3D Printing Scaffolds with Streamlined Cellularization

Anish Vaghela, George Feng, Richard Shen, Thomas Chedid, Narasimha Kuchimanchi, Anthony Yung

Problem: Currently, it is not possible to create more advanced cell structures such as a liver or a kidney using 3D-printing due to limitations to specify cell orientation in scaffolds and the inability to vascularize complex structures.

Need: A streamlined method for complex organ generation using 3D printing

Problem and Need Statement

Past, Present, and Future Past

- First successful kidney transplant 1954- During the 1960s - first successful pancrease transplant - During the 1980s - first successful lung transplant

Present - Approximately 500,000 Americans receive organ transplants each year

- About 108,000 individuals each year are placed on a waiting list for organ transplants, many of these individuals die - Tissue engineered scaffolds guide production of simple tissue such as knee cartilage and bone regeneration

Future - “Made to order organs” - Complex tissue regeneration in custom 3D printed tissue scaffolds

Current Scaffolding in Tissue Engineering

Scaffolds serve the following purposes:

● Allow cell attachment and migration

● Enable diffusion of nutrients and products

● Exert certain mechanical and biological influences to modify the behaviour of the cell phase

Current Scaffold Technology Decellularized scaffold with host cells seeded into it

- Scaffold taken from existing organ- Host cells seeded into scaffold to proliferate cell and tissue regeneration

Synthetic scaffold- Scaffold created from a variety of biocompatible polymers - Host cells seeded into synthetic scaffold for cell and tissue regeneration

3D printing- Scaffold printed using 3D printer for personalized scaffold creation - Personalized scaffold proliferated with host cells for tissue regeneration

Current State of 3D Printing in Tissue Engineering

I. Current 3D PrintingA. Combine image technique with regeneration medicine

study1. Such as CAD, MRI, RP and CT

B. Rapid Prototyping 1. Allow the fabrication of tailored conformation for

individual patients. (Winder 1999)2. Grants the production of complex scaffold with regulate

over scaffold features, properties, composition based on effective models.

Is 3D Organ printing a feasible technology?I. Must achieve:

A. development of a printer which can print cells and/or aggregates; B. demonstration of a procedure for the ‘layer by layer’, C. sequential deposition D. solidification of a thermo-reversible gel or matrix and demonstration of

fusion from closely placed cell aggregates into ring-like or tube-like structure within the gel.

Scaffold Vascularization - The Major Challenge

I. Vascular DensityA. The most crucial factor for adequate organ perfusion and supply of

oxygen and functioning. B. First Proposed by Danish Nobel Prize Laureate- August Krogh.

(Schmidt-Nielsen, 1994)

• 3D Printer with Multiple printheadso One printhead for scaffold printingo Set of printheads for cell printing

One nozzle for each cell type Number of cell printheads dependant on

complexity of tissue• Printer integrated inside incubator to control cell environment

o Printing platform submerged in culture medium to promote cell proliferation after seeding of cells

Platform lowers deeper into medium as each layer is printed

o Double temperature control Air above medium conditioned at room

temperature for matrix solidification Medium conditioned at physiological temperature

for cell proliferation

Approach

Z-Axis Motor X-Axis Motor, Printhead Holder, Printheads

Door

Observation Window

Printing Platform

Medium

Medium Heater

Medium Drainage/ Injector

Control Panel

Air Conditioner

Y-Axis Motor

Potential ProblemsProblem:

● Cells imbedded into the polymer matrix will begin to die without optimal temperature, humidity, and other conditions such as the carbon dioxide (CO2) and oxygen content. Nutrients must also be able to perfuse into the cells

Solution:• Printing will occur above an incubator which will control the in-vitro environment for

optimum tissue cell culture growth. o After each layer of the matrix is printed, the platform below will drop so the matrix

will be lowered into a media bath.

Problem:● Due to the slight fluctuation in pH in the media, as the individual layers are

printed on top of each other, the layers may break apart once the matrix is submerged.

Solution:• Submerge each preceding layer after the next layer has been printed above it and

adhered to

Potential Problems continuedProblem: • Organ matrices vary in strength and rigidity. The biomaterial used for the matrix must be able to

emulates the mechanical properties of the tissue being printed

Solution:• Various polymers can be used to emulate mechanical properties

o eg. PLA, PLGA, PGA,PMMA

Problem:● The compound used for the matrix must be biocompatible so to not cause an immune response

after implantation○ Usage of PLGA matrices may result in acidic micro-environments around the implant

causing an immune response or death of the surrounding cellsSolution:• Acidic environment can be neutralized with impregnation of basic ions into the matrix. • PLA provides adequate biodegradability, pore size, interconnectivity, bioactivity and mechanical

properties

● Decellularizing existing tissue is not a viable way of approaching tissue replacement therapy ○ Limited source of tissue○ Not cost effective compared to printing an artificial scaffold

● 3D printing ○ Printing a synthetic scaffold can eliminate the need for existing tissue

■ Elimination of the decellularization process■ Immuno-rejection is still avoided

Significance

Significance

● Control of cell proliferation and cell arrangement ○ Multiple nozzle system

■ Simultaneous printing of host cells in accordance with synthetic scaffold

○ Each specific layer of scaffold is seeded with host cells separately and then adhered to the next respective layer■ Allows the control of cell seeding

○ Potentially lead to printing complex organs■ Limitless supply of organs and tissue for regeneration therapy

Significance

● Printing occurs within incubation chamber○ Two layers of scaffold are adhered to one another

■ The former layer gets submerged into the media● Simultaneous incubation

○ Incubation occurs as soon as the layer is seeded with cells■ Speeds up the overall printing process■ Cell arrangement is controlled

Benchmarks For Success● Cell Viability

○ Stain cells with calcein-AM(live) and ethidium homodimer-1(dead)

○ Utilize epifluorescence microscope

● Rate of cell growth○ For proliferation to occur, it must be greater

than rate of cell death

● Monitor the cell differentiation ○ Examine the measurable changes in

morphological characteristics

Benchmarks for Success Cont.

● Create appropriate scaffold with minimal cost○ Must be worth the cost of production

● Scaffold customized for each patient ○ Absence of immune response○ Appropriate size and shape and porosity

• Biocompatibility o Polymer has optimal degradation rateo Polymer has optimal mechanical properties

Project Timeline

● Planning: ~6 Months○ Research current 3D printers on market

■ Identify best 3D printer to modify○ Determine the optimal characteristics of media○ Research which polymers are needed for each tissue/organ case for printing the scaffold

● Concept Development: ~1 year○ Develop concepts for:

■ 3D printer nozzles■ Media chambers■ Necessary software■ Additional hardware modifications needed to make incubating environment

Project Timeline Cont.

• System Design: ~6 months○ Combination of concepts○ Test preliminary concept combinations

● Detail Design ~6 months○ Finalize details for product testing

● Testing and Refinement; ~6 months○ Refine product and continue to test

● Production ramp up: ~ 1 year○ Market final prototype to potential buyers or venture capitalists to get funding○ Use funding to ramp-up production at a greater scale

Work Cited• Serra, J.A. Planell, M. Navarro, High-resolution PLA-based composite scaffolds via 3-D printing technology, Acta

Biomaterialia, Volume 9, Issue 3, March 2013, Pages 5521-5530, ISSN 1742-7061, http://dx.doi.org/10.1016/j.actbio.2012.10.041.

• Y. Yang, Y. Zhao, B. Chen, Q. Han, W. Sun, Z. Xiao et al. Collagen-binding human epidermal growth factor promotes

cellularization of collagen scaffolds, Tissue Eng Part A, 15 (2009), pp. 3589–3596• Biodegradable synthetic polymers for tissue engineering. P. A. Gunatillake, R. Adhikari Eur Cell Mater. 2003 May 20; 5: 1–

16.

• Winder J, Cooke RS, Gray J, Fannin T, Fegan T (1999) Medical rapid prototyping and 3D CT in the manufacture of custom

made cranial titanium plates. J Med Eng Technol 23 (1):26–28

• B. Schmidt-Nielsen, August Krogh and capillary physiology Int. J. Microcirc. Clin. Exp., 14 (1994), pp. 104–110

• NIH Fact Sheets - Regenerative Medicine

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• http://www.3ders.org/articles/20131101-researchers-developing-gelatin-bio-ink-to-3d-print-human-tissues-and-organs.html