3D Bioprinting for Cartilage and Osteochondral Tissue Engineering

TLDR: The adoption of biofabrication technology in musculoskeletal tissue engineering may make it possible to produce the next generation of biological implants capable of treating a range of conditions.

Abstract: Significant progress has been made in the field of cartilage and bone tissue engineering over the last two decades. As a result, there is real promise that strategies to regenerate rather than replace damaged or diseased bones and joints will one day reach the clinic however, a number of major challenges must still be addressed before this becomes a reality. These include vascularization in the context of large bone defect repair, engineering complex gradients for bone-soft tissue interface regeneration and recapitulating the stratified zonal architecture present in many adult tissues such as articular cartilage. Tissue engineered constructs typically lack such spatial complexity in cell types and tissue organization, which may explain their relatively limited success to date. This has led to increased interest in bioprinting technologies in the field of musculoskeletal tissue engineering. The additive, layer by layer nature of such biofabrication strategies makes it possible to generate zonal distributions of cells, matrix and bioactive cues in 3D. The adoption of biofabrication technology in musculoskeletal tissue engineering may therefore make it possible to produce the next generation of biological implants capable of treating a range of conditions. Here, advances in bioprinting for cartilage and osteochondral tissue engineering are reviewed.

Figures

Figure 5. Nucleic acid incorporation into 3D bioprinting. (A) Two-step incorporation of cells and nucleic acids into porous 3D printed constructs by direct loading. (B) One-step incorporation of nucleic acids into the 3D printing process through the introduction of the genetic material into the cell population due to the forces applied during inkjet printing. (C) One-step incorporation

Figure 2: 3D printing technology to control BMP-2 and VEGF delivery spatially and temporally to promote large-volume bone regeneration 131. (A) Outline of experimental groups. In group 3, region specific dual growth factor delivery was achieved by depositing the central portion of the scaffold with alginate/gelatin/VEGF and the outer portion of the scaffold was filled with collagen/BMP-2. This was compared to homogenous distributions of each growth factor (Group 1&2). (B) Analysis of vessel formation at different sites in the implanted constructs after 28 days in vivo. (a-c) Higher levels of vessel formation were observed in both central and peripheral regions of group 3 where VEGF was localised to the central region of the construct.

Figure 4: Biofabrication as a tool for joint resurfacing. (A) Tissue engineered cartilage templates for joint replacement were produced by seeding chondrocytes onto tibial shaped scaffolds, generated using FDM and particulate leaching 172. Pre-implantation, (c, d) SEM analysis, (e, f) Safranin-O/live-dead analysis and (g, h) immunohistochemical staining for collagen I, II demonstrated robust chondrogenesis within both systems. However, compared to particulate leaching, the scaffolds produced using FDM supported superior cell viability and matrix production in central regions. This is a key

Figure 1: Applications of biofabrication technology in cartilage tissue engineering. (A) Direct inkjet bioprinting of chondrocytes loaded in a photo-curable PEGMA bioink into a chondral defect 100. (B) Biofabrication of osteochondral tissue equivalents where microextrusion bioprinting was used to create a bi-layered construct containing chondrocytes in the cartilage layer and osteoprogenitors in the bone layer. Distinctive tissue formation was observed in the system in vivo as demonstrated by localised ALP staining in the bone region and collagen IV in the cartilage region 81. (C) Bioprinting of

Table 1: Summary of bioinks used for CTE Scoring system, Chondrogenic capacity = 1, defines a material that has no known inherent chondroinductive factors. Chondrogenic capacity = 2, defines material that can support chondrogenesis of MSCs without the addition of exogenous growth factors such as TGF-β3. Inherent printability = 1, defines a material that cannot be extruded in a reliable fashion for more than 1 layer with ease. Inherent printability = 2, defines a material that can be extruded into relatively simple shapes. Inherent printability = 3, defines a material that can be extruded into complex shapes with overhanging structures

Figure 3: Biofabrication of Composite Constructs for CTE (A) Dual bioprinting of composite, anatomically accurate, PCL/bioink constructs for auricular CTE 153. (B) Hybrid printing of mechanically improved constructs for CTE. The constructs were produced by inkjet bioprinting rabbit chondrocytes in a fibrin collagen bioink onto electrospun PCL microfibers in a layer-by-layer fashion. (C) (a) Biofabrication of soft network reinforced constructs for CTE. Graphical representation of MEW device and operating parameters, (b-d) SEM of crosshatch structure produced with 200 µm, 400 µm

Full text

3D Bioprinting for Cartilage and
Osteochondral Tissue Engineering
Andrew C. Daly
1,2,3
, Fiona F Freeman
1,2,3
, Tomas Fernandez Gonzalez
1,2,3,4
Susan E
Critchley
1,2,3
, Jessica Nulty
1,2,3
, Daniel J Kelly
1,2,3,4
1
Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin,
Ireland.
2
Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin,
Dublin, Ireland.
3
Department of Anatomy, Royal College of Surgeons in Ireland, Dublin, Ireland
4
Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and
Trinity College Dublin, Dublin, Ireland.
*Corresponding Author: Tel: +353-1-8963947, email: kellyd9@tcd.ie

Abstract
Significant scientific progress has been made in the field of cartilage and bone tissue
engineering over the last two decades. As a result, there is real promise that strategies to
regenerate rather than replace damaged or diseased bones and joints will one day reach the
clinic, however a number of major challenges must still be addressed before this becomes a
reality. These include vascularization in the context of large bone defect repair, engineering
complex gradients for bone-soft tissue interface regeneration and recapitulating the stratified
zonal architecture present in many adult tissues such as articular cartilage. Tissue engineered
constructs typically lack such spatial complexity in cell types and tissue organization, which
may explain their relatively limited clinical success to date. This has led to increased interest
in biofabrication technologies in the field of musculoskeletal tissue engineering. The additive,
layer-by-layer nature of bioprinting strategies makes it possible to generate zonal distributions
of cells, matrix and bioactive cues in 3D. The adoption of biofabrication technology in
musculoskeletal tissue engineering may therefore make it possible to produce the next
generation of biological implants capable of treating a range of conditions. Here we will review
advances in bioprinting for cartilage and osteochondral tissue engineering, from the
engineering of fiber reinforced tissues to biofabrication strategies for whole joint regeneration.
Furthermore, we will outline some of the key future research directions for this burgeoning
field, from bioprinting of vascularised constructs to the development of gene activated bioinks
for interface tissue engineering.

Contents
Abstract .................................................................................................................................. 2
1. Introduction: ................................................................................................................... 4
2. 3D Bioprinting in Cartilage and Bone Tissue Engineering: .............................................. 7
2.1 Cartilage Tissue Engineering........................................................................................ 7
2.1.1 Bioprinting Technology for Cartilage Tissue Engineering ................................... 7
2.1.2 General Bioink Requirements ............................................................................... 8
2.1.3 Bioink Development for Cartilage Tissue Engineering ........................................ 9
2.1.4 Bioprinting of Heterogeneous Cartilage Tissues ................................................ 12
2.2 Bioprinting in Bone Tissue Engineering .................................................................... 14
2.2.1 Developing bioinks for bone tissue engineering ................................................. 14
2.2.2 Bioprinting and vascularisation in BTE .............................................................. 16
2.3 Bioprinting of Composite Reinforced Tissues ............................................................... 19
2.3.1 Fused Deposition Modelling (FDM) ....................................................................... 19
FDM in Cartilage and Osteochondral Tissue Engineering .......................................... 20
FDM in Bone Tissue Engineering ............................................................................... 21
2.3.2 Bioprinting of Reinforced Constructs for Cartilage and Osteochondral Tissue
Engineering ....................................................................................................................... 22
2.3.3 Bioprinting of Reinforced Constructs for Bone Tissue Engineering ...................... 26
2.4 Whole Joint Resurfacing ................................................................................................ 26
3. Future Directions: ........................................................................................................ 30
3.1 Cartilage Tissue Engineering ................................................................................. 30
3.1.1 Biofabrication of Mechanically Functional Cartilage Tissues............................ 30
3.1.2 Bioprinting of Stratified Cartilage Tissues and Osteochondral Tissue Interfaces
...................................................................................................................................... 31
3.1.3 Towards Biofabrication of Anatomically Accurate Cartilage Tissues........... 33
3.2 Bioprinting of Vascularised Bone Tissue Engineering Constructs ............................ 34
3.3 Bioprinting of Gene Activated Bioinks ...................................................................... 37
3.3.1 Engineering Zonally Organised Interface Tissues Using Nucleic Acid Delivery
...................................................................................................................................... 40
4. Conclusions ...................................................................................................................... 41
5. References .................................................................................................................... 42

1. Introduction:
Osteoarthritis (OA) is a degenerative joint disease that affects millions of people
worldwide. In the USA alone, OA affects 37% of adults over 65 years old
1
. The disease is
characterised by progressive loss of hyaline cartilage in the synovial joints which leads to
significant joint pain, swelling and stiffness for sufferers. The disease is also a significant
economic burden with associated costs estimated to range from $3.4-13.2 billion per year in
the USA
2
. The current gold standard treatment option for OA is total joint arthroplasty where
the diseased cartilage and underlying bone are replaced with a metal and polymer prosthesis.
While the procedure is well established failures and complications are not uncommon
3,4
. For
example, ten year revision rates of up to 12% have been reported
5
. This has led to an increased
interest in the field of cartilage and osteochondral tissue engineering (TE) where de novo
tissues can be engineered to facilitate joint regeneration and hopefully prevent the onset of OA.
Significant progress has been made in the field of TE over the last two decades with
numerous studies demonstrating how combinations of biomaterials, cells and bioactive factors
can be used to engineer de novo cartilage and bone in vitro and in vivo
6–11
. In the case of
cartilage tissue engineering this has classically involved encapsulating chondrocytes, or stem
cells which can be differentiated along a chondrogenic linage, in a supportive matrix such as a
hydrogel or scaffold. The efficacy of such approaches for treating focal cartilage or
osteochondral defects have been demonstrated by a number of groups in large animal models
12–18
. In addition, a number of chondrocyte based therapies such as MACI (autologous cultured
chondrocytes on porcine collagen membrane) are available clinically and newer tissue
engineered cartilage products have entered the clinical trial stage, with some demonstrating
improvements in defect healing compared to existing treatment options such as microfracture

or autologous chondrocyte implantation
19
. However many products have also failed to
demonstrate efficacy and challenges remain in translating TE technologies into the clinic
19
.
Furthermore, existing approaches are designed to repair focal cartilage defects, but are not
suitable for treating osteoarthritic joints. The majority of TE products are typically formed
using mechanically weak hydrogels or scaffolds and are not suitable for treating the large areas
of degenerative joint surfaces associated with diseases such as OA. It is evident that a new
generation of more sophisticated tissue engineered cartilage and osteochondral grafts are
required to treat this more challenging patient population.
TE strategies typically aim to homogenously distribute biological factors such as cells
and growth factors throughout a biomaterial matrix. As a result, engineered tissues are often
homogenous in composition. However, articular cartilage is a highly anisotropic tissue whose
composition and organization varies greatly with depth. The tissue can be divided into three
zones, the superficial, middle and deep zone which are defined by gradients in collagen and
proteoglycan content and collagen fiber alignment
20–26
. These variations in ECM composition
and architecture in turn impart zonal biomechanical properties to the tissue
24,27
. It is generally
accepted that further progress in the field will require strategies that can better recapitulate the
spatial complexity of the native tissue and its interface with subchondral bone
28
. The next
generation of cartilage and osteochondral tissue engineered products should therefore
incorporate these considerations. Another major challenge in the field of cartilage TE is that
the mechanical properties of tissue engineered cartilage often fall below those of the native
tissue. Ideally tissue engineered cartilage would be able to withstand the high levels of
compressive and shear loading that will be present in an articulating joint upon implantation.
This is another key consideration for the next generation of tissue engineered cartilage
products, especially those designed to treat joints with large areas of degeneration.

Frequently asked questions

Q1. What have the authors contributed in "3d bioprinting for cartilage and osteochondral tissue engineering" ?

Here the authors will review advances in bioprinting for cartilage and osteochondral tissue engineering, from the engineering of fiber reinforced tissues to biofabrication strategies for whole joint regeneration. Furthermore, the authors will outline some of the key future research directions for this burgeoning field, from bioprinting of vascularised constructs to the development of gene activated bioinks for interface tissue engineering. 

Q2. What are the important rheological parameters to consider when designing bioinks?

The most important rheological parameters to consider when designing bioinks are viscosity, yield stress and shear thinning behaviour 74. 

Q3. What can be used to engineer biomimetic tissue gradients?

In a tissue engineering context, physicalgradients such as pore size and substrate stiffness, or biochemical gradients such as growth factor presentation, can be introduced into a construct. 

Q4. What is the role of gradients in biomimetic tissue engineering?

It has been demonstrated that biochemical gradients, such as the graded presentation of growth factors, can lead to the development of heterogeneous engineered tissues. 

Q5. What is the common use of FDM for TE 133?

As cells cannot be incorporated during the printing process due to high processing temperatures FDM is not technically considered bioprinting, however it is commonly used for producing porous scaffolds for TE 133. 

Q6. How did the bioink with bone marrow MSCs and BMP-2 bound collagen?

When compared to a bioink encapsulated with bone marrow MSCs alone and cultured in osteogenic medium, the bioink with bone marrow MSCs and BMP-2 bound collagen microfibers induced faster osteogenesis of MSCs compared to those cultured in the presence of osteogenic growth factors after 14 days in vitro 110. 

Q7. How much bone formation was observed in the TCP coated constructs?

At 12 weeks, the TCP coated constructs showed a trend towards increased bone formation however, new bone formation was <10% across the treatment groups 151 . 

Q8. What is the role of pericellular environment in the development of mesenchymal stem?

The pericellular environment regulatescytoskeletal development and the differentiation of mesenchymal stem cells anddetermines their response to hydrostatic pressure. 

Q9. What is the rate of degradation of the PCL fibers produced using FDM?

PCL fibers produced using FDM are usually large (>150 µm in diameter) and theporosity of the resultant scaffolds are usually less than 80%. 

Q10. How can endothelial cells be seeded in a TE construct?

Recentadvances in direct seeding techniques have shown that vessels as small as 20 µm in diameter can be seeded with endothelial cells 195. 

Q11. What are the limitations of traditional tissue engineering?

Although the described studies highlight the potential of spatial gene delivery for the regeneration of complex interface tissues, 3D bioprinting might solve the limitations of traditional tissue engineering associated with poor layer integration, the scalability of the approach and the tissue organization present in the repair tissue. 

Q12. What is the way to overcome limitations with residual PCL material?

Another way to overcome limitations with residual PCL material is to reduce the amount of the reinforcing polymer used by increasing the porosity of the reinforcing phase. 

Q13. What could be used to print auricular shaped constructs?

(a-f) Using patient specific data, anatomically accurate, auricular shaped constructs containing overhanging layers could be printed with the ink. (g-j) 

Q14. What is the common approach to co-extrude a stiff thermoplastic polymer?

The most common approach is to co-extrude a stiff thermoplastic polymer such as PCL using FDM, alongside a bioink containing cells using microextrusion bioprinting 32,53,62. 

Q15. What could be used to engineer more native like cartilage tissues?

Bioprinting technology could be used to further expand on these ideas to engineer cartilage tissues with a native like organisation. 

Q16. What could be used to print tissue templates in the geometry of the intervertebral disc?

The system could also be used to print tissue templates in the geometry of the intervertebral disc and nose, further demonstrating the versatility of the approach 91Osteoarthritis is a disease that can affect both the articular cartilage and the underlyingsubchondral bone. 

Q17. What is the main advantage of using melt-electrowriting?

This has led to an increased interest in the use of melt-electrowriting (MEW) which is an emerging technology that combines key aspects of melt-electrospinning and FDM. 

Q18. What is the promising approach to developing mechanically functional implants for jointresurfacing?

Another promising approach to developing mechanically functional implants for jointresurfacing could involve the development of tough bioinks based on interpenetrating network (IPN) hydrogels.