*3.1.6 Polyhydroxyalkanoates (PHA) as base material and its derivatives*

Unlike different biomaterials, *polyhydroxyalkanoates (PHA)* are the class of biodegradable biopolymer extracted by the harvesting of microbial cells. Despite the cost-effective synthesis and ease of processing, the hydrophobicity, brittleness and lack of antibacterial limits it random uses in biomedical applications such drug delivery, surgical suture and supporting matrices for tissue regenerations. Efforts are being made to improve its biocompatibility, mechanical strength and antimicrobial properties by blending or modifying the surface with bioactive and high-strength nanomaterials. One approach was used to develop the biocompatible collagen-immobilized porous 3D scaffold based on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [collagen/PHBV], which allows better growth and proliferation of the rat osteogenic cell line (UMR-106 cell line) than native PHBV scaffold [41]. A similar trend was also observed when hydroxyappetite was further incorporated into the collagen grafted PHBV composite. The modified scaffolds revealed better adhesion and growth of osteoblast cells [42]. In another study, a hyaluronic acid immobilized chitosan-grafted porous PHA membrane was fabricated and it exhibited better protein adsorption and improved adhesion and proliferation of L929 fibroblasts. The supportive matrix also showed excellent antibacterial activity against several bacterial strains [43]. The mechanically and biocompatibility challenged porous PHH-mixed PHB [PHBHHs] scaffolds showed significant growth and proliferation of chondrocytes isolated from rabbit articular cartilage (RAC) as compared to only PBHs or PHB scaffolds. With the increase of the PHBHH content in the mixture, both the mechanical and the cell compatibility increased

*Current Scenario of Regenerative Medicine: Role of Cell, Scaffold and Growth Factor DOI: http://dx.doi.org/10.5772/intechopen.94906*

dramatically, they have a potential impetus in tissue engineering applications [44]. Recently, a group of researchers has potentially formulated the carbonaceous or conductive nonmaterial such as polyaniline, graphane oxide modified PHA based 3D porous scaffolds for tissue engineering applications. The antimicrobial and cellstimulated active 3D constructs not only improved cell attachment, proliferation, but migratory behaviors were observed through interconnected porous networks. The magnetically active MRI scaffolds have tissue engineering applications controlled by significant bioimaging [45–47].

#### *3.1.7 Silk as base material and its derivatives*

Silk is a naturally occurring fibrous protein with biodegradability, biocompatibility, and mechanical durability that has utility in tissue engineering applications. In one study, silk fibroin-grafted polycaprolactone nanofibers were able to deliver dual growth factors such as bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-β), in the regeneration of bone tissue [48]. Li et al. also presented a similar type of biocompatibility while a PCL/silk 3D bioprinting scaffold was imposed to regenerate the meniscus tissue [49]. The computer-assisted 3D printed silk matrices have attracted significant attention and found to be improved the cell–cell and cell-matrix interaction and enable their activity in patient specific tissue architecture [50]. Similarly, the gelatin-silk composite was subjected to the fabrication of 3D bioprinting for cartilage tissue engineering in rabbit model [51].

The novel development of 4D printing hydrogel has gained significant attention in next generation biofabrication. The fabrication of 4D printing from 3D printing hydrogel was regulated by the modulation their interior or exterior properties with the proper controlled of expansion rate of the hydrogel in distilled water and salt water. The biocompatibility of assessment of the 4 D printing hydrogel was conducted in culture medium by shape change method as mentioned earlier. The results revealed the adhesion and growth of the PKH127 (green)-labeled human chondrocyte (hTBSCs) along with the deposition of cartilage extracellular matrix in the side of the construct. To verify the clinical applicability of the construct, the rabbit TBSC and chondrocytes-laden artificial 4D construct was implanted into the site of the rabbit trachea and the results of 8 weeks post-implantation revealed the regeneration of the respiratory epithelial layer and formation of neocartilage around the perichondrium. This findings proved the potential application of the cell laden 4D hydrogel in the recovery of respiratory organ, trachea regeneration [52]. A very recently, the approach of development of electrical simulation modulated polypyrrole/silk fibroin (PPy/SF) based conductive composite scaffold has been opened up the new avenue in the neuronal tissue regeneration [53]. The 3D printing electrospining method was used to fabricate for the alignment of silk fibrous, followed by the coating of polypyrrole (a mechanically stable conductive material) to get the desired silk fibroin (PPy/SF) composite scaffold as nerve guidance conduits (NGCs). Morphological tracking by SEM analysis exhibited the coreshell structure having interpenetrating PPy fibers on the smooth SF nanofibers with average diameter of 0.427 ± 0.083 μm. Resultant physicochemical properties such as mechanical stability (0.059 MPa) and conductivity (0.11446 ± 0.00145 mS/mm) of composite were comparable to ideal working in NGCs system, indicating the increase of mechanical property of the conduit by the coating of PPy. The ES controlled cell compatibility of the NGCs was evaluated with the seeding of Schwann cells (SCs) and it showed the significant growth, proliferation and migration of the cells with the expression of neurotrophic factors. Further, to investigate the effect of artificial NGCs on *in vivo* nerve tissue regeneration, the composite was implanted in defected sciatic nerve of rat and monitored for six months under ES regulation.

#### **Figure 5.**

*(A) (i) (a) 3D model of the designed octahedral type of scaffold with marked dimensions; (b) scaffold was loaded on the compression platform; (c) strain–stress curves; (d) elastic moduli for three groups of homogeneous scaffolds; (ii) (a-i) Fluorescent microscope images taken with a 4 × objective to show the 3 T3 fibroblasts distribution in the scaffold. (a-ii) Interaction of 3 T3 fibroblasts with the joint of scaffold. (b-i) optical and fluorescent microscope images taken with a 10× objective. (b-ii) Fibrous-like cell layer formed on the frame surface of scaffold. [Ref: [54], reproduced with permission from publishing authority]. (B) Interaction of A10 VSMCs with D-PHI porous scaffolds. SEM (a-h) and live-dead (i-viii) images of D-PHI-0 (a-d and i-iv) and D-PHI-5 (e-h and v-viii) porous scaffolds in the absence of cells and following a 3 day, 1 week, and 2 week culture period with A10 VSMCs at 0 μm depth (scaffold surface). Cell-free scaffolds (i and v) fluoresce red under the microscope. All SEM images were taken at 500 × original magnification. [Ref: [55], reproduced with permission from publishing authority].*

*Current Scenario of Regenerative Medicine: Role of Cell, Scaffold and Growth Factor DOI: http://dx.doi.org/10.5772/intechopen.94906*

Histochemical and microscopic analysis revealed the densely regenerated myelinated fibers and mylinated axon dispersed in the fibrous networks that promoted the regeneration of the pheripheral injured nerve. Since, the cellular activities in nervous system are regulated by the expression of various neurotrophic factors (BDNF and NT-4/5) and signaling pathways. The ES modulation may activate the MAPK in the cell microenvironment and promoted the growth of the axon, correlated with nerve regeneration. Therefore, although several reports have been undergone using silk for the construction of different tissue architectures but intensive research must be conducted to find potential validity in clinical trials.

#### *3.1.8 Different synthetic materials and their combination*

The synthetic materials based 3D hydrogel have also shown to mimic the native tissue stiffness while the optimum conditions for the 3D constructions are digitally controlled. The digital light processing (DLP) based printed poly(ethylene glycol) diacrylate (PEGDA) hydrogels exhibited nearly 60% of enhanced elastic modulus, suited for the support of 3 T3 cells adhesion and proliferation as shown in **Figure 5A** [54]. In one study, degradable, polar hydrophobic and ionic porous polyurethane scaffolds were synthesized using a lysine-based crosslinker. The scaffolds demonstrated (see **Figure 5B**) the comprehensive mechanical, swelling and biocompatible properties that support the adhesion and growth of muscle cells in vascular tissue engineering [55]. Apsite et al., reported the design and fabrication of polycaprolactone and poly(N-isopropylacrylamide) based multilayerd porous electrospun mats. The self-folding 4D bio-fabrication was found to act as good cells adhesion and viability, assigning as a new perspective in new generation tissue engineering [56]. In a paper, Kutikov explained how the integration of hydrophilic polyethylene glycol into hydrophobic polyester block copolymers changes the physicochemical properties of 3D matrices. The incorporation also demonstrated the different types of cell adhesion, growth, and tissue regeneration both *in vitro* and *in vivo* experiment [57]. Therefore, the deviations in the fabrication of 3D artificial support matrices using natural biopolymer or synthetic materials individually must be compensated by the chemistry of piling or surface modification to increase any physical properties that would satisfactory fill the gaps to improve the clinical applications.

### **4. Effect of growth factors on cell-matrix interaction**

Enormous studies have been thoroughly investigated on the interaction between cellular and bio-mimetic 3D matrices *in vitro* and *in vivo* tissue generation experiments that demonstrated the phenomena of adhesion, growth and differentiation of different cells. But, most of the study doesn't meet the pre-requisite for the successful clinical application due to the insufficient secretion of protein molecules that responsible for the biological and biochemical signaling between cell–cell and cell matrix cross-talk. The prominent small proteins that induce cell growth, proliferation, differentiation and regulate angiogenesis are encoded as growth factors. The emergence of versatility of different growth factors related to the reported mediated repair of damaged tissue tends to fall into various categories based on their functionality in tissue engineering. EGFs, NGFs, IGF, FGFs, PDGFs, interleukins etc. are the class of growth factors mainly disclosed for the cell–cell medicated trafficking of proliferation and actin-cytoskeleton in living tissue regeneration process.

Mechanically, the function of growth factors is to drive progenitor cells to its damaged target tissues by extracellularly mediated signaling pathways. In fact,

therapeutic molecules bind to the cell surface transmembrane receptor and then to the internalized receptor-protein complex through phosphorylation-mediated signal transduction that triggers down-regulation of cells, followed by reduction of overwhelming responses and stimulation at the cellular level to carry out biological functions. Furthermore, the non-diffusible method leads to the binding of growth factor to the cell surface without any major internalization or downregulation in the results of long-term biological activities, as shown in **Figure 6A(i&ii)** [58]. Mimicking the *in vivo* tissue environment, various approaches have been implicated using growth factors loaded 3D biocomposite for sustained release without any dysfunction of the protein molecules. Bone morphogenesis protein-2 and 7 (BMP-2 & BMP-7) are the part of transforming growth factors enable the proliferation and osteogenic differentiation of bone marrow derived mesenchymal stem cells. The removal of bio-signaling molecules has demonstrated the deregulation of cell proliferation, differentiation and alteration of bone tissue formation [59, 60]. Co-administration of TGF-b3 and BMP-2 via alginate-based scaffolds revealed a tendency for increased osteogenesis in *in vivo* bone formation tests. A similar type of output has been observed while TGF-1 and IGF-1 are used simultaneously for bone tissue engineering [61, 62]. In a study, Kim et al., demonstrated that the inhibition of epithelial growth factor predominantly affects the cell–cell and contact based cell proliferation. In addition, over-expression of cadhreine, a transmembrane-type cell surface protein limited cell-to-cell contact with the arrest of cell cycle, resulted in spatial cell rearrangement tuned to tumor formation. Therefore, the epithelial growth factor plays an active function towards the formation of epithelial cells in tissue engineering [63].

It has been a challenge to meet the need to develop a bio-mimetic tool for vascular tissue engineering. In contrast to various soft tissues, vascular tissue controls the supply of oxygen, essential cellular nutrients, the transport of waste products and stem cells as well as progenitor cells. Therefore, it is urgent to reconstruct the

#### **Figure 6.**

*(A) (i) Modes of action of growth factors. Growth factors interact with their receptors in a diffusible manner (e.g., by endocrine, paracrine, autocrine and intracrine pathways) or in a non-diffusible manner (e.g., by juxtacrine and matricrine pathways). Some growth factors are known to act in both ways. (A) (ii) Three main factors in tissue engineering: cells, growth factors and matrices (scaffolds). The conjugation of growth factors and matrices provides a new approach for generating biofunctional substrates for regenerative medicine [Ref-58, reproduced with the permission from publishing authority]. (B) (i/a): Schematic representation of proposed mechanisms for enhanced bone regeneration in vivo*. *(i/b): Optical images of retrieved specimens from representative critical-sized bone defects at 4 and 8 weeks. (i/c) Micro-CT analysis. (i/d) Quantitative analysis of bone regeneration. (i/e) Fibrous tissue thickness at the defect site at 4 and 8 weeks (\*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001). [Ref. [66], adapted with permission from publishing authority].*

*Current Scenario of Regenerative Medicine: Role of Cell, Scaffold and Growth Factor DOI: http://dx.doi.org/10.5772/intechopen.94906*

network of the neovascularization process with the initiation of adhesion, growth and differentiation of cells as a native tissue environment in artificial tissue engineering. Cao et al. demonstrated the therapeutic approaches of growth factors and their signal cascade that control neovascularization and the formation of neovessels using the spatiotemporally controlled 3D construct both *in vitro and in vivo* pathways. The literature also explained how vascular epithelial growth factor (VEGF) plays a critical role in vasculogenesis in stages of embryonic development from pre-existing blood vessels through consecutive signaling pathways [64, 65]. The multifunctional triple layered chitosan/poly (gamma-glutamic acid)/hydroxyapatite (CPH) hydrogels was formulated to regulate the release of platelet-rich fibrion (PRF) which was extracted from rat abdominal arota, into the site of damaged bone tissues. The PRF entrapped composite hydrogel was prepared through noncovalent electrostatic interaction and lyophilization technique to promote the osteoconductive mediated new bone tissue formation. The PRF entrapped composite hydrogel was prepared through noncovalent electrostatic interaction and lyophilization technique to promote the osteoconductive process in the formation of new bone. PRF is a combination of different growth factors such as TGF-β, PDGF, and IGF that have an abiity to induce the mineralization and the upregulation of various osteogenetic biomarkers in order to activate the osteoblast as seen in vitro and in vivo experiments. As a part of '*in vivo*' tissue regeneration study, rat calvarial defect models demonstrated the superior healing of the calvarial defect after 8 weeks post implantation period in presence of PRF/CPH composite than that of control experiment (see **Figure 6B**) [66]. In summary, the combined growth factors entrapped 3D supporting matrixes would bring a new avenue towards the cell–cell cross-talk mediated tissue generation with the advancement of bio-mimetic tools in finding the arrays of the artificial tissue engineering in the future.

### **5. Conclusions**

Understanding the mechanism and basic criteria in the process of tissue regeneration has unveiled the secret of the communication involved in cell–cell, cell-matrix interaction that enables healing in an artificial tissue environment such as native tissue repair processes. In fact, the biocompatibility of any fabricated 3D architecture plays an important role in adhesion, proliferation, migration, and differentiation of the cells of interest to biologically mimic the signaling cascade that triggers cellular activities. Several investigations have shown that 3D constructs comprising naturally extracted and synthetic materials having a porous and mechanically stable geometry promoted integrin ligand-mediated differentiation and tailored actin-cytoskeletal cell morphology in a better way. The studies also explained how the biological and biochemical performances of cells are influenced by the different growth factors mediated signaling pathways and the active function of the ECM components. Therefore, present review provided the core thinking behind the physicochemical features of supporting matrixes that significantly control the cell– cell and cell-matrix interaction towards the implementation of clinically approved artificial 3D biocomposite for the successful clinical tissue engineering applications.

#### **Acknowledgements**

Dr. Nilkamal Pramanik and Dr. Tanmay Rath acknowledge UGC, DST, SERB- DST (NPDF), Govt. of India, for their financial supports. Dr. Nilkamal Pramanik also gratefully acknowledges Dr. Tanmay Rath, Prof. Patit Paban Kundu (Department of Polymer Science and Technology) and Dr. Ranjan Kumar Basu (Department of Chemical Engineering), University of Calcutta for their kind guidance to carry the biomaterials based tissue engineering works.
