Bio Fabrication Techniques for Bone and Cartilage Tissue Regeneration

(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)


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Figure 1 Summary of the current fabrication techniques that have been used to produce 2D films and 3D polymeric scaffolds.

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.


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Figure 2 A schematic illustration of the fabrication procedure for PLGA scaffolds by the TIPS method; adapted from[3]

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]. 


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Figure 3 Summary of scaffold fabrication technologies adapted from[14]

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]. 


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Figure 4 illustrates the working principle of electrospinning. adapted from[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.

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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

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