3D bioprinting shows promise for applications in orthopaedics such as cartilage and bone regeneration. For cartilage, bioprinting can replicate the complex zonal structure of native tissue by printing cells and extracellular matrix layer-by-layer. For bone, combinations of biomaterials in hybrid scaffolds can mimic native bone properties. Bioprinting is also being explored for meniscus, intervertebral discs, and other orthopaedic tissues to address limitations of current treatment options.

1. THREE-DIMENSIONAL BIOPRINTING IN
ORTHOPAEDICS
DR. BIPUL BORTHAKUR
PROFESSOR, DEPT OF ORTHOPAEDICS,SMCH.

2. INTRODUCTION
 3-dimensional (3D) printing, is the process of engineering objects layer by layer to produce a
specific functional product.
 Since its inception in 1986, the process of 3D printing has expanded rapidly and has
positively impacted multiple fields of engineering and manufacturing .
 Use of 3D printing in orthopaedics includes the fabrication of custom casts and braces, the
production of surgical templates, the design and manufacture of patient specific arthroplasty
instrumentation, the design of custom total joint arthroplasty implants.

3. INTRODUCTION
 Three-dimensional-printing technology has evolved dramatically in the last 30 years from rudimentary
selective laser sintering printers to current printers that are able to create 3D products with living
components through a process called 3D bioprinting (3DBP).

4. 3DBP – BASIC PRINCIPLES
 The basis of 3DBP relies on concepts of biomimicry and biologic self-assembly.
 Biomimicry is the concept that a product should mimic the desired native tissue. Each specific cell and
tissue type must be accounted for, and proper cellular signaling is promoted by extrinsic environmental
factors that help to replicate the target tissue.
 Self-assembly is the concept that if the proper cells and components are in place, the desired tissue will
form as a result of cells and tissues having inherent mechanisms for maturation and development.
 Three-dimensional bioprinting can utilize a scaffold or be scaffold-free.

5. SCAFFOLD PRINTING
 Scaffold-type printing uses cells in an inert medium, which are seeded onto a premade scaffold.
 These mixtures of cells in an inert medium are known as bioinks, and they are the foundation of 3DBP9 .
 The inert media are usually hydrogels, which have various properties depending on the biomaterials that
are included.

6. SCAFFOLD PRINTING
 Hydrogels are defined as “water-swellable, water-insoluble, cross-linked networks” of polymers that can
provide multiple advantages in tissue engineering by supporting cell viability and differentiation.
 Scaffolds are appealing as they represent a volume-stable construct. They have an excellent ability to
control the spatial structure of tissues and create a robust extracellular matrix (ECM)

7. SCAFFOLD FREE PRINTING
 Scaffold-free techniques utilize microscopic tissue units, such as cell spheroids or pellets.
 These are deposited onto a substrate and mature to form functional tissue.
 Spheroids are produced by self-assembly processes through adhesion molecules in cell culture that mimic
the natural processes of embryogenesis, morphogenesis, and organogenesis.

8. Bioprintring processes and techniques
 The desired 3D digital file is created, usually from computed tomography (CT) or magnetic resonance
imaging (MRI), and exported into a 3D digital file.
 The production of the desired target tissue includes the selection of the type of printer to be used as well
as the type and combination of the components (bioink, scaffold versus scaffold-free, growth factors, and
cell types).

9. Bioprintring processes and techniques
 Bioprinter technology can be classified as
 inkjet/droplet,
 laser assisted, or
 extrusion-based.
 The selection of printer type is made with consideration of the target tissue that is to be replicated.

10. Inkjet Bioprinter
 Inkjet printers use either thermal or piezoelectric energy to create droplets of bioink that are propelled
through a nozzle.

11. Extrusion- based Bioprinter
 Extrusion-based bioprinting (EBB), the most basic method of bioprinting
 It involves building pressure in a reservoir and using either a screw or pneumatic cylinder to dispense the
bioink.
 These printers continuously dispense bioink onto a collecting surface to construct a final product.

12. Extrusion- based Bioprinter

13. Laser- assisted Bioprinter
 Laser-assisted bioprinting (LAB) is a method of bioprinting in which a laser source fires a direct and
focused beam of energy onto a ribbon that contains bioink.
 The ribbon contains a metal covering that absorbs the energy and transfers it to the bioink, forming a
bubble that propels the bioink.
 LAB is highly precise, and resolution as high as 1 cell per droplet has been reported, allowing for
structures of varying cell types to be printed in close proximity to one another.

14. Laser- assisted Bioprinter

15. Clinical applications: Articular cartilage
 Use of 3DBP represents an innovative approach for articular cartilage restoration; however, the unique
biology of this tissue provides challenges.
 Hyaline cartilage is on average 2 to 4 mm thick; it is avascular and aneural and is comprised of ECM and
chondrocytes.
 This ECM contains collagen, proteoglycans, and glycoproteins in varying concentrations and orientations
based on the depth of the cartilage. This unique composition allows for the retention and intrasubstance
movement of water, which is critical to the anisotropic and viscoelastic properties of cartilage.

16. Articular cartilage
 Injured articular cartilage is associated with an unpredictable and potentially disordered capacity to heal.
 Currently, there is one commercially available synthetic cartilage implant, CARTIVA SCI (cartiva), which
is an acellular molded polyvinyl alcohol hydrogel. CARTIVA was approved to treat patients with hallux
rigidus, but it has failed to achieve consistent clinical success.
 Recent advances in 3D printing have contributed techniques that will allow for the production of materials
that more closely replicate native cartilage.

17. Articular cartilage
 Prior cartilage tissue engineering methods involved printing or molding the ECM components in the
desired shape and then adding cells to the matrix; however, this does not mimic the native architecture.
 Now, bioprinters can print cells along with ECM-like material layer by layer to more closely replicate the
complex and unique zonal ultrastructure of cartilage.

19. Bone:
 Bone defects as a result of trauma, tumor resection, and infection represent a common treatment
challenge.
 At present, options for the management of bone defects are limited to the use of bone autografts and
allografts.
 Use of autogenous bone graft is potentially limited by the extent of bone that is available and donor-site
morbidity.
 Allogenic bone grafts are associated with the potential for disease transmission, and there is limited
availability and a high cost.

20. Bone :
 Multiple different materials (bioceramics such as calcium phosphate [cap], tricalcium phosphate [tcp], and
hydroxyapatite [hap]) that are currently used clinically as bone substitutes.
 These bone substitutes are osteoconductive given their chemical similarity to the apatite crystals of bone;
however, their lack of live cells means that they are not osteoinductive, and they have other issues, such as
partial disintegration when exposed to body fluids.
 Because of the limitations of each of these methods, bone restoration through tissue engineering has been
a focus of extensive research.

21. Bone :
 Although scaffold-free bioprinting seems ideal for bone tissue engineering because it allows for precise
control over porosity and deposition of cells and biomaterials, constructs often lack compressive strength.
 Given the initial structural integrity that can be provided by using scaffolds and the relative infancy of
scaffold-free bioprinting, much of the bone bioprinting research revolves around 3d-printed scaffolds that
are seeded with cells after production.
 Since neither bioceramics nor polymers are perfect for bone tissue engineering, many combinations of
materials have been combined as hybrid materials for scaffold formation, and some have been found to
have properties very similar to native bone

22. Bone :
 Bioinks made from polymers such as alginate, collagen, or gelatin have been the focus of scaffold-free
techniques because of their structural similarities to bone ECM.
 Poor compressive strength remains an issue, but the addition of other biomaterials to the base bioink, the
cross-linking of polymers, and post-processing compressive loading have been useful in transforming
them into more mechanically effective constructs.
 The addition of biomaterials can concurrently increase compressive strength and osteoconductivity (e.g.,
With the addition of HAp, TCP, and/or bioactive glass). Osteoinductive elements, including growth factors
like BMPs, also have been incorporated to guide differentiation of the precursor cells that are included in
the bio-ink.

23. Bone :
 Providing vascularity to support the synthetic graft once it has been implanted is a challenge in bone tissue
engineering, especially for the generation of larger grafts.
 To remedy this issue, researchers have created vessel-like structures that can be lined with endothelial
cells to promote vascularization.
 The introduction of endothelial cells can cause the formation of microcapillaries, and self-assembly of
capillaries in bioprinted bone has been accomplished through this technique.

24. Bone :
 One methodology includes coculture of human umbilical vein endothelial cells (HUVECS) with
mesenchymal stem cells (MSCs), along with both osteogenic growth factors and vasculogenic factors,
usually vascular endothelial growth factor (VEGF).
 Through this method, several investigators recently have produced osteogenic and vasculogenic niches
within the same printed tissue, and this process has been deemed “prevascularization”

26. Meniscus:
 Many meniscal tear patterns may be reparable; however, for patients with substantial meniscal tissue loss,
replacement through allogeneic transplantation is the gold-standard treatment option.
 Allograft transplantation is associated with limited tissue availability, immunogenicity, and impaired
remodeling capacity.
 There are 2 acellular meniscal substitute scaffolds that are commercially available in the united states;
however, their use is considered controversial.

27. Meniscus:
 These scaffolds are the collagen meniscus implant (CMI; stryker), made from bovine achilles tendon
collagen and gags, and the actifit (orteq sports medicine), made from a poly-«-caprolactone (PCL) and
polyurethane blend.
 Multiple authors have reported on bioprinting models of menisci that are based on MRI. The results have
shown variable cell viability within the tissue constructs.
 Filardo et al. Printed a bioink of collagen and MSCs using an inkjet printing technique and showed that
although cell viability was 50% after printing, likely due to processing temperatures, there was no
additional loss in viability.

28. Meniscus:
 Markstedt et al. Created a bioink from nanocellulose and alginate, and bioprinted it along with human
nasoseptal chondrocytes into the shape of sheep menisci, demonstrating 70% cell viability.
 Chansoria et al. Used alginate and human adipose stem cell (ASC) bioink with a novel technique of
ultrasound-assisted bioprinting to print meniscal tissue with 100% cell viability

29. Meniscus:
 Romanazzo et al. Described a biphasic meniscal tissue construct made with ECM from porcine menisci
that was seeded with infrapatellar fat-padderived stem cells with added connective tissue growth factor
(CTGF) for the periphery and TGF-ß3 for the inner meniscal tissue.
 After bioprinting, histologically the inner and peripheral meniscal tissue resembled the native meniscus in
terms of collagen and GAG expression.
 Compressive strength was low for the bioink alone, but when combined with PCL, a 100-fold increase in
strength was noted, giving the tissue a compressive modulus near the 0.1 to 1 MPa of native meniscal
tissue. Cell viability was 80% to 90% in the final constructs.

30. Meniscus:
STEPS IN 3D PRINTING.

31. Intervertebral disc:
 Degenerative disease of the intervertebral disc (IVD) of the spine is a common cause of back pain
 Surgical treatment options include spinal fusion, discectomy, and total disc replacement.
 All of these treatment options have advantages and disadvantages; hence, the potential to create a tissue-
engineered (TE)-IVD is a compelling area of research.

32. Intervertebral disc:
 Challenges encountered in creating a TE-IVD are replicating the highly aligned collagen organization of
the anulus fibrosus, matching the size and shape to the native disc, optimizing mechanical properties of
both the synthetic anulus fibrosus and nucleus pulposus tissue, and combining the synthetic anulus
fibrosus and the nucleus pulposus into a composite TE-IVD.
 Multiple different approaches have been utilized, including the use of electrospun nanofibrous PCL,
wetspinning and lyophilization of alginate/ chitosan, and contraction of anular collagen gels to create
scaffolds with architecture similar to the native anulus fibrosus

33. Intervertebral disc:
 PCL continues to be a popular polymer for anulus fibrosus tissue engineering due to its high young’s
modulus, which is similar to the native anulus fibrosus.
 Christiani et al. used 3D-printed scaffolds of PCL with differing angular orientations of polymer fibrils
and were able to create constructs that have axial compressive and circumferential tensile properties that
are similar to the native anulus fibrosus.
 Choy and chan showed that varying the number of cross-linked collagen lamellar rings in the scaffold
impacted biomechanical properties. They found that 10 anulus-like lamellae created a scaffold with elastic
compliance that was nearly identical to the native anulus fibrosus.

34. Intervertebral disc:
 Different combinations of biomaterials have been reported to create biphasic scaffolds that mimic the
anulus fibrosus and the nucleus pulposus of native IVD.
 Hyaluronic acid, fibrin-hyaluronan gel, alginate, and collagen-GAG coprecipitate all have been utilized to
create synthetic nucleus pulposus as part of biphasic synthetic IVD.

35. Intervertebral disc:
 Du et al. seeded a PCL/alginate biphasic IVD scaffold with rabbit anulus fibrosus and nucleus pulposus
cells and showed that the anulus fibrosus cells colonized the PCL scaffold while the nucleus pulposus
cells colonized the alginate hydrogel.
 Blanquer et al. Used 3d-printed scaffolds of poly(trimethylene carbonate) to mimic the anulus fibrosus
and then seeded them with human-adipose-derived stem cells and induced differentiation into anulus
fibrosus-like cells using TGF-ß3. They showed that these anulus fibrosus-like cells produced collagen and
GAG in a ratio comparable with human anulus fibrosus.

36. Intervertebral disc:
3D-PRINTING FOR DISECTOMY