1. INTRODUCTION:

Microtia is a congenital disorder, where the external ear is malformed or not completely developed. It has a strong influence and impact on the psychological and physiological health of children and a few adults. It is a deformity where the external ear is underdeveloped or even totally absent and this results in hearing impairment in children and adults^[5,27,35]. The successful reconstruction and regeneration of human ear-shaped cartilage using diversified tissue engineering and regenerative medicine applications represents a promising advancement in the field of auricular or ear reconstruction^[55].

In the contemporary world, tissue engineering has become the most dependable solution for auricular implants due to the advantages over other methods. A right combination of cell source and scaffolds are to be incorporated for the development of high fidelity auricular construct^[47]. Developing a functional and high fidelity human auricular relies on optimal cell culture environment, type of polymer, behavior of the seeding cells and the molecular interaction between the scaffolds and the cells^[43]. Besides, the auricular construct must be compatible and acceptable by the immunocompetent test case model and additionally should maintain long term integrity for successful beneficial application of auricular construct through tissue engineering.

Scaffolds play a major role in tissue engineering as they act as a template for regeneration of tissue by guiding them^[32]. 3D scaffolds are expected to be highly porous, have extensively well-interconnected pore networks and have consistent, adequate and enough pore size for cell migration and infiltration^[26]. A range of materials has been tested for ear construct, including natural polymers such as alginate, collagen and synthetic polymers such as poly-(lactic-co-glycolic acid) (PLGA), poly-glycolic acid (PGA), high-density porous polyethylene (HDPP)^[37], poly-ɛ-caprolactone (PCL)^[47]. The 3D scaffold fabrication through conventional methods like fiber bonding, particulate leaching, phase separation, solvent casting, membrane lamination, molding, and foaming had limitations. These techniques did not permit or allow enough control of scaffold architecture, pore network, pore size and interconnectivity^[50]. Thus the use of 3D printing in the scaffold construction showed great advantages over the conventional methods.

A number of three-dimensional (3D)-printing processes have been applied to tissue engineering The development and fabrication of scaffolds using a CAD system, which involves the direct 3D printing of porous biomaterials in which porosity changes in space with a specific gradient also known as functionally graded scaffolds. The different technologies of additive manufacturing including selective laser sintering (SLS), fused deposition modeling (FDM) etc., are used to create tissue constructs. The macro and micro attributes are looked upon precisely while developing the construct using such techniques^[51,52]. 3D printing technology used to prepare scaffolds provides optimum space for cell attachment and adaptation. Growing a well-designed living tissue remains a crucial task till date, due to the type of cells, organization of cells and the matrix components^[11]. Hence tissue engineering can provide a solution to develop a 3D tissue construct for auricular development. Also, the developed 3D tissue construct can be used for many other platforms like drug testing and biological studies^[38]. But the technological limitations and upcoming trends and prospects highlight the possibilities of future improvements and advancements for various new 3D-printing methodologies and techniques for tissue engineering.

2. 3D PRINTING OF SCAFFOLDS:

One of the most significant developments to tissue engineering came after the advent of 3D-printing for tissues and scaffolds. 3D-printing methods have since been used to fabricate customized scaffolds with a control over pore size and pore structure; thus overcoming previously existing drawbacks like-low control over pore size, pore network and scaffold architecture, leading to less than ideal 3D scaffolds. Bio-printing using 3D-printing technology has numerous advantages like precise and controlled deposition of cells, growth factors, and drugs etc., thus directing improved tissue regeneration. Until now several biomaterials including bone and cartilage tissues, neural, cardiac tissues and heart valve, liver, skin, retinal and tissue composites have been fabricated through this technology^[1]. Methods like fused deposition modeling (FDM), stereo-lithography, inkjet printing, selective laser sintering (SLS), and color-jet printing are the most extensively used ones out of over 40 3D-printing techniques developed², due to their ability to process plastics which is the most important case for Ear reconstruction with cartilage scaffolds^[3,6].

3D printing technology has been successfully applied for the fabrication of complex 3D scaffolds by using both direct and indirect techniques. Direct 3DP refers to the process in which the scaffold material is directly printed during the scaffold fabrication process. In this technique, the scaffold is constructed by stacking 2D patterns that represent the cross-section shape of the 3D scaffold. Solid freeform fabrication techniques (direct 3DP) are the most widely used technique in tissue engineering.

Figure 1: Overall process - scaffold fabrication by Indirect 3DP

Indirect 3DP techniques use a negative mold based on a scaffold design, onto which the desired biomaterial will be cast and then sacrificed to obtain the final scaffold (Figure 1). Techniques like stereo-lithography, fused deposition modelling, and selective laser sintering make use of direct 3D-printing techniques. Indirect 3D printing is used for the fabrication of ceramic and polymeric scaffolds of various biomaterials, such as poly (lacticco-glycolic) acid (PLGA), poly(L-lactide), collagen, poly(ε-caprolactone) (PCL), and chitosan-alginates^[36]. Indirect 3DP techniques enforces a solvent-based molding process for the fabrication of polymeric scaffolds. The actual biomaterial solution that is supposed to be injected into the mold cavity is prepared using an organic solvent, which leads to increased scaffold fabrication time since it requires additional time for dissolving biomaterial in solvent and for casting biomaterial inside the mold by solvent/non-solvent exchange or evaporation under atmospheric pressure/vacuum.

3. COMPUTER AIDED DESIGN (CAD):

There have been several methodologies for the design of 3D scaffold models, including CAD-based methods, image-based design, implicit surfaces and space-filling curves. To provide a well-organized, custom 3D construct for a specific organ or tissue defect, careful design is required. CAD-based modeling is the preferred method for scaffold fabrication using 3D printing, due to the simple yet powerful user-interfaces of commercial CAD software packages^[15]. The light controlling architectures and dimensions printed are commonly patterned using Computer Aided Design (CAD) software. CAD files of a designed object or CT scans of a 3D object are sliced into separate layers. The light source is directed using these layers to cure the polymer. This method allows for personalized medicine as imaging a patient’s tissue enables production of 3D replicas that are almost identical to the replaced tissue. The light source and imaging optics control the scaffolds (overall size and individual features) resolution, dimension, fidelity.

3.1 Slicing algorithm:

3D free-form structures are produced using 3D printing technique through vertical stacking of 2D patterns. These structures are generated from cross-sections of the 3D model designed using a native ear^[20]. The slicing algorithm calculates the outer boundary (contours) of the cross-sections of succeeding planes. The layer thickness is pre-determined and using imported STL data the contours are generated from intersections between the polygons and the planes.

3.2 Algorithm to generate printing paths:

The printing path for the frame is defined depending on the cross-section contours of the 2 cell-laden hydrogel parts, as follows. First, parallel lines are placed on the contours, determined by the line spacing^[20]. With each layer, the lines for hydrogel part-1 are perpendicular to those of hydrogel part-2, which reduces mixing between the cell-laden hydrogels. End points of the printing paths are determined by calculating the intersection between contours and lines. The lines on each layer are assigned into a single group of printing paths to define the frame. Likewise, the path for the support is calculated from the contours belonging to that part. Finally, the two cell-laden hydrogel’s printing paths are placed between the lines that make up the frame.

4. BIOPRINTING METHODS:

Bio-printing deals with three main types of methods including droplet-, laser-and extrusion-based bio-printing.

4.1 Droplet-based bio-printing:

It is based on thermal, piezo, or acoustic-driven mechanisms; and uses heat energy, electrical energy, and sound energy, respectively for generation of droplets of cell suspension in a high throughput fashion. These bio-printers have received much consideration owing to their simplicity, versatility, agility^[16] and high-throughput potential^[48] to allow a variety of biologics such as viable cells, growth factors, genes, and pharmaceutics^[41].

4.2 Extrusion-based bio-printing:

It is a rapidly growing technology printing various biologics. It is a hybrid of a fluid dispensing system and an automated robotic system for extrusion and bio-printing, respectively. These bio-printing systems use mechanical or pneumatic-driven systems and deposit viable cells in a filament form. In such systems, bioink is dispensed using a deposition in a computer-controlled manner which ensures an accurate dispensing of viable cells encapsulated in a filament (cylindrical)^[34].

4.3 Laser-based bio-printing:

Viable cells from a donor-slide to a receiver-slide are dispensed without the assistance of a nozzle using laser energy in a laser-based bio-printing system. This modality offers several advantages in dispensing a variety of biologics such as biomaterials, live cells, growth factors, drugs and genes and vectors^[33].

5. BIO-PRINTING PROCESS:

The process of bio-printing occurs in three steps/phases (Figure 2). The first phase or the pre-processing phase includes the planning details that precede production of bio-printed tissue^[4]. This phase includes CT/MRI imaging to analyze the target tissue’s anatomical structure and subsequent CAD to convert the imaging data into a blueprint for bio-printing. Specialized software programs like AutoCAD, SOLIDWORKS, and CATIA transform imaging data into cross-sectional layers of appropriate scale so that the bio-printing device will be able to add them in a layer-by-layer fashion. The processing phase is the second phase. It includes steps involved in the actual construction and manufacturing of the bio-printed tissue. There arises a complexity in choosing a specific printing method and formulating a combination of materials (bio-ink, scaffold, and other additives). Each selection has the ability to alter the interaction of the individual components and to affect the final tissue product as a result. The final phase or the post-processing phase includes steps that must occur before bio-printed tissue is fully mature and ready for in vivo use. For most 3D bio-printing applications, this usually happens within a bioreactor. While bioreactors have certainly played a pivotal role in bio-printing, more refinement of the bioreactor technology is needed^[4]. Current bioreactors are not able to appropriately recreate the in vivo environment for many tissue types leading to loss of tissue viability during the maturation period.

Figure 2: Bio-printing process flowchart

6. SOLID FREEFORM FABRICATION IN TISSUE ENGINEERING:

Solid freeform fabrication (SFF) is a direct and rapid prototyping method which add materials in the form of layers. The indirect methods cannot easily create complex structures and is time consuming. The SFF is most predominantly used in tissue engineering for producing various constructs. Factors like pore size and porosity play a major role in cell proliferation and differentiation after seeding cells onto the scaffolds^[22]. Such factors are easily manufactured in a controlled way using SFF than subtractive methods. Each of the techniques in solid freeform fabrication involves making a 3D construct layer by layer using different processes and suitable materials.

6.1 Stereolithography apparatus (SLA):

It is a photo-polymerization-based technology that involves irradiation of liquid resin. Microscale features are precisely constructed directly from a computer model using this method^[9]. In this method, the captured 3D image is fed into the computer and the image is sliced into layers. The laser prints computer generated layer in the vat. At each point in the vat, where power absorption from the laser takes place, polymerization occurs. Many photopolymers including polyvinyl alcohol, polyisoprene, poly (D, L-lactide) poly (propylene fumarate), polyvinyl cinnamate, polyamides, polycaprolactone (PCL) based materials are used for making the scaffolds^[42].

6.2 Fused deposition modelling (FDM):

It is a prototyping technology that produces novel scaffolds with controllable porosity, pore size and interconnected channel network^[53]. The FDM can build both solid and honeycomb style porous structures by extruding heated material through a nozzle^[29]. Many polymers including polyfumarates, polyorthoesters, polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid (PLA), copolymers of PLA and PGA (PLGA), polyanhydrides and polycarbonates are found suitable for FDM^[28]. The success in producing a functional construct lies in the choice of scaffolds, seeding cells and 3D printing technique employed.

6.3 Selective laser sintering (SLS):

It involves selective solidification of a range of fine powders^[29]. The fabrication takes place in a chamber at a temperature maintained below the melting point of the powder. Several biocompatible polymers including bioceramics such as Hydroxyapatite (HA), Polycaprolactone (PCL), Polyetheretherketone (PEEK), Poly(L-lactic acid) (PLLA), Poly (vinyl alcohol) (PVA) are used for SLS technique^[44].

6.4 Three-dimensional printing (3D-P):

It involves layering of precursor powder by spraying binders through printer nozzles^[29]. The printed part is lowered and a layer of powder is spread after printing every layer. Many synthetic polymers including poly (l-lactic acid), poly (ε-caprolactone) and polylactide–coglycolide are used along with organic solvent binder and natural polymers including gelatin, dextran, and starch are used along with water as a binder^[7].

7. EAR REGENERATION WITH CARTILAGE SCAFFOLDS:

High fidelity auricular reconstruction is one of the challenging factor for the surgeons. In the current treatment method hand-carved autologous costal cartilage grafts and porous polyethylene implants are being used for the construct. The focus has been shifted to the field of tissue engineering and regenerative medicine, advancing different ways to solve problems. One of the key factor in developing a high fidelity human auricular depends on optimal cell culture environment, type of polymer, performance of the cells and the interaction between the cells and scaffolds. Also the developed construct must be acceptable in immunocompetent test case model and maintain long-term integrity^[43]. Hence there is a need to develop a therapeutic strategy that satisfies the above all factors. Many natural polymers including collagen, alginate etc., are used as hydrogels for tissue-engineering^[11].

The natural polymers support cell attachment, proliferation and differentiation. While the use of synthetic polymers including Polyglycolic acid (PGA), Polylactic acid (PLA), Polyethylene glycol (PEG), Poly (lactic-co-glycolic acid) (PLGA) and Poly-ɛ-caprolactone (PCL), provide control over mechanical and degradation properties^[17]. It has been stated that PEG is widely used in soft-tissue engineering, though it can maintain the cell position precisely, it is non-biodegradable. PCL degrades slowly, PLA involves high temperature for printing the scaffold. But at the same time natural polymers contribute to low mechanical strength and also shows faster degradation^[17]. Therefore, developing the right combination of cells and materials gives rise to a successful creation of auricular implants.

7.1 Seeding cell options for ear construct:

Two of the most clinically used cell sources for ear reconstruction are MSCs and chondrocytes[^25]. The chondrocyte cells and stem cells can be easily isolated from the auricle (elastic cartilage) or nasal septum (hyaline cartilage), without causing any functional damage to the donor tissue^[12]. The cartilage engineered from stem cells is unstable and inclined to specified osteogenesis in the subcutaneous or intramuscular implantation site of the ear or in general the head and the neck regions^[10]. For the auricular reconstruction for patients with microtia, the deformed ear cartilage, which is usually discarded, provides an ideal or a perfect seeding cell source without harming the surrounding healthy cartilage^[21,30]. Chondrocytes have low proliferation ability. However, researchers have found that chondrocytes isolated from the nasal septum or auricle were more proliferative than previously expected. Also, the engineered cartilage formed by the chondrocytes maintains stable phenotype and functions without shrinkage or osteogenesis in a subcutaneous environment. Although they are proliferative, however, these cells still have dedifferentiation issues^[54].

But then again chondrocytes have limitations like the issue of dedifferentiation, where cells regress to a simpler state from a specialized function, cytokine stimulation, which leads to matrix degradation, and reduced oxygen-tension. To overcome these boundaries of chondrocytes, MSCs have been used as a cell source as they do not affect cartilage activity. They have the capability to sustain multi-potency after many number of expansions. Moreover MSCs have immunosuppressive properties which makes allogenic application possible. Hence, the co-culture of chondrocytes with mesenchymal stem cells may prove to be a more reasonable seed cell strategy.

7.2 Scaffold options for ear construct:

Both hydrogel and solid scaffolds are used for 3D bio-printing. Using combinations of hydrogels or with other types of scaffolds are being encouraged. To inherit the merit of different scaffolds, combined application of naturally derived material with a synthetic scaffold became a positive approach^[24].

7.2.1 Collagen type I hydrogel:

3D structure of normal ear was digitalized and then converted to physical form, using CAD/CAM technique combined with digital photogrammetry^[37]. The ear anatomy was scanned from a female, aged 5 years, noise was removed and then digitally fabricated. The ear was constructed using collagen type I hydrogel and were seeded with bovine auricular chondrocytes. It was inspected that the size of the acellular implant significantly reduced in a short period of time, while the cellular part was retained, maintaining their fidelity after implantation. The auricle was developed to be biomechanically and histologically similar to the native auricle, even after a long period of implantation.

7.2.2 Fibrin hydrogel:

Xu et al. investigated chondrocyte-seeded fibrin hydrogel. The construct was externally supported by a stent for the early 6 weeks of implantation. After 12 weeks of implantation cartilage formation was observed and the size, shape and fidelity of the model was evaluated which showed satisfactory results^[49].

7.2.3 Silk alginate copolymer:

A 3D cell-copolymer human ear construct was implanted and evaluated in an immunocompetent rabbit model^[43]. The Mesenchymal progenitor cells were cultured and expanded into chondrocytes which were seeded onto an alginate and silk polymer. After 2 months of implantation, the cells and the scaffolds of the construct was collected and analyzed in terms of size and histology. The copolymer scaffold reduced in size but sustained the shape and fidelity of the ear model. Through histological study, tissue cartilage formation and extracellular matrix components of the copolymer construct was seen.

7.2.4 Hyaluronic hydrogel:

Hyaline cartilage is primarily made up of type II collagen and chondroitin sulphate. They are also found in elastic cartilage. It has been shown that a scaffold made up of hyaluronan with methacrylated hyaluronan [HAMA] could be used to construct cartilage in-vitro^[11]. UV light was used to cross-link the polymer and HAMA. When bovine chondrocytes were seeded onto the scaffold, the cells showed 98% viability after 7 days of culture. The UV light did not seem to affect cell viability, nor did HAMA seemed to be toxic.

7.2.5 Cage construct by poly-ɛ-caprolactone:

Three diverse constructs were tried (i) a fibrin/hyaluronic acid (FB/HA) hydrogel, (ii) a FB/HA hydrogel combined with a collagen I/III scaffold, and (iii) a cage construct containing FB/HA combined with a collagen I/III scaffold, surrounded by a 3D-printed poly-e-caprolactone (PCL) mold^[47]. Polycaprolactone scaffolds have spherical and random pores produced by 3D laser printing^[56]. The solution containing hydrogel is simply trapped between frames of biodegradable polymer. Polymers other than PCL used are poly lactic acid (PLA) and poly lactic-co-glycolic acid) (PLGA). The frame or the cage provides effective support to the printed shape than a construct containing only hydrogel^[20]. CT images were segmented and utilized in image-based CAD to create porous structures. Different combinations of seeding cells were used including chondrocytes and perichondrocytes and adipose-derived mesenchymal stem cells. All the combinations were evaluated after implantation. It was found that for contraction free ear cartilage, 3D printed cage construct can be used for a range of cell combinations.

7.2.6 Polyglycolic acid and Polypropylene complex:

A three polyglycolic acid layer construct surrounded by polypropylene was constructed by Enjo et al. onto which chondrocytes were seeded^[13]. After 5 weeks of grafting, a thick cartilage was obtained with a slower release of basic fibroblast growth factor (bFGF). The polymer complex induced angiogenesis and greater cartilage regeneration. Hence a complex of absorbable and non-absorbable material, polyglycolic acid and polypropylene respectively, is very useful in cartilage regeneration.

7.2.7 Pluronic F127:

Saim et al. investigated a hydrogel scaffold named Pluronic F127, which is a mixture of polypropylene oxide and polyethylene oxide. The investigation was carried out to find a scaffold combination to avoid foreign body reactions^[39]. The hydrogel scaffold was seeded with chondrocytes and no immune reaction was detected. After 10 weeks of implantation, the formation of extracellular matrix was observed and the histological results were consistent with the auricular cartilage.

7.2.8 Polyhedral oligomeric silsesquioxane poly (carbonate-urea) urethane (POSS-PCU):

POSS-PCU is a nanocomposite used as an auricular scaffold. Nayyer et al. aimed at inducing desirable cellular interactions and reduce extrusion rate using the nanocomposite as a scaffold^[31]. The fibroblast ingrowth and proliferation was supported by POSS-PCU. Also the collagen production was higher in POSS-PCU than those on polyethylene scaffold. The nanocomposite scaffold can be a promising alternative to other synthetic and natural polymers for auricular reconstruction.

8. DECELLULARIZATION OF EAR TISSUE SCAFFOLDS:

Scaffolds are extensively used to reconstruct cartilage. The current major challenge is to fabricate and develop a scaffold with a favorably efficient and a highly organized microenvironment that substantially resembles the original native cartilage. Scaffolds derived from non-cellular extracellular matrices are usually able to provide such a favorable microenvironment. Decellularization is the process of removing cellular content from a tissue or organ while retaining the extra cellular matrix structure and proteins. Thus, decellularization of full thickness ear cartilage still remains an unsolved challenge.

According to a report, decellularized ear cartilage scaffolds were prepared and characterized extensively^[46]. Cartilage decellularization was optimized in such a way to remove cells and cell debris or remains from the elastic cartilage. After the removal of nuclear material, the generated scaffolds retained their original collagen and elastin contents and also their architecture and shape. Their observations from high magnification scanning electron microscopy showed not much difference in the matrix density after decellularization of the scaffold. But, apparently glycosaminoglycan content was extensively reduced, resulting in a loss of the visco-elastic properties of the cartilage scaffold. Also when in contact with the cartilage scaffolds the human bone-marrow-derived mesenchymal stem cells (BM-MSC) remained viable and were able to differentiate towards the chondrogenic lineage when cultured in vitro. These results, including the ability to decellularize whole human ears highlight the clinical potential of decellularization as an improved cartilage reconstruction strategy^[46].

Majority of the tissues for ear cartilage reconstruction are animal-derived because of the high availability of the tissue. The decellularization process reduces immune response, which is largely responsible for acute rejection of xenografts. After decellularization, the neural and other stem cells can be seeded into the decellularized extra cellular matrix to induce differentiation and subsequent in vivo engraftment into damaged ear. The decellularized extracellular matrix can then be used as a mechanical and biochemical scaffold for the induction of stem cells and other cells toward a targeted tissue phenotype. Such induced cells can be used for tissue and organ transplants in preclinical animal and human clinical applications^[40]. Hence through decellularization, the current challenges like maintaining the biomechanical integrity of the scaffold, the recellularization potential while reducing immune response of the individual, and extra cellular matrix disruption-all of which affect cell repopulation and structural integrity of the cartilage upon implantation, can be overcome.

9. FUTURE PROSPECTS AND TRENDS:

Cartilage construction has achieved some good progress in the past few years. But there is a need for insight into the mechanical and biological properties of the construct. There are many limitations associated with current engineered auricular construct, including degradation, antigenicity, inflammation, mechanical instability, foreign body reaction, fibrosis and calcification^[19]. The mechanical properties should match and integrate with the nearby cartilage^[25]. The ear must bear the load for a long period of time and must not degenerate. The construct must be highly biocompatible to avoid any undesirable immune response. Moreover the complete structural and functional property of the construct must match with the native ear. Hence there is a need for critical collaboration of experts from different fields like developmental biology, material science and surgeons to have a deep scientific understanding of the tissue reconstruction for effective expansion in the future use. According to a report, fabrication of pre-seeded alginate construct printed into simple geometrics showed great retention of their shape after almost 20 weeks of culturing^[8]. The process was successfully encapsulated with bovine chondrocytes while maintaining cell viability and sterility. Using this approach, large implants could be fabricated that would have otherwise been difficult to seed the cells onto the scaffolds due to the limitations of transportation throughout the scaffold. Further studies on re-seeded scaffold fabrication can give new insights into tissue reconstruction.

3D printing technology is presently used in the medical field for various purposes. But since the cost of fabrication is high it is necessary to find an alternative to reduce the cost and time of fabrication. Soft tissue such as artificial ear can be fabricated using a method known as Scanning Printing Polishing Casting (SPPC^)[18]. The 3D scanner scans the anatomy, then a casting mold is designed on computer and then printed with desktop printer. Later a polishing method is followed to bring in a smooth surface. Finally, a medical grade casting material is cast into the mold. Then the soft cast is removed from the mold. Hence such technique can be used to make an ear construct with a natural polymer to which the cells can be seeded upon. One of the major challenges faced presently is to position and culture multiple types of cells in a single process at a defined location. Although researchers have obtained initial success in printing heterogeneous tissues, these were printed in separate compartments and did not replicate the microstructure of the native tissue^[14,23]. Further research on this area would be of much significance. Also developments in the field of 3D bio-printing has led to the emergence of the 4D bio-printing concept^[45]. 4D printing refers to the 3D printing of programmable materials; since the printed part gradually transforms in shape over the post-printing period, the fourth dimension refers to time.

10. ACKNOWLEDGEMENT:

Our sincere thanks to our faculty Dr. Manjubala I for guiding us throughout the project and the Vellore Institute of Technology management for presenting us with the opportunity.

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Received on 07.04.2018 Modified on 15.05.2018

Research J. Pharm. and Tech 2018; 11(9): 4179-4186.

DOI: 10.5958/0974-360X.2018.00767.9