**3. Collagen electrospinning**

Collagen (COL) is the most widely distributed class of proteins in the human body. The use of COL-based biomaterials in the field of tissue engineering applications has been growing strongly during the last decades. It is for this reason that multiple cross-linking methods have been investigated and different combinations with other biopolymers have been explored to improve tissue function. The COL has a great advantage, as it is biodegradable, biocompatible, readily available, and highly versatile. However, since COL is a protein, it remains difficult to sterilize without alterations in its structure [18].

We have investigated the possibility of preparing COL-inspired nanofibers by electrospinning aqueous suspension of telopeptide-free COL molecules avoiding organic solvents and blends with synthetic and natural polymers. The results underscored the need for a basic atmosphere between the needle and the ground collector in order to increase the pH of the environment during auto-assembly of COL molecules along the electrostatic force lines in order to prepare a biomimetic component of reinforcement of new biomaterials for medical and surgical use [19].

It has been reported that he designed a durable sandwich wrap preparation system with high liquid absorption, biocompatible, and with antibacterial properties. For this purpose, various weight ratios of the COL solution to chitosan were used to immobilize on the polypropylene nonwoven fabric, which was pre-grafted with acrylic acid or N-isopropyl acrylamide to construct a durable wound sandwich liner membrane with high water absorption, easy removal, and antibacterial activity in an animal skin model. The results indicated that tissue immobilized with N-isopropyl acrylamide and COL/chitosan/PP/N-isopropyl acrylamide-COL/chitosan) showed a better healing effect than COL/chitosan immobilized polypropylene tissue. The poly (propylene)/N-isopropyl acrylamide/COL/chitosan-treated wound showed an excellent remodeling effect on histological examination with respect to the construction of the vein, epidermis, and dermis at 21 days post-cutaneous lesion. The water absorption values and water diffusion coefficient for polypropylene/N-isopropyl acrylamide/COL/chitosan were higher than those of polypropylene /acrylic acid/COL/chitosan under a weight-volume ratio of COL/ chitosan. Both polypropylene/N-isopropyl acrylamide/COL/chitosan and poly (propylene)/ acrylic acid/COL/chitosan showed antibacterial activity [20].

Since GEL undergoes a gelation process, Furuike et al., used a new dry spinning process for GEL. In this case, the nonwoven GEL fabrics were electrospun by applying dry spinning principles. The diameter of the fibers, the viscosity, and flow rate of the solution depended directly on the GEL concentration. Spunted nonwoven fabrics with a concentration of 25% (w/w) GEL exhibited a nanoscale diameter. In order to improve the properties of the nonwoven fabrics, they were cross-linked with glutaraldehyde vapor after spinning by the addition of N-acetyl-dglucosamine to the GEL solution before spinning followed by heating of these fibers. Nonwoven fabrics cross-linked by glutaraldehyde vapor exhibited improved mechanical properties compared to those without cross-linking or cross-linking of N-acetyl-d-glucosamine. Swelling and water absorption did not produce morphological changes in glutaraldehyde cross-linked fibers. The thermogravimetric analysis (TGA) thermogram did not confirm any phase change in the composite structure. In addition, in vitro cytocompatibility studies using human mesenchymal stem cells showed the compatible nature of the developed nonwoven fibers, where

they demonstrated that these nonwoven fibers could be useful in medical care [16].

release by varying the cross-linking time and the pH of the release medium [17].

**3. Collagen electrospinning**

18 Tissue Regeneration

ficult to sterilize without alterations in its structure [18].

Delivery of hydrophobic drug into the hydrophilic polymer matrix as a carrier is usually a challenge. Therefore, in one study, the synthesis of GEL nanofibers by electrospinning was presented, which were evaluated as a potential carrier for the oral system of hydrophobic drugs, piperine. GEL nanofibers were cross-linked by exposing to saturated glutaraldehyde vapor, to improve their water-resistive properties. An exposure of only 6 min was not only adequate to control early degradation with intact fiber morphology, but also significantly marginalized any adverse effects associated with the use of glutaraldehyde. The results illustrated good compatibility of the hydrophobic drug in GEL nanofibers with promising patterns of controlled drug

Collagen (COL) is the most widely distributed class of proteins in the human body. The use of COL-based biomaterials in the field of tissue engineering applications has been growing strongly during the last decades. It is for this reason that multiple cross-linking methods have been investigated and different combinations with other biopolymers have been explored to improve tissue function. The COL has a great advantage, as it is biodegradable, biocompatible, readily available, and highly versatile. However, since COL is a protein, it remains dif-

We have investigated the possibility of preparing COL-inspired nanofibers by electrospinning aqueous suspension of telopeptide-free COL molecules avoiding organic solvents and blends with synthetic and natural polymers. The results underscored the need for a basic atmosphere between the needle and the ground collector in order to increase the pH of the environment during auto-assembly of COL molecules along the electrostatic force lines in order to prepare a biomimetic component of reinforcement of new biomaterials for medical and surgical use [19]. It has been reported that he designed a durable sandwich wrap preparation system with high liquid absorption, biocompatible, and with antibacterial properties. For this purpose, various weight ratios of the COL solution to chitosan were used to immobilize on the polypropylene Electrospinning is a process that is used to create nanofibers, which have the potential to be used in many medical and industrial applications. The molecular structure of the raw material is an important factor in determining the structure and quality of the electro-chip fibers. COL has been extracted from a cold-water hoki species (*Macruronus novaezelandiae*), and this was prepared in several different molecular formats (triple helical CO, denatured whole chains, denatured atelocollagen chains, and GEL) for electrospinning. When denatured COL chains were used, 10% acetic acid proved to be an aqueous solvent effective to produce uniform fibers. This information is useful for the development of a nontoxic aqueous solvent system suitable for the industrial enlargement of the electro-silting process [21].

Nerve tissue engineering is one of the most promising methods in nerve tissue regeneration. The development of combined scaffolds of COL and glycosaminoglycans can potentially be used in many soft tissue-engineering applications. In a study by Timnak *et al*. developed two types of randomized and aligned electro-alloying. Their cellular tests showed that the scaffold acted as a positive factor to support the growth of connective tissue cells. These results suggested that scaffolding of nanostructured porous COL-glycosaminoglycans is a potential cell carrier in nerve tissue engineering [22].

On the other hand, COL and hyaluronic acid are the main components of the extracellular matrix naturally and have been successfully used in the electrospinning. In this case, a solution of COL/hyaluronic acid polymer was electrospun creating a scaffold for patients with osteoporosis who have reduced bone strength. The membranes were cross-linked to render them insoluble and conjugated to gold nanoparticles to promote biocompatibility. Their results showed that COL/hyaluronic acid scaffolds were insoluble in aqueous solutions and promoted cell fixation that could be used as a tissue engineering framework to promote cell growth [23].

Zulkifli *et al*. [24] focused on the degradation behavior of nanofibrous scaffolds composed of HEC/PVAL (alcohol hydroxyethyl cellulose/polyvinyl alcohol) and HEC/PVAL/COL as potential substrates for the engineering of cutaneous tissues in two media (PBS) and Dulbecco's modified Eagle's medium (DMEM) for a period of 12 weeks of incubation. Once the scaffolds were characterized, the HEC/PVAL/COL scaffolds showed a slower degradation rate in both media compared to the HEC/PVAL blend nanofibers. All fibers showed irregular and rough surfaces toward the final week of incubation in PBS and DMEM solution. As the degradation time increased, there were few changes in the chemical structure determined by the FTIR spectra, while the thermal studies revealed that the melting and crystallinity temperatures of the scaffolds were slightly shifted to a lower value. Both HEC/PVAL and HEC/PVAL/COL fibers showed a significant decrease in Young's modulus and tensile stress during the 12 weeks of degradation. Their results demonstrate that these nanofibrous scaffolds showed degradation behavior that meets the requirements as a degradable biomaterial potential for dermal replacement.

thus being a promising approach as a model for the chondrogenic differentiation of mesenchymal stem cells human beings. This is why Pustlauk *et al.* [28] investigated their potential for joint cartilage repair. They studied the expression of the COL 2 gene and found that its expression was comparable in all scaffold types examined. However, the COL 2/COL 1 ratio was higher for pure alginate disks and alginate-cell suspension scaffolds compared to alginate-embedded stem cells. In addition, they found that the secretion of sulfated glycosaminoglycans was comparable in the suspension of alginate cells and cells embedded in alginate scaffolds. They conclude that hybrid COL constructs of jellyfish and alginate support the chondrogenic differentiation of stem cells and provide more stable constructs

Gelatin and Collagen Nanofiber Scaffolds for Tissue Engineering

http://dx.doi.org/10.5772/intechopen.73316

21

Angarano *et al.* [29] synthesized GEL and COL cross-linked fibers by the reactive electrospinning technique using a mixture of nontoxic solvents: acetic acid, ethyl acetate, and water (5, 3, and 2 w/w/w), eliminating fluorinated solvents, which require post-treatment and purification by the implementation of glyoxal, represented an easy, versatile, and one-step procedure. Enabling the expansion and fabrication of synthetic fabrics of COL based on nanofiber cross-linked GEL in situ. This in situ cross-linking renders the water soluble GEL fibers water resistant without adversely affecting the hydrophilicity, excellent wetting of fibers, cell compatibility, reabsorption, cell adhesion, and proliferation typical of COL nonwoven nanofibers cross-linked.

Tylingo *et al*. [30] prepared and characterized new porous scaffolds composed of chitosan, COL, and GEL for the preparation of GEL and COL scaffolds isolated from fish skin with various physicochemical properties. All biomaterials obtained showed homogeneous porosity. The type of protein polymer determined the rheology and mechanical properties of the preparation of the preparations. The use of protein polymers decreased the swelling ability of the materials by about 30% compared to the materials obtained from chitosan. GEL-containing materials showed the highest solubility (approximately 30%). Scaffolds obtained in 100% chitosan were found to be harder than COL materials by an average of 30% and less flexible more than twice. In addition, materials containing protein polymers showed good antioxidant properties.

In **Table 1**, other studies with the electrospinning technique are summarized.

Cell regeneration in fibroblasts (BJ-5ta) and human embryonic kidney cells (HEK 293 T)

regeneration in fibroblasts (3 T3)

Tissue engineering

GEL (type A porcine) Tissue engineering Cell

**Polymers Application Characteristics References**

Food industry Soft nanofibers without

pearl formation

cell proliferation

glutaraldehyde.

Nanofibers with up to 90%

Nanofibers cross-linked in

[9]

[14]

[5]

compared to pure hydrogels.

GEL

GEL

(type B porcine)

(type B porcine)

**4. Electrospinning with the GEL/COL system**

The development of biomaterials with the capacity to induce the healing of cutaneous wounds is a great challenge in biomedicine. In one study, COL sponges were developed from tilapia skin and electro-nylon nanofibers for wound dressing. It was found that nanofibers could significantly promote the proliferation of human keratinocytes and stimulate epidermal differentiation through the expression of regulated genes involved, filaggrin and type I transglutaminase in human keratinocytes. In addition, COL nanofibers could also facilitate the regeneration of rat skin, in one study, electrolyzed nanofibers of COL were prepared from biomimetic tilapia skin and were shown to have a good bioactivity and could accelerate the healing of wounds from rat quickly and efficiently. These biological effects can be attributed to the structure of the biomimetic extracellular matrix and to the multiple amino acids of the COL nanofibers. Therefore, tilapia COL nanofibers could be used as a new wound dressing, effectively avoiding the risk of transmitting diseases in future clinical application [25].

Another study using the double-extrusion electrospinning technique prepared with multilayer 3D scaffolds stacking poly-lactic-co-glycolic acid (PLGA) microfiber membranes alternately to micro- /nano-mixed fibrous membranes of PLGA and COL. The density of the COL fibers in multilayered scaffolds obtained was able to control the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 cells. Demonstrating that the homogeneous dispersion of glutamic acid-modified hydroxyapatite nanoparticles (nHA-GA) in the COL solution improved the osteogenic properties of the multilayer scaffolds fabricated. In addition, it found that PLGA-COL-HA micro-nano fibrous scaffolds were highly bioactive compared to pristine microfibrous PLGA and PLGA and COL micro/nano-mixed fibrous platforms [26].

The development of biomimetic scaffolds represents a promising direction in the engineering of bone tissue. This was demonstrated by Ma *et al.* [27], when they designed a two-step process to prepare a type of biomimetic hybrid hydrogels that were composed of COL, hydroxyapatite, and alendronate, the latter as anti-osteoporosis drug. These hybrid hydrogels of collagen, hydroxyapatite, and alendronate exhibited remarkably improved mechanical properties (G: 38–187 kPa storage modulus), higher gel contents, and lower swelling proportions compared to hydrogels prepared from COL only under similar conditions. In addition, they showed degradable behaviors against collagenase. The hybrid hydrogels of COL-hydroxyapatitealendronate well supported the adhesion and growth of MC3T3-E1 osteoblastic cells. Such resistant but enzymatically degradable hybrid hydrogels hold the potential as scaffolds for bone tissue engineering.

The hybrid constructs from marine organism material for porous scaffolds of COL, such as fibrillated jellyfish and alginate hydrogel, mimic the two major components of cartilage, thus being a promising approach as a model for the chondrogenic differentiation of mesenchymal stem cells human beings. This is why Pustlauk *et al.* [28] investigated their potential for joint cartilage repair. They studied the expression of the COL 2 gene and found that its expression was comparable in all scaffold types examined. However, the COL 2/COL 1 ratio was higher for pure alginate disks and alginate-cell suspension scaffolds compared to alginate-embedded stem cells. In addition, they found that the secretion of sulfated glycosaminoglycans was comparable in the suspension of alginate cells and cells embedded in alginate scaffolds. They conclude that hybrid COL constructs of jellyfish and alginate support the chondrogenic differentiation of stem cells and provide more stable constructs compared to pure hydrogels.
