues, and Organs

3D-Printing Technologies for Craniofacial Rehabilitation, Reconstruction,


and Regeneration
ETHAN L. NYBERG,1 ASHLEY L. FARRIS,1 BEN P. HUNG,1 MIGUEL DIAS,1 JUAN R. GARCIA,2
AMIR H. DORAFSHAR,3 and WARREN L. GRAYSON 1,4
1
Department of Biomedical Engineering, Translational Tissue Engineering Center, Johns Hopkins University School of
Medicine, 400 N. Broadway, Smith 5023, Baltimore, MD 21231, USA; 2Department of Art as Applied to Medicine, Johns
Hopkins University School of Medicine, Baltimore, MD, USA; 3Department of Plastic and Reconstructive Surgery, Johns
Hopkins University School of Medicine, Baltimore, MD, USA; and 4Department of Material Sciences & Engineering, Johns
Hopkins University School of Medicine, Baltimore, MD, USA
(Received 10 February 2016; accepted 31 May 2016)

Associate Editor Amir Abbas Zadpoor oversaw the review of this article.

Abstract—The treatment of craniofacial defects can present ABBREVIATIONS


many challenges due to the variety of tissue-specific require-
ments and the complexity of anatomical structures in that 3D Three dimensional
region. 3D-printing technologies provide clinicians, engineers ABS Acrylonitrile butadiene styrene
and scientists with the ability to create patient-specific
BMPs Bone morphogenetic proteins
solutions for craniofacial defects. Currently, there are three
key strategies that utilize these technologies to restore both CNC Computer numerical control
appearance and function to patients: rehabilitation, recon- CT Computed tomography
struction and regeneration. In rehabilitation, 3D-printing can ECM Extracellular matrix
be used to create prostheses to replace or cover damaged FDM Fused deposition modeling
tissues. Reconstruction, through plastic surgery, can also
MRI Magnetic resonance imaging
leverage 3D-printing technologies to create custom cutting
guides, fixation devices, practice models and implanted PCL Polycaprolactone
medical devices to improve patient outcomes. Regeneration PDMS Polydimethylsiloxane
of tissue attempts to replace defects with biological materials. PDGF-BB Platelet-derived growth factor BB
3D-printing can be used to create either scaffolds or living, PEEK Polyetheretherketone
cellular constructs to signal tissue-forming cells to regenerate
PMMA Polymethyl methacrylate
defect regions. By integrating these three approaches, 3D-
printing technologies afford the opportunity to develop PLA Polylactide
personalized treatment plans and design-driven manufactur- SLA Stereolithography
ing solutions to improve aesthetic and functional outcomes SLS Selective laser sintering
for patients with craniofacial defects. VSP Virtual surgery planning

Keywords—Facial prosthetics, Craniofacial implants, Tissue


engineering, Regenerative medicine, 3D-printing, Scaffolds. INTRODUCTION

Craniofacial defects arise as a direct result of trau-


ma, oncological resection, or congenital differences.
They cause soft tissue or bone deficits, or a combina-
tion of both leading to non-healing composite tissue
Address correspondence to Warren L. Grayson, Department of
Biomedical Engineering, Translational Tissue Engineering Center,
wounds. Defects in the craniofacial region in particular
Johns Hopkins University School of Medicine, 400 N. Broadway, are difficult to treat because of the emphasis on posi-
Smith 5023, Baltimore, MD 21231, USA. Electronic mail: tive aesthetic outcomes and the number of tissue types
wgrayson@jhmi.edu (bone, cartilage, muscle, and skin) and structures (au-
Ethan L. Nyberg and Ashley L. Farris contributed equally to this ricle, orbit, nose, oral cavity) in close proximity. The
work.

 2016 Biomedical Engineering Society


NYBERG et al.

current options for reconstructive surgery to treat these magnetic resonance imaging (MRI), or light scanning
defects include grafts, local tissue rearrangement which and then 3D-modeled digitally to create useful 3D-
fills defects with adjacent healthy tissue, microsurgical printed products. The particular method of 3D-print-
tissue transfer whereby one area of the body is trans- ing affects the print outcome and may be selected
ferred with its blood supply to another area,9,23 and based on the particular applications (Table 1). The
vascularized composite allotransplantation whereby a primary methods for 3D-printing include fused depo-
portion of the body containing skin, muscle and/or sition modeling (FDM), stereolithography (SLA),
bone is transplanted from one patient to another.18 selective laser sintering (SLS), direct-ink writing, in-
However, the major challenges with using traditional kjet bioprinting, extrusion bioprinting, and laser as-
reconstructive surgery to treat large craniofacial de- sisted bioprinting, which have been reviewed
fects are donor-site morbidity and procuring sufficient extensively.50,56 Briefly, in FDM, molten material is
donor tissue with the same properties as the sur- extruded layer-by-layer onto a bed; once the material
rounding recipient tissue to restore normal anatomic cools and solidifies, it serves as the foundation for the
structure and primary organ functions. These proper- layer above it. While this method is easily applied to
ties include skin color, quantity and contour of bone, many materials—any material that can be melted and
and quantity and quality of subcutaneous tissues. extruded—it requires support structures for printing
The challenge of integrating the various tissues of overhangs. SLA uses a laser to solidify photocurable
the face while maintaining or improving aesthetics liquid polymers in a layer-by-layer fashion.56 In con-
motivates collaboration between the fields of pros- trast, SLS creates structures by sintering a powder bed
thetic rehabilitation, craniofacial reconstruction, and layer-by-layer. The powder that is not sintered there-
regenerative medicine. Prosthetic Rehabilitation refers fore serves as the support structure. A variation of this
to the use of custom-made facial prosthetics to restore method, direct-ink writing, also uses a powder bed, but
normal facial appearance (Fig. 1a). Reconstruction of uses a chemical binder instead of a laser to bind the
the craniofacial region can be performed using a particles together. Inkjet bioprinting, uses acoustic,
variety of plastic surgery techniques to replace struc- thermal, or electromagnetic forces to eject hydrogel
tures and is aided by the precise manufacture of cutting droplets, which could contain cells or biological mo-
guides, fixation devices, practice models and implanted lecules, onto a platform in an additive fashion, onto a
medical devices (Fig. 1b). Regeneration aims to stim- clean print bed or a binding solution.56 Extrusion
ulate regrowth of damaged or malformed craniofacial bioprinting is similar to inkjet printing, but uses
tissues using stem cells and biologically active scaffold pumps, screws, or pneumatic systems to extrude cell
materials (Fig. 1c). For a particular defect, these slurries with viscosities too high for inkjet printing.
approaches may be employed individually or in con- Finally, laser-assisted bioprinting consists of a laser
junction with one another. However, a common thread source, a glass ‘‘ribbon’’ covered with a layer of cells in
is the need for patient-specific treatments that fit a hydrogel solution, and a receiving substrate. The laser
particular defect site to achieve both aesthetic cosmesis vaporizes a small portion of the hydrogel solution,
and functional replacement. As such, 3D-printing which forms a bubble that can then fall as a droplet
techniques that can create highly complex craniofacial onto the platform below.56
geometries with high fidelity are well-suited for In this review, we examine how recent developments
addressing particular needs. in 3D-printing enable more effective personalized
Anatomical geometries can be captured using treatment of complex craniofacial defects. We high-
medical imaging such as computed tomography (CT), light advances in 3D-printing as applied to prosthetic

FIGURE 1. Examples of rehabilitation, reconstruction, and regeneration. (a) Custom PDMS midfacial and ocular prosthesis. (b)
Cutting and placement guides for auricular autogenous reconstruction. (c) 3D-printed maxilla, porous PCL scaffold.
TABLE 1. Summary of 3D-printing technologies used for treating craniofacial deformities.

Treatment type 3D printing applications Printing methods Materials Qualities of printed material

Prosthetic rehabilitation  Molds for casting duplicates  Direct-ink writing PDMS The material should be lightweight, non-
(Improve patient aesthetics)  Printed prostheses  FDM ABS degradable, flexible, and fit the ana-
 Auricle, orbit, and nose rehabilitation PMMA tomic shape of the missing tissue or
create a rigid mold that can be used to
case PDMS or acrylic parts.
Reconstruction (Tissue grafting)  Surgical positioning and cutting guides  FDM Titanium The material should be rigid, non-
 Custom metal implants  Stereolithography PEEK degradable, and biologically inert so
 Bone reconstruction  SLS Polypropylene that the surgeon can manipulate the
 Direct-ink writing Bioglasses graft into place.
PLA
ABS
PMMA
Hydroxyapatite
Tissue regeneration  Scaffold generation Acellular PCL The material should be degradable and
(Recapitulate native tissue  Cellular constructs  SLS Calcium Phosphate porous. Bioactivity, mechanical prop-
structure and function)  Bone, cartilage, skin, muscle  FDM erties, and anatomical shape should
 Composite craniofacial tissues mimic healthy native tissue.
Bioprinting Fibrinogen The material should be degradable.
 Inkjet Gelatin Bioactivity and 3D cell organization of
 Extrusion Alginate the native tissue should be mimicked.
 Laser-assisted
3D-Printing Technologies for Craniofacial Rehabilitation

 Stereolithography
NYBERG et al.

FIGURE 2. (a) Orbital mold 3D model obtained through a fully digital workflow. (b, c) Resulting 3D-printed 3-piece mold that can
be used for casting PDMS prosthesis. (d) The final PDMS prosthesis can be colored and provide satisfactory cosmesis. Photos
used with author’s permission. (Perry, R. The Development of an Orbital Prosthesis Workflow Using Advanced Digital Tech-
nologies, A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Arts,
Baltimore, Maryland October, 2015).

rehabilitation, surgical reconstruction, and tissue achieved through digitally analogous methods (Fig. 2).
regeneration for non-healing defects in the craniofacial Only one study to date has reported a clinically viable
region and identify avenues for further research. workflow for directly 3D-printing these devices.31 It is
still limited, however, as it does not result in a fully
colorized prosthesis with physical properties similar to
3D-PRINTING FOR PROSTHETIC the PDMS devices typically made by traditional
REHABILITATION methods. An alternate approach has been to 3D-print
a negative multiple-piece mold that can be used for
Recapitulation of patient specific coloring, texture, casting the final PDMS prosthesis. Advanced digital
stiffness, and shape for prostheses is currently a labor- technologies and additive manufacturing techniques
intensive process, which could be streamlined using can thus be leveraged in craniofacial cases to increase
3D-printing. Prosthetic rehabilitation may be used in the quality of outcomes for prosthetic rehabilitation.
cases where successful surgical reconstruction is not a Future development of methods to directly print fully
viable option due to factors such as poor prognosis, colorized PDMS prosthetics could significantly im-
co-morbidities, compromised healing due to poor prove manufacture time and costs for craniofacial
vascularization,83 and patient refusal of further surgi- prostheses. A number of companies are developing
cal interventions.2 Further, the economic burden and technologies to directly print PDMS46,62,70 and new
treatment time for prosthetic rehabilitation is lower techniques to precisely color complex and soft con-
than that of surgical reconstruction.76 Typical sites for structs (such computational hydrographic printing81)
craniofacial prosthetic rehabilitation include oral, or- offer exciting methods to fully recapitulate the
bital, nasal, and auricular regions.51,74 Prosthetic appearance of the prosthetic.
rehabilitation can also serve as an interim strategy
during the period of treatment planning for a later
surgical reconstruction.72 Besides providing an aes- 3D-PRINTING FOR SURGICAL
thetic solution to covering an affected area, prosthetic RECONSTRUCTION
devices are considered medically necessary due to the
functional benefits they offer to warm incoming air, 3D modeling and manufacturing tools can provide
maintain humidity of moisture filled cavities, protect aid in the personalized, surgical reconstruction of
fragile tissue, modulate speech, and provide support complex craniofacial defects by precisely cutting tis-
for corrective eyeglasses. sues according to preoperative plans, decreasing the
Treatment of craniofacial defects with prostheses total time and cost of surgery, and planning the shape
traditionally involves the creation of a custom made of alloplastic and metal materials. Furthermore, such
device generally made of polydimethylsiloxane tools have helped to improve precise shaping and
(PDMS) to replace missing tissue and cover underlying positioning of the newly incorporated tissues and im-
tissue.51,74 The workflow for creating these devices has proved the cosmetic and functional outcome of
gone relatively unchanged since the 1970s. However, reconstructive operations47 and are useful for patient
the use of advanced 3D imaging techniques (including education.14 Tools that are used transiently in the
surface laser scanning and stereo photogrammetry) reconstruction process, such as placement or cutting
combined with 3D-printing is changing what was once guides, are produced using FDM or SLA out of sterile
a traditionally based workflow to include several facets and bioinert materials such as acrylonitrile butadiene
3D-Printing Technologies for Craniofacial Rehabilitation

FIGURE 3. Synthes Pro Plan Virtual Surgery Plan and 3D-printing Cutting and Placement Guides. (a) Pre-operative CT Scan of the
right fibula. Graft pieces are labeled beginning 6.6 cm from the distal end of the fibula. (b) Planned cutting guide superimposed
over the fibula. (c) Planned fibular flaps in the context of the remaining zygoma, using the positioning guides and exact graft
pieces. (d) 3D-printed parts delivered to the surgeon include an anatomic guide, the fibula cutting guide, and positioning guides.

styrene (ABS), poly(methylmethacrylate) (PMMA), or planning aids intraoperative precision and efficiency
polypropylene.55 Implanted products additionally re- of the surgery to match the preoperative design.
quire long-term biocompatibility and mechanical Models of the defect site and the transferred bone
strength and are often laser sintered from titanium or segments can be manufactured to practice position-
bioglass. Both types of products are often accurate to ing, fixation, and evaluate aesthetic outcomes.68 Such
the millimeter scale. planning segues easily into precise, custom cutting
and placement guides, increasing cosmesis and
reducing ischemia and total surgery time. Consider
Virtual Surgery Planning and Guides
the clinical standard for reconstruction of mandibu-
Advances in 3D imaging and manipulation of the lar bone, the free fibular flap60: the fibula and the
resulting datasets have enabled surgeons to plan defect site are first scanned using CT (Fig. 3a), then
surgeries using computer models of the patient, vir- cuts are made in the fibula to adequately position
tually moving bones and other tissue to assess dif- the grafted bone into the defect site (Fig. 3c). To aid
ferent approaches, options, and outcomes (Fig. 3). in the precision of harvesting and repositioning the
This virtual surgery planning, together with rapid pieces of fibula, cutting and placement guides are
physical modeling of the defects and custom cutting designed and rapidly manufactured, often through
and positioning guides, has vastly improved preop- FDM (Fig. 3d). Finally, custom surgical guides have
erative planning techniques compared to more tra- been essential in enabling the advent of facial
ditional approaches, and has significantly aided the transplants—in addition to the planning and guide
surgeon in his or her approach to complex cranio- fabrication, 3D-printing is essential in preparing an
facial reconstruction.22,65 3D modeling and virtual exact fit for the donated face.8
NYBERG et al.

Pre-fitting Implants and implanted into an 83-year-old patient. The patient


was able to speak and swallow the same day, and
Rapid prototyped models of the defect, or the pre-
exhibited excellent restoration of facial aesthetics.28
dicted defect, are also used to pre-bend generic off-the-
While titanium is the industry standard in orthopedic
shelf implants such as reconstruction plates and tita-
implants, the cost of materials, unknown long term
nium meshes to fit the specific anatomy of the patient.
efficacy, and manufacturing remain limiting. There is
Such precise and methodical pre-bending can result in
particular concern of implant exposure and infection
improved functional and aesthetic outcomes,5
over time as there is often only a thin layer of soft
decreased subjectivity,29 and reduced surgery and
tissue covering the implant.
ischemia time.65 Stereolithographic models of the de-
As the intersection of 3D imaging, manipulation,
fect site have also been used to mold PMMA to fash-
design, and manufacturing develops further, these
ion an alloplastic bone-graft alternative.15 In addition,
tools for surgeons will broaden from individual case
3D-printing models of ideal and patient-specific anat-
studies to common practice. The past decade of
omy produced by mirroring a normal contralateral
developing these tools apace with the maturation of
side has been used to press fit a composite titanium and
3D technology will likely revolutionize surgical stan-
porous polyethylene implant, and then guide the sur-
dards, just as 3D-printing has revolutionized transra-
gical placement in order to reconstruct the orbital floor
dial prostheses.19,20 Increased efficiency and accuracy
after facial trauma.63 These methods allow for the
provided by these tools will be driving factors of their
customization of patient implants without significantly
widespread adoption while regulatory, biocompatibil-
changing the manufacturing process of the device,
ity, and reimbursement challenges remain.42 Innova-
which is a major regulatory and production hurdle.
tion stimulated and facilitated by these 3D
technologies will also continue, leading to techniques
Materials for Patient Specific Implants as impressive as the recent total face and jaw trans-
plants.8,18
Non-resorbable implants can be designed and
manufactured specific to individual patients and can
used in lieu of autologous tissue.58 Many materials 3D PRINTING FOR CRANIOFACIAL BONE
including metals, bioglasses, and bio-inert plastics can REGENERATION
be used in a number of manufacturing processes and
maintain biocompatibility over time. For example The goal of a 3D-printed construct for regeneration
polyetheretherketone (PEEK) has strong biocompati- is to fill the defect with biological tissue. To accomplish
bility, mechanical strength, and radiographic translu- this, an appropriately shaped construct can be pro-
cency and can be 3D fabricated into patient specific duced that is populated uniformly with tissue-forming
implants through laser sintering or Computer cells that are signaled to regenerate tissue. This can be
Numerical Control machining.39 In addition, patient- accomplished in two ways: printing of acellular scaf-
specific titanium mesh can be manufactured via direct folds that can be populated with cells prior to
metal laser sintering to hold grafted bone in place and implantation or the printing of living, cellular con-
re-create contours and structures of the facial bone.69 structs, termed ‘bioprinting’.
Bioglasses (such as S53P4, 6P53B, and 13-93) have
been widely used in craniofacial surgery as a bone graft
Acellular Printing
substitute due to their biocompatibility, strong
mechanical strength, and osteoconductivity.1,75 Bio- Several key parameters should be considered and
glass structures can be manufactured by mixing glass optimized for scaffold development: (1) macro-geom-
particles into a solution, cold-printing in a layer-by- etry (Fig. 1c), (2) micro-architecture, (3) bioactivity,
layer fashion, and then dehydrating at high tempera- and (4) mechanical properties (Fig. 4). The strengths
tures to sinter the glass particles together and remove and weaknesses of these currently investigated printing
the solution.24,33 Others have reported formulations of approaches to achieving the four considerations out-
bioglass (such as 13–93 which has the composition lined above are discussed below.
53SiO2, 6Na2O, 12K2O, 5MgO, 20CaO, 4P2O5; wt%) Incorporating micro-architecture, which encom-
which can be laser sintered into anatomic shapes.44 passes pore geometry and pore size, is critical for
Hydroxyapatite (the main component of bone) im- uniform cell distribution and cell migration into the
plants, via a resin carrier, can be produced through scaffold; interconnected pores can improve integration
SLA and have been used to reconstruct large of regenerated tissue with native tissue.48 For bone
(>20 cm2) defects with resolutions less than 0.4 mm.7 tissue engineering in vivo, higher porosity has been
Finally, in 2012 a titanium mandible was laser sintered correlated with increased bone ingrowth into scaf-
3D-Printing Technologies for Craniofacial Rehabilitation

FIGURE 4. 3D-printed scaffolds. (a) The scaffold should have appropriate micro-architecture, encompassing pore size and
porosity. Using direct-ink writing of a ceramic powder in a viscoelastic solution, different well-defined pore geometries were
manufactured and visualized under scanning electron microscopy. Scale bars represent 500 lm. Adapted from Ref. 66. (b) Cells
residing within the scaffold should be signaled appropriately to regenerate tissue. Sintered tricalcium phosphate scaffolds were
implanted in critically sized iliac defects in sheep. Bone formation by resident cells, denoted by the red stain, is evident when
compared against other osteoinductive materials (bone morphogenetic protein and autologous bone graft). Adapted from Ref. 80.
(c) The mechanical properties of the scaffold must be appropriate for the tissue being regenerated. Selective laser sintering of
polycaprolactone was used to fabricate a porous cylinder, which was tested mechanically to result in a stiffness of 15 MPa, within
the range of trabecular bone. Adapted from Ref. 21.

folds.36 Designing pore architecture results in higher polycaprolactone (PCL) with incorporated tricalcium
pore connectivity and uniform cell distribution com- phosphate particles in FDM.67 In addition, incorpo-
pared to random architecture resulting from salt- ration of bioactive molecules, such as bone morpho-
leaching methods, despite similar porosity, pore size, genetic proteins (BMPs), have been investigated;
and surface area.52 Pore size and interconnectivity also however, given most 3D-printing methods rely on high
improves nutrient diffusion into and waste diffusion temperatures, up to 1300 C for sintering methods,71
out of scaffolds.61 Scaffold vascularization, a critical use of growth factors in 3D-printing remains a chal-
component of tissue survival, has been shown to in- lenge. Chemical binding methods have the distinct
crease with increasing pore size; pore sizes between 160 advantage of printing at room temperature, creating
and 270 lm resulted in extensive vessel formation in potential for application of the method to growth
both mathematical and experimental models.3,12 Os- factor incorporation, though careful choice of binder is
teoblast proliferation and migration through collagen– required to prevent pH-related damage. A second
glycosaminoglycan scaffolds also depends on pore size, approach is to load growth factors onto a scaffold
with larger pores around 300 lm resulting in higher post-printing, which circumvents these issues but adds
cell numbers throughout the scaffold.54 In the context another step to scaffold manufacturing.
of 3D-printing, some methods are better suited to Finally, in the replacement of craniofacial bone, the
creating defined pores. For instance, FDM relies on scaffold must provide structural support for both res-
rapid cooling of an extruded molten material, resulting ident cells and for transduction of mechanical forces
in well-defined scaffold struts and well-defined pores.73 through the craniofacial skeleton. Target scaffold
In contrast, chemical binding-based approaches rely stiffness depends on anatomical location, with the
on dispensing a liquid binder onto a powder substrate elastic modulus of human trabecular bone within the
and result in pore sizes less than 100 lm due to binder mandibular condyle ranging between 120 and
flow.38 450 MPa or within the mandible from midline to ra-
The scaffold should also provide biological signals mus ranging from 112 to 910 MPa.32 Many current
to resident cells to form tissue. For bone, the most 3D-printed scaffolds have achieved stiffness within the
widely used strategy is incorporation of mineral phases 10–100 MPa range.32,37,38,82 Testing mechanical prop-
in scaffolds for osteoinductivity4; similar strategies erties of polymeric scaffolds under physiological con-
have been investigated with 3D-printed scaffolds. For ditions is crucial as groups have shown changes in
example, a phosphoric acid binder was used to bind compressive moduli at different temperatures and in
calcium phosphate together, creating a mineralized aqueous media.37 It should be noted that increased
structure that can house cells.38 Another method used porosity leads to lower mechanical properties—a study
NYBERG et al.

using sintered PCL reported that the stiffness of prin- fidelity resulted in a scaffold tailored to the specific
ted porous scaffolds was around 15 MPa, compared to defect. The ability to incorporate bioactive factors into
300 MPa for a solid PCL piece.21 As such, the the printed scaffold was also demonstrated. Finally,
importance of porosity for cellular ingrowth and pro- the choice of PCL as a printable biomaterial illustrated
liferation must be balanced against the importance of the ability to print mechanically appropriate scaffolds
structural scaffold properties for mechanical support for load-bearing craniofacial regions. In addition to
and force transduction. the group featured in this case study, other groups
The importance of these four criteria is clearly have commercialized FDA-approved PCL scaffolds
demonstrated in the clinical regeneration of soft and fabricated by FDM.57
osseous tissue holding the left mandibular cuspid in A relatively underexplored area of 3D-printed
place.64 Using the patient’s CT scan the exact macro- scaffolds involves printing biological and mechanical
scopic geometry of the scaffold was determined. The gradients. For example, printing scaffolds with
scaffold was printed using SLS of PCL containing 4% hydroxyapatite gradients could improve bone forma-
hydroxyapatite for osteoinductivity. In addition, the tion with exterior areas having more mineral to
scaffold was designed to release platelet-derived encourage growth of compact bone and interior areas
growth factor BB (PDGF-BB), a factor known to having more diffuse mineral to mimic trabecular bone.
support vascularization and mineralization,30,34 in a While printing with growth factors has been a chal-
burst manner from pre-formed channels. Due to the lenge due to printing conditions for many techniques
high printing temperatures associated with sintering, surpassing biological pH and temperatures at which
the scaffold was first printed without growth factor these molecules are stable, several groups have printed
and immersed in PDGF-BB solution for 15 minutes bioactive ceramics or extracellular matrix (ECM).35,49
after printing. The use of PCL as the main biomaterial The incorporation of ECM enhanced scaffold bioac-
was justified from a mechanics standpoint: the stiffness tivity, but high ECM concentrations decreased scaffold
of PCL scaffolds manufactured using SLS has been mechanics. Printing ECM proteins in 3D spatial gra-
reported to be ~15 to 300 MPa, depending on poros- dients has been achieved by using mask-based SLA to
ity,21 values that fall within the reported range for stimulate assembly of genetically engineered photoac-
human trabecular bone. tive proteins, though this was used as a surface modi-
The scaffold porosity or micro-architecture was not fication for tissue culture rather than an
reported, though the lack of interconnected pores was implantable 3D construct.77 Another group used inkjet
noted as a limitation of the approach. The implanta- printing to create gradients of laminin and used their
tion of the scaffold was successful—the image-based materials to study cell alignment.10
geometry fit the defect well—and the printed channels Printing mechanical gradients by varying pore
for growth factor release successfully dispensed structure and size could also assist with building tissue
PDGF-BB in a burst manner.64 As a shortcoming, the that mimics native function, particularly in the bone
patient presented with scaffold exposure and wound example. One group has recently demonstrated that
failure past 13 months post-implantation. Upon re- gradient pore sizes created by FDM can slightly im-
moval of the scaffold, histological analysis indicated a prove both chondrogenesis17 and osteogenesis,16 al-
preponderance of connective tissue formation and little though they did not investigate different geometries.
bone regeneration, suggesting the lack of internal mi- By designing scaffold pore sizes and geometries based
cro-architecture prevented the infiltration of regener- on biological mechanical requirements, these
ative and vascular cells and therefore precluded improvements may be further enhanced.
regeneration. Combined with the slow-degrading
properties of PCL, the authors concluded that the
Bioprinting
scaffold’s low porosity served to block tissue regener-
ation. As such, while the macro-geometry and Bioprinting differs from the traditional tissue engi-
mechanical properties were appropriate (over the 13- neering approach of seeding cells onto preformed
month period, the scaffold did not fail mechanically scaffolds by depositing cell and scaffold simultane-
despite being in a region of load), the lack of micro- ously, forming a predesigned composite structure.25
architecture inhibited the bioactive and regenerative Bioprinting is the computer-aided deposition of living
properties of the scaffold. cells into 3D patterns. It is currently performed with
This example of the clinical application of 3D- micron-scaled precision.50 As cell viability must be
printing scaffolds for craniofacial regeneration high- maintained during the printing process, the methods
lights strengths and necessary improvements. The used for bioprinting differ from those used for tradi-
combination of image-based extraction of craniofacial tional 3D-printing. Important parameters of 3D-bio-
geometry and the ability to 3D-print shapes with high printing scaffolds include (1) cell positioning and (2)
3D-Printing Technologies for Craniofacial Rehabilitation

mechanical strength. In many cases, the type of bioink nally, the majority of bioinks were natural hydrogel
used and the required resolution dictates the optimal polymers, particularly alginate and fibrin, which when
printing technique for a particular application. printed have compressive moduli of approximately
Bioprinting offers a key advantage over the tradi- 5 kPa.11 Human bone and cartilage typically have
tional approach of seeding cells into 3D-printed scaf- moduli of about 100 MPa–20 GPa and 700 kPa,
folds: digitally designing layer-by-layer deposition of respectively.26 In order to print tissues having similar
cells to precisely regulate 3D cell distribution. This is load-bearing capacities to native bone and cartilage,
advantageous when designing vascularized soft tissue, PEG-based hydrogels have been printed with com-
as adequate nutrient and oxygen supplies are necessary pressive moduli between 300 and 350 kPa.27 Another
during tissue regeneration.43 For example, Kolesky method used to improve mechanical strength is inte-
et al. developed a bioprinter that could print up to four grating acellular and cellular bioprinting. Merceron
cell types simultaneously and created complex 3D et al. used a combination of FDM and extrusion bio-
patterns of fluorescently labeled human dermal printing to print two thermoplastic polymers along
fibroblasts and human umbilical vein endothelial with C2C12 and NIH/3T3 cells to create a 3D-printed
cells.45 However, there are also several challenges muscle–tendon unit53 and Kang et al. integrated FDM
associated with cellular printing. and extrusion bioprinting to print vascularized bone,
A disadvantage of bioprinting compared to acellu- muscle, and cartilage.40 Printing hybrid scaffolds with
lar printing is that the mechanical strength of bioinks is cellular and acellular components may be one way to
typically lower than thermoplastic polymers. Origi- improve mechanical strength of bioprinted scaffolds.

FIGURE 5. Bioprinting for engineering skin and bone tissues. Full thickness dermal wounds after 4 weeks of healing with (a)
Apligraf applied, denoted by the purple circle and (b) 3D-bioprinted skin applied denoted by the blue circle. Severe wound
contraction and scaffold drying took place in the Apligraf scaffold compared to the bioprinted scaffold with microvessels (c–e).
H&E stains of (c) Apligraf, (d) no treatment, and (e) 3D-bioprinted skin scaffold. (f) A higher magnification image of 2 weeks of
healing following application of 3D bioprinted skin shows lumenized vessels, denoted with arrows. Adapted from Ref. 79. (g)
Schematic of laser-assisted bioprinting directly into mouse calvarial defect. nHA slurry refers to a nano-hydroxyapatite suspended
in a glycerol solution for printing. (h) H&E stain 3 months after calvarial defects were made. Bone healing observed in the area
where the 3D bioprinted scaffold was applied (denoted by the star) and minimal bone healing in the no scaffold control (denoted
by the arrow). (g) and (h) adapted from Ref. 41.
NYBERG et al.

This limitation is one of the reasons that bioprinting X-ray micro-tomography at the group observed con-
has not yet been used to regenerate craniofacial tissues siderable variation in bone formation between indi-
in human patients. vidual mice and did not provide quantitative data for
Of the tissues necessary for craniofacial recon- bone ingrowth. Though these results are preliminary,
struction, skin bioprinting is the nearest towards clin- they do show that bioprinting in vivo is possible and
ical translation, with studies conducted in vivo using may have potential for clinical use with the proper
mice and pigs. One study of note compared bioprinted bioink and cell source.
scaffolds to a commercially available engineered skin The precise patterning of biological molecules and
graft (Apligraf).79 A current limitation of engineered cells through bioprinting may be useful in creating tis-
skin grafts such as Apligraf is that they lack sues with complex spatial orientations. Though the field
microvasculature to maintain cell viability over time is young, promising results have been achieved for skin
and instead rely upon diffusion to transport oxygen and bone engineering in vivo. Studies have investigated
and nutrients to cells. Bioprinting can overcome this cartilage,13 muscle,53 and adipose59 tissue engineering
limitation by precisely patterning microvascular using bioprinting, though these have not yet advanced to
structures for skin grafts. Bioprinted scaffolds were in vivo studies. The expensive specialized equipment
trilayered with the top layer composed of collagen and necessary to use bioprinting technologies and the added
printed keratinocytes, the middle layer composed of regulatory burden of incorporating cells into a bioma-
fibrin and endothelial cells, and the bottom composed terial, acellular printing may be the preferred regenera-
of collagen and fibroblasts. Apligraf is a bilayered tive method for treating craniofacial defects.
material cast with two collagen layers: one containing Bioprinting could be further improved by widening the
dermal fibroblasts and the other containing ker- selection of available bioinks, decreasing print time,
atinocytes.78 The group found that wound contraction, increasing print resolution, and moving more studies
which if excessive can be a marker for joint contrac- towards in vivo models.
tion, malfunction, and poor aesthetic outcomes,
decreased in the bioprinted scaffolds compared to
Apligraf and no treatment, which were statistically CONCLUSION
similar. Additionally, the mice with printed grafts
healed between 14 and 16 days, whereas those with no Craniofacial deformities, when they arise, are par-
grafts or with Apligraf healed within 21 and over ticularly debilitating as they impact emotional, psy-
28 days, respectively. Histologically, the printed chosocial, and functional well-being of the affected
groups showed microvessel formation by implanted individual. They are difficult to treat due to the geo-
human endothelial cells in the printed scaffolds. metrical requirements and multiplicity of tissue types
Macroscopic images of skin regeneration in Apligraf that are impacted. However, recent advances in 3D-
and bioprinted groups can be seen in Figs. 5a–5f. printing technologies hold tremendous promise for
Patterning endothelial cells to form lumenized advancing treatment options available to patients.
microvessels to improve graft viability could allow for The requirements of 3D-printed products differ
scale up in terms of graft thickness and area by depending on the size and severity of the defects,
reducing oxygen and nutrient diffusion limitations. which together with patient-specific factors determine
Binder et al. have developed a promising in situ bio- whether the primary treatment modality is prosthetic
printer for skin, but initial preclinical tests in pigs rehabilitation, surgical reconstruction, or regenera-
demonstrated unsatisfactory healing outcomes in tion. For rehabilitation, the use of 3D-printing tech-
wound closure rates, which the authors suggested was nologies to either directly create PDMS prosthetics or
due to an insufficient cell density (2.0 9 105 cells/ print molds has the potential to significantly stream-
cm2).6 While the bioprinted materials have improved line the associated workflows for this process. The
skin wound healing in terms of decreasing wound prostheses are flexible, non-degradable, and need to
contraction and healing time in vivo is a promising incorporate patient-specific skin tones. They differ
advance for skin bioprinting, but such methods are still considerably from 3D-printed guides or alloplastic
inferior or comparable to cell spraying techniques.6 implants used in reconstructive surgeries. Perhaps the
Bioprinting of bone has also moved forward, with most transformative applications, of 3D-printing lie in
some preliminary bioprinting studies conducted in vivo. the realm of tissue regeneration. This area remains
Of particular import is a pilot study conducted by relatively nascent to date and significant research ef-
Keriquel et al. that investigated the use of laser assisted forts are being dedicated to its continue rapid
bioprinting to manufacture hydroxyapatite scaffolds advancements that include the development of
directly into a calvarial defects in mice, as seen in biodegradable scaffolds as well as bioinks used for
Figs. 5g, 5h.41 When bone formation was measured by printing live cells. The successful implementation of
3D-Printing Technologies for Craniofacial Rehabilitation
9
these technologies clinically will expand the treatment Broyles, J. M., N. B. Abt, S. M. Shridharani, B. Bojovic, E.
options available to patients. D. Rodriguez, and A. H. Dorafshar. The fusion of cran-
iofacial reconstruction and microsurgery. Plast. Reconstr.
Surg. 134:760–769, 2014.
10
Cai, K., H. Dong, C. Chen, L. Yang, K. D. Jandt, and L.
ACKNOWLEDGMENTS Deng. Inkjet printing of laminin gradient to investigate
endothelial cellular alignment. Colloids Surf. B. Biointer-
This work was supported by an NIH Biomedical faces 72:230–235, 2009.
11
Engineering Training Grant (ELN), an NSF graduate Catelas, I., N. Sese, B. M. Wu, J. C. Y. Dunn, S. Helger-
research fellowship (ALF), an NIH pre-doctoral fel- son, and B. Tawil. Human mesenchymal stem cell prolif-
lowship (BPH), and grants from the Maryland Stem Cell eration and osteogenic differentiation in fibrin gels in vitro.
Tissue Eng. 12:2385–2396, 2006.
Research Fund and the Department of Defense (WLG). 12
Chiu, Y. C., M. H. Cheng, H. Engel, S. W. Kao, J. C.
Larson, S. Gupta, and E. M. Brey. The role of pore size on
vascularization and tissue remodeling in PEG hydrogels.
Biomaterials 32:6045–6051, 2011.
CONFLICT OF INTEREST 13
Cui, X., K. Breitenkamp, M. G. Finn, M. Lotz, and D. D.
The authors declare no conflict of interest. D’Lima. Direct human cartilage repair using three-dimen-
sional bioprinting technology. Tissue Eng. Part A 18:1304–
1312, 2012.
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ETHICAL STANDARDS D’Urso, P. S., R. L. Atkinson, M. W. Lanigan, W. J.
Earwaker, I. J. Bruce, A. Holmes, T. M. Barker, D. J.
No animal or human studies were carried out by the Effeney, and R. G. Thompson. Stereolithographic (SL)
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