A Review on Some Natural Biopolymers and Their Applications in Angiogenesis and Tissue Engineering

Tissue engineering is a reparative means which merges biomaterials, cells and functional environmental factors to induce growth, proliferation and differentiation signals for promotion of tissue repair or functional regeneration.1,2 This interdisciplinary field faces many limitations which some of them are listed here3-5: • Proper selection of bio-compatible and bio-active materials for the purpose of repairing or regenerating the target tissue; • Optimizing mechanical properties to produce a welldeveloped scaffold suitable for soft or under load tissues; • The technique used to construct the appropriate engineered scaffold or structure; • Identification of angiogenic factors in each tissue; • Utilizing factors that can stimulate the production and secretion of angiogenic factors in target tissue; and • How to transfer these factors to the tissue in absence of the native factors. Natural polymers are biologically suitable materials to use as structures in tissue engineering applications (Figure 1).6 In this study, we focused on some polymers that used in research works in angiogenesis and other tissue engineering applications in different tissues and organs. The studied polymers are collagen, gelatin, chitosan, silk fibroin and fibrin. The aforementioned polymers have the ability of mimicking many properties of the extracellular matrix (ECM). Therefore, they can potentially induce the attachment, growth, migration, organization and differentiation of cells during regeneration of tissue or wound healing.7,8 The biological process that pre-existing vessels form new ones is called angiogenesis. As mentioned, angiogenesis is one of the most important issues in regeneration of a tissue.9 Vessels deliver nutrients and take away wastes and their lack is one of the problems for scaffolds or engineered constructions implanted in body.10 One of the current purposes in tissue engineering is to solve this concern by utilizing biomaterials that can act as a suitable environment to provide a condition for enhancing angiogenesis during tissue regeneration. These materials have also the potential of homing and releasing other angiogenic factors or effective elements while used as engineered constructs. Induction of endothelial cell homing, stimulation and activation of pro-angiogenic factors such as osteopontin, Forkhead box protein C2 (FOXC2), basic A Review on Some Natural Biopolymers and Their Applications in Angiogenesis and Tissue Engineering


Introduction
Tissue engineering is a reparative means which merges biomaterials, cells and functional environmental factors to induce growth, proliferation and differentiation signals for promotion of tissue repair or functional regeneration. 1,2This interdisciplinary field faces many limitations which some of them are listed here [3][4][5] : • Proper selection of bio-compatible and bio-active materials for the purpose of repairing or regenerating the target tissue; • Optimizing mechanical properties to produce a welldeveloped scaffold suitable for soft or under load tissues; • The technique used to construct the appropriate engineered scaffold or structure; • Identification of angiogenic factors in each tissue; • Utilizing factors that can stimulate the production and secretion of angiogenic factors in target tissue; and • How to transfer these factors to the tissue in absence of the native factors.Natural polymers are biologically suitable materials to use as structures in tissue engineering applications (Figure 1). 6In this study, we focused on some polymers that used in research works in angiogenesis and other tissue engineering applications in different tissues and organs.The studied polymers are collagen, gelatin, chitosan, silk fibroin and fibrin.The aforementioned polymers have the ability of mimicking many properties of the extracellular matrix (ECM).Therefore, they can potentially induce the attachment, growth, migration, organization and differentiation of cells during regeneration of tissue or wound healing. 7,8he biological process that pre-existing vessels form new ones is called angiogenesis.As mentioned, angiogenesis is one of the most important issues in regeneration of a tissue. 9essels deliver nutrients and take away wastes and their lack is one of the problems for scaffolds or engineered constructions implanted in body. 10One of the current purposes in tissue engineering is to solve this concern by utilizing biomaterials that can act as a suitable environment to provide a condition for enhancing angiogenesis during tissue regeneration.These materials have also the potential of homing and releasing other angiogenic factors or effective elements while used as engineered constructs.Induction of endothelial cell homing, stimulation and activation of pro-angiogenic factors such as osteopontin, Forkhead box protein C2 (FOXC2), basic fibroblast growth factor (bFGF), Tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1) and vascular endothelial growth factor (VEGF), inducing proliferation and migration of endothelial cells, improving the mitogenic response to angiogenic factors and overexpression of angiogenic genes are the effects of elements such as silicon, phosphorus, copper, magnesium and europium used in natural-based constructs to improve angiogenesis in tissue engineering approaches. 1 In the following part, some natural polymers and their applications will be reviewed.

Collagen
2][13] Therefore, it is considered as a proper matrix or scaffold.Collagen interaction with connective tissue cells transduces crucial signals for the regulation of cell adhesion, survival, proliferation, migration and differentiation. 14ollagen has many types but type I collagen is the most studied for tissue engineering and biomedical uses. 15ts properties including high mechanical strength, low antigenicity, good biocompatibility and ability of being cross-linked, make collagen an ideal biomaterials for tissue engineering applications. 16,17Laiva et al developed a scaffold with pro-angiogenic gene-activated properties to influence angiogenesis by producing paracrine angiogenic factors in wound healing.They combined nanoparticles of polyethyleneimine containing stromal derived factor-1 alpha (SDF-1α) gene with a collagen-chondroitin sulfate scaffold.They evaluated the effect of this structure on mesenchymal stem cells (MSCs) angiogenic potential.The MSCs on scaffold showed SDF-1α mRNA over-expression associated with VEGF and CXCR4 activation which are angiogenic factors.They conclude that combination of SDF-1α gene with collagen-based scaffolds can provide a suitable condition for angiogenic response during wound closure. 18Some of these applications are summarized in Table 1.

Gelatin
Gelatin is a natural polymer derived from collagen which is widely used for medical applications and pharmaceutical due to its biocompatibility and biodegradability in physiological environments. 49,50In addition, gelatin has low antigenicity in comparison to collagen which is shown antigenicity because of its animal origin.Gelatin is a denatured protein received by collagen processing using alkaline and acid. 51,52The alkaline process affects asparagine and glutamine amide groups and hydrolyses these group into carboxyl ones.Acidic treatment has little influence on the amide groups.Therefore, acidic treatment is electrically different from alkaline-processed gelatin.The different conditions of gelatin processing allow polyion complexation flexibility of a gelatin scaffold, carrier or hydrogel with either negatively or positively charged biomolecules and cells. 49,53Because of mentioned properties of gelatin, it is been used in engineering of various tissues and drug delivery applications.We summarize many of these applications in table.Nemati et al used alginate-gelatin microcapsules to provide an appropriate microenvironment for human umbilical vein endothelial cells (HUVECs) during a 5-day period.In vitro studies revealed promotion of HUVECs proliferation and cell survival.The expression of VEGFR-1, VEGFR-2, Tie-1 and Tie-2 were enhanced however it was not significant.They investigate the potential of the encapsulated cells in angiogenesis by implanting it in immune-compromised mouse model for 7 days.The result showed an increase of encapsulated cells angiogenic response in comparison to non-capsulated ones.They claimed that angiogenic response could be promoted by alginate-gelatin encapsulation. 54In Table 2, some of gelatin applications in tissue engineering are summarized.

Chitosan
Chitosan is a chitin-obtained cationic polymer consists of copolymers of N-acetyl-D-glucosamine and β (1→4)-glucosamine.Chitin as a natural polysaccharide found in the cuticles of insects, shell of crustacean and fungi cell walls.Chitosan is the partially or fully deacetylated form of chitin 71 .The chitosan deacetylation degree is usually a range between 70% and 95%, and the molecular weight is also between 10 to 1000 kDa. 72Its application in tissue engineering and drug delivery fields is wide ranging from cartilage, bone, vascular grafts and skin to substrates for cell culture.Biologically renewable, biocompatible, biodegradable, nontoxic and non-antigenic properties of chitosan make it a bio-functional useful biomaterial. 52,73,74In addition, hydroxyl and amino groups of chitosan can be modified chemically to provide a high chemical diversity.It also has bio-adhesive properties. 72Chitosan exhibits different behaviors at various pH levels.It doesn't dissolve at high pH while it is soluble at lower pH ranges. 75This property makes chitosan a suitable tool for delivery applications.The summarized applications of chitosan in tissue engineering exhibited in Table 3. Cheng et  al carried out a study to construct a thermo-sensitive hydrogel of chitosan/gelatin composition that is suitable for angiogenic applications by sustained release of adipose-derived stem cells which is a result of gelatin gradual degradation.In vitro studies revealed a significant concentration of VEGF in the supernatant of chitosan/gelatin hydrogels containing adipose-derived stem cells.Tube-like structures were formed in co-culture of the encapsulated adipose-derived stem cells and SVEC4-10 endothelial cells.This result demonstrates the potential of chitosan/gelatin hydrogel in inducing angiogenesis. 76Recent applications of chitosan are summarized in Table 3.

Silk
Silk is a protein biopolymer produced by spiders, silkworms, mites, scorpions and flies. 98Spider silk is an interesting biomaterial that is weightless, elastic and strong that is comparable to the best fibers synthetized by new technology in terms of mechanical properties. 99It is also a bio-degradable material and environmentally safe.Because of the limited amount of spider silk, silk fibroin as a natural polymer which produced by silkworms is a good alternative. 100,101Sericin and fibroin are the major components of it.Fibroin, a fibrous protein creating the silk core, is composed of composed of fibroin Light chain, fibroin heavy chain and fibrohexamerin. 102xcellent mechanical properties, biocompatibility and slow degradability make this material interesting 103,104 .Recently, silk is used as a biomaterial in corneal tissue engineering due to its transparency potential.Some of applications are mentioned in Table 4. Liu et al fabricated a Porous scaffold composed of basic FGF-immobilized silk fibroin.Proliferation and growth of L929 cells were improved on bFGF-immobilized silk fibroin scaffolds.In vivo studies contained implantation of the scaffold into the skin defect of rat that displayed significant re-epithelialization and skin regeneration.Formation of new vessels and deposition of collagen after 4-week treatment, showed the potential of these scaffold in angiogenesis and tissue regeneration. 105Table 4 shows silk fibroin applications in regenerative medicine studies.

Fibrin
The applications of this natural polymer are well developed because of its innate ability to cellular interaction induction and scaffold remodeling in comparison to synthetic scaffolds. 135Fibrin-based materials biochemical specifications make them ideal for drug and cell delivery.It can also be harvested autologous that provides an immuno-compatible carrier for drug, cell and active biomolecules delivery. 136,137][140] It is also very used in chondrocytes encapsulation for tissue engineering of cartilage.Rapid degradation of fibrin can be considered as a disadvantage in tissue engineering.2][143] An investigation was performed by Dohle et al to establish co-culture system of primary osteoblasts and outgrowth endothelial cells within injectable platelet-rich fibrin matrices.They were in an effort to determine the effect of platelet-rich fibrin on angiogenesis and wound healing by activating endothelial cells in this system.Histological studies indicated vessel-like structures formation after 7 days culturing.Expression of the VEGF, as a pro-angiogenic factor, was increased on the mRNA and protein levels.Therefore, platelet-rich fibrin might be ideal for wound healing through promoting angiogenesis. 144Applications of fibrin-based constructs in tissue engineering are summarized in Table 5.

Conclusion
We reviewed different usages of some natural polymers in tissue engineering based on previous researches.Among the materials, polymers have been widely applied and have excellent potential to regenerate tissues due to their flexible features. 166As mentioned one of the issues in the tissue regeneration is to provide blood supplying. 1For this purpose, the quality of the regenerated vascular network is more important than the quantity.In other words, amount of perfused blood through a vascular network is the criterion, not just the number of vessels.Therefore, the importance of the vascular structure organization and maturation is clear.On the other hand, over-stimulating of angiogenesis leads to the creation of many unorganized vessels which are poorly perfused and have inefficient performance. 167Organization of vascular structures is not the only factor that determines the success chances of engineered tissues, but it seems to be a basic principle. 168Tissue engineering is in an effort to focus on vascular cells patterning in the target tissue to control the organization and maturation of vascular structures.Incorporation of active bio-molecules like growth factors, angiogenic factors and elements is a novel strategy that is highly useful to improve tissue regeneration by improving angiogenesis, 169 but achieving a tissue with normal and functional vascular structures is still a challenge.

Figure 1 .
Figure 1.Using Natural Polymers as Hydrogel in Bone Tissue Engineering.

Table 4 .
Silk fibroin applications in tissue engineering