Cellulose Bio – ink on 3D Printing Applications

Cellulose Bio – ink on 3D Printing Applications

                                                    Abstract

Cellulose naturally can be found from nature in living plants, wood and even agricultural refuse. Its function in many plant cell walls has enabled research and studies to be carried out to try and maximize its uses. Study and research on Cellulose have recognized it as a polymer that has many derivatives. From cellulose polymers, one can obtain Nanocellulose like Nanofibils(CNF), Nanocrystals (CNC), Bacterial nanocellulose, Cellulose Nanoryan, Most of this derivatives have shaped the medical field in a more significant way. Many biomedical practices that were not able to be carried out initially can now be performed. Tissue engineering and regenerative medicine(TERM), Organ transplants, Wound dressing and healing are all being carried out due to studies of Cellulose. Bionink Printing has made this possible.3D bioink Printing has made significant strides in biomedical applications and engineering field. This review focuses mostly on how Bioink 3D printing has made use of Cellulose possible in various fields. The subsequent chapters of this writing details the derivatives of nanocellulose, cellulose modifications and applications, How bioink works together with Cellulose to achieve strides in biomedical applications and bioengineering fields. It also goes further to elaborate on the materials used in Bioinks and types of bioinks. It finally concludes by emphasizing the roles played by 3D nanocellulose bioink printing in the Treatment of cancer and its application in TERM.

KEY  : CNC,CNF,AGU,TERM,BIOINK,CA,LIFT,DIW,DP,FDM,ECM,SLA.

                                                Introduction

1. Cellulose is a polymer, sources, and Structure

Cellulose (C₆H₁₀O) is an organic compound Linear in Structure and a long-chained carbohydrate polymer with glucosidic linkages. It is an essential component in the plant cell wall and used as indigestible fibre in the human diet. It is a polysaccharide in nature since it is made up of sugar molecules. As a polymer naturally, Cellulose is contained in wood, paper, and cotton with wood and cotton containing fibrous Cellulose (Trache et al.2017). The first synthetic polymers were made from Cellulose (Yim et al.2017). These synthetic polymers include cellulose nitrate, cellulose acetate, and rayon.

Cellulose is considered the best biopolymer in nature, also as a replacement for petrochemical products due to being a renewable, eco-friendly resource (Lauer et al.2019). Its modifications have resulted in products that have durable mechanical power. Most are biodegradable in the environment, making them be in high demand in the current market. Chemical change of Cellulose has resulted in the manufacturing of multipurpose materials. This Modification is through the esterification of the hydroxyl groups –OH- in the Cellulose (Dong et al, .2018). Chemical Modification of Cellulose leads to the production of Nanocrystals (NCC), Nanofibres (CNF), and many other thermoplastics of Cellulose.

Cellulose is vital in cell walls of higher plants. Its also a natural organic polymer that of late has many used in paper industries, textile and pharmaceutical applications. It has to lead to research and further studies in it to maximize its uses as a green environmental-friendly producer. Cellulose is obtained mostly from plants and wood. (Tyagi et al,2016).It can also be obtained from food waste. Majorly Cellulose constitutes plant fibre that provides mechanical strength to woody stems. Its pure form can be found in cotton seeds where its 90 wt %, also as lignified Cellulose, Hemicellulose and pectins. All of this can be extracted and processed. ( Menon et al,2017) .isolation of Cellulose must occur before its production, Treatment or Modification has occurred.

1. Modification of Cellulose and applications

Cellulose is a linear polymer with repeating D-glucopyrase units, also known as AGU ( anhydroglucose unit). Glucosidic bonds link this AGU’s. Degree of polymerization (DP) varies depending on origin and extraction methods. It can also vary with the amount of wood pulp ranging from 800-10000 which can contain either 300 or 1700 AGU’s Usually DP is average, and for each, three hydroxyl groups are present. After polymerization, they rearrange themselves with fibrils of high orders in them influencing characteristics like texture, density and other properties. ( Feng et al,2019) . Studies on cellulose modification didn’t start yesterday but rather in the 1880s. During Gunpowder warfare, cellulose nitrate was made accidentally by treating Cellulose with nitrate. Used as explosives. Later on, celluloid replaced cellulose nitrate because of the dangers the later posed. Cellulose acetate (CA) later in 1865, replaced the two.  It has led to broad spread research and study on its modifications, improvement and applications(Gumrah Dumanli, 2017).

For instance, Cellulose from wood is isolated using a technique called pulping. Here process like Kraft/sulfate process and or Sulfite process are done. Cellulose is separated from lignin and hemicellulose. Pulps of Various strength and property cellulose are obtained. Paper pulp cellulose for making paper can be obtained here, Also Dissolving gride pulp cellulose for regenerated Cellulose and its derivatives can be obtained. ( Kargarzadehet al,2017)Here the derivatives of Cellulose can be used in making pharmaceutical products and the making of polymers that can be used in bioink Printing. ( Sharip et al,2019)

Before Modification, Its must be clear that Cellulose is insoluble in water and organic solvents because of its macromolecular nature with high DP. Also, due to its thermodynamics and hydroxyl groups that make its C and H atoms to have low hydrophobic interactions, .derivatizing and non-derivatizing solvents are hence used to treat the Cellulose. Cellulose applications depend on cellulose modification. Reactive groups in Cellulose are the hydroxyl groups. They are suitable for a variety of modification techniques. Chemical Modification is one of the techniques commonly used. It results in different cellulose derivatives with many applications. The reaction can occur at C atoms of AGU’s by nucleophilic displacement.

Oxidation of Cellulose to form carboxyl groups provides a source for making cellulose derivatives. Some of the nanocellulose derivatives are nanocrystals (NCC), nanofibrils (CNF) bacteria nanocellulose(Zhang et al., 2019). Materials produced depends on the source and Treatment of Cellulose. The availability of thermoplastics in Nanocellulose has led to research and studies that have helped in biomedical applications like 3D bio-ink since the Nanocellulose can be used as modifiers for inks (Sharma et al., 2019).  The Nanocellulose has muscular mechanical strength, their ability to support cells and prevent pore damage attributes that also aid in implants modifications and regeneration (Bacakova et al., 2019).

On the other hand, Nanofibres (CNF) consisting of NFC (Nanofibrilliated cellulose) and Nanofibrils are obtained by mechanical distortion and dismantling of regions of cellulose chains in its chemical Structure (Squinca et al, 2020). Nanocellulose is also used in cancer therapy treatment. Though still in advanced stages of research and studies, medial practices like tissue engineering, skin grafting, and regeneration and the use of bacterial Cellulose as drug carriers are some of the processes taking place in cancer treatment. (Anirudhan, et al., 2019) a significant mile storm in the medical field to try and eliminate this monster called cancer.  The breakthrough though not complete, has borrowed the uniqueness of Nanocellulose and 3D bio-ink Printing to help in biomedical application.

Nanocellulose can be obtained from cellulose sources of wood, plants, paper, and cotton through a top-down process where different varieties of Nanocellulose are obtained because Cellulose has qualities that differentiate itself from other synthetic polymers, such as polyfunctionality, chain stiffness, and sensitivity. Several research pieces have been carried out on Cellulose to understand its functionality and properties. Done by Hermann Staudinger, who tried to isolate Cellulose through acetylation and deacetylation. In his findings, he found out that the glucose linkage in Cellulose was attached repeatedly giving the Cellulose its polymer properties (Caloni et al .2020)

Micro fibrillated Cellulose and bacterial Cellulose are useful in the production of bio – nanocomposites. This nanocellulose can be modified using several mechanical processes. They have low density and are biodegradable. Equally, they have high mechanical strength. The only disadvantage nanocellulose has is its automatic extraction from Cellulose, and they also have high energy consumption (Leszczyńska et al., 2018) Pretreatment of the microfibrillated Cellulose helps in overcoming such disadvantages. The surface modification reduces the mechanical problems of the microfibril cellulose.

Nonocelluloses that include cellulose nanocrystals and nano fibrillated celluloses with low as well as high aspect ratio, are promising bio-founded materials that are prepared from plant cellulose such as wood through mechanical cropping in water in absence or presence of pretreatments. However, its extraction is limited due to being hydrophilic, thus restricting its usage. Enzymatic surface modification is done on Nanocellulose to help make it perfect even with its hydrophilic properties.

Chemical Modification of nanocellulose is done to improve the properties of Cellulose through grafting in or grafting off of substrates made of either nanocrystals or nanowhiskers. The grafting process starts with the initiator, mostly monomer concentrations. Here the acid hydrolysis enables the hydrolysis groups to cover the upper surfaces of the Cellulose. When the surface of the Cellulose occurs, the Cellulose’s mechanical properties facilitate the compatibility of Cellulose (Vineethet al.2016).

In a surface modification of nanocellulose, two main procedures are done, namely compatibilization and polymerization. In the former, a reactive reagent is attached at the surface of Cellulose. A mobility agent that needs two functional groups is attached to the cellulose surface during polymerization. One of the functional group reacts with the hydroxyl group. In contrast, the other group attaches to the covalent bonds of the polymer matrix, thus helping in the mechanical strength of the nanocellulose when doing its function. Polymerization can be done in different ways like graftings, radical reactions, or organometallics (Shahnaz, Padmanaban, & Narayanasamy, 2020.)

Reagents used in the Modification of nanocellulose are succinic anhydrous, Phenyl isocyanate, and isoprene.  Nanocelluloses have many applications due to their remarkable mechanical properties since they can be changed into polymerics using methods like melt-compounding, solvent casting, and in-situ polymerization. Of the three methods, solvent casting is the preferred method for research purposes. It mixes with polymer matrix in a suitable solvent.  Then the mixture is cast in a recipient forming a nanocomposite film through evaporation of the solution.

However, the significant setback of processing CNCs through thermal-mechanical compounding causes low thermal stability due to sulfate groups present on the surface. These groups generate corrosive species upon heating, which induce cellulose chain degradation. Calling for an alternative method that facilitates the production of high-content Cellulose nanostructured composites. However, combining a high fraction of the CNF network with a hydrophobic matrix has been mostly unexplored. Yet, with the advancement in technology, this can be cracked. The CNF network in the form of nano paper can be repaired by spraying the CNF liquid suspension followed by a solvent exchange and supercritical carbon dioxide (CO₂) suspension. This process allows the removal of the solvent without cellulose degradation as a critical temperature, and the CO₂ pressure is 31 ° C and 7.4 MPa, respectively. Hence preserving the CNF network, which has an internal surface area as high as 480 m2 g-1. (Yao et al .2020).

Nanocellulose CNF is the most important and widely used Cellulose since the processes of     Modification of Cellulose perfectly fits them. They are also easily obtained from Cellulose either through chemical modification or surface treatment using suitable solvent materials. Its availability has resulted in many breakthroughs in the medical field as some of its uses like TERM (Tissue engineering and regenerative medicine and biomedical applications, Cartilage regeneration, tissue repair, skin grafting, wound dressing).

1. Ideal bioinks 3D printables and characteristics of 3D Printing    

Bio ink materials can be used to make artificial living tissues by using a 3D printing technique. The cells being used here contain extra materials that seal them. The ink being used is usually composed of the cells being made. Some of the bioink techniques in use are selective laser sintering(SLS), Injet bioprinting, direct ink writing(DIW), fused deposit modelling(FDM)and laser infused forward transfer(LIFT), stereolithography(SLA) etc. (Gopinathan et al,2018).

Currently, there are more advanced methods of 3D Printing that is being applied in Printing of tissues and organs(Panwar et al,2016). Acoustic Printing, Microwave bioprinting, Electro-hydrodynamic printing, pneumatic bioprinting are some of the methods used. Scientists have been exploring ways of using 3D bioink Printing which increases printing speed at the same time retaining cell properties. ( Zhao et al,2016) .This scientist found out that specific requirements are to be in place for a 3D bioink printing these requirements go hand in hand with the characteristics and properties of right 3D bioink materials.

Bioink is affected by many factors that determine its characteristics. This technique is dependent on single material printing, though, with current research, many materials have been recommended and are in use already. These materials are usually called biomaterials. Some of these materials are natural, while others are not. Natural biomaterials have the upper hand over synthetic materials over time most of the characteristics mentioned in the table above are obeyed by natural biomaterials(Sheikh et al 2017)

Comparison between Natural Biomaterials and Synthetic Biomaterials

• Advantages of Natural Biometrics over Synthetic Biometrics

• Advantages of Synthetic Biomaterials over Natural biomaterials

. A challenge with crosslinked printable materials or generally Synthetic biomaterials is that they should be regulated at body temperatures of between 37^o c and below to minimize side effects on cells during biomedical applications (Sultan et al., 2017). The year 2016 led to a range of commercial inks that were compatible with commercial printers, Inks containing natural polymers like chitason, collagen, and gelatine have been in use with positive results so far. (Bernal, et al., 2019)Further findings and research in the fields have helped in making it easier for bioinks to give these beneficial results in biomedical applications (Chimene, et al., 2016) Tissue engineering, regenerative medicine (TERM), Treatment of cancer, and some other biotechnology that add insight on bio-ink. According to Gao et al (2017), 3D bioink printing is in demand in biotechnology, particularly in the regeneration of tissues, organs, or cells to facilitate the restoration of normal biological functions.

Bio ink has a high reproductivity and better control of fabricated and modified materials than other techniques like the use of thermoplastics (Habib et al.2019). After their deposition, they can still be maintained by removing them through small openings of the printers in the form of filaments. These filaments can even be used again. Natural deviations must be bioactive. Bioinks are also utilized in the construction of collagen and cartilage, restructuring and creation of skin patches, and many other body parts (Choi et al.2016). Our focus is on nanocellulose that is best in chemical acid hydrolysis.

Previously, skin grafting, regeneration, and some advanced therapy were never in existence, but with the use of 3D bio-ink printing, this has been made possible. 3D technology allows the production of many complex structures through the bottom-up method (Jessop et al., 2018),  3D bio-ink allows biocompatibility and linkages between the gel mixed with the living cells. When this bioink is printed, it is said to be 3D Printing. Bioink materials determine their applications and uses. There are many bioink materials in use today (Ahadian et al,2017). Some of the materials are shown in the table below.

Other examples of bioink based materials are; Fibril based bioinks, Cellulose based bioinks, Silk based bioinks, Extracellular matrix (ECM) based bioinks, cell aggregation based bioinks, synthetic-based bioinks. (Mozetic et al.2017).This study deals with Cellulose-based bioinks. As stated earlier, Cellulose based bioink materials have led to the production of Nanocrystals, Nanofibrils and Nanocellulose. The table below shows a  summary of the Nanocellulose polymers based biomedical materials.

Applications of different Nanocellulose in Biomedical applications

• Cartilage treatment and growth

Nanocellulose in wood that contains 2.5% CNF having been alginate with CaCl₂ for Cartilage tissue growth. As it is known, cartilage problems, when allowed to be severe, can cause serious clinical problems. Traditionally, it was difficult to detect and treat cartilage problems. With cell growth and tissue maturity being a requirement in growth, Biolink made it possible to use alginate CaCl₂ combined with Nanocellulose to regenerate cells are for grafting and Treatment. A milestone in the medical field as cartilage replacement makes this possible as Alginate-nanocellulose combination can allow printability. Several advanced research on how to improve on already discovered techniques of cartilage repair and growth using 3D bioink printers (Nguyen et al.2017).

• Auricular cartilage regeneration

Happens through tissue engineering or what is known as a TERM (Tissue engineering and regenerative medicine). Here, regeneration of cartilage to treat the disease is done even in clinical cases by restoring and repairing the damaged cartilage. Autologous cartilage grafting to re-establish unique biological and functional properties of tissues. Multipurpose stem cells MSCs are used from either bone marrows or adipose tissues (Ávila et al., 2016). It is worth noting that more research in this field is ongoing. The stem cells of MSCs have chondrogenic potentials and proliferative growth and regeneration capacity. They also have transformed growth factors of bone morphogenetic proteins (BMPs) that play a part in this process. Earlier 3d bioink had scanty or no information on auricular cartilage regeneration. However, as current easiness to harvest BMP and adipose stem cells (ASCs) derived from the stem cells has made it possible for the reconstruction of auricular cartilage (Oh et al .2020)

• Wound dressing

Traditionally wound dressing was no challenge as it was a standard treatment done in our homes even with the most underqualified relatives, family members, and friends, however, with the world wars. Deep wounds needed special care, and they needed faster Treatment because the wounded needed to go back into the battlefield. 3D burning printing came in handy to solve this problem. This advancement was not until the late stages of the 19^th century and fast-tracked in the 20^th century (Kim et al., 2018). A 3D printer can b deposit skin layers covering large parts of the wound or affected area, then the Bioink of the 3D printer fast tracks the healing process of the injury. Here, MSCs cells that promote skin regeneration and growth and, at the same time, reducing scarring of the skin. Initially, it was through skin grafting, where one part of the skin would be removed and grafted on the part of the skin, which led to differences in texture, colour, and complications of the surface copied to other parts of the skin. With the current wound healing method, the MSCs show similarity in the biological and physical properties of the area on Treatment. 3D bioinks are essential in wound dressing of more significant parts of the body that develops at a faster rate (Smandri et al., 2020). It’s important to note that 3D bioink Printing helps in Tissue engineering where advancements in TERM have made it possible for organ transplants and tissue regeneration.

 Use of Cellulose 3D Bioink Printing in Cancer treatment

Cancer is a complex disease that up to date research about it is to try and fully understand it. Cancer showing heterogeneous cellular composition cannot show all its stages of initiation, development, metastasis, cell-cell interactions, and extracellular matrix interactions (ECM) in its 2D or 3D models. That is why the importance of 3D bioink Printing came into play because in 2D, and 3D extent of tumour and stages. 3D bioink Printing helps in creating high-resolution images that help in the study and treatment of cancer. It helps in the study of cancer genesis, growth, and response to cancer drugs (Serrano et al.2018). 3D structures imitate an entire tumour by coping with the cells with properties for the type of cancer and stage of the disease. In doing this, the 3D helps in cancer screening and therapy (Swaminathan et al.2019). The 3D models that are a prototype of ECM cancer cells based on hydrogel, gels with tumour cells. The hydrogels are from collagens like nano collagen. Advantageous from using other synthetic polymers because the 3D hydrogels pick the exact tumour cell to use. Therefore, they show similarity in the biological, physical properties of the tumour cells from the affected area.

Conclusion

It is now clear and beyond a reasonable doubt that 3D bioinks is the future of biomedical applications and biomedical engineering. Its ability to produce and regenerate tissues and organ structures has opened further studies in this field. Currently, commercialization of bioinks is taking place at a rapid rate with state of the art patient-specific 3D structures for their urgent medical needs produced at a faster unique way.3D structures of numerous advantages like flexibility, improved mechanical strength, controlled biodegradability, and user-specific have made it possible to transplant, regenerate and cushion any loopholes in the medical field. The materials are also unique. The hydrogel is best in this scenario. Use of hydrogel, selection of different bioinks and their availability is a detailed format in the above chapters.

The use and development of bioinks are still in progress just as the saying education and knowledge never ends or never expires. This knowledge has enabled the introduction of 4D bioinks that are currently in use in some parts of the world.3D bioinks has also made strides in cancer treatment and therapy. The 3D bioinks has enabled the introduction of more unique and easy to understand models structures of cancer cells, enabling researchers to be able to identify tumour cells, development and nearly all stages of cancer. ECM-based bioinks, decellularized bioinks, cell aggregates or spheroids that showed promising results are being used in the development of functional tissues or organs using 3D bioprinting technology. This techniques though requires a large number of specific cells on top of many characteristics and properties. Development of advanced bioprinters which are cheap with high resolution has enhanced prospects further. Appropriate bioinks that satisfactorily meet bioprinting requirements with regards to the mechanical, rheological, and biological properties are scanty to date. Creation of new bioink materials and the engineering of novel bioink formulations are currently significant areas of interest. This document has tried to blend the already known information about bioinks with work in progress discoveries and new ideas in the biomedical field, particularly bioink Printing.

It shouldn’t go unnoticed on how bioink has played a more significant part in the biomedical field, with TERM and cancer treatment making significant strides. Since science is evolving daily, many expectations are held particularly on how 3D bioinks will shape the medical field further. Each day with the discoveries of new diseases, new measures counter the alarming trend. Many scientists world over are doing their best to incorporate 3d technology in their practices. Now the medical practice has been made possible and faster since the inception of 3D bioink Printing. The introduction of 4D Printing has even taken these expectations much higher.