3D bioprinting is defined as “the construction of tissue constructs using a set of techniques that transfer biologically important materials onto a substrate with computer-aided, specialized 3D printers.”

The arena of bioprinting, even though young and evolving, has established a niche for itself across various branches of science and technology, including but not limited to materials sciences, tissue engineering, biomedicine, regenerative therapeutics, prosthetics, dentistry, microfluidics, and space technology to name a few. 

So, is bioprinting different from 3D printing?

The answer is yes and no! The principle involved in bioprinting is like that of 3D printing. For example, in the case of 3D printing, we design a model that takes physical shape and is printed layer by layer using a polymer/resin/thermoplastic material. When we investigate bioprinting, we replace this resin/polymer with a living cell suspension which gets printed. To ensure that the printed model serves its purpose (in the field of regenerative/reparative/restorative therapy) certain conditions such as maintenance of sterile printing and post-printing environment, temperature control, etc., must be ensured and enforced. 

There are three main stages of bioprinting that are employed across multiple fields listed above1. In the context of organ/tissue printing, these stages can be divided into substages as shown in figure 1.

1. Preprocessing – to create a display model of the desired tissue to be printed.
2. Processing/Printing – Printing process with the help of bio-inks.
3. Post-processing – Ensuring the stability of the printed biological material through chemical and mechanical stimulation.

During the preprocessing stage, the role and contribution of computer-aided systems (design and manufacturing—CAD/CAM) cannot be undervalued. The history of CAM dates to the 1950’s when the first commercial numeric control programming system was developed named PRONTO (Program for Numerical Tooling Operations) was developed. The credit for the development of CAD goes to Dr. Ivan Sutherland from the Massachusetts Institute of Technology (MIT), and the first-ever CAD software was known as Sketchpad. The impact of these inventions laid the foundations of modern-day CAD/CAM technology that makes 3D printing (bioprinting) a reality. CAD/CAM technologies have greatly impacted and benefitted the manufacturing industries, which further paved the way for its expansion into Additive Manufacturing (AM)/ 3D Printing (3DP) technologies. Today, AM encompasses various forms such as fused deposition modeling (FDM), stereolithography (SLA), laser sintering (SLS), electron beam melting (EBM), and material jetting (MJ) to name a few2. This further manifested into the field of bioprinting focused on on-body applications. The early 1990’s saw the first successful research output which involved the use of CAD technology to bio print 3D scaffolds3. This research by Langer and Vacanti, was a breakthrough in the field of regenerative tissue engineering and despite the cons of the scaffolds in terms of porosity and lack of intricate nutrient infiltration networks, served as the inspiration for a decade long such endeavors within the scientific community. Such tremendous progress due to advancements in 3DP has made precise control of bio-printed scaffolds a reality today in addition to creating a flourishing market for a wide spectrum of bio-inks to print them⁴. Throughout this journey, CAD/CAM technologies provided a supporting platform to bring about extensive and exhaustive improvements to the design and development processes⁵. 

As the field of bio printing evolved, the need to visualize and capture the models created through CAD/CAM in 3D rose steadily. This led to development and fine tuning of multiple technologies in the arena of medical imaging, such as, computed tomography (CT), positron emission tomography (PET), sonography, magnetic resonance imaging (MRI), to name a few. By utilizing 3D imaging modules, these techniques paved way for us to visualize broken bones and observe blood vessels for adequate blood flow patterns⁶. Thus, patient specific 3D geometry data is generated for better understanding of the complexities of the human body. In the direct context of bio printing, the relationship of 3D printing and biomedical imaging can be described in a fourfold manner (figure 2) as published by Squelch (2018)⁷. 

Today, there are still substantial challenges to overcome with regards to establishing ideal data processing methodologies, establishing model accuracies and in mimicking the scanning processes at a cellular level⁸.

The actual bio printing process is made possible with the help of bio inks. The origin of the term bio ink dates to early 2000’s where it was used as a complementary term with bio paper. Bio paper was mainly a hydrogel material that required the insertion of living cells or tissue constructs as the ‘bio ink’ during the printing process. The definition of bio-ink could be stated as the cellular component that was deposited within the 3D space of the design model tissue to be printed9.  Bio inks could thus be aptly called the foundation of the bioprinting process. Careful considerations need to be taken into account while choosing the appropriate bio-ink material, which drastically influences the cell viability, lifespan of the printed construct and its mechanical stability post-implantation. However, till date, there is no single bio ink that could be universally adapted for versatile applications as the mechanical strength and requirements vary drastically between our biological tissues. A wide spectrum of natural and synthetic materials have been investigated to serve as bio inks^4,10. Some of them are mentioned below. 

Table 1 : A brief overview of biomaterials that have been extensively investigated as bio inks for bioprinting.

There are some properties that these bio ink candidates need to fulfil to be accepted for commercialization. These properties can be classified under four broad disciplines namely: chemical, rheological, mechanical and biological¹¹. In the next few articles, we aim to provide our readers with more insights on the most desirable properties, studies showing current applications of these bio inks and present a few commercial bio ink materials that are out in the market to this date.

Moving on to the post processing stage, bioreactors are essential tools in making organ printing a reality. In simple terms, organ printing is the biomedical application of additive manufacturing processes, enabled largely by “computer-aided robotic layer by layer additive bio fabrication of functional living human tissue constructs”12. The fundamental principle behind organ printing revolves around tissue fusion which is accomplished to a large extent on a small scale with bioreactors and tissue spheroids. Post-processing plays an important part in the whole bioprinting process in conferring stability and ensuring that the printed construct is mechanically viable for the intended application. In this regard, different types of bioreactors dominate tissue and organ regeneration arena today, each applicable for a particular organ of interest, for instance: Cartilage tissues prefer a compression-based bioreactor¹³.The most commonly used bioreactors in the field of bioprocessing are:

• Stirred Tank Bioreactors
• Bubble Column Bioreactors 
• Airlift Bioreactors 
• Fluidized Bed Bioreactors 
• Packed Bed Bioreactors 

Each of these have innate advantages and concerns in context to desired tissue construct that can be grown in it which are beyond the scope of this current article. However, in addition to choosing the right type of bioreactor, finding the optimal parameters to tailor such as hydrostatic pressure, shear stress, growth factor control plays a predominant role in finally defining the outcome of the process¹⁴. Moreover, the outcome of bioprinting a tissue in a bioreactor is not always the final product. Accelerated maturation of the bio printed construct with the help of a pre-defined concoction of chemical and physical factors is carried out. These factors are known as maturogens or maturogenic factors. Till date, there are quite a few matrix materials that have been established as maturogenic factors – TGFβ, Serotonin, Periostin, Transglutaminase, Fibronectin to name a few¹⁵. 

Having understood the basic processes and terminologies involved in various stages of printing an organ/tissue construct, let us continue our discussion on the different types of bioprinters in the upcoming article. Stay Tuned!

References:

1. Soleimani, E., Khorsand Movaghar, R., Ghasemzadeh, N., Shirafkan, S. & Chabok, H. R. A Study of State of Art Technologies Used in Skin Regeneration. (2017).

2. Wimpenny, D. I., Pandey, P. M. & Kumar, L. J. Advances in 3D printing & additive manufacturing technologies. (Springer, 2017).

3. Langer, R. & Vacanti, J. P. Tissue engineering. Science 260: 920-926. TISSUE Eng. UNION Biol. Eng. 98, (1993).

4. Chopra, H., Kumar, S. & Singh, I. Bioinks for 3D printing of artificial extracellular matrices. in Advanced 3D-Printed Systems and Nanosystems for Drug Delivery and Tissue Engineering 1–37 (Elsevier, 2020).

5. Alghazzawi, T. F. Advancements in CAD/CAM technology: Options for practical implementation. J. Prosthodont. Res. 60, 72–84 (2016).

6. Abdullah, K. A., McEntee, M. F., Reed, W. & Kench, P. L. Development of an organ‐specific insert phantom generated using a 3D printer for investigations of cardiac computed tomography protocols. J. Med. Radiat. Sci. 65, 175–183 (2018).

7. Squelch, A. 3D printing and medical imaging. J. Med. Radiat. Sci. 65, 171–172 (2018).

8. Lau, I. & Sun, Z. Three‐dimensional printing in congenital heart disease: A systematic review. J. Med. Radiat. Sci. 65, 226–236 (2018).

9. Mironov, V. Printing technology to produce living tissue. (2003).

10. Janarthanan, G. & Noh, I. Recent trends in bioinks for 3D printing. Biomater. Res. 22, (2018).

11. Lee, H. J. et al. A new approach for fabricating collagen/ECM‐based bioinks using preosteoblasts and human adipose stem cells. Adv. Healthc. Mater. 4, 1359–1368 (2015).

12. Esmond, R. W. & Sterling, D. Bioprinting: The Intellectual Property Landscape BT  – 3D Printing and Biofabrication. in (eds. Ovsianikov, A., Yoo, J. & Mironov, V.) 1–28 (Springer International Publishing, 2016). doi:10.1007/978-3-319-40498-1_18-1.

13. Elitok, M. S., Gunduz, E., Gurses, H. E. & Gunduz, M. Tissue engineering: towards development of regenerative and transplant medicine. in Omics Technologies and Bio-Engineering 471–495 (Elsevier, 2018).

14. Rosser, J. & Thomas-Vazquez, D. Bioreactor processes for maturation of 3D bioprinted tissue. in 3D Bioprinting for Reconstructive Surgery: Techniques and Applications 191–215 (2018). doi:10.1016/B978-0-08-101103-4.00010-7.

15. Hajdu, Z. et al. Tissue spheroid fusion-based in vitro screening assays for analysis of tissue maturation. J. Tissue Eng. Regen. Med. 4, 659–664 (2010).

About the Author:

Vidya Chamundeswari Narasimhan , Ph.D.

Dr VIDYA CHAMUNDESWARI NARASIMHAN (Scientist) 3DHEALS COMMUNITY MANAGER SAN FRANCISCO  

Dr. Vidya is a Research Scientist at an upcoming cultivated meat company based in Berkeley, California. She received her Ph.D from Nanyang Technological University(NTU), Singapore in 2018. Her research focused on developing bioactive polymeric scaffolds for musculoskeletal tissue regeneration applications.

Over the last two years, Vidya spearheaded various academia-industrial collaborative projects in Singapore. At 3DHEALS, she is our Community Manager of San Francisco Bay Area and is also actively involved in STEM mentor ship as well as in tutoring University and Junior college students for various competitive examinations.  

Vidya has been actively involved in research pertaining to sustainability within the agro-food and biomaterials sector, and is passionate about emerging technologies in the field of 3D bioprinting. She hopes to actively share and spread this interest amongst like minded professionals in the field of  regenerative medicine and tissue engineering. Currently Vidya has contributed to a couple of expert column blogs on bone tissue engineering and is the co-instructor of an online course on Bioinks used for Bioprinting available on Udemy!

Related Articles:

An Introduction to Scaffolds for Tissue Engineering of the Bone and Cartilage

3D Bioprinting Bone – One Defect At A Time

Patent Protection for Medical 3D Printing & Bioprinting Technologies

Control your 3D Bioprinting Hydrogels

Bio Fabrication Techniques for Bone and Cartilage Tissue Regeneration

The post An Introduction to 3D Bioprinting appeared first on 3DHeals.

(About the photo above: Artist Amy Karle’s Spine Morphologies Smithsonian Institution Collaboration)

A biomaterial is “any substance or a combination of substances either natural or synthetic in origin, which can be used for any period of time, as a whole or as a part of any system which treats, augments, or replaces any tissue, organ or function of the body”. Synthetic scaffolds belong to the subclass of polymeric biomaterials. The goal of a scaffold is to provide a 3D environment for cells to grow on. For a biomimetic scaffold to form bone or cartilage, the model tissue has to mimic the native extracellular matrix (ECM).

ECM of bone and cartilage are composed primarily of Col I and Col II fibrils respectively, which are mineralized and form the basic building blocks of the tissue. In recent times, advancements in biofabrication techniques have developed novel scaffolds to promote both bone and cartilage growth. However, issues such as the cost of production and control over scaffold properties attract researchers to utilize film-like 2D models and sheets for preliminary understanding of release patterns of bioactive molecules and in in vitro studies. The table (Table 1) below summarises important phases involved in tissue engineering a bone or cartilage scaffold.

Table 1 Six phases of Classification of Tissue Engineering of Bone and Cartilage.

1. Fabrication of bioresorbable scaffold.
2. Seeding of osteoblasts/chondrocytes populations into the scaffold.
3. Growth of premature tissue in a dynamic environment.
4. Growth of mature tissue in a physiological environment.
5. Surgical transplantation.
6. Tissue-engineered transplant assimilation/remodeling.

The objectives stated in the table are interdisciplinary. Beginning with the raw polymer material chosen, to the scaffold architecture to the cell-seeded in it and the transplantation process, each step must be planned carefully to accomplish the final goal. The focus of my research was on synthetic polymers as they have a long history of use as degradable sutures for surgery: 

• Biocompatibility and FDA approved for human use. 
• They degrade by non-enzymatic hydrolysis processes by ester bond breakage. The degradation products of PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone) and PLGA(poly(lactic-co-glycolic) acid) are not toxic and the body eliminates them as carbon dioxide and water. 

By altering parameters such as chemical composition (polymer ratios), molecular weight and crystallinity can be varied in addition to the degradation rates of these polymers. Despite extensive research on synthetic polymers, there still exists a dire need to improve their functionality to expand their applications. For developing the scaffold, choosing the right cells for seeding is an important aspect of achieving an engineered tissue.

My research focuses on MSCs (mesenchymal stem cells) as they are multi-potential cells with the ability to differentiate into both bone and cartilage in response to the cues provided to them. Their marked plasticity enables researchers to use them to differentiate into multiple cell lineages and to fabricate a multi-phasic tissue. MSCs are derived from the bone marrow and do not pose any ethical concerns in terms of usage. They can be easily expanded in vitro and there are vast amounts of literature that provide strong evidence for their differentiation in vitro and in vivo.

To accommodate MSCs, biomaterial scaffolds exhibit several qualities for serving as carriers, providing good mechanical integrity and a 3D ECM mimicking environment.   Several academic studies produced fibers, sponge-like, and gel-based structures using synthetic biodegradable materials for culturing MSCs from different species. The focus of this article is to take a sneak peek into the various fabrication techniques available for researchers in the field of bone and cartilage tissue regeneration. (See Figure 1 below)

The basic working principle, advantages, and disadvantages of each of these are briefly explained below. 

1. Thermally Induced Phase Separation (TIPS) 

TIPS methods were first applied to tissue engineering of scaffolds in the 1990s and it is a five-step process. It involves polymer dissolution, phase separation, extraction of solvent, freezing and freeze-drying. Several research labs have fabricated 3D nano-fibrous structures using this technique. In a polymer like PLGA (Poly-l-glycolic acid), under ambient conditions, the polymer-rich phase forms the nano-fibrous matrix while the polymer-deficient phase is extracted with the solvent resulting in nanofibers in 50-500 nm regimes[1-3].

The advantages of TIPS include highly porous and narrow pore size distribution in the resulting scaffold combined with high reproducibility and simplicity of the process. The disadvantages include the need for additives and the formation of a dense skin layer on the scaffold with remnant solvent remaining in the end product.

2. Solvent casting and particulate leaching

This technique involves the addition of salt particles of a specific diameter to a polymer solution to form a uniform suspension. The steps include the incorporation of salt to polymer-solvent evaporation of the solvent followed by vacuum drying. This scaffold is then immersed in water to leach off all the salt thus inducing the formation of pores. Porosity values of about 86% have been achieved through this technique with pore sizes exceeding 100 µm. While attaining high porosity is an advantage of this technique, it is limited in terms of use as only thin membranes (or 3 mm thick scaffolds) can be made by this method[4].

3. Melt molding

This process involves filling a mold with polymer melt or powder and obtaining the shape of the mold. There are two variations of this technique including compression molding and injection molding. In the work published by Thompson et al, compression molding was employed, and Teflon was the mold used. PLGA particles and gelatin microspheres were added to the mold and heated above the glass transition temperature of PLGA, simultaneously applying pressure to the mixture. This led to the particles to bond together.

Once the mold has cooled down, gelatin was leached out by immersion in water and the scaffold was dried. Poor mechanical integrity and lack of structural stability limit the application of this technique in the tissue engineering domain despite its ability to achieve uniformly porous structures[5].

4. Gas foaming

Gas foaming includes the aspects of both melt molding and particulate leaching. High porosities of about 95% can be achieved by this technique with pore sizes in the range of 200-400 µm[6]. Open porous scaffolds can be fabricated by this technique. The fabrication process involves the addition of ammonium bicarbonate to the polymer solution in an organic solvent such as chloroform. The resulting mixture possesses a high viscosity and is shaped using a mold. This is followed by solvent evaporation and finally vacuum drying or immersion in hot water.

The undesirable surface skin layer obtained in other techniques is not observed in gas foaming and this technique can be used in the future for various tissue engineering applications. The fabricated scaffold exhibits high porosity combined with good cell viability. However, the mechanical properties of the scaffold fabricated through this method need to be tailored for clinical usage[7].

5. Freeze drying

In this technique, the scaffold material is frozen and surrounding pressure is increased along with the addition of heat to allow the water content in the frozen scaffold to sublime. This results in a direct transfer of a solid phase to a gaseous phase. Yannas et al have developed a collagen scaffold by freezing a solution of collagen followed by freeze [8]. The pore sizes can be tailored by changing the freezing temperature and also with changes in pH. Lack of structural stability is a drawback of this technique.

6. Fiber bonding

This technique involves the casting of a solution of one polymer over fibers of another polymer mesh. Solvent evaporation takes place and the result is a non-bonded second polymer deposited on a mesh-like network of the first polymer. The remnant solvent is selectively removed from polymer 1. This method is considered as a post-treatment for fibers that are fabricated through other techniques and is not suitable for cases that require precise control over porosity and cannot be used for polymers that dissolve in the same solvent[9].

While all the above methods have been extensively studied by the scientific community, it is a well-known fact that the cellular interaction with the ECM is both a dynamic and a demanding process. Hence, the next generation of tissue engineering scaffolds focuses on the advancements in designing scaffolds with precision and good mechanical integrity for use as bone and cartilage replacements. Currently, the three major fabrication techniques that strive to fulfill this promise for bone and cartilage tissue engineering are 3D printing, Self-Assembly, and Electrospinning.

7. 3D Bioprinting/Additive Manufacturing

3D Bioprinting constitutes a family of processing techniques that rely on additive manufacturing and Computer-Aided Design (CAD) models for making scaffolds. There are several techniques that fall under the umbrella. They result in the formation of a 3D scaffold/pattern in a layer-by-layer process based on the virtual design or CAD model. These techniques were restricted to the field of surgical planning in the bone and cartilage tissue regeneration until Griffith and co-workers reported utilizing one of the additive manufacturing techniques called Stereo-Lithography (SLA) to fabricate ceramic components.

SLA uses a UV laser to photo crosslink and polymerize a photocurable monomer. The complexity of the steps involved and limitations in choice of photocurable raw materials are concerns posed by this technique. The advantages include the relative ease to achieve minute features in the resulting scaffolds. Complex scaffold architecture with a vast array of polymeric materials can be designed using any of these techniques or a combination of additive manufacturing techniques. 3D printing (3DP) – Binder jetting was first introduced by Bredt et al.

The advantages of 3DP includes independent control over pore size and porosity. It can achieve pore sizes in the range of 45 -500 µm, high surface area to volume ratio and there are many choices of materials that could be used for this process. However, having to use toxic organic solvents and lack of mechanical strength over-shadowed the benefits posed by this technique until recently where a plethora of alternatives to organic solvents have been developed. Selective Laser Sintering (SLS) requires high processing temperatures to sinter heat fusible powders to a CAD geometry model one layer at a time. While this processing technique boasts of complete control over pore geometry, is solvent-free and gives good compressive strength, the heat involved in the sintering process makes it undesirable for loading growth factors or any bioactive molecules for tissue engineering.

Fused deposition modeling (FDM) uses a moving nozzle to extrude a polymeric fiber from which a physical model is built layer by layer. Macro shape control, solvent-free fabrication and high porosity are the benefits of FDM, however, it is limited in terms of material type availability, high processing temperatures, and inconsistent pore openings. Lastly, the Inkjet printing technique which utilizes temperature-sensitive inks. Cells are directly sprayed onto the solidifying thin layer of the polymer solution. Although the current technique highlights concerns in limited material choice and cell aggregates within droplets [10-13], this poses significant research opportunities for innovation and will hopefully bridge the gap between supply and demand of cell-laden scaffold for tissue regeneration[10-13]. 

8. Self-Assembly

Self-assembly involves the spontaneous organization of individual components into an ordered and stable structure with pre-programmed non-covalent bonds. Only a few polymer configurations such as diblock, tri blocks and dendrimers are fabricated using this technique as it is a complex laboratory procedure. This complexity combined with low productivity limit the large-scale utilization of this technique[15]. 

9. Electrospinning

The first description of electrospinning dates back to 1902 when J F Cooley filed a United States patent entitled ‘Apparatus for electrically dispersing fibers’. However, major commercialization of this process began only in the 1930s after the reports of Anton Formhals and Norton. The theoretical basis for electrospinning was developed in the 1960s by Sir Geoffrey Ingram Taylor through the Taylor cone model. The concept of electrospinning is not a new one and it received much attention after integrating it with the field of nanotechnology in the late 1990s. It is a simple, reliable, robust and scalable technique that finds wide applicability in research labs and in industries.

The combination of factors such as tailorable mechanical properties, a wide array of choice of polymeric materials, cost-effectiveness and formation of structurally diverse nanofibers have added on to the increased interest in this fabrication process. However, the current limitations of this technique include the need for organic solvents and the complex interplay of various processing parameters on the final product. Further control over thickness and composition along with the porosity of the fibrous meshes can be achieved using a simple experimental setup. [16-18]. 

Stay tuned for my next article to find out more about my go-to fabrication technique! You are welcome to share your thoughts and experiences working with any of the above techniques pertaining to bone and cartilage research. 

#Jointhetribe 
#3Dscaffolds
#bonetissueregeneration

References:

1. Liu, M., et al., Formation of microporous polymeric membranes via thermally induced phase separation: A review. Frontiers of Chemical Science and Engineering, 2016. 10(1): p. 57-75.

2. Do Kim, H., et al., Effect of PEG–PLLA diblock copolymer on macroporous PLLA scaffolds by thermally induced phase separation. Biomaterials, 2004. 25(12): p. 2319-2329.

3. Cao, Y., et al., Production and surface modification of polylactide-based polymeric scaffolds for soft-tissue engineering, in Biopolymer methods in tissue engineering. 2004, Springer. p. 87-111.

4. Huang, R., et al., Preparation of tissue engineering porous scaffold with poly (lactic acid) and polyethylene glycol solution blend by solvent-casting/particulate-leaching. Materials Research Express, 2014. 1(4): p. 045403.

5. Thomson, R., et al., Biodegradable polymer scaffolds to regenerate organs, in Biopolymers Ii. 1995, Springer. p. 245-274.

6. Yoon, J.J. and T.G. Park, Degradation behaviors of biodegradable macroporous scaffolds prepared by gas foaming of effervescent salts. Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials, 2001. 55(3): p. 401-408.

7. Nam, Y.S., J.J. Yoon, and T.G. Park, A novel fabrication method of macroporous biodegradable polymer scaffolds using gas foaming salt as a porogen additive. Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials, 2000. 53(1): p. 1-7.

8. O’Brien, F.J., et al., Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds. Biomaterials, 2004. 25(6): p. 1077-1086.

9. Chen, G., T. Ushida, and T. Tateishi, Scaffold design for tissue engineering. Macromolecular Bioscience, 2002. 2(2): p. 67-77.

10. Yang, S., et al., The design of scaffolds for use in tissue engineering. Part II. Rapid prototyping techniques. Tissue engineering, 2002. 8(1): p. 1-11.

11. Sachs, E., et al., Three dimensional printing system. 1998, Google Patents.

12. Chim, H., et al., A comparative analysis of scaffold material modifications for load-bearing applications in bone tissue engineering. International journal of oral and maxillofacial surgery, 2006. 35(10): p. 928-934.

13. Melchels, F.P., J. Feijen, and D.W. Grijpma, A poly (D, L-lactide) resin for the preparation of tissue engineering scaffolds by stereolithography. Biomaterials, 2009. 30(23-24): p. 3801-3809.

14. Nigam, R. and B. Mahanta, An overview of various biomimetic scaffolds: Challenges and applications in tissue engineering. Journal of Tissue Science & Engineering, 2014. 5(2): p. 1.

15. Gu, F., et al., Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers. Proceedings of the National Academy of Sciences, 2008. 105(7): p. 2586-2591.

16. Li, D., et al., Electrospinning: a simple and versatile technique for producing ceramic nanofibers and nanotubes. Journal of the American Ceramic Society, 2006. 89(6): p. 1861-1869.

17. Bhardwaj, N. and S.C. Kundu, Electrospinning: a fascinating fiber fabrication technique. Biotechnology advances, 2010. 28(3): p. 325-347.

18. Reneker, D.H., et al., Bending instability of electrically charged liquid jets of polymer solutions in electrospinning. Journal of Applied physics, 2000. 87(9): p. 4531-4547.

About the Author:

Vidya Chamundeswari Narasimhan , Ph.D.

Dr. Chamundeswari completed her Doctorate by Research from Nanyang Technological University, Singapore; her Ph.D. work focused on developing bioactive polymeric scaffold systems for bone and cartilage tissue regeneration applications. She has interned at reputed institutions such as MIT-Harvard and National Chemical Laboratory, Pune India. She was the title winner of the Young Persons’ World Lecture Competition, in Australia (2017), and a finalist at the Falling Walls Lab Competition in Singapore (2019). 

Over the last couple of years, Vidya has been actively involved in a wide spectrum of research projects ranging from designing medical devices, oral and scaffold-based drug delivery systems all the way to the extraction of bio-resins from food waste, to name a few.  She is currently a Community Manager with 3DHeals, San Francisco and is passionate about applying her knowledge to drive R&D progress pertaining to healthcare and regenerative medicine.

Related Articles:

An Introduction to Scaffolds for Tissue Engineering of the Bone and Cartilage

Manufacturing of Functional Tissues In Vitro Using Bioprinting and Bioreactors

3D Bioprinting Bone – One Defect At A Time

3D Bioprinting Substrate Stiffness – Often Overlooked, But Always at Work

Stem Cell Considerations for Bioprinting or Tissue Engineering

The post Bio Fabrication Techniques for Bone and Cartilage Tissue Regeneration appeared first on 3DHeals.

(Photo Credit: NewAtlas)

The field of healthcare is being revolutionized by the concept of artificial tissues. Tissue engineering (TE) applies the principle of biology and engineering to the development of functional substitutes for damaged tissue. It holds immense potential for replacement therapy where damaged tissues and organs such as liver, connective tissues, bone, cartilage, and muscles can be regenerated or replaced if they are beyond repair. The design of bone scaffolds has emerged as a victorious alternative to bone substitutes including autografts, allografts, and xenografts. TE of the bone and cartilage has evolved over the last few decades with the primary aim to overcome the limitations of all conventional treatments offered. The clinical applications in the field of bone and cartilage tissue engineering constitute a wide spectrum ranging from bone and cartilage regeneration, tumors, reconstructive surgeries, and arthritis treatments. The general strategy in creating a functional bone or cartilage tissue requires the incorporation of living cells into matrices or scaffolds, as structural support for cell adhesion, while influencing the fate of immature cells through bioactive molecules and/or physical cues. The key considerations to be considered while designing scaffold materials for bone and cartilage are biocompatibility, biodegradability, mechanical requirements in accordance with the tissue, architecture of the scaffolds and the fabrication technology. One of the primary goals of TE lies in developing methods to construct organs in the laboratory that can be used subsequently in medical applications. Thus, the major requirements for bone and cartilage tissue engineering are the perfect synergy between osteogenic/chondrogenic signals, suitable scaffolds, and cells (Figure 1).

• The signals constitute various biochemical cues, growth factors or bio-molecules that are essential for the differentiation into the desired lineage. 
• Cells constitute osteoblasts, chondroblasts or mesenchymal stem cells (MSCs) which direct the differentiation process into the respective lineage. MSCs are quite easily available and the protocols for their extraction and culture are well documented in the literature. They can be characterized by their ability to develop into osteogenic or chondrogenic lineages in response to external stimuli and expression markers that can be supplemented. 
• Lastly, scaffolds play a vital role in supportive matrix information of a tissue-engineered bone or cartilage. There is insufficient evidence to show that grafts could fully heal injuries, without causing issues like an increased inflammatory response, antigenic reactions, fixation site failure and lack of long term biocompatibility which has limited their application in treating bone and cartilage regeneration. The design of scaffolds has received a lot of attention, as it has emerged as an alternative to bone substitutes. For instance, nano-fibrous structures have multifarious applications in the nano-bioengineering domain based on their morphology and processing techniques. 

Moreover, due to variations in structure within a bone (compact and spongy) and cartilage (lamellae and trabeculae), the definition of an ideal scaffold is difficult for these applications.

The key factors to consider while fabricating an ideal scaffold (Figure 2) are 

(a) pore size [macro- >100 µm), micro- < 20 µm] and interconnected open porosity for in vivo tissue in-growth; 

(b) Mechanical Properties: mechanical properties combined with controlled degradation kinetics, 

(c) ability to survive through all the physical forces in vivo; and 

(d) maintenance of a sterile environment condition for cell seeding onto the scaffold. 

Three-dimensional(3D) porosity with interconnected pores is essential to facilitate cell growth, transport of nutrients and removal of metabolic wastes. Biocompatibility and bio resorption with controllable degradation rates are required to be in accordance with the cell model of interest. Conducive surface properties for cell proliferation, attachment and detachment are essential followed by commendable mechanical properties to ensure proper load transfer.

Classification of Scaffolds:

Scaffolds, in the context of musculoskeletal tissue regeneration, are temporary support structures that induce the formation of the desired lineage. They also act as carriers of drugs and other growth factors and bioactive molecules that enable osteogenesis and/or chondrogenesis to happen. To this end, scaffolds are broadly classified into three categories namely ceramics, polymers and metals (Figure 3). 

Demineralized bone matrix, collagen composites, fibrin, calcium phosphate, polylactide (PLA), poly(lactide-co-glycolide)(PLGA), polylactide-polyethylene glycol(PLA-PEG), hydroxyapatite(HA), dental plaster and titanium are few of the currently approved materials used extensively for musculoskeletal tissue regeneration approaches.

Metals and ceramics pose the issue of lack of biodegradation in a biologic environment and possess limited processability and tenability. 

Polymeric scaffolds, on the other hand, have gained significant attention as they can be tailored to closely mimic the extracellular matrix (ECM) of the native tissues. They are biodegradable and exhibit excellent design flexibility, which in turn relies on the material chosen, composition and the ratios of the polymers to be tailored for different applications. Polymers used in bone and cartilage TE are required to be biocompatible thus avoiding any immune responses from the host tissue. Their degradation rate can be controlled to suit the rate of tissue regeneration and degradation products are excreted through metabolic pathways.

Polymers are sub-classified into naturally-derived (eg: collagen and fibrin), and synthetic polymers (eg: poly (lactic acid) (PLA), poly (glycolic acid) (PGA), and their copolymers (PLGA)).

Natural polymers exhibit immunogenicity and do not provide flexibility over mechanical properties. In addition, batch to batch consistency, risk of pathogen infection and biodegradability are issues of concern. Synthetic polymers are reproducible in large quantities without any variations arising from different batches. They possess negligible immunogenicity and properties such as microstructure, degradation rate, and mechanical strength can be tailored according to user needs.

Various composite materials, with an optimal concoction of natural and synthetic polymers, have also been developed. Synthetic hydrogels are being explored widely for devising an injectable biodegradable scaffold system, with significant efforts focused on improving their mechanical strength.

A plethora of advancements in this arena indicates that engineered tissues to possess added clinical applicability and how it repositions itself as a viable therapeutic option for those who would benefit from the life-extending benefits of musculoskeletal tissue regeneration.

What are your thoughts on your preferred choice of cell-type, scaffold and signals for growing a bone tissue?

Stay tuned to know more about various fabrication technologies currently used for musculoskeletal tissue engineering in the next article!

References:

1. Mooney, D.J. and J.P. Vacanti, Tissue engineering using cells and synthetic polymers. Transplantation Reviews, 1993. 7(3): p. 153-162.

2. Stevens, M.M., Biomaterials for bone tissue engineering. Materials today, 2008. 11(5): p. 18-25.

3. Ferrone, M.L. and C.P. Raut, Modern surgical therapy: limb salvage and the role of amputation for extremity soft-tissue sarcomas. Surgical oncology clinics of North America, 2012. 21(2): p. 201-213.

4. Hoo, S.P., et al., Preparation of a soft and interconnected macroporous hydroxypropyl cellulose methacrylate scaffold for adipose tissue engineering. Journal of Materials Chemistry B, 2013. 1(24): p. 3107-3117.

5. O’brien, F.J., Biomaterials & scaffolds for tissue engineering. Materials today, 2011. 14(3): p. 88-95.

6. Meinel, L., et al., Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Annals of biomedical engineering, 2004. 32(1): p. 112-122.

7. Bader, K.F. and J.W. Curtin, A successful silicone tendon prosthesis. Archives of Surgery, 1968. 97(3): p. 406-411.

8. Bose, S., M. Roy, and A. Bandyopadhyay, Recent advances in bone tissue engineering scaffolds. Trends in biotechnology, 2012. 30(10): p. 546-554.

9. Rouwkema, J., N.C. Rivron, and C.A. van Blitterswijk, Vascularization in tissue engineering. Trends in biotechnology, 2008. 26(8): p. 434-441.

10. Cordonnier, T., et al., Biomimetic materials for bone tissue engineering–state of the art and future trends. Advanced Engineering Materials, 2011. 13(5): p. B135-B150.

11. Harakas, N.K., Demineralized bone-matrix-induced osteogenesis. Clinical orthopaedics and related research, 1984. 188: p. 239-251.

12. Liu, X. and P.X. Ma, Polymeric scaffolds for bone tissue engineering. 2004.

13. Thomson, R., et al., Biodegradable polymer scaffolds to regenerate organs. Biopolymers Ii, 1995: p. 245-274.

14. Swetha, M., et al., Biocomposites containing natural polymers and hydroxyapatite for bone tissue engineering. International journal of biological macromolecules, 2010. 47(1): p. 1-4.

15. Dai, N.-T., et al., Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin. Biomaterials, 2004. 25(18): p. 4263-4271.

16. Shang, Q., et al., Tissue-engineered bone repair of sheep cranial defects with autologous bone marrow stromal cells. Journal of Craniofacial Surgery, 2001. 12(6): p. 586-593.

17. Kenley, R., et al., Osseous regeneration in the rat calvarium using novel delivery systems for recombinant human bone morphogenetic protein‐2 (rhBMP‐). Journal of Biomedical Materials Research Part A, 1994. 28(10): p. 1139-1147.

18. Kuo, C.K. and P.X. Ma, Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties. Biomaterials, 2001. 22(6): p. 511-521.

About the Author:

Vidya Chamundeswari Narasimhan , Ph.D.

Dr. Chamundeswari completed her Doctorate by Research from Nanyang Technological University, Singapore; her Ph.D. work focused on developing bioactive polymeric scaffold systems for bone and cartilage tissue regeneration applications. She has interned at reputed institutions such as MIT-Harvard and National Chemical Laboratory, Pune India. She was the title winner of the Young Persons’ World Lecture Competition, in Australia (2017), and a finalist at the Falling Walls Lab Competition in Singapore (2019). 

Over the last couple of years, Vidya has been actively involved in a wide spectrum of research projects ranging from designing medical devices, oral and scaffold-based drug delivery systems all the way to the extraction of bio-resins from food waste, to name a few.  She is currently a Community Manager with 3DHeals, San Francisco and is passionate about applying her knowledge to drive R&D progress pertaining to healthcare and regenerative medicine.

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