HAFS Think-Tank Ultimatum Final Report

Myocardium Bioprinting

November 23, 2022

HANKUK ACADEMY OF FOREIGN STUDIES

International
Leader: Sooan Lee (10137)
Member: SeokWon Jung (10142)

Ⅰ. Abstract
 Despite the high demand for cardiac organs, insufficient supply caused millions to

die due to cardiovascular diseases (CVD), cardiomyopathy, and coronary artery disease

(CAD) every year. Modern medicine has developed technologies to 3D print organoids that

effectively resemble the cardiac organ which might be a breakthrough in curing cardio-

related diseases. A technological explanation will be given on the three types of

bioprinters; inkjet-based, microextrusion, and laser-assisted, sourced by different types of

cells, proteins, hydrogels, and other biomaterials. In the process of manufacturing cardiac

organoids, cultivating the myocardium itself is critical. We will explain the biological and

chemical process of inducing natural fibroblasts into pluripotent stem cells (PSCs) and

reprogramming them to cardiomyocytes. Unlike CRISPR genetic modification where (cis-

9) enzymes interfere with the polymerase process, iPSC modifies a certain part of the

histone protein which activates gene transcription. When we get to the cardiomyocyte

mechanism, additional information on iPSC’s Chromatin remodeling will be discussed.

Ⅱ. Introduction

Starting from the 1980s and being popularized in 2009, bioprinting has been around for

quite some time. From printing photosensitive resin layer by layer to printing stem cells on

collagen bases, this field has progressed to develop correlation and accuracy day by day. There

are three 3D printing methods developed so far, each has its own pros and cons. The materials

used in these printers have also developed to a point where the cells are just identical, and with

these technological developments, the future of 3D bioprinting can be said to be innovative.

Diving into the mechanisms of iPSC and histone methylation, here’s the flow. Before iPSCs are

ready to be modified by transcription factors in the transcription start site (TSS), they go

through histone methylation by TET1. This process is called Histone post-translational

modification. After the transcription factors bind the gene expressions, additional protein kinase

is necessary to regulate tissue-specific genes. This protein interacts with the Extra Cellular

Matrix (ECM) to reinforce cardiomyocytes to proliferate and attenuate CVDs along with other

cardiac diseases.
Ⅲ. Bioprinting

1. About Bioprinting

Bioprinting is a three-dimensional printing technology for biomedical parts. It is

capable of producing living parts such as tissues, blood vessels, bones, and in further

potential, organs. Bioprinting requires an appropriate bioink to operate. Bio ink consists

of biomaterials such as living cells, proteins, nanomaterials, and other bioactive agents.

Decellularized extracellular matrix (dECM) also works as a critical component of a

bioink for it provides a microenvironment and encapsulates the biomaterials. Non-

cellular materials (or dECM) used in bioink should satisfy several requirements to

compose a bioink. Those non-cellular materials should dissolve in a real body and

protect the biomaterials. Hydrogel, collagen, alginate, and gelatin methacryloyl

(GelMA) are some non-cellular materials commonly used for bioink.

2. Types of Bioprinting

There are three different types of 3D bioprinting: inkjet-based, microextrusion, and

laser-assisted bioprinting. Inkjet-based bioprinting is a printing process in which the

nozzle dispenses the droplets of dilute and precise bioink solution without contact.

Microextrusion bioprinting (also known as pressure-assisted bioprinting) uses the

method of extruding biomaterials by pressure in the form of continuous filament that

contains bioink through a micronozzle or a microneedle. Laser-assisted bioprinting uses

a laser as the energy source to deposit biomaterials onto a substrate.

3. Potential Advantages and Developments Through Bioprinting

Bioprinting is a future-oriented technology that is constantly showing growth and

potential for further applications in the medical bioengineering field. If bioprinting

reaches the capability of producing “real–like” organs, bioprinted organs would serve
 as the alternative to animal testing. Instead of testing animals for new drug research,

testing bioprinted organs would be more ethical and beneficial. Using bioprinted organs

also leads to higher effectiveness and accuracy in testing since the test subject

(bioprinted organ) shows higher similarity to actual human organs. Bioprinted organs

are also open to the possibility of more accurate and personalized (or patient-specific)

cures. If the bioink contains the patient’s own living cells, proteins, and other

biomaterials, the bioprinted organ would react similarly to the patient’s actual organ,

thus ending up with minimizing side effects and maximizing the effectiveness of the

cure remedy. Bioprinting also contributes to personalized organ-transplant. According

to the Life Source, “Each day, 17 people die waiting for a life-saving organ transplant

and a new name is added to the transplant waiting list every 9 minutes.” As can be

inferred, an organ for transplant has a high demand but a relatively less supply, thus

ending up in the loss of so many lives. The main reason why it is so hard to get an

organ transplant is that in order to transplant an organ, there is a list of particular

requirements such as the size, blood type, et cetera. However, if bioprinted organs

develop to become more real-like organs, it is possible to transplant a personalized

artificial organ to the patient. This process also involves the patient’s own cells and

proteins, so this transplanted organ can reduce immune rejection.

Ⅳ. Myocardium Bioprinting

1. About Myocardium and Myocardial Diseases

Myocardium is a specialized muscle tissue that forms the muscle of the heart. It is the

middle layer of the four heart walls in each heart chamber. The heart muscle tissues

including myocardium are responsible for keeping the heart pumping blood by

involuntarily contracting and releasing. Therefore, the myocardium plays a vital role in

cardiac functions, and thus myocardial diseases are extremely problematic since it is

intimately associated with cardiac dysfunction. The primary cause of most cardiac

diseases is the coronary artery obstruction. Coronary artery obstruction is a

phenomenon that occurs when the plaques(atherosclerosis) block the coronary artery

and gradually narrow the artery, disabling the blood and oxygen supply. When the
 blood is not regularly supplied, the cardiomyocytes (cells responsible for the

contraction of the heart), and even the whole tissue, are destroyed. Once the

cardiomyocytes and the tissue are destroyed, the heart cannot contract, ultimately

resulting in diseases such as cardiomyopathy, myocardial ischemia, and myocardial

infarction. These diseases result in the loss of the ability to pump and circulate blood

throughout the body, which is fatal to human health.

2. Application of Bioprinted Myocardium to Myocardial Disease Cure

The obstruction of a coronary artery can be resolved by stents (a medical procedure that

enlarges the blood vessels). However, the destroyed myocardial tissue needs to be

replaced by new healthy tissue since the heart does not regenerate the cardiomyocytes.

This is when the bioprinted myocardium is needed. The bioprinted myocardium will

replace the destroyed natural myocardium, helping the heart to recover its original

function—a regular contraction and relaxation—by providing force as muscle tissue.

As previously mentioned, transplantation of bioprinted tissues or organs is beneficial

for personalized cure and minimization of immune rejection. Therefore, myocardium

bioprinting is highly prospective and significant technique in cardiology field.

Ⅴ. Cardiomyocyte Mechanism
1. Myocardium modeling through iPSC

The objective of this mechanism is to reprogram a fibroblast into immature

cardiomyocytes (CM) through the genetic modification process of Induced Pluripotent

Stem Cell (iPSC). Fibroblasts go through Chromosomal modifications by Histone

methylation.

TET1 protein, which belongs to the α-ketoglutarate oxygenase, is a

5-methylcytosinehydroxylase that binds the regions of 5’-CpG-3’ dinucleotides. It is

the key factor in maintaining the differentiation pluripotency of embryonic stem cells.

TET1 converts 5-methylcytosine(5mC) bonds, which represses transcriptional activity,

to 5-hydroxymethylcytosine(5hmC) which are associated with active transcription.

Chromatin remodeling occurs by Histone H3 lysine 4 demethylation (H3K4me2).

Its modification enriches in cis-regulatory regions of the transcriptional start sites (TSSs).

Before RNA Polymerization II, these epigenetic modifiers rearrange the chromatin from a

condensed state to a transcriptionally accessible state from CTCF, MII3/4, TF enhancer to

P300/CBP stage. Fourth, OSKM iPSC transcription factors start binding proteins to control

gene expression.
2. Cardiomyocyte Proliferation

As shown in the diagram below, histone post-translational modification is where tissue-

specific gene regulation is identified, in this case, Glycogen synthase kinase 3(GSK-3)

directs the cell to mature into a CM.

GSK-3 protein kinase phosphorylates glycogen synthase and activates enzymes that

catalyze the transfer of glucose from Uridine diphosphate glucose

(UDPG/C15H24N2O17P2) to glycogen, which traffics glycogen metabolism and cell

signaling. As a result, GSK-3 interacts with the extracellular matrix (ECM) while

trafficking vesicular structures which attenuates cardiac or ventricular dysfunction and

promotes cardiomyocyte proliferation.

VI. Conclusion

All things considered, myocardium bioprinting is beneficial and requires association

with highly advanced technologies such as iPSC. It is therefore predictable that myocardium

bioprinting would lead the cardiology field and ultimately benefit many patients suffering from

cardiac diseases and desperately waiting for the transplant. However, there are still limitations in

creating a real-like artificial cardiac muscle tissue. Prevalent limitations in the current state are

immunogenicity (the ability of a foreign substance to provoke an immune response in the body),

toxicity, and degeneration of biomaterials. Since 3D myocardium bioprinting’s main goal is to

make an alternative to the natural dysfunction of cardiac muscle tissue, the adverse reaction and

the construction of the microenvironment are also significant factors to be considered. Research

and experiments of myocardium bioprinting are constantly going on to supplement the

limitations of the current state and step out further to reach greater achievements that would

eventually improve the medical healthcare field

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