Tissue Engineering: New Paradigm of Biomedicine

Tissue engineering is a multidisciplinary field of biomedicine that is being used to develop a new tissue or restore the function of diseased tissue/organ. The main objective of tissue engineering is to overcome the shortage of donor organs. Tissue engineering is mainly based on three components i.e. cells, scaffold and growth factors. Among these three components, scaffold is a primary influencing factor that provides the structural support to the cells and helps to deliver the growth factors which stimulate the proliferation and differentiation of cells to regenerate a new tissue. The properties of a scaffold mainly depend upon types of biomaterial and fabrication techniques that are used to fabricate the scaffold. Biofabrication facilitates the construction of three-dimensional complex of living (cells) and non-living (signaling molecules and extracellular matrices polymers etc.) components. Biofabrication has potential application especially in skin and bone tissue regeneration due to its accuracy, reproducibility and customization of scaffolds as well as cell and signaling molecule delivery. In this review article, different types of biomaterials and fabrication techniques have been discussed to fabricate of a nanofibrous scaffold along with different types of cells and growth factor which are used for tissue engineering applications to regenerate a new tissue. Among different techniques to fabricate a scaffold, electrospinning is simple and cost effective technique that has been mainly focused in the review to produce nanofibous scaffold. On the other hand, a tissue might be repair itself and restore to its normal function inside the body by applying the principle of regenerative medicine.

The failure or loss of a tissue or organ by severe disease, trauma and surgical interventions, is a frequent and devastating challenge in the health care industry.Organ or tissue transplantation is the first choice to restore or maintain the function of damaged tissue/organ 1 .According to a medical survey, more than 8 million surgical treatments are performed to replace damage tissue or organ and the healthcare cost for transplantation is estimated more than $400 billion per year 1 .Autograft, allograft and xenograft are conventional implants used for organ or tissue transplantation 2 .Although autografts are used more frequently for tissue defects, but often these are limited for donor sites.Alternatively, allografts and xenogarft can be obtained in abundance, but these are related to high risk of disease transmission and immune rejection 3 .Thus, there is an intense need of a potential solution which can overcome the limitations of conventional therapies.Biomedicine is the application of biology and physiology to clinical medicine.Tissue engineering is now emerging as a promising alternative approach of biomedicine to treat injured tissues or to replace the damaged tissue/organs 4 .
The term tissue engineering was initially described in National Science Foundation meeting in 1988 5 .Later in 1993, Langer and Vacanti defined the early developments in tissue engineering and explained it as "an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve tissue or organ function" 6 .Tissue engineering is mainly based on the three important components: cells, scaffold (3D polymeric matrix) and growth factors (signaling molecules) 7 .These three components interact with each other under suitable environmental conditions and regenerate a new tissue or organ.The components of tissue engineering are illustrated in Figure 1.
Among these three components, scaffold is one of the most significant factor which accommodates the cells and creates favorable environment by supplying nutrients and growth factors for cells to proliferate and differentiate into specific tissue 8 .A tissue engineered scaffold should be biodegradable, biocompatible, mechanically strong and mimic the morphological structure and chemical composition of extra cellular matrix (ECM), so that cells can adhere to the scaffold surface, proliferate and differentiate into new tissue 6 .Some characteristic features of a tissue engineering scaffold are explained in Table 1.

Biomaterials used for scaffold fabrication
The selection of biomaterials for scaffold fabrication plays a pivotal role in tissue engineering.A wide range of biomaterials are used to fabricate the scaffold which usually includes natural polymers, synthetic polymers, composites and ceramics (Table 2).Naturally originated polymers are being widely used for scaffold fabrication owing to their similarities with ECM such as enzyme-controlled degradability, inherent cellular interaction and good biological performance.Collagen is a natural polymer and a major component of ECM of bone, tendon, skin, blood vessels, cartilage and heart valve 16 .Due to this reason, collagen has been widely exploited for different types of tissue engineering applications.The attributes of a tissue engineering scaffold, discussed in Table 1, depend upon the selected biomaterial for fabrication of the scaffold.
For example, bilayered collagen gels seeded with human fibroblasts and human keratinocytes have been used as the 'dermal' matrix of an artificial skin product under the name of Apligraf® 17 .Further, collagen scaffold in the form of gel, sponge and electrospun nanofibers have been used for skin 18 , cartilage 19 and bone 20 tissue engineering.Besides this, gelatin 21 , silk [22][23][24][25] , chitosan 26 , alginate 27 and chondroitin sulphate 28 etc. have also been applied for scaffold fabrication to regenerate various kind of tissues.However, there are some drawbacks of natural polymers which include low mechanical strength, batch to batch variation, immune rejection and risk of pathogen transmission 29 .Synthetic polymers offer several notable advantages over natural polymers and thus have potential use in tissue repair.Synthetic polymers can be tailored for specific applications due to controllable properties 19 .They exhibit a wide range of mechanical strength and physical properties such as degradation rate, tensile strength and elastic modulus 30 .Because to these properties, a variety of synthetic polymers such as PLA 31 , PGA 32 , PCL 33 , PVA 34 and PLGA 35 etc. have been applied for various kind of tissue repair and regeneration.Apart from several beneficial properties, synthetic polymers also associated with some drawbacks, include lack of cell recognition site for cell attachment and proliferation, and generate acidic products in degradation procedure which produce inflammatory effect on surrounding tissues.However, the disadvantages associated with synthetic and natural polymers can be overcome by using two or more polymers (Synthetic/ natural) in combination in order to afford higher mechanical strength, excellent cell attachment and tunable degradation 36 .Therefore, various types of composite materials have been applied for fabricating the scaffold for tissue regeneration.Composite scaffold of hydroxyapatite/chitosan 37 and PLGA/collagen 38 were used for the treatment of osteochondral defects.PCL/gelatin/collagen type I 39 , PCL/gelatin 40 and PLGA/collagen 41 composite scaffolds were used for skin and cartilage tissue regeneration respectively.Bioactive ceramics such as hydroxyapatite 42,43 , calcium phosphate 44 Fig. 1.Tissue engineering components and their interaction and bioactive glass provoke osteoconductive and osteoinductive 14,45 , thus these are the attractive candidates for bone tissue engineering.However, they have limited biocompatibility and biodegradability which is not sufficient for any tissue regeneration.These problems are overcome by blending the ceramic with natural or synthetic polymers which improved the scaffold properties for tissue regeneration 46 .Further, several metallic materials are also frequently used for implantation in orthopedic and dental surgery to provide support for healing bones or replace damaged bone.Metallic materials such as stainless steel 316 L (ASTM F138), Co based alloys (mainly ASTM F799 and ASTM F75), magnesium alloys (AZ31, AZ91, WE43 and LAE442) and titanium alloys (Ti-6Al-4V, F136 and ASTM F67) are used widely in joint implants and bone defects 47,48 .Moreover, besides all above discussed biomaterials, decellularized matrix is also being focused by scientists due to its bio-origin which mimic architecture of native ECM [49] and consists of various kinds of biopolymers 50, 51 required for tissue regeneration.Decellularized matrix has been exploited for different tissue engineering applications e.g., small intestinal submucosa 52 , heart valves 53 and urinary bladder [54] etc. Generally used biomaterials for tissue engineering scaffold are summarized in Table 2.

Fabrication techniques for tissue engineering scaffold
The success of a scaffold for tissue regeneration primarily depends upon two parameters i.e. composition (synthetic or natural origin) and architecture (designing) of scaffold.The complexity of scaffold architecture includes porosity, pore size, interconnectivity, and surface topography etc. which influence cell proliferation properties over the scaffold, greatly depends upon the scaffold fabrication technique.There are various technique for scaffold fabrication, e.g.,'foaming' 55 , 'phase separation' 56 , 'solvent casting and particulate leaching' 57 , 'self assembly' 58 , 'solid free form fabrication technique' 59 , freeze drying 60 and 'electrospinning' 61,62,63 .Nowadays, a novel area called as "biofabrication" in which latest 3D printing technologies are exploited with the aim to spatially incorporate different cells, biomaterials and molecules into a matrix to form an artificial tissue.In our body the native ECM mainly composed of nanoscale collagen fibers.Therefore, creation of a nano-featured environment is believed to be one of the promising conditions for efficient cell attachment and proliferation, and differentiated into new tissue 1 .There are only few techniques to produce the nanofibrous scaffold which are discussed in Table 3.

Electrospinning technique for nanofibrous scaffold fabrication
Among the above mentioned techniques, only elctrospinning is a simple and cost effective technique which produce nanofibrous scaffold that mimic the structural similarity with the native ECM of our body.It produces the continuous nanofibers with control over the fiber orientation 1,69 .First time, the technique was introduced by Zelency in 1914 70 and patented by Formhals in 1934 71 .The electrospinning technique is governed by many parameters which include flow rate of polymeric solution, electric field, viscosity, distance between tip of needle to collector, surface tension, conductivity, humidity and temperature 72 .Each of these parameters significantly affect the morphology and diameter of nanofibers fabricated during electrospinning process and by proper manipulation of these parameters, nanofibers of desired morphology and diameters can be obtained 73 .In brief, the electrospinning apparatus consists of mainly three components: high voltage power supply, a syringe pump holding a syringe with a metallic needle and electrically grounded collector (Figure 2).Polymeric solution is filled in the syringe for its electrospinning.The electric field is applied on the needle of the syringe to produce charges in the polymer droplet which held at the tip of needle with its surface tension.The droplet present at the tip of needle become elongate as the intensity of electric field is increased, and forms a conical shape which is recognized as Taylor cone.
At a critical value of electric field, the surface tension of polymer droplet is overcome by repulsive electrostatic force overcomes and a charged jet is evicted from the tip of Taylor cone.The evicted jet undergoes an instability and elongation process, which convert the jet into very long and thin.During the jet travelling, the solvent becomes evaporate and it collects on the collection target in the form of nanofibers 74 .Electrospinning is emerged as attractive approach for tissue engineering application because it produce nanofibrous scaffold which illustrate high porosity with tremendous pore interconnectivity, high surface area to volume ratio and mimic nano-feature environment similar to native ECM.These are the some favorable characteristics which influence cellular growth and function over the scaffold 1 .Therefore, electrospun nanofibrous scaffolds from different biomaterials are being widely explored for various kinds of tissue engineering applications.Some examples of electrospun nanofibrous scaffold used in tissue engineering applications are illustrated in Table 4.

Surface functionlization of nanofibrous scaffold
Sometimes, scaffold fabricated by electrospinning is not sufficient for providing all desirable properties to the cells for regenerating into a specific tissue.There is often necessary to modify the scaffold to enhance the some specific properties, for better cell attachment and proliferation to regenerate a new specific new tissue.Several techniques have been used to modify the nanofibrous scaffold but physical adsorption and covalent surface bonding are commonly applied techniques to modify the scaffold 86 .A variety of biomolecules have been used to modify the nanofibrous scaffold to increase their bioactivities.Collagen, laminin and many other molecules have been used for coating synthetic nanofibrous scaffold by physical absorption method which reflected the enhanced cell attachment, spreading, viability, and phenotype preservation 87 Similarly, many researchers have also used physical absorption method to coat the nanofibrous scaffold by hydroxyapatite to stimulate the expression of osteogenic genes by osteoblastic cells 88,89 .Besides physical adsorption, many biomolecules are bounded covalently to the surface of nanofibrous scaffolds to enhance the surface properties.It has been observed in a study that covalent bonding of laminin and collagen to nanofibrous scaffold improved the suitability of the scaffold for neural tissue 87 .In some cases, both covalent bonding and physical absorption can also be used to biofunctionalize the scaffold surface.Chen et al. modified the surface of PLLA electrospun scaffold by SBF coating using physical adsorption method and then, further functionalized the scaffold by hydrolysis of PLLA scaffold in NaOH aqueous solution 88 .In addition to surface functionalization, biomolecules can also be incorporated directly into the polymer solution during electrospinning.Antibiotics have been incorporated into electrospun scaffold during electrospinning process for their sustained release and bioactivity retention 90 .Vitamins 91 and nanoparticles 92 have been loaded directly into the polymer solution to functionalize the scaffold.

Cells used for tissue engineering
The cells selected for new tissue regeneration should be able to respond to surrounding environment, differentiate into new tissue and integrate with native tissue.For the treatment of injured tissues or to regenerate a new tissue, cells are taken from same patient or another individual.On the basis of cell source, the cells can be differentiated into autologous, allogenic and xenogenic cells.Autologous cells are isolated from the same individual while if the cells are isolated from another individual of same species are called allogenic cells.Xenogenic cells are obtained from the individual of another species.Over the last decade, adult stem cells and embryonic stem cells have been investigated as a potential source to regenerate or repair damaged tissues 93,94 .Embryonic stem cells are derived from pre-implantation embryo and have the capacity to differentiate into any tissue or organ 95,96 but the use of embryonic stem cells is restricted due to some ethical and political considerations.Adult stem cells such as mesenchymal stem cells (MSC) have capability to differentiate into a wide range of tissue includes, cartilage, bone, muscle, tendon, fat and connective tissue, furthermore these cells are less problematic in terms of ethical issues which make them very attractive and promising source for tissue engineering applications 97 .

Growth factors and nutrients for tissue engineering
Growth factors are polypeptides signaling molecules which transmit signals to modulate cellular activities such as cellular adhesion, migration, proliferation, differentiation, and gene expression 98 .Commonly used growth factors which influence the tissue regeneration are insulin-growth factor I (IGF I), bone morphogenetic proteins (BMPs), transforming growth factor-â (TGF-â), basic fibroblast growth factor (FGF-2),Plateletderived growth factor (PDGF), polypeptide growth factor etc. For tissue engineering applications, these signaling molecules are used singly or in combine form to enhance tissue regeneration.
Normally, Dulbecco's modified Eagle's (DMEM) media supplemented with FBS, penicillin and streptomycin antibiotics are used for cell.

ConClusions
Tissue engineering is one of the most exciting research areas which are growing exponentially with time to overcome the shortage of donor organ.The success of tissue engineering mainly depends upon the chemical composition and surface architecture of scaffold that can obtain specific, desired and timely responses from surrounding cells and tissues in a host.A wide range of natural polymer, synthetic polymer and ceramics have been used to fabricate a scaffold.Nowadays, more than one biomaterial is being used to fabricate a scaffold sothat it can mimic the chemical composition of native tissue.Surface functionalization strategies are being further applied to improve the surface properties of scaffold.In addition, highly optimized nanofibrous scaffold fabrication technologies are being applied to synthesize a more efficient tissue engineering scaffold.Recently, biofabrication techniques are offering a definite and structured strategy for tissue generation.However, there are still considerable constraints with biofabrication for the development of clinically appropriate constructs.Thus, further advances in material designing and biofabrication techniques are increasing the possibility of production of good quality implants.

Fig. 2 .
Fig. 2. Schematic representation showing major components of the electrospinning setup; pictures of the syringe pump and high voltage power supply has been taken from the laboratory electrospinning setup

Table 1 .
Properties of tissue engineering scaffold

Table 2 .
Various biomaterials used for tissue engineering Scaffold

Table 3 .
Techniques employed to fabricate nanofibrous scaffold for tissue engineering applications