New Benefits of 3D Printing Essay example

The purpose of this paper is to explore the process of ossification and the development of three-dimensionally (3D) printing prosthetics and live cells, whilst focussing on the particular field of 3D printing bone and its cells. This field of research is becoming increasingly vital to human survival rates due to the high incidents of cancer within the population (15). This is creating an impetus to develop the technology to be able to 3D print tissues. 

Bone is one of the two primary supporting connective tissues in the human body, the other being cartilage. One of the key functions of bone is to provide structure to the body and to protect soft tissues, such as tendons, ligaments, muscles, and nerves. Because of the collagen fibres within bone tissue’s extracellular matrix (ECM) bone is impervious to being bent, compressed or stretched and can resist tensile stress (1). The ECM of bone is secreted by osteoblasts. These cells line the surface of a bone and developed from osteoprogenitor cells (the proclaimed ‘stem cells’ of bone). The matrix deposited by osteoblasts soon becomes calcified and traps osteoblasts in the organic matrix, where they gradually become osteocytes (cells that maintain bone tissue) (12). Bone is a hard tissue due to the process of calcification, which is the accumulation of calcium salts in a body tissue and is, therefore, able to withstand immense pressure. Naturally, ossification (the formation of bone) occurs in two ways: intramembranous ossification and endochondral ossification (2). Both intramembranous and endochondral ossification start with the formation of mesenchymal condensations. During endochondral ossification, these ‘condensations’ form a cartilage matrix, whereas, during intramembranous ossification, these mesenchymal condensations differentiate directly into osteoblasts (3).

Intramembranous ossification is where a bone is formed by direct replacement of mesenchyme, which is a somewhat organised embryonic tissue that can ultimately develop into skeletal and connective tissues. This process is involved in the formation of the flat bones of the skull, the mandible and the clavicle (4). Endochondral ossification is where cartilage is systematically replaced by bone to form the growing skeleton (4). This action is the mechanism responsible for the formation of all long bones of the axial skeleton, for example, vertebrae and ribs, and the appendicular skeleton, such as limbs (5). However, although bone has an inherent capacity for regeneration and repair, in some cases, it is necessary to artificially replace bone.

For example, presently, a major Australian research project is working to radically advance the way physicians surgically treat tumours and bone cancer. This research involves the use of 3D implants and robotic surgery to remove the tumour and print a replica of the missing bone. In this work, specialised imaging is used to scan the bone and plan the surgery. The acquired dimensional data from the scan is then interpreted to guide the robotic-assisted surgery and remove the tumour. Following this operation, another scan is performed on the removed diseased bone in order to 3D print an implant to precisely fit the vacant space left in the bone (6). This protocol of 3D printing for bone repair is proving to be a revolutionary, with evidence suggesting incidences of reduced reliance on limb amputation and thus is effectively decreasing the long-term impacts of bone cancer.

This comprehensive success will greatly benefit modern society due to the increased likelihood of contracting cancer (from one in three chance to one in two) (15). This has been attributed to the increase in life expectancy (having increased by approximately 6 years since the year 2000) which has resulted in an ageing population (15) and therefore a larger number of people being susceptible to the disease.

The process of printing structures such as bone can be a timely task due to the complex procedure for printing live cells. Firstly, the cells need to be printed under compatible conditions for live cells. However, for standard additive manufacturing methods, such as Fused Filament Fabrication (FFF), the printing process relies on using plastic filaments and high melting temperatures which would be unsuitable for the cells. Therefore, whilst printing with cells, one should endeavour to use printers designed for cell inclusion such as the Bioscaffolder 3.1. This 3D printer model is an example of a bioprinter, of which the first one was created in 2003 by Thomas Boland (16).

In order for the cells to be printed, they must first be isolated and grown to a high cell density. This is done by the process of isolating individual living cells from a tissue for the generation of ‘primary cell cultures.’ Next, a bioink paste is prepared and the cells are collected via centrifugation (a separation process which implements the action of centripetal force to promote accelerated settling of particles in a solid-liquid mixture) and counted- using a haemocytometer to discern the cell number to be added per gram of material. The cells are subsequently re-suspended in a medium (a substance designed to support the growth of micro-organisms).

There are many growth mediums which can be employed during cell culture, such as vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs) and bone morphogenic proteins (BMPs), which are important in bone tissue engineering. VEGF is an angiogenic protein which regulates endothelial cell proliferation. In which, angiogenesis is the formation of new blood vessels, a process which involves the migration, growth and differentiation of endothelial cells that line the inner wall of blood vessels (14). Whereas, FGFs are a group of proteins essential for the FGF signalling pathway that provokes angiogenesis through endothelial and osteoblast cell proliferation (7). BMPs, however, induce ossification through osteoprogenitors and mesenchymal stem cell (MSC) differentiation to osteoblasts or via binding to collagen (8, 9, 10). 

Once the cells in the solution have been counted, a defined volume is pipetted into the pre-prepared bioink paste, often a hydrogel, to be loaded into a cartridge with a plunger placed on top and a tip (with a determined internal diameter) tightened to the bottom. The cartridge containing the material and cells is then loaded into the 3D printer and attached to a gas line (often nitrogen due to its near inert state and its high abundance, which renders it less expensive than other applicable gases - like noble gases which are truly inert). Once ready, the gas will exert pressure and slowly force the hydrogel out through the tip. After attaching the gas line, a digital file is generated by the software or a file is uploaded. The printer’s software interface has many print parameters that can be adjusted to the user’s liking. For example, the amount of pressure exerted onto each cartridge, the height of the structure and the size of the pores in the material. The pores are essential for cell growth, nutrients, and oxygen delivery. However, low mechanical strength is a major challenge in porous scaffolds (10). The effects of which can be combatted by decreasing the pore size and volume.

An alternative example to tackle the known porosity related strength issue was evidenced by Tarafder et al. who used an effective densification approach achieved using microwave sintering (sintering is when the density of a material is increased by melting, which forms bridges between particles (13)) compared to conventional heating. The effects of this sintering approach demonstrated an improvement to the mechanical properties of 3D-printed TCP (tricalcium phosphate) scaffolds (8). However, sintering cannot be achieved on cell-laden scaffolds without damage to cells due to the high temperatures required to form the bridges between particles. Researchers have also encountered another problem concerning getting sufficient oxygen into 3D scaffolds to produce clinically applicable tissues/organs. One potential strategy that has been investigated to overcome this limitation is to use an oxygen releasing scaffold (11). Currently, this method is proving to be highly successful and compatible with the surrounding cells, as it is prolonging the survival of the aforementioned cells within the scaffold. This procedure works by preparing hollow microparticles (HPs) that are loaded with an emulsion of an oxygen carrier known as perfluorooctane for the timely supply of oxygen to surrounding cells (11). 

After tweaking the scaffold design using the software interface, the printing starts and, layer by layer, the structure is printed. Immediately following the print process, the deposited gel is commonly cross-linked. Crosslinking is a treatment used to set and harden materials. Crosslinking can be performed via a variety of methods including the use of a change to temperature or pH or via the addition of ionic compounds, the process used is dependent on the material type. Usually, hydrogel materials are used for printing because they are biodegradable, injectable, and printable. Additionally, the high water content of hydrogels mimics that of the natural extracellular matrix (bone’s extracellular matrix is 25% water (1)). Hydrogels are water-swollen polymeric materials that can be synthetic or natural. The problem with using natural hydrogels is that the body may reject the materials due to immune rejection. 

Immune rejection can cause many problems in biomaterial implantation. Rejection occurs when the new material triggers the host’s immune system, and subsequently causes a mass infiltration of lymphocytes (white blood cells), called the foreign body reaction. This response can cause fibrosis and is where connective tissue thickens and encapsulates the implant, rendering it unreachable for blood supply and nutrient exchange. However, a negative immune response can be combatted by the careful selection of biomaterials as well as the use of a patient’s cells within the scaffolds to limit the chance of rejection. For example, CaP ceramics (calcium phosphate ceramics) are widely used in bone tissue engineering due to their excellent compatibility as well as bioactivity (the effect of an agent on a living tissue or organism), osteoconductivity (the ability to grow ‘bony tissue’ into the structure of an implant or graft (17)) and similarity in composition to bone (10). 

In summary, because of a steadily increasing likelihood within the population for contracting cancer, the development of 3D printing bone and other tissues is progressively becoming an issue of importance for human survival and our continued health into old age. However, despite the necessity for the success of 3D printed tissues and organs, many challenges within this field remain to be overcome. Such is highlighted in the quotation by Dr Anthony Atala, a professor and director for the Wake Forest Institute of Regenerative Medicine, who stated that “One of the biggest challenges in bioprinting so far has been getting printed tissue to survive long enough to form blood vessels and nerves and otherwise fully integrate with the body in which it is implanted. ’Whilst there is evidence to indicate that a solution to the problems highlighted by Dr Atala is nearing a conclusion, it is clear that challenges remain. Despite this, the problems that have arisen within 3D printing body parts thus far should not undermine the continuing success that these studies have demonstrated.