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Scaffold-free: A developing technique in field of tissue engineering
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Scaffold-free: a developing technique in field of tissue engineering Adel Alblawi , Achalla Sri Ranjani , Humaira Yasmin , Sharda Gupta , Arindam Bit , M. Rahimi Gorji PII: DOI: Reference:
S0169-2607(19)31697-9 https://doi.org/10.1016/j.cmpb.2019.105148 COMM 105148
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Computer Methods and Programs in Biomedicine
Received date: Revised date: Accepted date:
2 October 2019 17 October 2019 20 October 2019
Please cite this article as: Adel Alblawi , Achalla Sri Ranjani , Humaira Yasmin , Sharda Gupta , Arindam Bit , M. Rahimi Gorji , Scaffold-free: a developing technique in field of tissue engineering, Computer Methods and Programs in Biomedicine (2019), doi: https://doi.org/10.1016/j.cmpb.2019.105148
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Highlights We proposed a cell sheet based bioprinting technique where mesenchymal stem cells (MSCs) on the surface of thermoresponsive polymer were subjected to mechanosensing either by introducing acoustic energies or stress created by polymeric strain energy function. Mechanosensing stimulus will trigger the intracellular signal transduction pathway leading to differentiation of the MSCs into desired cells.
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Scaffold-free: a developing technique in field of tissue engineering Adel Alblawi1, Achalla Sri Ranjani2, Humaira Yasmin3, Sharda Gupta2, Arindam Bit *2, M. Rahimi Gorji*4 1
Mechanical Engineering Department, College of Engineering, Shaqra University, Dawadmi, P.O. 11911, Ar Riyadh, Saudi Arabia, Email: [email protected] 2
Department of Biomedical Engineering, National Institute of Technology, Raipur, India, [email protected] (Arindam Bit)
3
Department of Mathematics, College of Science, Majmaah University, 11952, Saudi Arabia, Email: [email protected] 4
Faculty of Applied Sciences, Ton Duc Thang University, Vietnam. * Correspondence to: [email protected]
Abstract Scaffold-free tissue engineering can be considered as a rapidly developing technique in the field of tissue engineering. In the areas of regenerative medicine and wound healing, there is a demand of techniques where no scaffolds are used for the development of desired tissue. These techniques will overcome the problems of rejection and tissue failure which are common with scaffolds. Main breakthrough of scaffold free tissue engineering was after invention of 3-D printers which made it possible to print complex tissues which were not possible by conventional methods. Mathematical modeling is a prediction technique is used in tissue engineering for simulation of the model to be constructed. Coming to scaffold-free technique, mathematical modeling is necessary for the processing of the model that has to be bio-printed so as to avoid and changes in the final construct. Tissue construct is developed by use of non-destructive imaging techniques i.e. computed tomography (CT) and magnetic resonance imaging (MRI).In this review, we discussed about various mathematical models and the models which are widely used in bioprinting techniques such as Cellular Potts Model (CPM) and Cellular Particle Dynamic (CPD) model. Later, developed of 3-D tissue construct using micro CT scan images was explained. Finally, we discussed about scaffold free techniques such as 3-D bioprinting and cell sheet technology. In this manuscript, we proposed a cell sheet based bioprinting technique where mesenchymal stem cells (MSCs) on the surface of thermoresponsive polymer were subjected to mechanosensing either by introducing acoustic energies or stress created by 2
polymeric strain energy function. Mechanosensing stimulus will trigger the intracellular signal transduction pathway leading to differentiation of the MSCs into desired cells.
Key words: 3-D bio-printing, mathematical modeling, micro CT, cell-sheet technology 1. Introduction Tissue Engineering is one of the most vital sciences for regeneration of tissues and wound healing.
It is multidisciplinary science which thrives for the development of bio-artificial
organs/tissues to regenerate or heal or enhance its function [1].Scaffold can be considered as backbone of tissue engineering as it serves the crucial providing support to the regenerating tissue. Scaffolds are devices for tissue engineering with perfect chemistry and architecture which will develop the target cell or tissue. Initially, scaffolds were widely used in tissue engineering. Later, after development of various advanced techniques like 3-D bio printing, new methods of tissue engineering without the help of scaffolds came into light. These kinds of procedures without the involvement of scaffolds are considered as scaffold-free tissue engineering methods. Scaffold based methods include using of scaffold as back-bone support on which the cells are grown. They are usually made of biomaterials providing elasticity and mechanical strength to the tissue .The biomaterial will eventually will degrade and the cells grown on the surface will grow normally and form a tissue. The major characteristics to be taken into consideration are porosity, mechanical strength, biocompatibility, thickness etc [2]. The major issues with scaffolds are degradation, adjustment to the environment, immunological response, toxicity of the material used to the surrounding tissues. To overcome these problems, scaffold-free techniques are used as alternative source. The limitation of the scaffolds and ECM based materials has led to the development of alternative method where scaffolds interaction with cell- cell and extracellular matrix cannot result in adverse host responses [2].Advancement in techniques like 3-D techniques gave rise to scaffold-free techniques. These techniques will help in development of 3-D constructs. The most important and widely used
methods in scaffold-free constructs include 3-D printing and cell
sheath technology. Scaffold-free techniques made easy by minimizing the time for the construct
3
to get adjusted to the surrounding environment as there is no other biomaterial used to support its growth which consumes time to degrade and adjust to the atmosphere. A Mathematical model serves as prediction tool which will help in prediction of bio-printing process. Before designing any tissue engineering construct, mathematical modeling of that particular construct is desirable. Mathematical modeling of any tissue engineering construct is used to predict the working and mechanical properties of the tissue engineered construct [3]. It is kind of simulation in which we can mimic the working of the construct. After implantation, there might be effect of stress on construct which can be evaluated using mathematical modeling. Scaffold-free tissue engineering methods also need a strategy for printing process in order to avoid the post processing where there are chances of change in the model and properties of the construct. Many imaging techniques are available such as Ultra sound, MRI, X-ray imaging, microscopy. Among imaging techniques used in tissue engineering, MRI and Micro CT plays major role. One technique uses magnetic resonance and another uses X-rays. Among these, micro CT is considered the user friendly and prominent technique that scans the data and also allows the formation of 3D images of the desired sample [4]. This technique is mainly used in bone tissue constructs. Micro CT is used in construction of finite element models (FEM) of bone tissue constructs helping in perfect geometry and imaging of the bone implants [5]. 2. Mathematical models in Tissue Engineering Mathematical modeling can be addressed as simulation of the construct to be developed. It helps in analyzing its outcome both numerically and analytically [6]. Mathematical modeling can be implemented in various fields of tissue engineering such as tissue-regeneration, wound healing. Radisic et al (2004) [7] has developed a model for evaluating the distribution of oxygen in tissue engineering constructs of cardiac tissue. They had constructed a cardiac tissue using porous scaffolds. Hydraulic permeability was checked by inserting cells isolated from rat ventricles on the tissue. Various tissue engineering experimental models were designed using different parameters such as pH, temperature, flow rate etc and all the models were compared to get overall idea how oxygen is distributed across the scaffold while forming tissue.
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Shear stress and viscosity were calculated. Re and Pe numbers were formulated for all geometries and velocities to simplify the mathematical model. The velocity
can be
mentioned by the equation (1). )
(1)
Where = viscosity of the media, =fluid velocity of the media and
= radius of channel.
Chen et al (2011) [8] proposed mathematical model to evaluate the degradation of bulk polymers, which are applicable in scaffolds constructed by tissue engineering. In this hydrolysis reaction is explained by fundamental stochastic process and also explained in detail the autocatalytic affect by concentration of carboxylic acid by continuous diffusion equation. Here it was assumed that for polymers and co-polymers of PLA, PGA (scaffold building block materials), the water penetration speed would be higher than hydrolysis rate. So, it is assumed that the polymer matrix is completely saturated in initial state i.e. (t=0) where the concentration of the products degraded will be zero. Finally, a degradation model with both fundamental hydrolysis and autocatalysis was defined by the equation 2. (
is fundamental hydrolysis without any auocatalysis,
)
(2)
is probability density function,
are contributions of fundamental hydrolysis and autocatalysis respectively. to manage contribution of autocatalysis match the original data and
and
is constant used
is the concentration of
monomer. Whittaker et al. (2009) [9] proposed a model for fiber-enhanced perfusion in a tissue engineered bio-reactor. Here they have taken porous scaffold in which the culture medium flows through the bioreactor. The main principle of this mathematical modeling is Darcy’s law which is used to calculate distribution of nutrients and shear stress around the scaffold. Darcy’s law is explained in the equation 3. (3) 5
where
is Darcy velocity,
is constant,
is dynamic viscosity and
is pressure gradient
vector. Mathematical models to analyze the tissue-engineered angiogenesis has been developed [10] using cell-trafficking model. Here it discusses about the volume of the scaffold to be maintained, other parameters to be maintained so the angiogenesis will happen efficiently. The cells that are seeded on the scaffold are called as seeded cells. The seeded cells are expected to die or survive depending on the concentration of the available oxygen in the implant. All the mathematical models that have been explained here are used in tissue engineered constructs using scaffolds. It also has to be implemented in scaffold-free tissue engineering to predict and develop broad view of the techniques. The expected outcomes and problems can also be analyzed. Majorly used mathematical models in scaffold-free process are Cellular Potts Model (CPM) and Cellular Particle Dynamic (CPD) model in bio-printing techniques. Cellular Potts Model (CPM) Glazier-Graner- Hogeweg (GGH) model [11] is part of CPM majorly based on cell sorting experiment [12-14]. The main application of this modeling is designing the soft tissues in this model. Each cell is considered as domain sharing a cell index σ. In biological cells, to access the characteristics of cell, cells are considered as clusters [15]. In this model, cells are considered as → , each cell contains cell types,
identical indices patches
energy between the spins of cell-types
. Where
denotes
and . The equation representing this model is explained
in the equation 4. ∑
( ((→)))
( (→))
( (→)
(→)) +∑ (4)
+Δ Here,
is considered as Lagrange multiplier which specifies the strength of constraint,
denotes area of the cell
, and
is the target area.
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is the total change in
effective energy,
represents change in effective energy of chemotatxis and
is change in effective energy of the cell. Stochastic modified metropolis algorithm [16] was used in selected target sites. The neighboring cells
→ and
→ having probability
→→
→ is defined by Boltzmann acceptance
{
(5)
function. ( →→
→)
Here ΔH is change in effective energy membrane. Whereas
⁄
denotes amplitude of the fluctuations in cell
gives the fluctuations amplitude in boundary of the cell [15].
This model is widely used in simulation and prediction of various systems such as tumor growth [17], angiogenesis [18], wetting [19] etc. Cellular particle dynamics (CPD) model This model was introduced by Newman [20] for developing multicellular system where interacting cellular particles (CP) were introduced. In this method N number of cells were considered which are composed by elements M. Each cell is labeled as “i” and element in cell as . When considered that there is lack of chemical signaling, the element will change in time according to dynamics fluctuations, and biomechanical intercellular forces [20]. Taking into consideration, the above factors, interaction of CPs can be determined by equation 6. ( ) Here r is distance,
( )
(6)
is energy necessary to divide the CPs and σ is diameter of CP. Here potential
of intercellular interaction can be calculated by the equation 7. (7) In the same way, intracellular potential is calculated by equation 8. (8) 7
Here k is elastic constant, θ(r) is Heaviside step function. Initially, with assumption of the inertia that is negligible, motion of the ith CP in nth cell can be denoted by the equation 9 (9) Here
is fluctuation and
is friction factor.
Work done on various mathematical models was present in table1. Table1. Table summarizing various mathematical modeling used for tissue engineered constructs. Author
Mathematical model
Radisic et al ,2004
Distribution of oxygen in tissue engineered cardiac tissue
Model to evaluate degradation of
Chen et al., 2011
bulk polymers
Fiber-enhanced perfusion in a
Whittaker et al.,2009
tissue engineered bio-reactor Analysis of tissue engineered
Lemon et al.,2009
angiogenesis
3. Development of 3-D construct using reconstructing technique The immediate requirement of developing any 3-D construct is to have the detailed understanding of the structure and components of the particular organ or tissue. In this regard, imaging acts as major step in development of a construct. The most commonly used noninvasive techniques in tissue engineering are CT and MRI. They help in development of 3-D 8
constructs providing with better understanding about the structure and other aspects of the tissue or organ. Computed tomography (CT) can be defined as 3-D imaging technique that uses X-rays that are projected through the object at many angles to get a tomographic image. The images obtained through this method are made of voxels. Micro Computed-Tomography has become popular in construction of 3D tissue engineering constructs. Micro CT is one of the non-destructive and user friendly methods [21]. Initially X-rays were used for the interpretation of bone and skeletal muscles which was shown only in two dimensions. The imposition of third dimension into the 2D construct was overcome with the launch of Micro CT where the visualization is clear among the bone constructs [22].The image that is obtained by micro CT will be between 500 3 voxels and 2003 voxels. Once the data from the Micro CT is available, the next task is development of 3-D tissue constructs. During this development of tissue constructs, there are several steps involved such as analysis of the sample of interest by Micro CT, Software to convert the Micro CT data to CAD, selection of region of interest, thresholding, analyzing and assessment of porosity of the scaffold [21]. The sample of interest whether it may be bone, cartilage or any other tissue is first analyzed using microtomograph. Depending upon the scanner used the parameters such as resolution of the sample, rotation angle, quality of the output image is set and the data is collected. This data collected will be converted into CAD using software [21]. Although many types of software are developed by the investigators according to their convenience, there are many commercially available software which can make the task easy. Some of the softwares available in the market are Amira, Analyze, and open-source programs such as Drishti and Image J. MIMICS software is also one of the widely used ones .The CT scan images obtained are processed
using MIMICS (Materialize) software and the images are
transformed into CAD to know the internal structure and geometry of the image. The algorithm of software is explained in the figure 1
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Figure1. Flow chart of software algorithm of MIMICS software.
Within the Region of Interest, the volume of the sample is analyzed. The volume is usually mentioned in percentage. Once the volume is obtained, porosity and interconnectivity of the pores are calculated. Micro CT is very successful method used for bone and cartilage constructs. But, coming to soft tissues, Micro CT is not the better option. Micro CT provides better image quality for object having higher atomic weight elements such as bones. In case of soft tissue, Optical coherence tomography (OCT) is used for the better resolution of the tissues. OCT is one of the imaging technique that is noninvasive and results in high resolution 3-D imaging [23]. This imaging technique is similar to ultrasound, but near infrared rays are used instead of sound [23].OCT is mainly used for soft tissues like cornea [24], measuring the thickness of corneal tissue , cartilage tissues [25]. 4. Process of Bio-printing
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Scaffold-free tissue engineering is the emerging technique in the field of tissue engineering and regenerative medicine. Many effective methods and techniques for scaffold-free tissue engineering have emerged. Of them, most efficient techniques are 3-D bio-printing, Cell sheet technology. Literature regarding scaffold-free techniques is mentioned in table2. Table2. Details of authors using various techniques for scaffold-free tissue engineering. Author
Technique used
Taniguchi et.al.,2018
3-D bio-printing
Norotte et.al.,2009
3-D bio-printing
Y.S.Zhang et.al.,2018
3-D bio-printing
Murphy et.al.,2014
3-D bio-printing
Shimizu et.al ,2018
3-D bio-printing
Haraguchi et.al.,2012
Cell-sheet technology
Elloumi et.al.,2010
Cell-sheet technology
J.S.Tchan et.al.,2008
Cell-sheet technology
Matsuda et.al .,2007
Cell-sheet technology
Nishida et.al.,2004
Cell-sheet technology
With advances in computer aided design (CAD) and fabrication technologies, there is rapid advancement in the field of 3-D printing. The term 3-D bio-printing can be described as the process of layering the cells and other biological factors to regenerate a tissue. 3-D bio-printing when compared with other conventional methods holds great relation with the tissue and also have wide applications in regenerative medicine [26]. Norotte et al(2009) [2] introduced cells as Bio-Ink directly using the 3-D bio printers First of all desired cells were collected and cultured under appropriate conditions to prepare cell spheroids. Those multi-cellular spheroids are analyzed using microscopy by adding related dye. Once they are visualized and analyzed, immune-histochemical tests are performed are printed using bioprinters.
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Taniguchi et al( 2018) [27] developed a procedure for scaffold-free trachea regeneration by 3-D bio-printing. Cell spheroids were grown and they are printed using 3-D bio printing and that printed spheroids are made to grow in bioreactor and that is implanted in the place of trachea. There are 3 phases of bio-printing such as pre-processing phase, processing phase and postprocessing phase. Pre-processing phase includes all the details of the tissue to be printed which includes the imaging part (CT, MRI) to analyze the structure and anatomy of the target that has to be printed and subsequent CAD from which the image data is processed for bio-printing [28].Specialized softwares are used which transfer the image data into layers of proper scale so that the printing device will print the target in layer-layer method [29]. Final phase of bio-printing is Post –processing. It involves the procedures to be followed before the t bio-printed tissue is further used for in-vivo testing. This procedure usually takes place in bioreactor [30]. Bio-reactors on which the printed tissues are grown have to be much precised to provide in-vivo environment. Lack of those abilities in the bioreactor effects the viability of the tissues at the time of maturation [31]. The main point to be taken into consideration is that how far the cells will go well with in-vivo environment [32]. Tissues are prepared either by directly printing the cells of interest along with supporting cells or stem cells are printed which will further differentiate [33].Once the model is ready to be printed, it will go for 3-D Bio-printing. Major two types of 3-D bio-printers, Laser based Bio-printer and Laser-free bio-printers [31]. Laser –free bio-printing Laser-free bio-printing techniques include ink-jet bio-printers which use bio-ink for the deposition [34].These printing techniques using ink-jet are two types, thermal inkjet and piezoelectric ink-jet [35],where thermal ink-jet uses heating circuit to reach high temperature and eject the bio-ink whereas piezoelectric ink-jet uses piezoelectric element. Laser free printing techniques are highly preferable as there will be no exposure of cell to laser light and cell viability can be assured. 12
Inkjet bio-printing In the initial stages of 3-D bio-printing, 2D inkjet printers were used by using some modifications to print the bio ink [36].These kinds of printers are also called as drop-on-demand printers. They use technique of non-contact in which they may utilize either electromagnetic or thermal or piezo-electric forces for depositing the bio-ink on the target. The printed tissue using this technique will be the replica of the CAD design [37]. The main concern regarding this inkjet bio-printing is that the cell viability may be affected due to high temperature up to 300°C at the nozzle. Although the duration of exposure is extremely short this may result in increase of 4-1°C of the bio-ink [37]. Working process of laser-free bio-printers is shown in figure 2. Later, studies revealed that the increase in these temperatures will not affect the cell viability [38,39].Main advantages of inkjet bio-printing are high speed, availability and low cost whereas the drawbacks of this method include droplet size, need of low viscous bio inks and clogging of the bio-ink near the nozzle.
Figure 2. Ink- jet bio printers where in thermal based printing heater provides the temperature which releases bio-ink in form of droplets and piezoelectric bio-printer where piezoelectric actuator is used for the cell deposition.
Micro-extrusion 3D bio-printing Micro-extrusion 3D printing is the most common method used for printing in the present time [31].It can also print highly viscous bio-inks such as spheroids, polymers [40,41]. Other advantage of this technique is its ability to print cells with high densities to form tissue [42]. 13
These bio-printers use mechanical forces to release bio-ink from the nozzle using a specific pattern that is computer generated [43]. One disadvantage with this technique is deformation of the cell shape and low cell viability due to the pressure formed while releasing the bio-ink. When used high extrusion pressure, the cell viability is as low as 40% showing the inverse relationship of cell viability with the extrusion pressure [44].The method of printing is explained in the figure 3.
Figure3. Diagrammatic representation of Micro-extrusion 3D bio-printer [45].The cells were loaded in the reservoir were printed on the platform using pneumatic or mechanical pressure.
Laser Based Bio-printing A new technique has been developed called as biological laser printing (BioLp) [46] which is a fine technique without orifice and deposits the cells with high accuracy. Matrix-assisted pulsed laser evaporation direct write (MAPLE DW) was the technique used initially for laser based printing [47] which is deposition technique without contact and no contamination. But, limitation of this technique is its specification to only certain biomaterials and thickness. Cell preparation of cells in this technique is prepared by suspending the cell culture grown in sodium alginate solution [48]. BioLp has overcome these difficulties by depositing the cells layer by layer attaining 3- D model. The major drawback of laser based bio-printing is that, cell viability will not be 100% due to exposure of the cell to laser light and can print only one cell type at a time which will be a disadvantage to scaffold-free techniques. The principle involved in this process is explained in figure 4.
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It is a non-contact, nozzle free technique initially used for patterning of metals in high resolution and fabrication of computer chip [49,50]. This technique passes laser light through the ribbon containing bio-ink which is supported by titanium or gold layer which helps in transferring the energy to the ribbon. Major advantage of this method is resolution that can be achieved. The maximum resolution that can be maintained is one cell per droplet [51].
Figure 4.Diagram representing Laser-based bio-printing [52].The laser rays were focused on absorbing layer creates pressure pushing the cells onto the substrate.
Snyder et al (2011) [53] has developed a cell printing and microfluidic system which was to study the conversion of drug and protection from radiation in liver tissues. They have bio-printed cell laden matrigel which is used to test the radiation sheating amifostine of the liver cells. Bio-printing has good use in bone tissue engineering. Pietrabissa A (2016) [54] discussed about the printing of 3-D models which are further used in surgical and bio-medical applications. Pati et al (2015) [55] mentioned about 3-D scaffolds with use of mesenchymal stem cells to produce ECM similar to bone. A technique was developed to print nerve guidance conduits (NGC) using PEG to be used for studies on peripheral nerve repair [56].Various components and aspects of bio-printing techniques are explained in the table 3. Table 3 Various techniques of 3-D bio-printing.
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Inkjet
Micro-Extrusion
Laser-based
Cell density
Low density
No limit
Medium
Resolution
High resolution
Medium resolution
High resolution
Speed of printing
Fast
Medium speed
Fast
Cost
Low
Medium
High
Cell Sheet technology Cell sheet technology is one of the prominent methods of tissue engineering without the use of scaffolds which will be degraded later. The major principle involved in this technique is the attachment and detachment of the cell to a thermo responsive culture dish [57]. The cells of interest are collected, harvested and extracted as complete sheet along with the Extra cellular Matrix (ECM).The important aspect in this method is the extraction of the whole cells in form of a sheet without disturbing and without using any chemicals or enzyme treatment [58].The main principle in this cell sheet technology is the uses of thermo-responsive substrate. The polymer used for easy adhesion and detachment of the cell is PPIAM i.e. poly (Nisopropylacrylamide). It is a thermo responsive polymer. It is having lower critical solution temperature (LCST) of around 32°C when in an aqueous media. This polymer shows hydrophilic properties when temperature goes below the LCST [34]. Once the temperature of the polymer is below the LCST, cells gets detached from the surface. The polymer is coated on the surface of tissue culture plates and cells were grown on its surface at 37°C. Once the cells are grown properly, the temperature is reduced to 20°C and along with water supply; cells are removed from the plates [59]. Since cell sheets formed along with extracellular matrix (ECM) can be united with other cell sheets, layering of the cell sheets can be easily made which helps in the construction of 3-D tissues. The diagrammatic representation of cell sheath technology is shown in figure 5.
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Figure 5: Diagrammatic representation of process of cell sheath technology. a) At 37°C, the polymer PPIAM was loaded with cells which formed ECM. b) With the fall of the temperature to 20°C, the cells sheet along with ECM starts detaching from the polymer. c) Polymer after detachment of cell sheet.
Cell sheet technology has many applications in tissue engineering and regenerative medicine. The major applications are mainly in development of heart tissues [60], corneal tissue reconstruction [61], for pancreatic tissues in the treatment of diabetes [62], periodontal tissue regeneration [62,63] sealing of air leaks in the lungs [64,65], for ulcers in the esophagus [66]. Discussion and Conclusion Scaffold-free tissue engineering is one of the fast growing techniques due to its advantages and increased cell viability. Advancement in the 3-D printing technologies has made this technique more applicable. Along with the printing techniques, the most crucial aspect is mathematical modeling which helps the concept of scaffold free tissue engineering to understand and evaluate each and every step which helps in assuming the possible results of the construct making easy simulation of the construct. The role of mathematical modeling in designing the tissue engineering construct was mentioned. Mathematical modeling is a cost effective simulation tool which helps in prediction of the construct. It provides mechanical properties of the implant which helps in avoiding tissue failure. The next major step for developing the construct is imaging. The imaging techniques we have 17
discussed is Microtomography (micro CT) which is considered very easy and non-destructive method using x-rays as the source. Optical coherence tomography (OCT) is the technique used in case of soft tissues. The images from the imaging techniques will be used for the development of 3-D construct. The software MIMICS used for the development of 3-D construct by converting the image into CAD files. Once the 3-D tissue construct has been designed, the major part is to print the designed 3-D construct. The two major techniques for printing of 3-D constructs are 3-D bio-printing and cell sheath technology. In this review, discussion of various types of 3-D bio-printers and their advantages along with its applications is covered. A new technique with combination of both 3D bio printing and cell sheet technology was introduced by [67] where they used cell sheet based bio ink for 3-D bio printing. In this process, they have developed cell sheet of hums skin fibroblast cells (HSF) on thermo-responsive polymer. Bio-ink was prepared for 3-D printing by disrupting the cell sheet into cell aggregates. This technique has several uses in case of shape and reproducibility. We have proposed a new technique of scaffold free tissue engineering, where the direct mechanosensing for attached cells is created in the microenvironment on the surface of thermoresponsive polymer. This mechanosensing will trigger the signal transduction pathway in the Mesenchymal stem cells (MSCs) anchored on the polymer. MSCs will be differentiated into desired type of the cells by the signal transduction pathway. The two ways to induce mechanosensing is by sending acoustic waves through the polymer and by stretching the polymer in order to create stress. The signal transduction pathway which triggers the differentiation of MSCs will be integrin dependent. When a mechanical stimulus is produced, mechanoreceptors gets activated triggering signaling pathway that is integrin –dependent. With increase in the mechanical loading, subunits of integrin α2, α 5, β1 and β3 were expressed .Further these subunits will form clusters of α 5β1 and αvβ3 at the deformation site. These clusters will activate the cytoplasmic proteins which further triggers the cascade of reactions leading signal transduction pathway. Choi, Park, & Jeong, (2016) [68] used sound waves on bone marrow derived MSCs which induced neural differentiation by Pyk2 activation and ryanodine receptor-induced calcium. The 18
MSCs were differentiated at a particular frequency of 1 kHz and 81 dB. In this study, intracellular ca2+ induced by sound waves was increased mediated by L-type ca2+ channels and RYRs channels resulting in phophorylation of ERK and CREB promoting neural differentiation. The mechanism of the signalling pathway is explained in the figure 6.
Figure 6: Proposed technique of scaffold free tissue engineering construct shows (a) acoustic chamber (petri- plate) containing the culture media, thermo-responsive polymeric base, and printed cells (Cell type-1, and cell type-2); thermoresponsive polymer is fixed with load cell and a fixator via silk thread, producing tensile force within the polymer; a piezoelectric bed transmitter at the bottom of the chamber creates mechanical waves within the chamber. These two phenomena create micro-environmental mechanosensing stimulus on extracellular surfaces of the seeded (printed cells) at the cell-polymer interaction surface. (b) Cell type-1 MSCs under the influence of micro-environmental mechanosensing trigger gives rise to osteogenesis pathways of MSCs to form osteogenic stem cells, whereas (c) Cell type2 MSCs under similar but different graded mechanosensing stimulus triggers neurogenesis pathways to form neural stem cells
X. Chen, He, Zhong, & Luo, (2015) [69] worked on BM-MSCs where they showed that acoustic vibration of 800Hz was appropriate for osteogenic differentiated by increasing activator RUNX2 expression. ERk1/2 signalling pathway might be involved in the process of osteogenesis [70]. Therefore, the proposed cell printing technique will handle the differentiation of MSCs into different lineages while printing over thermoresponsive polymer. The proposed idea is unique, and it will explore a new horizon of cell printing to form tissues. Nowadays medicine and engineering problems are related together which some recent publications can prove this claim [71-82]. 19
"Compliance with Ethical Statements" Conflict of interest The authors have not any conflict of interest. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors. Acknowledgement This study supported by the grant from Department of Science and Technology (ECR/2017/001115, INT/RUS/RFBR/P-332 and DST/BDTD/EAC/2018) New Delhi, India, and Global Innovation & Technology Alliance (2018TW0208004). References [1] Nerem, R. M., Ph, D., Sambanis, A., & Ph, D. (1995). Tissue Engineering : From Biology to Biological Substitutes, 1(1). [2] Norotte, C. (2009). Scaffold-Free Vascular Tissue Engineering Using Bioprinting. Biomaterials, 30(30), 5910–5917. https://doi.org/10.1016/j.biomaterials.2009.06.034. [3] Neumaier, A. (2004). Mathematical Model Building (pp. 37–43). Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-0215-5_3 [4] Lemon, G., Sjöqvist, S., Lim, M. L., Feliu, N., Firsova, A. B., Amin, R., … Macchiarini, P. (2015). The use of mathematical modelling for improving the tissue engineering of organs and stem cell therapy. Current Stem Cell Research & Therapy, 0(0), 1–12. https://doi.org/10.2174/1574888X10666151001115942 [5] Jaecques, S. V. N., Van Oosterwyck, H., Muraru, L., Van Cleynenbreugel, T., De Smet, E., Wevers, M., … Vander Sloten, J. (2004). Individualised, micro CT-based finite element modelling as a tool for biomechanical analysis related to tissue engineering of bone. Biomaterials, 25(9), 1683–1696. https://doi.org/10.1016/S0142-9612(03)00516-7 [6] Marion, G., Scotland, S., & Daniel Lawson, by. (2008). An Introduction to Mathematical Modelling. Retrieved from https://people.maths.bris.ac.uk/~madjl/course_text.pdf
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Scaffold-free: a developing technique in field of tissue engineering Adel Alblawi , Achalla Sri Ranjani , Humaira Yasmin , Sharda Gupta , Arindam Bit , M. Rahimi Gorji PII: DOI: Reference:
S0169-2607(19)31697-9 https://doi.org/10.1016/j.cmpb.2019.105148 COMM 105148
To appear in:
Computer Methods and Programs in Biomedicine
Received date: Revised date: Accepted date:
2 October 2019 17 October 2019 20 October 2019
Please cite this article as: Adel Alblawi , Achalla Sri Ranjani , Humaira Yasmin , Sharda Gupta , Arindam Bit , M. Rahimi Gorji , Scaffold-free: a developing technique in field of tissue engineering, Computer Methods and Programs in Biomedicine (2019), doi: https://doi.org/10.1016/j.cmpb.2019.105148
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Highlights We proposed a cell sheet based bioprinting technique where mesenchymal stem cells (MSCs) on the surface of thermoresponsive polymer were subjected to mechanosensing either by introducing acoustic energies or stress created by polymeric strain energy function. Mechanosensing stimulus will trigger the intracellular signal transduction pathway leading to differentiation of the MSCs into desired cells.
1
Scaffold-free: a developing technique in field of tissue engineering Adel Alblawi1, Achalla Sri Ranjani2, Humaira Yasmin3, Sharda Gupta2, Arindam Bit *2, M. Rahimi Gorji*4 1
Mechanical Engineering Department, College of Engineering, Shaqra University, Dawadmi, P.O. 11911, Ar Riyadh, Saudi Arabia, Email: [email protected] 2
Department of Biomedical Engineering, National Institute of Technology, Raipur, India, [email protected] (Arindam Bit)
3
Department of Mathematics, College of Science, Majmaah University, 11952, Saudi Arabia, Email: [email protected] 4
Faculty of Applied Sciences, Ton Duc Thang University, Vietnam. * Correspondence to: [email protected]
Abstract Scaffold-free tissue engineering can be considered as a rapidly developing technique in the field of tissue engineering. In the areas of regenerative medicine and wound healing, there is a demand of techniques where no scaffolds are used for the development of desired tissue. These techniques will overcome the problems of rejection and tissue failure which are common with scaffolds. Main breakthrough of scaffold free tissue engineering was after invention of 3-D printers which made it possible to print complex tissues which were not possible by conventional methods. Mathematical modeling is a prediction technique is used in tissue engineering for simulation of the model to be constructed. Coming to scaffold-free technique, mathematical modeling is necessary for the processing of the model that has to be bio-printed so as to avoid and changes in the final construct. Tissue construct is developed by use of non-destructive imaging techniques i.e. computed tomography (CT) and magnetic resonance imaging (MRI).In this review, we discussed about various mathematical models and the models which are widely used in bioprinting techniques such as Cellular Potts Model (CPM) and Cellular Particle Dynamic (CPD) model. Later, developed of 3-D tissue construct using micro CT scan images was explained. Finally, we discussed about scaffold free techniques such as 3-D bioprinting and cell sheet technology. In this manuscript, we proposed a cell sheet based bioprinting technique where mesenchymal stem cells (MSCs) on the surface of thermoresponsive polymer were subjected to mechanosensing either by introducing acoustic energies or stress created by 2
polymeric strain energy function. Mechanosensing stimulus will trigger the intracellular signal transduction pathway leading to differentiation of the MSCs into desired cells.
Key words: 3-D bio-printing, mathematical modeling, micro CT, cell-sheet technology 1. Introduction Tissue Engineering is one of the most vital sciences for regeneration of tissues and wound healing.
It is multidisciplinary science which thrives for the development of bio-artificial
organs/tissues to regenerate or heal or enhance its function [1].Scaffold can be considered as backbone of tissue engineering as it serves the crucial providing support to the regenerating tissue. Scaffolds are devices for tissue engineering with perfect chemistry and architecture which will develop the target cell or tissue. Initially, scaffolds were widely used in tissue engineering. Later, after development of various advanced techniques like 3-D bio printing, new methods of tissue engineering without the help of scaffolds came into light. These kinds of procedures without the involvement of scaffolds are considered as scaffold-free tissue engineering methods. Scaffold based methods include using of scaffold as back-bone support on which the cells are grown. They are usually made of biomaterials providing elasticity and mechanical strength to the tissue .The biomaterial will eventually will degrade and the cells grown on the surface will grow normally and form a tissue. The major characteristics to be taken into consideration are porosity, mechanical strength, biocompatibility, thickness etc [2]. The major issues with scaffolds are degradation, adjustment to the environment, immunological response, toxicity of the material used to the surrounding tissues. To overcome these problems, scaffold-free techniques are used as alternative source. The limitation of the scaffolds and ECM based materials has led to the development of alternative method where scaffolds interaction with cell- cell and extracellular matrix cannot result in adverse host responses [2].Advancement in techniques like 3-D techniques gave rise to scaffold-free techniques. These techniques will help in development of 3-D constructs. The most important and widely used
methods in scaffold-free constructs include 3-D printing and cell
sheath technology. Scaffold-free techniques made easy by minimizing the time for the construct
3
to get adjusted to the surrounding environment as there is no other biomaterial used to support its growth which consumes time to degrade and adjust to the atmosphere. A Mathematical model serves as prediction tool which will help in prediction of bio-printing process. Before designing any tissue engineering construct, mathematical modeling of that particular construct is desirable. Mathematical modeling of any tissue engineering construct is used to predict the working and mechanical properties of the tissue engineered construct [3]. It is kind of simulation in which we can mimic the working of the construct. After implantation, there might be effect of stress on construct which can be evaluated using mathematical modeling. Scaffold-free tissue engineering methods also need a strategy for printing process in order to avoid the post processing where there are chances of change in the model and properties of the construct. Many imaging techniques are available such as Ultra sound, MRI, X-ray imaging, microscopy. Among imaging techniques used in tissue engineering, MRI and Micro CT plays major role. One technique uses magnetic resonance and another uses X-rays. Among these, micro CT is considered the user friendly and prominent technique that scans the data and also allows the formation of 3D images of the desired sample [4]. This technique is mainly used in bone tissue constructs. Micro CT is used in construction of finite element models (FEM) of bone tissue constructs helping in perfect geometry and imaging of the bone implants [5]. 2. Mathematical models in Tissue Engineering Mathematical modeling can be addressed as simulation of the construct to be developed. It helps in analyzing its outcome both numerically and analytically [6]. Mathematical modeling can be implemented in various fields of tissue engineering such as tissue-regeneration, wound healing. Radisic et al (2004) [7] has developed a model for evaluating the distribution of oxygen in tissue engineering constructs of cardiac tissue. They had constructed a cardiac tissue using porous scaffolds. Hydraulic permeability was checked by inserting cells isolated from rat ventricles on the tissue. Various tissue engineering experimental models were designed using different parameters such as pH, temperature, flow rate etc and all the models were compared to get overall idea how oxygen is distributed across the scaffold while forming tissue.
4
Shear stress and viscosity were calculated. Re and Pe numbers were formulated for all geometries and velocities to simplify the mathematical model. The velocity
can be
mentioned by the equation (1). )
(1)
Where = viscosity of the media, =fluid velocity of the media and
= radius of channel.
Chen et al (2011) [8] proposed mathematical model to evaluate the degradation of bulk polymers, which are applicable in scaffolds constructed by tissue engineering. In this hydrolysis reaction is explained by fundamental stochastic process and also explained in detail the autocatalytic affect by concentration of carboxylic acid by continuous diffusion equation. Here it was assumed that for polymers and co-polymers of PLA, PGA (scaffold building block materials), the water penetration speed would be higher than hydrolysis rate. So, it is assumed that the polymer matrix is completely saturated in initial state i.e. (t=0) where the concentration of the products degraded will be zero. Finally, a degradation model with both fundamental hydrolysis and autocatalysis was defined by the equation 2. (
is fundamental hydrolysis without any auocatalysis,
)
(2)
is probability density function,
are contributions of fundamental hydrolysis and autocatalysis respectively. to manage contribution of autocatalysis match the original data and
and
is constant used
is the concentration of
monomer. Whittaker et al. (2009) [9] proposed a model for fiber-enhanced perfusion in a tissue engineered bio-reactor. Here they have taken porous scaffold in which the culture medium flows through the bioreactor. The main principle of this mathematical modeling is Darcy’s law which is used to calculate distribution of nutrients and shear stress around the scaffold. Darcy’s law is explained in the equation 3. (3) 5
where
is Darcy velocity,
is constant,
is dynamic viscosity and
is pressure gradient
vector. Mathematical models to analyze the tissue-engineered angiogenesis has been developed [10] using cell-trafficking model. Here it discusses about the volume of the scaffold to be maintained, other parameters to be maintained so the angiogenesis will happen efficiently. The cells that are seeded on the scaffold are called as seeded cells. The seeded cells are expected to die or survive depending on the concentration of the available oxygen in the implant. All the mathematical models that have been explained here are used in tissue engineered constructs using scaffolds. It also has to be implemented in scaffold-free tissue engineering to predict and develop broad view of the techniques. The expected outcomes and problems can also be analyzed. Majorly used mathematical models in scaffold-free process are Cellular Potts Model (CPM) and Cellular Particle Dynamic (CPD) model in bio-printing techniques. Cellular Potts Model (CPM) Glazier-Graner- Hogeweg (GGH) model [11] is part of CPM majorly based on cell sorting experiment [12-14]. The main application of this modeling is designing the soft tissues in this model. Each cell is considered as domain sharing a cell index σ. In biological cells, to access the characteristics of cell, cells are considered as clusters [15]. In this model, cells are considered as → , each cell contains cell types,
identical indices patches
energy between the spins of cell-types
. Where
denotes
and . The equation representing this model is explained
in the equation 4. ∑
( ((→)))
( (→))
( (→)
(→)) +∑ (4)
+Δ Here,
is considered as Lagrange multiplier which specifies the strength of constraint,
denotes area of the cell
, and
is the target area.
6
is the total change in
effective energy,
represents change in effective energy of chemotatxis and
is change in effective energy of the cell. Stochastic modified metropolis algorithm [16] was used in selected target sites. The neighboring cells
→ and
→ having probability
→→
→ is defined by Boltzmann acceptance
{
(5)
function. ( →→
→)
Here ΔH is change in effective energy membrane. Whereas
⁄
denotes amplitude of the fluctuations in cell
gives the fluctuations amplitude in boundary of the cell [15].
This model is widely used in simulation and prediction of various systems such as tumor growth [17], angiogenesis [18], wetting [19] etc. Cellular particle dynamics (CPD) model This model was introduced by Newman [20] for developing multicellular system where interacting cellular particles (CP) were introduced. In this method N number of cells were considered which are composed by elements M. Each cell is labeled as “i” and element in cell as . When considered that there is lack of chemical signaling, the element will change in time according to dynamics fluctuations, and biomechanical intercellular forces [20]. Taking into consideration, the above factors, interaction of CPs can be determined by equation 6. ( ) Here r is distance,
( )
(6)
is energy necessary to divide the CPs and σ is diameter of CP. Here potential
of intercellular interaction can be calculated by the equation 7. (7) In the same way, intracellular potential is calculated by equation 8. (8) 7
Here k is elastic constant, θ(r) is Heaviside step function. Initially, with assumption of the inertia that is negligible, motion of the ith CP in nth cell can be denoted by the equation 9 (9) Here
is fluctuation and
is friction factor.
Work done on various mathematical models was present in table1. Table1. Table summarizing various mathematical modeling used for tissue engineered constructs. Author
Mathematical model
Radisic et al ,2004
Distribution of oxygen in tissue engineered cardiac tissue
Model to evaluate degradation of
Chen et al., 2011
bulk polymers
Fiber-enhanced perfusion in a
Whittaker et al.,2009
tissue engineered bio-reactor Analysis of tissue engineered
Lemon et al.,2009
angiogenesis
3. Development of 3-D construct using reconstructing technique The immediate requirement of developing any 3-D construct is to have the detailed understanding of the structure and components of the particular organ or tissue. In this regard, imaging acts as major step in development of a construct. The most commonly used noninvasive techniques in tissue engineering are CT and MRI. They help in development of 3-D 8
constructs providing with better understanding about the structure and other aspects of the tissue or organ. Computed tomography (CT) can be defined as 3-D imaging technique that uses X-rays that are projected through the object at many angles to get a tomographic image. The images obtained through this method are made of voxels. Micro Computed-Tomography has become popular in construction of 3D tissue engineering constructs. Micro CT is one of the non-destructive and user friendly methods [21]. Initially X-rays were used for the interpretation of bone and skeletal muscles which was shown only in two dimensions. The imposition of third dimension into the 2D construct was overcome with the launch of Micro CT where the visualization is clear among the bone constructs [22].The image that is obtained by micro CT will be between 500 3 voxels and 2003 voxels. Once the data from the Micro CT is available, the next task is development of 3-D tissue constructs. During this development of tissue constructs, there are several steps involved such as analysis of the sample of interest by Micro CT, Software to convert the Micro CT data to CAD, selection of region of interest, thresholding, analyzing and assessment of porosity of the scaffold [21]. The sample of interest whether it may be bone, cartilage or any other tissue is first analyzed using microtomograph. Depending upon the scanner used the parameters such as resolution of the sample, rotation angle, quality of the output image is set and the data is collected. This data collected will be converted into CAD using software [21]. Although many types of software are developed by the investigators according to their convenience, there are many commercially available software which can make the task easy. Some of the softwares available in the market are Amira, Analyze, and open-source programs such as Drishti and Image J. MIMICS software is also one of the widely used ones .The CT scan images obtained are processed
using MIMICS (Materialize) software and the images are
transformed into CAD to know the internal structure and geometry of the image. The algorithm of software is explained in the figure 1
9
Figure1. Flow chart of software algorithm of MIMICS software.
Within the Region of Interest, the volume of the sample is analyzed. The volume is usually mentioned in percentage. Once the volume is obtained, porosity and interconnectivity of the pores are calculated. Micro CT is very successful method used for bone and cartilage constructs. But, coming to soft tissues, Micro CT is not the better option. Micro CT provides better image quality for object having higher atomic weight elements such as bones. In case of soft tissue, Optical coherence tomography (OCT) is used for the better resolution of the tissues. OCT is one of the imaging technique that is noninvasive and results in high resolution 3-D imaging [23]. This imaging technique is similar to ultrasound, but near infrared rays are used instead of sound [23].OCT is mainly used for soft tissues like cornea [24], measuring the thickness of corneal tissue , cartilage tissues [25]. 4. Process of Bio-printing
10
Scaffold-free tissue engineering is the emerging technique in the field of tissue engineering and regenerative medicine. Many effective methods and techniques for scaffold-free tissue engineering have emerged. Of them, most efficient techniques are 3-D bio-printing, Cell sheet technology. Literature regarding scaffold-free techniques is mentioned in table2. Table2. Details of authors using various techniques for scaffold-free tissue engineering. Author
Technique used
Taniguchi et.al.,2018
3-D bio-printing
Norotte et.al.,2009
3-D bio-printing
Y.S.Zhang et.al.,2018
3-D bio-printing
Murphy et.al.,2014
3-D bio-printing
Shimizu et.al ,2018
3-D bio-printing
Haraguchi et.al.,2012
Cell-sheet technology
Elloumi et.al.,2010
Cell-sheet technology
J.S.Tchan et.al.,2008
Cell-sheet technology
Matsuda et.al .,2007
Cell-sheet technology
Nishida et.al.,2004
Cell-sheet technology
With advances in computer aided design (CAD) and fabrication technologies, there is rapid advancement in the field of 3-D printing. The term 3-D bio-printing can be described as the process of layering the cells and other biological factors to regenerate a tissue. 3-D bio-printing when compared with other conventional methods holds great relation with the tissue and also have wide applications in regenerative medicine [26]. Norotte et al(2009) [2] introduced cells as Bio-Ink directly using the 3-D bio printers First of all desired cells were collected and cultured under appropriate conditions to prepare cell spheroids. Those multi-cellular spheroids are analyzed using microscopy by adding related dye. Once they are visualized and analyzed, immune-histochemical tests are performed are printed using bioprinters.
11
Taniguchi et al( 2018) [27] developed a procedure for scaffold-free trachea regeneration by 3-D bio-printing. Cell spheroids were grown and they are printed using 3-D bio printing and that printed spheroids are made to grow in bioreactor and that is implanted in the place of trachea. There are 3 phases of bio-printing such as pre-processing phase, processing phase and postprocessing phase. Pre-processing phase includes all the details of the tissue to be printed which includes the imaging part (CT, MRI) to analyze the structure and anatomy of the target that has to be printed and subsequent CAD from which the image data is processed for bio-printing [28].Specialized softwares are used which transfer the image data into layers of proper scale so that the printing device will print the target in layer-layer method [29]. Final phase of bio-printing is Post –processing. It involves the procedures to be followed before the t bio-printed tissue is further used for in-vivo testing. This procedure usually takes place in bioreactor [30]. Bio-reactors on which the printed tissues are grown have to be much precised to provide in-vivo environment. Lack of those abilities in the bioreactor effects the viability of the tissues at the time of maturation [31]. The main point to be taken into consideration is that how far the cells will go well with in-vivo environment [32]. Tissues are prepared either by directly printing the cells of interest along with supporting cells or stem cells are printed which will further differentiate [33].Once the model is ready to be printed, it will go for 3-D Bio-printing. Major two types of 3-D bio-printers, Laser based Bio-printer and Laser-free bio-printers [31]. Laser –free bio-printing Laser-free bio-printing techniques include ink-jet bio-printers which use bio-ink for the deposition [34].These printing techniques using ink-jet are two types, thermal inkjet and piezoelectric ink-jet [35],where thermal ink-jet uses heating circuit to reach high temperature and eject the bio-ink whereas piezoelectric ink-jet uses piezoelectric element. Laser free printing techniques are highly preferable as there will be no exposure of cell to laser light and cell viability can be assured. 12
Inkjet bio-printing In the initial stages of 3-D bio-printing, 2D inkjet printers were used by using some modifications to print the bio ink [36].These kinds of printers are also called as drop-on-demand printers. They use technique of non-contact in which they may utilize either electromagnetic or thermal or piezo-electric forces for depositing the bio-ink on the target. The printed tissue using this technique will be the replica of the CAD design [37]. The main concern regarding this inkjet bio-printing is that the cell viability may be affected due to high temperature up to 300°C at the nozzle. Although the duration of exposure is extremely short this may result in increase of 4-1°C of the bio-ink [37]. Working process of laser-free bio-printers is shown in figure 2. Later, studies revealed that the increase in these temperatures will not affect the cell viability [38,39].Main advantages of inkjet bio-printing are high speed, availability and low cost whereas the drawbacks of this method include droplet size, need of low viscous bio inks and clogging of the bio-ink near the nozzle.
Figure 2. Ink- jet bio printers where in thermal based printing heater provides the temperature which releases bio-ink in form of droplets and piezoelectric bio-printer where piezoelectric actuator is used for the cell deposition.
Micro-extrusion 3D bio-printing Micro-extrusion 3D printing is the most common method used for printing in the present time [31].It can also print highly viscous bio-inks such as spheroids, polymers [40,41]. Other advantage of this technique is its ability to print cells with high densities to form tissue [42]. 13
These bio-printers use mechanical forces to release bio-ink from the nozzle using a specific pattern that is computer generated [43]. One disadvantage with this technique is deformation of the cell shape and low cell viability due to the pressure formed while releasing the bio-ink. When used high extrusion pressure, the cell viability is as low as 40% showing the inverse relationship of cell viability with the extrusion pressure [44].The method of printing is explained in the figure 3.
Figure3. Diagrammatic representation of Micro-extrusion 3D bio-printer [45].The cells were loaded in the reservoir were printed on the platform using pneumatic or mechanical pressure.
Laser Based Bio-printing A new technique has been developed called as biological laser printing (BioLp) [46] which is a fine technique without orifice and deposits the cells with high accuracy. Matrix-assisted pulsed laser evaporation direct write (MAPLE DW) was the technique used initially for laser based printing [47] which is deposition technique without contact and no contamination. But, limitation of this technique is its specification to only certain biomaterials and thickness. Cell preparation of cells in this technique is prepared by suspending the cell culture grown in sodium alginate solution [48]. BioLp has overcome these difficulties by depositing the cells layer by layer attaining 3- D model. The major drawback of laser based bio-printing is that, cell viability will not be 100% due to exposure of the cell to laser light and can print only one cell type at a time which will be a disadvantage to scaffold-free techniques. The principle involved in this process is explained in figure 4.
14
It is a non-contact, nozzle free technique initially used for patterning of metals in high resolution and fabrication of computer chip [49,50]. This technique passes laser light through the ribbon containing bio-ink which is supported by titanium or gold layer which helps in transferring the energy to the ribbon. Major advantage of this method is resolution that can be achieved. The maximum resolution that can be maintained is one cell per droplet [51].
Figure 4.Diagram representing Laser-based bio-printing [52].The laser rays were focused on absorbing layer creates pressure pushing the cells onto the substrate.
Snyder et al (2011) [53] has developed a cell printing and microfluidic system which was to study the conversion of drug and protection from radiation in liver tissues. They have bio-printed cell laden matrigel which is used to test the radiation sheating amifostine of the liver cells. Bio-printing has good use in bone tissue engineering. Pietrabissa A (2016) [54] discussed about the printing of 3-D models which are further used in surgical and bio-medical applications. Pati et al (2015) [55] mentioned about 3-D scaffolds with use of mesenchymal stem cells to produce ECM similar to bone. A technique was developed to print nerve guidance conduits (NGC) using PEG to be used for studies on peripheral nerve repair [56].Various components and aspects of bio-printing techniques are explained in the table 3. Table 3 Various techniques of 3-D bio-printing.
15
Inkjet
Micro-Extrusion
Laser-based
Cell density
Low density
No limit
Medium
Resolution
High resolution
Medium resolution
High resolution
Speed of printing
Fast
Medium speed
Fast
Cost
Low
Medium
High
Cell Sheet technology Cell sheet technology is one of the prominent methods of tissue engineering without the use of scaffolds which will be degraded later. The major principle involved in this technique is the attachment and detachment of the cell to a thermo responsive culture dish [57]. The cells of interest are collected, harvested and extracted as complete sheet along with the Extra cellular Matrix (ECM).The important aspect in this method is the extraction of the whole cells in form of a sheet without disturbing and without using any chemicals or enzyme treatment [58].The main principle in this cell sheet technology is the uses of thermo-responsive substrate. The polymer used for easy adhesion and detachment of the cell is PPIAM i.e. poly (Nisopropylacrylamide). It is a thermo responsive polymer. It is having lower critical solution temperature (LCST) of around 32°C when in an aqueous media. This polymer shows hydrophilic properties when temperature goes below the LCST [34]. Once the temperature of the polymer is below the LCST, cells gets detached from the surface. The polymer is coated on the surface of tissue culture plates and cells were grown on its surface at 37°C. Once the cells are grown properly, the temperature is reduced to 20°C and along with water supply; cells are removed from the plates [59]. Since cell sheets formed along with extracellular matrix (ECM) can be united with other cell sheets, layering of the cell sheets can be easily made which helps in the construction of 3-D tissues. The diagrammatic representation of cell sheath technology is shown in figure 5.
16
Figure 5: Diagrammatic representation of process of cell sheath technology. a) At 37°C, the polymer PPIAM was loaded with cells which formed ECM. b) With the fall of the temperature to 20°C, the cells sheet along with ECM starts detaching from the polymer. c) Polymer after detachment of cell sheet.
Cell sheet technology has many applications in tissue engineering and regenerative medicine. The major applications are mainly in development of heart tissues [60], corneal tissue reconstruction [61], for pancreatic tissues in the treatment of diabetes [62], periodontal tissue regeneration [62,63] sealing of air leaks in the lungs [64,65], for ulcers in the esophagus [66]. Discussion and Conclusion Scaffold-free tissue engineering is one of the fast growing techniques due to its advantages and increased cell viability. Advancement in the 3-D printing technologies has made this technique more applicable. Along with the printing techniques, the most crucial aspect is mathematical modeling which helps the concept of scaffold free tissue engineering to understand and evaluate each and every step which helps in assuming the possible results of the construct making easy simulation of the construct. The role of mathematical modeling in designing the tissue engineering construct was mentioned. Mathematical modeling is a cost effective simulation tool which helps in prediction of the construct. It provides mechanical properties of the implant which helps in avoiding tissue failure. The next major step for developing the construct is imaging. The imaging techniques we have 17
discussed is Microtomography (micro CT) which is considered very easy and non-destructive method using x-rays as the source. Optical coherence tomography (OCT) is the technique used in case of soft tissues. The images from the imaging techniques will be used for the development of 3-D construct. The software MIMICS used for the development of 3-D construct by converting the image into CAD files. Once the 3-D tissue construct has been designed, the major part is to print the designed 3-D construct. The two major techniques for printing of 3-D constructs are 3-D bio-printing and cell sheath technology. In this review, discussion of various types of 3-D bio-printers and their advantages along with its applications is covered. A new technique with combination of both 3D bio printing and cell sheet technology was introduced by [67] where they used cell sheet based bio ink for 3-D bio printing. In this process, they have developed cell sheet of hums skin fibroblast cells (HSF) on thermo-responsive polymer. Bio-ink was prepared for 3-D printing by disrupting the cell sheet into cell aggregates. This technique has several uses in case of shape and reproducibility. We have proposed a new technique of scaffold free tissue engineering, where the direct mechanosensing for attached cells is created in the microenvironment on the surface of thermoresponsive polymer. This mechanosensing will trigger the signal transduction pathway in the Mesenchymal stem cells (MSCs) anchored on the polymer. MSCs will be differentiated into desired type of the cells by the signal transduction pathway. The two ways to induce mechanosensing is by sending acoustic waves through the polymer and by stretching the polymer in order to create stress. The signal transduction pathway which triggers the differentiation of MSCs will be integrin dependent. When a mechanical stimulus is produced, mechanoreceptors gets activated triggering signaling pathway that is integrin –dependent. With increase in the mechanical loading, subunits of integrin α2, α 5, β1 and β3 were expressed .Further these subunits will form clusters of α 5β1 and αvβ3 at the deformation site. These clusters will activate the cytoplasmic proteins which further triggers the cascade of reactions leading signal transduction pathway. Choi, Park, & Jeong, (2016) [68] used sound waves on bone marrow derived MSCs which induced neural differentiation by Pyk2 activation and ryanodine receptor-induced calcium. The 18
MSCs were differentiated at a particular frequency of 1 kHz and 81 dB. In this study, intracellular ca2+ induced by sound waves was increased mediated by L-type ca2+ channels and RYRs channels resulting in phophorylation of ERK and CREB promoting neural differentiation. The mechanism of the signalling pathway is explained in the figure 6.
Figure 6: Proposed technique of scaffold free tissue engineering construct shows (a) acoustic chamber (petri- plate) containing the culture media, thermo-responsive polymeric base, and printed cells (Cell type-1, and cell type-2); thermoresponsive polymer is fixed with load cell and a fixator via silk thread, producing tensile force within the polymer; a piezoelectric bed transmitter at the bottom of the chamber creates mechanical waves within the chamber. These two phenomena create micro-environmental mechanosensing stimulus on extracellular surfaces of the seeded (printed cells) at the cell-polymer interaction surface. (b) Cell type-1 MSCs under the influence of micro-environmental mechanosensing trigger gives rise to osteogenesis pathways of MSCs to form osteogenic stem cells, whereas (c) Cell type2 MSCs under similar but different graded mechanosensing stimulus triggers neurogenesis pathways to form neural stem cells
X. Chen, He, Zhong, & Luo, (2015) [69] worked on BM-MSCs where they showed that acoustic vibration of 800Hz was appropriate for osteogenic differentiated by increasing activator RUNX2 expression. ERk1/2 signalling pathway might be involved in the process of osteogenesis [70]. Therefore, the proposed cell printing technique will handle the differentiation of MSCs into different lineages while printing over thermoresponsive polymer. The proposed idea is unique, and it will explore a new horizon of cell printing to form tissues. Nowadays medicine and engineering problems are related together which some recent publications can prove this claim [71-82]. 19
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