Three-Dimensional Bioprinting for Bone Tissue Regenerationcssharma/assets/pdf/43.pdf · Three-Dimensional Bioprinting for Bone Tissue Regeneration Shiva Kalyani, Nandini Dhiman, Anindita

Accepted Manuscript

Three-Dimensional Bioprinting for Bone Tissue Regeneration

Shiva Kalyani, Nandini Dhiman, Anindita Laha, Chandra S. Sharma, SeeramRamakrishna, Mudrika Khandelwal

PII: S2468-4511(17)30021-1

DOI: 10.1016/j.cobme.2017.03.005

Reference: COBME 12

To appear in: Current Opinion in Biomedical Engineering

Received Date: 6 March 2017

Accepted Date: 8 March 2017

Please cite this article as: S. Kalyani, N. Dhiman, A. Laha, C.S. Sharma, S. Ramakrishna, M.Khandelwal, Three-Dimensional Bioprinting for Bone Tissue Regeneration, Current Opinion inBiomedical Engineering (2017), doi: 10.1016/j.cobme.2017.03.005.

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Three-Dimensional Bioprinting for Bone Tissue Regeneration

Shiva Kalyani,a Nandini Dhiman,b Anindita Laha,c Chandra S. Sharma,d Seeram Ramakrishnae and Mudrika Khandelwala,*

a Natural and nature inspired material for society Laboratory Department of Materials Science & Metallurgical Engineering,

Indian Institute of Technology, Hyderabad, Kandi-502285, INDIA

b Department of Biomedical Engineering, Indian Institute of Technology, Hyderabad, Kandi-502285, INDIA

c Department of Chemical Engineering, Indian Institute of Technology, Hyderabad, Kandi-502285, INDIA

d Creative & Advanced Research Based On Nanomaterials Laboratory,

Department of Chemical Engineering, Indian Institute of Technology, Hyderabad, Kandi-502285, INDIA

e Department of Mechanical Engineering, National University of Singapore, Singapore

Abstract

Three-dimensional bioprinting can prove to be a promising technology for bone tissue

regeneration as it facilitates good spatio-temporal distribution of cells in scaffold. The feed

for bioprinting is bioink, which comprises of cells incorporated in the scaffold material.

Progress has been made on the incorporation of growth factors in the bioink, which not only

enables efficient regeneration but at the same time proves the feasibility of large constructs.

Important parameters which determine the suitability of bioink have been discussed here.

Lack of vascularization limits the success of this technology in its present form. Advances in

inducing vascularization and growth factors have also been discussed. Towards the end,

challenges and opinions in the area of bioprinting of bone tissue regeneration have been

presented.

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• Corresponding author: Email: [email protected]

Keywords: Bioink; Growthfactors; Controlled release; Angiogenesis; Osteogenesis;

Introduction

Three-dimensional (3D) printing has advanced the field of bone regenerative medicine by

overcoming engineering challenges of biomimicking biological tissues or organs. The

conventional 3D printing approach involves the predefined layered printing of scaffolds

followed by cell seeding and perfusing the construct before implantation. However, this

method suffers from a lack of uniform spatial and temporal distribution of cells and growth

factors in the construct. A new class of 3D printing called bioprinting – printing along with

the cells – promises to overcome all these limitations.

In this mini review, we report the most recent advances in the field of 3D bioprinting of bone

with respect to methods, bioink properties and growth factors/drug aided vascularization in

the constructs. Later, we present our opinion and the challenges needed to overcome, to

advance the field of bone bioprinting.

Bioprinting Techniques and Bioinks

Current focus has been to develop novel bioinks which can be 3D printed in cell-compatible

conditions to fabricate a cell-laden 3D structure. Bioink comprises of cells embedded in a

printable material which aids proliferation of cells by maintaining a supply of nutrients,

oxygen and growth factors (GFs). Bioinks can be in the form of hydrogels, viscous fluids or

micro-carriers [1]. Polymeric hydrogels are preferred as they mimic native extracellular

matrix (ECM) and facilitate cell adhesion and matrix integration [2]. Selection of suitable

combination of bioprinting method and bioink is very essential for a successful fabrication of

tissues [3]. The widely employed bioprinting techniques include drop based or inkjet, laser-

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assisted and extrusion bioprinting [4], some of which are summarized in Table 1. The

essential properties required for bioink vary with different bioprinting method employed

(Table 2).

Table.1 Additive Manufacturing Techniques for bone tissue scaffold (conventional and bioprinting)

Technique Resolution (µm)

Pore size (µm)

Porosity (%)

Advantages Disadvantages Ref.

1. Photo polymerization based Techniques Stereo lithography (SLA)

14-150

20-1000

<90

Complex internal features; GFs and cell loading possible

Only for photopolymers; Toxic photoresins; shrinkage issue; Need of support structure

[5]

Micro-stereolithography (MSLA)

0.5-10

100-300

<90

Digital light processing

40

500

<90

2. Powder based techniques Selective laser sintering (SLS)

50-1000

30-2500

<40

Solvent free, fast operation; No need of support structure; No post processing required;

Expensive; Surface Powder finishing; Difficult to remove blocked powder; high temperature involved; Resolution depends on diameter of laser beam

[6]

3D Printing

50-300 45-1600 45-60

Mild process conditions

Poor mechanical properties

[7]

3. Extrusion based techniques Fused deposition modeling

100-150 100-2000 <80%

Good mechanical integrity;

Limited filament materials;

[8]

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Solvent and support structure not needed;

material exposed to high temperature; Scaffold with small pore size is difficult to fabricate.

Low temperature deposition

300-500 200-500 <80%

Broad range of material usage; Able to incorporate growth factors;

Solvent usage; Freeze drying is required;

Pressure assisted micro-syringe

10-1000 10-600 70% Very fine resolution

Printable viscosities are limited in range

Robocasting / Direct Ink writing

100-450 5-100 <90%

Independent and customized 3D nozzle movement with high resolution; Highly viscous suspensions can be used; Support is not required; 1mm structures;

Expensive; Optimization of Bioink properties is crucial;

4. Bioprinting Method and material

Resolution (µm)

Droplet size (µm)

Cell viability

Advantages Disadvantages Ref.

Droplet based bioprinting For hydrogels

~50µm 50-300 <85%

Compatible with narrow viscosity range; Compatible with various cells and GFs; Suitable to deposit cells on microarrays or organ-on-a-chip Inexpensive, flexible, and commercially

Non-uniform droplet size; Nozzle blocking in fibrous bioink and in high cell densities; Cross contamination while printing multiple bioinks simultaneously

[9,10]

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available Micro extrusion based Bioprinting Liquids, pastes and gels

5µm - 1mm wide

100µm-1mm

40-80%;

Compatible with wide range of viscosities; Enables printing of scaffold free bioink of tissue spheroids;

Shear stress of highly viscous bioink, tiny nozzle diameter, and large dispensing pressure causes significant cell damage; Not useful for high-throughput bioprinting of tissues; Low resolution limits the microchannel incorporation for vascularization. Limited control on cell–cell and cell– matrix interactions

Laser Assisted Bioprinting Cell suspension;

100-600µm >20µm >95%

Nozzle free technology enables less cell damage; High precision (1cell/droplet); Supports vascular channels

Laborious; Expensive; difficult to print hetero cellular scaffolds; Limited commercial viability

Table 2. Properties for bioink for different bioprinting methods [11]

Parameter Dropwise or Inkjet Laser assisted Extrusion

Viscosity Low (<100 mPa-s) Medium (1-300 mPa-s ) Higher (>6 -108 mPa-s)

Surface Tension Medium Low Low

Gelation Rapid (Sec) Rapid (Sec) Rapid (Sec to min)

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Fabrication speed medium low high

Ink behaviour Rheopectic High viscoelasticity Thixotropic

Adhesion Low Adhesion within layers Low

Natural polymers are preferred over synthetic polymers because of cell affinity and

resemblance to the ECM. However natural polymers undergo uncontrollable degradation and

possess poor mechanical stability. Crosslinking and polymer blending can control the

scaffold degradation kinetics [3,4,12]. Mechanically superior biocompatible synthetic

polymers are used with growth factors (to improve cell adhesive properties) provide better

control over cell specific stiffness and elastic modulus [3,13]. A tabular presentation of

different polymers used as a bioink has been presented in Table 3.

Table 3. Recently reported hydrogel based bio-ink for 3D-bioprinting of bone tissue

Types of

bioink

Biomaterials Bioink compositions Ref.

Natural

hydrogel

based bioink

Alginate Alginate based (low : high molecular weight

1:2) bioinks for fibroblast cell-printing

[14]

Agarose a) Agarose/cell suspension (2%; 20 million

ml−1)

Bone marrow derived mesenchymal stem

cells bioprinting

b) fibroblasts L929 cell-printing using

thermoresponsive agarose- agarose-collagen

composite hydrogel

[15,16]

Collagen Higher concentration (10−20 mg/ mL) [17]

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collagen hydrogels for tissue printing

applications

Chitosan Polyelectrolyte gelatin-chitosan hydrogel

bioink is used to print fibroblast by extrusion-

based printer

[18]

Fibrin Micro-valve bioprinting of fibrin [19]

Gelatin gelatin/sodium alginate/carboxymethyl

chitosan (Gel/SA/CMCS) hydrogel

combining with bone mesenchymal stem

cells (BMSCs) for 3D bioprinting

[20]

Hyaluronic Acid

(HA)

Shear-thinning supramolecular HA-hydrogels

were used as both as an ink and support

material for bioprinting

[21]

Matrigel Extrusion-based bioprinting of matrigel and

collagen

[2]

Synthetic

hydrogel

based bioink

Pluronic F-127 Shear thinning hydrogel Pluronic F-127 is

used as bioink

[22]

Methacylated

gelatin

MG63 osteoblast-like cells loaded Gelatin

methacryloyl (GelMA) hydrogel is bioprinted

by extrusion-based printer

[23]

Poly(ethylene

glycol)

Endothelial and stem cells loaded GelMA,

alginate, and PEGTA hydrogel bioink is used

in multilayered coaxial nozzle bioprinter

[24]

Multimaterial

Bioinks

Sodium alginate

hydrogel-polylactic

Stem cells loaded hydrogel for better

survivability

[25]

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acid (PLA)

nanofiber

PLA nanofibers mimic ECM

HA, Gelatin

methacrylate,

gellan gum

Gum improved viscosity, printing fidelity and

stability of bioink

[10]

Alginate/PVA Micro and macro porous scaffold with protein

and growth factor fabricated in mild

conditions

[26]

Few key material parameters for bioink involve printability, biocompatibility,

biodegradability, mechanical integrity and biomimicry [4,12]. The rheological behavior

(viscosity and shear thinning), surface tension, swelling properties, and gelation kinetics are

significant aspects for printability [2]. Properties of bioink are also dependent on the printing

method. Drop wise nozzle based or inkjet 3D printing can only handle viscosity less than 100

mPa-s [10], due to clogging whereas laser-assisted and extrusion bioprinting can handle

relatively viscous materials. Higher viscosity (due to a higher concentration, molecular

weight or crosslinking) ensures a better post printing structural stability but at the same time

lowers the cell viability due to increased shear force [27]. Thus, optimizing viscosity and

crosslinking is essential to prepare a bioink which is cell-compatible and provides high-

fidelity scaffold structure. Biocompatibility and biomimicry are necessary to mimic the tissue

microenvironment and aid cell viability and proliferation. Multi-material bioinks are

emerging in the past three years, which provide a better structural stability and cell

survivability (Table.3). However, it is important to avoid polymers with a large difference in

swelling ratio to ensure mechanical stability [4,12].

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Vascularization in 3D Bone Constructs

Pre-incorporation of cells in the hydrogel [28], electrospun fibres [29] and microspheres [30]

and, later making a composite with 3D printed scaffold has yielded satisfactory cell

survivability and differentiation in small bone constructs. In vivo, the osteocytes are located

closer than 100 µm from a capillary in the interosseous vasculature, and the natural blood

vessel growth in the implants tends to be very slow (less than 1mm/day) [19]. Although the

small-scale 3D printed tissue constructs are relatively easy to perfuse, physiologically

relevant larger bone constructs cannot survive merely on the diffusion-based supply of

growth nutrients and oxygen due to hypoxia and poor nutrient supply associated with

deprived vascularization [31]. Therefore, it is essential to direct the research efforts on

including vascularization into the design which would help in removing degraded biological

products and preventing necrotic core formation.

In this regard, research has mainly focused on 3D printing of different combination of cells,

like stem cells with the endothelial cells, along with the scaffold material and finding out

ways to control the supply of growth factors [27,31]. Blinder et al. [32] used 3D engineered

vascular tissue constructs to investigate and understand the development of new blood vessels

using live confocal microscopy and showed that neovascular formation happens through a

multi-stage morphogenic process. Kolesky et al. [33] described a method (see figure 1A (i-

iv)) for printing thick 3D vascularized tissues (>1 cm) containing multiple types of cells that

are perusable for a relatively longer time (more than six weeks). Various inks containing

human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts were

printed onto a tailored ECM embedded with vasculature lined with human umbilical vein

endothelial cells (see Figure 1A (v)). The final integrated tissue was then perfused with

growth factors to successfully differentiate hMSCs into bone cells.

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Growth Factors and Delivery Systems for Enhanced Osteo- and Angiogenesis

To enhance angiogenesis and osteogenesis, the focus is now on culturing stem cells with

osteogenic and angiogenic GFs. Angiogenic GFs include vascular endothelial GF (VEGF)

and platelet-derived GF (PDGF) while bone morphogenic factors such as BMP-2 & BMP-7

induce mitogenesis of mesenchymal stem cells (MSCs) and circulating osteoprogenitor cells,

and promote their differentiation into osteoblasts [34]. However, the clinical translation of

GFs is limited due to burst release and rapid clearance from the implanted site in the

conventional delivery system. Also, to achieve minimum effective concentration at the

injured site, GFs are used at supraphysiological levels which lead to heterotopic bone

formation and certain types of cancers. As a result, research has been motivated to design

better delivery systems to reduce dose and control spatiotemporal release [35]. In native

bone, GFs are encrypted in ECM which protects them from hydrolytic and enzymatic

degradation. Hence, maintaining native protein conformation and bioactivity all through the

scaffold and for the duration of in vivo release is a critical challenge in scaffold-based GF

delivery [36]. Recombinant GFs [37] and osteoinductive drugs such as alendronate [38] have

also been used for osteoconduction.

Various methods (physical entrapment, using micro/nanoparticles, hydrogel systems with the

current trend heading towards hybrid approaches dual growth factor delivery and stimuli-

responsive carrier) have been discovered to incorporate growth factors, drugs and peptides

into 3D polymeric and composite scaffold systems [37]. Alendronate loaded 3D printed

tricalcium phosphate (TCP) coated with Polycaprolactone (PCL), showed mechanical

integrity and controlled release of alendronate up to 6 weeks [38]. Poldervaart et al, showed

VEGF loaded gelatin micro-particles in a 3D printed construct promoted rapid

vascularization with a controlled delivery of VEGF up to 3 weeks [39]. In another study, as

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can be seen in Fig. 1(B), 3D printing was used to spatially and temporally control BMP-2 and

VEGF delivery for large-volume bone regeneration by printing mesenchymal stem cells with

BMP-2 in slow releasing collagen hydrogel on periphery and VEGF in a rapid releasing

alginate/gelatin blend in centre (where hypoxic zone forms). Early vasculogenesis and fast

bone regeneration even in the core of a large structure was observed [40]. Sustained release

of VEGF was observed when loaded to porous 2�N, 6�O�sulfated chitosan coated PLGA

hierarchical scaffolds [41]. Haitao Cui et al. achieved highly osteogenic bone construct with

organized vascular networks by alternative layering of biodegradable PLA fibers and cell-

laden gelatin methacrylate (GelMA) hydrogels and immobilizing growth factors (VEGF and

BMP-2) in preselected regions [42,43]. Bose at al. [44] reported a significant increase in

osteogenesis and angiogenesis in vivo by the sustained release of magnesium and silicon ions

from the 3D P TCP scaffolds with three different sizes of interconnected pores.

Going Beyond Three-Dimensions

4D bioprinting has emerged in recent times where, “smart” 3D constructs could be

programmed to alter their shapes or functionalities in the presence of various external stimuli

such as temperature [41], light [45], magnetic [33], and ultrasound [46]. Sawkins et al.

proposed thermoresponsive microparticulate material based on poly (lactic-co-glycolic acid)

for bioprinting mesenchymal stem cells to produce constructs at ambient conditions with

mechanical properties comparable to cancellous bone. Model protein Lysozyme showed

sustained release up to fifteen days and was bioactive till nine days [47]. Recently, Kang et

al. [48] reported a new type of printing technology – integrated organ-tissue printer (ITOP) -

which controls the shape of the tissue construct by translating clinical image data into the

movements of the nozzle. They demonstrated ITOP’s capabilities of successfully fabricating

mandible and calvarial bone, cartilage and skeletal muscle by co-printing cell-laden

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hydrogels and a matrix made of PCL. The bio-printed construct, which overcomes previous

bioprinting limitations on the size, shape, structural integrity, also includes micro-channels to

supply necessary nutrients, oxygen and growth factors. This novel bioprinting technique will

open up a host of new and exciting possibilities of developing patient-specific customizable

tissues or organs and will inspire the future commercialized bio-printers (Figure.2).

Current Opinion and Future Prospects

How much native complexity must be recreated while designing bone tissues remains an

open question in the field of tissue engineering. While 3D bioprinting offers so much in this

regard, designing suitable bioinks remains a challenge. Ideally, the 3D printed bone scaffold

should be able to release the angiogenic and osteogenic growth factors, placed in the central

region and peripheral region respectively, in a biphasic manner with an optimum degradation

rate of the scaffold. Angiogenic growth factors should be released rapidly for the initial few

days to augment the healing of necrotic tissue. As soon as the capillaries develop, release of

osteogenic growth factors should start in the scaffold in a controlled manner. Burst release of

osteogenic growth factors should be closely controlled to prevent the ectopic bone formation.

These ideal characteristics of an engineered bone are big challenges and directions for future

research. Multi-material hydrogels including interpenetrating networks, nanoparticle

composites and supramolecular hydrogels based bioinks pave the future direction to provide a

facile and effective method for attaining desirable bioink characteristics such as printability

and high structural fidelity, high post-printing mechanical strength, biodegradability and cell

viability. Smart-release systems or 4D bioprinting would be the future direction for the

biphasic release to prevent denaturation during printing and aid spatio-temporal release of

bio-actives. Determining the minimal standards necessary for successful integration of a 3D

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bioprinted tissue will need extensive optimization with regard to cells, shapes, and

mechanical integrity as well as pre-clinical research.

Figure 1. (A) Schematic illustration of the vascularized three-dimensional tissue

manufacturing process. (i) Fugitive ink acting as a vascular ink and cell-laden inks are printed

inside a 3D perfusion chip. (ii) ECM material (containing, fibrinogen, gelatin, thrombin,

cells, and TG) is then cast over the printed inks. (iii) Fugitive ink liquefies when cooled and

is evacuated, leaving behind a vascular network, which is (iv) seeded with endothelial cells

and perfused using an external pump. (v) Schematics of a printed tissue construct containing

hMSCs and hNDFs along with vascular channels contained within a perfusion chamber.

Reproduced from Kolesky et al., [33]. (B) Structure designs. Group 1: DPSCs printed

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structure using 2% collagen. Group 2: DPSC/BMP-2 printed structure using 2% collagen.

Group 3: DPSC/dual growth factors printed structure using 2% collagen and 10%

alginate/10% gelatin blend. Reproduced from Park et al., [40].

Figure 2. The future of 3D Bone Bioprinting: ITOP (Kang et. al. [48]) and methods to control

spatio-temporal release of growth factors are currently paving the future of patient-specific

bone tissue engineering. The shape and size of the defect is modeled using clinical imaging

data and a template for 3D printing is developed. Bioink contains hydrogel containing cells, a

supporting material, and sacrificial material for making vascular channels. Growth factors

and drugs may be added in the bioink to aid osteogenesis and angiogenesis in the 3D printed

bone construct.

References

1. Ouyang L, Yao R, Zhao Y, Sun W: Effect of bioink properties on printability and cell

viability for 3D bioplotting of embryonic stem cells. Biofabrication 2016,

8:035020.

2. Chimene D, Lennox KK, Kaunas RR, Gaharwar AK: Advanced bioinks for 3D printing:

A materials science perspective. Annals of biomedical engineering 2016, 44:2090-

2102.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

3. Guvendiren M, Molde J, Soares RM, Kohn J: Designing biomaterials for 3D printing.

ACS Biomaterials Science & Engineering 2016, 2:1679-1693.

4. Mandrycky C, Wang Z, Kim K, Kim D-H: 3D bioprinting for engineering complex

tissues. Biotechnology advances 2016, 34:422-434.

5. Scalera F, Corcione CE, Montagna F, Sannino A, Maffezzoli A: Development and

characterization of UV curable epoxy/hydroxyapatite suspensions for

stereolithography applied to bone tissue engineering. Ceramics International

2014, 40:15455-15462.

6. Mazzoli A, Ferretti C, Gigante A, Salvolini E, Mattioli-Belmonte M: Selective laser

sintering manufacturing of polycaprolactone bone scaffolds for applications in

bone tissue engineering. Rapid Prototyping Journal 2015, 21:386-392.

7. Shirazi SFS, Gharehkhani S, Mehrali M, Yarmand H, Metselaar HSC, Kadri NA, Osman

NAA: A review on powder-based additive manufacturing for tissue engineering:

selective laser sintering and inkjet 3D printing. Science and Technology of

Advanced Materials 2015, 16:033502.

8. Mota C, Puppi D, Chiellini F, Chiellini E: Additive manufacturing techniques for the

production of tissue engineering constructs. Journal of tissue engineering and

regenerative medicine 2015, 9:174-190.

9. Peng W, Unutmaz D, Ozbolat IT: Bioprinting towards physiologically relevant tissue

models for pharmaceutics. Trends in biotechnology 2016, 34:722-732.

10. Murphy SV, Atala A: 3D bioprinting of tissues and organs. Nature biotechnology 2014,

32:773-785.

11. Hospodiuk M, Dey M, Sosnoski D, Ozbolat IT: The bioink: A comprehensive review

on bioprintable materials. Biotechnology Advances 2017.

12. Garreta E, Oria R, Tarantino C, Pla-Roca M, Prado P, Fernández-Avilés F, Campistol JM,

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

Samitier J, Montserrat N: Tissue engineering by decellularization and 3D

bioprinting . Materials Today 2017.

13. Zhang H-B, Xing T-L, Yin R-X, Shi Y, Yang S-M, Zhang W-J: Three-dimensional

bioprinting is not only about cell-laden structures. Chinese Journal of

Traumatology 2016, 19:187-192.

14. Park J, Lee SJ, Chung S, Lee JH, Kim WD, Lee JY, Park SA: Cell-laden 3D bioprinting

hydrogel matrix depending on different compositions for soft tissue engineering:

Characterization and evaluation. Materials Science and Engineering: C 2017,

71:678-684.

15. Daly AC, Critchley SE, Rencsok EM, Kelly DJ: A comparison of different bioinks for

3D bioprinting of fibrocartilage and hyaline cartil age. Biofabrication 2016,

8:045002.

16. Tan YJ, Tan X, Yeong WY, Tor SB: Hybrid microscaffold-based 3D bioprinting of

multi-cellular constructs with high compressive strength: A new biofabrication

strategy. Scientific Reports 2016, 6.

17. Rhee S, Puetzer JL, Mason BN, Reinhart-King CA, Bonassar LJ: 3D bioprinting of

spatially heterogeneous collagen constructs for cartilage tissue engineering. ACS

Biomaterials Science & Engineering 2016, 2:1800-1805.

18. Ng WL, Yeong WY, Naing MW: Polyelectrolyte gelatin-chitosan hydrogel optimized

for 3D bioprinting in skin tissue engineering. International Journal of Bioprinting

2016, 2.

19. Stegen S, van Gastel N, Carmeliet G: Bringing new life to damaged bone: the

importance of angiogenesis in bone repair and regeneration. Bone 2015, 70:19-27.

20. Huang J, Fu H, Wang Z, Meng Q, Liu S, Wang H, Zheng X, Dai J, Zhang Z: BMSCs-

laden gelatin/sodium alginate/carboxymethyl chitosan hydrogel for 3D

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

bioprinting . RSC Advances 2016, 6:108423-108430.

21. Highley CB, Prestwich GD, Burdick JA: Recent advances in hyaluronic acid hydrogels

for biomedical applications. Current opinion in biotechnology 2016, 40:35-40.

22. Zhang YS, Yue K, Aleman J, Mollazadeh-Moghaddam K, Bakht SM, Yang J, Jia W,

Dell’Erba V, Assawes P, Shin SR: 3D bioprinting for tissue and organ fabrication.

Annals of biomedical engineering 2017, 45:148-163.

23. McBeth C, Lauer J, Ottersbach M, Campbell J, Sharon A, Sauer-Budge AF: 3D

bioprinting of GelMA scaffolds triggers mineral deposition by primary human

osteoblasts. Biofabrication 2017, 9:015009.

24. Jia W, Gungor-Ozkerim PS, Zhang YS, Yue K, Zhu K, Liu W, Pi Q, Byambaa B,

Dokmeci MR, Shin SR: Direct 3D bioprinting of perfusable vascular constructs

using a blend bioink. Biomaterials 2016, 106:58-68.

25. Narayanan LK, Huebner P, Fisher MB, Spang JT, Starly B, Shirwaiker RA: 3D-

Bioprinting of Polylactic Acid (PLA) Nanofiber–Algi nate Hydrogel Bioink

Containing Human Adipose-Derived Stem Cells. ACS Biomaterials Science &

Engineering 2016, 2:1732-1742.

26. Luo Y, Luo G, Gelinsky M, Huang P, Ruan C: 3D bioprinting scaffold using

alginate/polyvinyl alcohol bioinks. Materials Letters 2017, 189:295-298.

27. Datta P, Ayan B, Ozbolat IT: Bioprinting for Vascular and Vascularized Tissue

Biofabrication . Acta Biomaterialia 2017.

28. Neufurth M, Wang X, Schröder HC, Feng Q, Diehl-Seifert B, Ziebart T, Steffen R, Wang

S, Müller WE: Engineering a morphogenetically active hydrogel for bioprinting

of bioartificial tissue derived from human osteoblast-like SaOS-2 cells.

Biomaterials 2014, 35:8810-8819.

29. Yu Y, Hua S, Yang M, Fu Z, Teng S, Niu K, Zhao Q, Yi C: Fabrication and

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

characterization of electrospinning/3D printing bone tissue engineering scaffold.

RSC Advances 2016, 6:110557-110565.

30. Levato R, Visser J, Planell JA, Engel E, Malda J, Mateos-Timoneda MA: Biofabrication

of tissue constructs by 3D bioprinting of cell-laden microcarriers. Biofabrication

2014, 6:035020.

31. Tang D, Tare RS, Yang L-Y, Williams DF, Ou K-L, Oreffo RO: Biofabrication of bone

tissue: approaches, challenges and translation for bone regeneration.

Biomaterials 2016, 83:363-382.

32. Blinder YJ, Freiman A, Raindel N, Mooney DJ, Levenberg S: Vasculogenic dynamics in

3D engineered tissue constructs. Scientific reports 2015, 5:17840.

33. Kolesky DB, Homan KA, Skylar-Scott MA, Lewis JA: Three-dimensional bioprinting

of thick vascularized tissues. Proceedings of the National Academy of Sciences

2016, 113:3179-3184.

34. Hankenson K, Gagne K, Shaughnessy M: Extracellular signaling molecules to promote

fracture healing and bone regeneration. Advanced drug delivery reviews 2015,

94:3-12.

35. Samorezov JE, Alsberg E: Spatial regulation of controlled bioactive factor delivery

for bone tissue engineering. Advanced drug delivery reviews 2015, 84:45-67.

36. Martino MM, Briquez PS, Maruyama K, Hubbell JA: Extracellular matrix-inspired

growth factor delivery systems for bone regeneration. Advanced drug delivery

reviews 2015, 94:41-52.

37. Quinlan E, López-Noriega A, Thompson E, Kelly HM, Cryan SA, O'Brien FJ:

Development of collagen–hydroxyapatite scaffolds incorporating PLGA and

alginate microparticles for the controlled delivery of rhBMP-2 for bone tissue

engineering. Journal of Controlled Release 2015, 198:71-79.

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38. Tarafder S, Bose S: Polycaprolactone-coated 3D printed tricalcium phosphate

scaffolds for bone tissue engineering: in vitro alendronate release behavior and

local delivery effect on in vivo osteogenesis. ACS applied materials & interfaces

2014, 6:9955-9965.

39. Poldervaart MT, Gremmels H, van Deventer K, Fledderus JO, Öner FC, Verhaar MC,

Dhert WJ, Alblas J: Prolonged presence of VEGF promotes vascularization in 3D

bioprinted scaffolds with defined architecture. Journal of Controlled Release 2014,

184:58-66.

40. Park JY, Shim J-H, Choi S-A, Jang J, Kim M, Lee SH, Cho D-W: 3D printing

technology to control BMP-2 and VEGF delivery spatially and temporally to

promote large-volume bone regeneration. Journal of Materials Chemistry B 2015,

3:5415-5425.

41. Yu Y, Chen J, Chen R, Cao L, Tang W, Lin D, Wang J, Liu C: Enhancement of VEGF-

mediated angiogenesis by 2-N, 6-O-sulfated chitosan-coated hierarchical PLGA

scaffolds. ACS applied materials & interfaces 2015, 7:9982-9990.

42. Cui H, Zhu W, Nowicki M, Zhou X, Khademhosseini A, Zhang LG: Hierarchical

Fabrication of Engineered Vascularized Bone Biphasic Constructs via Dual 3D

Bioprinting: Integrating Regional Bioactive Factors into Architectural Design.

Advanced healthcare materials 2016, 5:2174-2181.

43. Cui H, Zhu W, Holmes B, Zhang LG: Biologically Inspired Smart Release System

Based on 3D Bioprinted Perfused Scaffold for Vascularized Tissue Regeneration.

Advanced Science 2016, 3.

44. Bose S, Tarafder S, Bandyopadhyay A: Effect of chemistry on osteogenesis and

angiogenesis towards bone tissue engineering using 3D printed scaffolds. Annals

of biomedical engineering 2017, 45:261-272.

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45. Gao G, Yonezawa T, Hubbell K, Dai G, Cui X: Inkjet�bioprinted acrylated peptides

and PEG hydrogel with human mesenchymal stem cells promote robust bone

and cartilage formation with minimal printhead clogging. Biotechnology journal

2015, 10:1568-1577.

46. Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG: Improved

Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted

Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation. Scientific

Reports 2016, 6.

47. Sawkins MJ, Mistry P, Brown BN, Shakesheff KM, Bonassar LJ, Yang J: Cell and

protein compatible 3D bioprinting of mechanically strong constructs for bone

repair . Biofabrication 2015, 7:035004.

48. Kang H-W, Lee SJ, Ko IK, Kengla C, Yoo JJ, Atala A: A 3D bioprinting system to

produce human-scale tissue constructs with structural integrity . Nature

biotechnology 2016, 34:312-319.

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Title of the Manuscript: Three-Dimensional Bioprinting for Bone Tissue Regeneration

Highlights:

1. 3D printing using bioinks for bone tissue regeneration

2. Incorporation of growth factors in bioink

3. Advances in inducing vascularization are promising

4. Multifacette challenges but enormous potential to develop tissue/organs in compliance

with patient need