Intraoperative bioprinting, Laser based bioprinting, Droplet based bioprinting, Extrusion based bioprinting, Oxygen generating biomolecules, Current developments in IOB, Automation of IOB, bioink, surgical settings.

1. Intraoperative
Bioprinting
REPAIRING TISSUES AND ORGANS IN A SURGICAL SETTING
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By Hasnat Tariq and
Zubia Shahid

2. Contents
Advances from in-vitro bioprinting to Intraoperative bioprinting.
Current developments.
Considerations and future outlooks for IOB.
Limitations of current biopinting modalities.
Compatibility of bioinks to surgical settings.
Automation of IOB.
Vascularization in Intraoperatively bioprinted tissue.
IOB of composite tissues and their translational potential.
Concluding remarks.
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3. Abbreviations
• IOB = Intra-Operative Bioprinting
• LBB = Laser Based Bioprinting
• DBB = Droplet Based Bioprinting
• EBB = Extrusion Based Bioprinting
• ECM = Extracellular Matrix
• DOF = Degree Of Freedom
• HIF = Hypoxia Inducible Factors
• OGB = Oxygen Generating Biomolecules
• CMF = CranioMaxilloFacial
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4. Intraoperative Bioprinting
In vivo bioprinting refers to the bioprinting process performed on a live subject
in a surgical setting, in which
• defect imaging,
• data processing,
• process planning, and
• bioprinting are performed consecutively in a single process.
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5. Advances from invitro to invivo
• In the last decade, bioprinting has considerably advanced and a
large number of studies have shown attractive outcomes in
different applications
• Engineering of various organs for regenerative medicine (i.e., skin,
cartilage, and bone )
• Tissue vascularization.
• Drug discovery, and disease modeling.
• However, the majority of bioprinting endeavors have been
conducted in vitro and, in some cases, further validated in vivo.
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6. Advances from invitro to invivo (conti..)
• Bioprinted tissue substitutes, especially hydrogels, have weak initial
mechanical strengths due to the fluid-rich nature, which makes them difficult
to handle with surgical tools. In contrast, the real-time design of the graft in
IOB is an effective process, the defect can be repaired with minimum risk of
contamination.
• Intraoperatively repaired defects are surrounded by native tissue, endogenous
cells could be directed by proper biochemical and biophysical cues to migrate
into the bioprinted constructs and differentiate into target tissue-specific
lineages.
• In addition, compared with manual injection of biomaterials into defect sites,
IOB enables the precise deposition of cells, genes, or cytokines with localized
control and anatomical biomimicry.
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7. Advances from invitro to invivo (conti..)
• For tissues that are heterocellular and formed of zonally
stratified arrangement of extracellular matrix (ECM)
• IOB is a powerful technology to precisely reconstitute
multiple layers that is quite challenging using manual
approaches.
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8. Current developments in IOB
• Handheld devices have been developed for cartilage repair in a sheep model.
• Skin repair in murine and porcine models .
• Although the deposition was performed manually, it has come up with a well-
developed strategy to miniaturize the number of external equipment for
crosslinking of a cell-laden hydrogel.
• In terms of bone repair, IOB in the mouse calvaria defect model using laser-
based bioprinting (LBB) system. In their follow-up studies, IOB gave rise to
organized microvascular networks and bone regeneration in calvaria defects.
• Currently, the most successful cases on IOB are mostly associated with skin
repair due to its ease of access and regenerative potential.
• Most recently, a mobile skin bioprinting system with integrated imaging
technology for on-site management of murine and porcine full-thickness skin
wound models has been demonstrated. 8

9. Figure: Examples
for Intraoperative
Bioprinting (IOB).
(A) IOB using the
biopen for
treatment of a full-
thickness chondral
defect in a sheep.
(B) Laser-based IOB
for skull repair in a
mouse calvarial
defect.
(C) Droplet-based
IOB for skin repair
in a porcine model. 9

10. Considerations and future outlook
• Different modalities available for IOB
• Limitations of current biopinting modalities.
• Compatibility of bioinks to surgical settings.
• Automation of IOB.
• Vascularization in Intraoperatively bioprinted tissue.
• IOB of composite tissues and their translational potential.
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11. Modalities availbale for bioprinting
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12. Limitations of modalities
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DROPLET BASED: EXTRUSION BASED: LASER BASED:
Droplet-based
bioprinting(DBB), in is
its resolution superior
than that of EBB.
Affordable,
commercially available.
Extrusion-based bioprinting (EBB) can be
considered an appropriate for IOB since
manual injection of biomaterials has
already been clinically applied for
decades and current surgical robots, can
be easily reconfigured to hold EBB tips.
Has a high resolution
similar to DBB. High
precision and accurate
fabrication due to laser
guidance.
Bioink deposition through a small orifice
results in higher risk of nozzle clogging,
which increase duration of surgery and
brings complications.
However, the print tip might interfere
with defect periphery since it is a
contact-based technology
Miniaturization of LBB
is a major challenge
due to complex setup
(light source, optical
instruments)
Limited by poor structural and
mechanical integrity of bioprinted
constructs, DBB is more suitable for
bioprinting thin tissues such as skin.
Low resolution prevents bioprinting of
stratified layers such as skin.
Which constrains its accessibility to
internal tissues.

13. Compatibility of bioinks to surgical setting
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Bioinks
Solution of a biomaterial or a mixture of several
biomaterials in the hydrogel form, usually
encapsulating the desired cell types, used for creating
tissue constructs. This bioink can be cross-linked or
stabilized during or immediately after bioprinting to
generate the final shape, structure, and architecture of
the designed construct

14. Compatibility of bioinks to surgical setting
• Since the deposition is performed directly into a defect in
physiological conditions, an ideal bioink should not only meet the
general bioink requirements but also possess some features specific to
IOB. In particular, such bioinks are expected to be:
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BIOINKS

15. Compatibility of bioinks to surgical setting (cont.)
• Most of the popular biomaterials (e.g. polyurethane, polylactic acid etc.)
relying on volatile organic solvent and high melting temperature, become
inappropriate.
• Among hydrogels, although collagen transitions to gel state at 37°C, its
slow gelation hinders its use in IOB.
• Fibrinogen is also popular due to its rapid crosslinking when it is
interchangeably bioprinted with thrombin into a defect.
• Photo-crosslinkable bioinks, such as gelatin methacrylamide , hyaluronic
acid methacrylate , and poly(ethylene glycol), have been commonly used
in bioprinting.
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16. Compatibility of bioinks to surgical setting (cont.)
• Since exposing UV light directly to a live subject can be dangerous one
practical example is to expose UV light towards the side of a transparent
nozzle during bioink extrusion.
• Recently, visible light photo-initiating systems have become popular,
which avoided the use of UV light.
• Newer materials may facilitate retention of the bioprinted constructs at
the desired site without the utilization of a support dressing/scaffold,
such as a vacuum assisted closure device. The manufacture of rapidly
integrable bioinks would expand the feasibility of bioprinting into
enclosed cavities (e.g., abdomen, thorax)
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17. Automation of IOB
• Imaging of defects during IOB should be performed in a minute timescale after
an excision since the prolonged exposure can increase surgical complications.
• To match this requirement, scanners based on 3D photogrammetry provide a
supreme solution to obtain raw data of the defect.
• Several models of portable photogrammetric scanners (e.g., Artec Space Spider
and CREAFORM HandySCAN) are able to reach a resolution up to ~30 μm, with
an extremely short scan time.
• To create a model of tissue constructs, image processing is necessary, including
segmentation and data mining During IOB, all these processes should be
completed in a few minutes. Hence, segmentation software is vital for
convenient extraction of the region of interest from 3D images.
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18. Automation of IOB
• Artificial intelligence and robotics can be incorporated to reduce the process time
and variation caused by operators.
• Machine learning can be used to automatically generate optimal bioprinting
strategies
• Commercially available bioprinters with three-axis coordinates are usually not
sufficient for IOB for irregular-shaped defects, which necessitates the use of
bioprinters with higher degrees of freedom (DOF), where robotic arms can be the
ideal solution.
• Although BioAssemblyBot with a six-axis robotic arm is currently the only
commercially available bioprinter with higher DOF, there are many companies
specialized in robotic surgeries (e.g., Intuitive Surgical, MAKO Surgical Corp, etc.) with
the potential of integrating bioprinting capabilities into surgical robots in the future.
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20. Vascularization in intraoperatively bioprinted
tissues
•Vascularization is crucial for maturation of bioprinted constructs. Although
bioprinted constructs with an embedded microvasculature have been
described, it is not feasible to directly anastomose them to the recipient
vasculature.
• To drive microvascularization within intraoperatively bioprinted tissues,
there are multiple ways, such as bioprinting of endothelial progenitor cells,
hypoxia-inducible factor (HIF), or vascular endothelialgrowth factor (VEGF).
• Oxygen-filled microparticles or oxygen-generating biomaterials (OGBs) can
be bioprinted within the bioink, which are expected to feed the cells until
capillaries can actively transfer the blood.
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21. Vascularization in intraoperatively bioprinted
tissues
• Oxygenation rate of OGBs is the key parameter for viable outcome
of organs.
• Involvement of porous internal structures is an advantage of
bioprinting and another option to facilitate infiltration of blood
from host tissue.
• The ultimate goal is to create a bioprinted construct that includes
an embedded microvasculature with a continuous anastomosable
artery and vein.
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22. IOB of coposite tissue
• Reconstruction of tissues with multiple components such as
craniomaxillofacial (CMF) defects (skin, bone, and muscle),
osteochondral defects (cartilage and bone), and musculoskeletal
defects (bone,muscle,tendon, and skin) possesses several
limitations.
• Different tissues exhibit variations in terms of physiological,
anatomical, and histological aspects and precise layer-by-layer
stacking is not trivial.
• Compartmentalization necessitates the precision and effective use
of stem cells and differentiation factors, since differentiating stem
cells into multiple lineages is crucial.
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23. IOB of coposite tissue:
• IOB of composite tissue should allow rapid defect information with
minimum manual interventions, enabling personalized
reconstructions in an anatomically accurate manner.
• Composite tissues are usually thick, vascularization is particularly
vital to enhance the proper tissue regeneration.
• Currently, free-flap surgery is the standard of care for the repair of
composite segmental defects. But free-flap surgery is limited by
donor supply site morbidity. Although the goal is to replace like
tissue with like tissue, this is often impossible.
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24. IOB of coposite tissue
• Therefore, it may be better to combine the principles of
reconstructive microsurgery and IOB. The surgeon configures the
recipient vasculature to perfuse an adjacent bioprinted construct by
direct anastomosis.
• It would eliminate the concerns of donor supply and morbidity,
providing exact match of replacement tissue.
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25. IOB of coposite tissue
• So far, animal models for IOB are almost non-load-bearing, such as skin and
calvarial defects.
• IOB for repairing load-bearing defect models, such as segmental bone defects
in long bones and joint defects, will gain more attention in the future.
• Mechanical stiffness of constructs depends on their inherent properties
determined by the bioink, which can be resolved by developing new materials
or integrating mechanically strong thermoplastics.
• In addition, postoperative care and rehabilitation are still necessary (as in
conventional surgeries) until intraoperatively bioprinted tissue restores
sufficient mechanical strength.
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26. Concluding remarks
• IOB has shown promising results for regeneration of
tissues, including cartilage, skin, and bone, in animals.
• However, regeneration of composite tissues, which are
composed of hard and soft tissues, and the interface layers
in between, have hardly been explored.
• Seeking a practical way to facilitate vascularization in vital.
• It is appealing to combine IOB with vascular anastomosis to
repair composite segmental defects.
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27. Concluding remarks
• In the long term, automation of IOB not only relies on the
integration of sequential processes, but also requires a large amount
of clinical cases to optimize the bioprinting strategies.
• Beyond scientific considerations, clinical studies will be impeded by
ethical and regulatory issues.
• Significant efforts will be required to address all these issues, we do
not doubt that IOB will be a game changer in regenerative surgical
care.
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28. References
• Wu, Y., Ravnic, D. J., & Ozbolat, I. T. (2020). Intraoperative Bioprinting:
Repairing Tissues and Organs in a Surgical Setting. Trends in
Biotechnology.
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29. Thank You
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