Nanoparticle Based Collagen Biomaterials for Wound Healing

Abstract

Wounds and infections are extremely common cases that are dealt with in the medical field. Their effective and timely treatment ensures the overall well-being of patients in general. Current treatments include the use of collagen scaffolds and other biomaterials for tissue regeneration. Although the use of collagenous biomaterials has been tested, the incorporation of nanoparticles into these collagenous biomaterials is a fairly new field, whose possibilities are yet to be explored and discovered. The current chapter explores the applications of the amalgamation of collagenous biomaterials with nanoparticles, which themselves are known to be effective in the treatment and prevention of infections.

1. Introduction

An injury that occurs in a quick manner, which often leaves the skin torn, cut, or punctured, or wherein the skin or any other tissues of the body undergoes acute trauma resulting in a contusion, is defined as a ‘wound’. This is when the body’s repair mechanism works to repair the damage by replacing the damaged tissue with newly synthesized tissue. This is characterized by a cascade of highly coordinated reactions that occur at the tissue damage region, working to restore normal tissue, which is called wound healing mechanism. This process requires nutrients and amino acids in adequate amounts to ensure the smooth repair of damaged cells, the supplementation of which has been viewed as a possible solution to augment the process and provide better strength and elasticity to the newly developing tissue [1].

It is known that collagen, being an integral part of most tissues in the body, plays an important role in the structural stability, elasticity, and tensile strength. It is therefore unsurprising that collagen is vital for restoring the structural integrity of the wounded tissue. It has been observed that, formation of scar tissue is an integral part of wound healing in most cases, with epidermal wounds being the exception. This scar tissue is composed primarily of collagen. This makes collagen synthesis an extremely crucial part of the wound healing process [2]. It is therefore practical to employ collagen supplements to augment and speed up the process of hound healing, and even enhance the tensile strength and other innate properties of the tissue. Through a study conducted by Felician et al., it was proven that collagen obtained from a species of jelly fish was indeed effective in escalating the pace of wound healing, making it a potential product that could be used in treating major wounds [3]. There is growing interest in the applications of collagen powder derived from marine sources to treat wounds effectively and reducing the possibility of a scar on the skin along with many other biomedical applications [4]. However, it must be understood that collagen powder is not the only form of collagen supplement for treatment of wounds and other tissue replacement procedures. There are a variety of forms, in which collagen is used as a biomaterial, for wound treatment [5].

Collagen derived from various sources is fabricated into various scaffolds, which can be implanted or grafted into the region of tissue damage, to act as an effective substrate for the attachment of precursor cells and allow their proliferation, thereby increasing the chances of tissue repair effectively. These precursor cells are multipotent adult stem cells which have the ability to differentiate to form various cells depending on the environment they are in, or the stimuli they receive for differentiation. These scaffolds can also be in the form of hydrogels, or fibers, and not just solid in nature. The use of collagen has proven to be effective for wound healing, due to the fact that it is an integral part of the extracellular matrix (ECM) on which most tissues are constructed [6]. Nanotechnology is a field of science that has been explored for its possible applications in the biomedical sector. Many nanomaterials such as nanoparticles and fibers are known to possess antimicrobial activities, which could be effective in the wound healing mechanism for the prevention of further infection. It is thereby prudent that the nanomaterials should be tried and tested along with those of collagen in order to come up with innovative methods to treat major wounds effectively. This chapter aims to summarize the importance of collagen and nanoparticles, synthesis of nano collagen in order to benefit from the wound healing properties of both nanoparticles and collagen, along with the areas of wound healing in which nano collagen is currently being used.

2. Nanotechnology

Nanotechnology is the branch of science and engineering that involves design, construction, and characterization of materials by restructuring the atoms and molecules with the size range of 1–100 nm in one or more dimensions [7, 8]. The engineered materials are nanomaterials that show distinct chemical and physical properties compared to the bulk materials due to the synthesis and assembly at the molecular level that can be exploited for commercial use [9]. Nanomaterials can be of different shapes mainly based on their dimensions i.e., nanoparticles of zero dimension, nanorods of one dimension, and nanosheets of two dimensions [10]. Nanoparticles, due to their small size have the ability to penetrate the bacterial cell wall, and though the cells metabolic pathway cause changes to the cell structure and function. Nanoparticles are also known to interact various components of the bacterial cell, such as lysosomes, enzymes, and ribosomes, thereby leading to oxidative stress, altered permeability of the cell membrane, protein deactivation, and altered gene expression, eventually causing cell death among the bacteria. Thus, it can be said that the Nanoparticles have antibacterial properties, which can be exploited for sterilization of larger wounds, thereby preventing infections from occurring during the wound healing process. When compared to the conventional wound healing drugs certain nanoparticles exhibit greater penetration of cell membrane [9]. Nanoparticles, nanocomposites, coatings, and scaffolds are the main nanomaterials used for wound healing (as shown in Figure 1). Nanoparticles can be (i) inorganic metal or non-metal (ii) organic non-polymeric or polymeric. Nanocomposites are made of porous materials, colloids, copolymers, or gels. Coating and scaffolds include hydrogels, nanofibers, films, and coatings [11]. Different classes of nanoparticles are involved for the treatment of wounds. They are discussed below:

Figure 1.

Types of nanomaterials used for treatment of wounds. The figure is reproduced with permission from [11].

2.1 Metallic nanoparticles

The antimicrobial property of metallic nanoparticles is exploited in wound management and can be used as a nanocarrier. The surface area to volume ratio of metallic nanoparticles is high. The small size enables them to cross barriers and penetrate the underlying layers of thick tissues like skin. These features make them ideal for drug delivery and to treat wounds. Some of the widely used metallic nanoparticles includes—silver nanoparticles (Ag NPs), gold nanoparticles (AuNPs), zinc oxide nanoparticles (ZnO NPs), iron oxide nanoparticles (IONPs), and titanium dioxide nanoparticles (TiO₂ NPs) [12].

2.2 Polymeric nanoparticles (PNPs)

Polymeric nanoparticles include polymer nanospheres and polymer nano capsules. Biologically active molecules such as drugs, genes, and fluorophores are absorbed on the surface of polymer nanospheres forming antibiotic incorporated nanoparticles (NPs). Griseofluvin (GF), one such NP, is known to function as an effective carrier of biologically active entities [12, 13]. The polymer nano capsules are vesicles where the core contains bioactive agents surrounded by polymeric shell. The polymers used in the preparation can be natural polymers like starch, polypeptides, albumin, sodium alginate, chitin, cellulose, gelatin, polyhydroxy alkanoates (PHAs) or artificial polymers like polyethylene glycol (PEG), poly lactic acid co-glycolic acid (PLGA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene etc. They show higher encapsulation efficiency and high stability of encapsulated active substance that helps them in the effective delivery of drugs to targeted sites [13].

2.3 Nano emulsions

Nano emulsions shows small droplet size and high surface area that makes them a suitable vehicle for drug delivery to treat wounds. A unique feature of these nano emulsions is their ability to deliver hydrophobic drugs [14]. They also have long shelf life, and are easily formulated [12]. The components of nano emulsions include different oil types, emulsifying agents like sodium deoxycholate, sodium dodecyl sulphate, antioxidants, chelating agents, preservatives etc. [15].

2.4 Solid-lipid nanoparticles (SLNs)

Solid-lipid nanoparticles are used as drug vehicles in case of inflamed or damaged skin. They are efficient and non-toxic carriers of both lipophilic and hydrophilic drugs. The structure is made up of long-fatty acid chains of palmitic acid, stearic acid or arachidic acid taurocholate, emulsifiers, and water.

2.5 Nanofiber scaffolds/mats

Nanofiber scaffolds/mats, considered as a substitute to damaged ECM, are mainly used in the wound dressing due to its healing power and unique structure. As the scaffolds are applied on the wound there will be attachment of fibroblasts and formation of matrix that acts as ground substrate and aid in faster wound recovery. Manufacturing of nanofibrous scaffolds involves electrospinning that produces uniform nanofibers [16].

2.6 Nanogels

Hydrogels are used as delivery vehicles for wound treatment due to their properties such as high porosity which keeps the wound environment moist, and the presence of 3D polymeric matrix that absorbs the wound exudates allowing for proper permeation of oxygen [12]. While nanogels demonstrates some advanced features compared to those of hydrogels such as stability, ease of synthesis, quick response to stimulus, an adjustable size that can be exploited for drug delivery, controlled release of drugs, and tumor imaging. Nanogels are made up of chemical polymers and biomolecules. The nanogels of amino acids and polypeptides are easy to synthesize and modify and show higher biocompatibility [17].

3. Collagen

The word ‘collagen’ is derived from a Greek term ‘kolla’, which means ‘Glue’. Collagen is essentially a matrix, which holds the connective tissue together, making it a major component of the ECM, and connective tissues, and is rightfully called the most abundant protein in the animal kingdom [18]. Collagen is a major component of the ECM, which provides mechanical support for cell growth and their integrity. Collagen represents an entire superfamily of glycoproteins, having, a polypeptide sequence signature with [Gly-X-Y]_n as the repeating amino acid unit, wherein X and Y are proline and hydroxyproline respectively. Another salient feature of these glycoproteins is their noteworthy quaternary structure with the right-handed triple helix structure composed of three left-handed polyproline chains of uniform length. The chains in the triple helix can either be identical, forming homotrimers as seen in collagen II, or be different from each other, forming heterotrimers, as seen in collagen IX. Presence of glycine is invariant in collagen and is known to stabilize the collagen structure. It has been found that the absence of glycine or any mutations to the same is known to cause disruption in the hydrogen bonds formed in collagen and distort the structure [19].

The presence of collagen and collagenous structures throughout the animal kingdom indicates its importance in biological structures. Collagen is expressed in all life forms classified under the animal kingdom. Right from sponges, the simplest multicellular animal which expresses genes for the formation of at least two types of collagens, to the various vertebrates, in which collagen is a major component of various connective tissues, thereby accounting for roughly a quarter of the whole-body protein in humans [20]. The basic triple helical pattern has been partially carried over into the architectures of other complex molecules in higher organisms, with complex physiologies. Evolutionary branching which was partially driven by chromosomal duplication has resulted in a plethora of collagen types, which are genetically distinct. There are 29 types of collagens that have been identified so far [21]. Although the exact function of many types of collagens is yet to be confirmed, the role and presence of collagen throughout the body is unmistakable. However, it is known that collagen types I, II and III represent the majority (approx. 80–90%) of the total body collagen. They are known to provide mechanical and tensile strength to the skin and various other organs. The ability of fully developed collagen to integrate hydroxyapatites and undergo mineralization to amalgamate with solid structures such as bones and teeth, combined with its nature of elasticity and strength makes it a very desirable candidate to be used as a primary component of biomaterials with various applications [22]. Biomaterials are defined as synthetic components that may be transplanted into body tissue as a part of a medical device. Biomaterials can also be employed to replace an organ or a part of it, thereby aiding it in its physiological and mechanical functions [23].

Despite the wide range in the types of collagens, only a handful of them are actually utilized for the production of collagen-based biomaterials. Fibril forming collagens, such as type I, which also happens to be the most abundant collagen in mammals, is often employed for construction of collagen-based biomaterials for various purposes such as wound healing and tissue engineering, and even 3D bioprinting of collagen-based structures or scaffolds [24]. Collagen can be extracted from any animal’s tissue including vertebrate’s skin and tendons, porcine skin, gut, bladder mucosa, rat tails, as well as invertebrates’ sponges and corals. The extracted collagen can show a slight difference in some characteristics, depending on the source of the animal, and the tissue. It has been found that the use of collagen from marine sources [25, 26, 27, 28] has advantages over those obtained from terrestrial organisms, such as being environmentally sustainable, high production of collagen, non-toxicity, and ease of absorption thanks to its lower molecular weight. However, occurrence of allergies and transmission of disease can hamper the use of collagen obtained from animal sources, thereby the application of recombination technology was duly suggested, wherein yeast and Escherichia coli were transfected to produce recombinant collagens [29].

3.1 Collagen and biomaterials

Biomedicine is currently seeing an increase in the use and integration of collagen-based scaffold and biomaterials in its applications. The technology aids the creation of biomaterials which exhibit biomimicry of the complex native tissues and organs. Decellularized collagen and refined scaffolds are the two categories into which collagen-based biomaterials are categorized. While the decellularized collagen structures retain most of the structural and functional properties of the tissue from which it is derived from, refined scaffolds are mostly obtained from the purification and polymerization of collagen. Decellularized collagen exhibits biomimicry the best [30]. Tissue grafts for tissue engineering, self-assembled hydrogels, freeze dried sponges, collagen films and tubes are some commonly used collagen-based biomaterials.

Tissue grafts are one of the most commonly used collagen scaffolds. Due to their resemblance to the native tissues, along with the ability to promote cell attachment and spatiotemporal organization of the cells, tissue grafts have been demonstrated as the most convenient and effective implantable devices [31]. Self-assembled hydrogels are generally used in the form of cell carriers, and injectables. They are often reliable for soft tissue treatment, for they resemble the structures on polymerization to form a fibrillar hydrogel structure, which is held together by ionic and hydrophobic bonds, thus aiding the entrapment of fluids, making it conducive for the exchange of ions and metabolites in the environment created [32]. Collagen type I hydrogels in combination with the appropriate precursor cells have been extensively used for the repair and as a structural and mechanical support for the attachment and stable growth of tissues such as skin to treat burns [33], cardiac myocytes [34], neurons [35], ocular tissues [36], etc. Collagen type I and type II hydrogels have often been used in combination for the treatment and repair of osteochondral tissues, and cartilage [37, 38]. Collagen scaffolds that can be easily used as grafts for various clinical purposes are created by the freeze-drying technique, wherein, collagen on undergoing freezing in a controlled environment, is trapped within the ice crystals formed, and is porous enough to facilitate cell migration, attachment, and growth [39]. So far, a variety of cell populations have been used to improve the bioactivity of the collagen sponge, and the experiments performed have shown encouraging results both in vivo and in vitro. Collagen scaffolds, integrated with glycosaminoglycan and fibrin networks have demonstrated their ability to enhance osteogenesis, and induce osteogenic and chondrogenic differentiation [40]. It was also established that these scaffolds are also used to aid in bone regeneration [41], vascularisation [42], and skin wound healing [43]. As discussed earlier, collagen and its biomaterials have already been well established in the biomedical field for their potential bioactivities. The integration of such collagen, with nanoparticles, which in itself has found extensive applications in the field of treatment and drug delivery, has piqued the interest of the scientific community for their potential synergistic activity to enhance wound healing. The synthesis and current application of this amalgamation is discussed further.

4. Nano collagen synthesis

Nano collagen is the term used to describe collagen brought down to the nanoscale range. This substance has the desirable properties of both nanoparticles, such as a high ratio between the surface to volume of the particle, and collagen, with its wound healing properties of biomaterials, and their functions simultaneously. The downscaling of the size of the collagen fibers, is beneficial in terms of the penetration, and wound accessibility to initiate wound healing [44]. Nano collagen is produced through various chemical, physical, and self-assembly methods, such as emulsification, complex coacervation, phase separation, nano spray drying, desolvation and many other techniques. The following section explains briefly the most popular techniques employed. Nano collagen fibers are produced through the following techniques: (a) electrospinning (b) nano emulsion (c) electrospray deposition (d) milling (as shown in Figure 2, Table 1).

Figure 2.

(A) Electrospraying—after applying a high voltage to the protein solution, a liquid jet stream is released via a nozzle (coaxial needle), generating an aerosolized droplet. To ensure that the polymer solution comes out of the syringe as NP, a high voltage is provided to it. (B) Electrospinning—at a high voltage and low current in the spinneret, collagen polymer solution added dropwise. The Taylor cone is formed at such conditions. The columbic forces also cause the dehydration of the ejected polymer thereby resulting in thin and dry fibers of nano collagen. (C) Milling—the application of mechanical energy through the spinning of a milling bowl breaks down a polymer substance into finer NPs. Milling balls are used to conduct high-energy mechanical impacts to break down polymers utilizing centrifugal force. (D) Nanoemulsion—the emulsion is formed by the mechanical agitation of two immiscible liquid phases, one of which has the protein, and the other in which the drug is dissolved. Figures A, C, and D are reproduced with permission from [11]. Figure B is reproduced with permission from [45].

4.1 Electrospinning

Electrospinning is one of the methods used to create nano collagen fibers, wherein nanofibers are created from polymeric solutions in the presence of an electrostatic field. Electrospinning is achieved by charging a spinneret to high voltages and low current, and then adding droplets of the polymeric solution. As a result, the surface becomes highly charged, and elongates to form a conical shape, which is called the Taylor cone. The conical form is a result of the electrostatic repulsion between the charged droplet surface and columbic forces from the spinneret. At a specific threshold of the electric field, the electrostatic forces are strong enough to overcome the surface tension holding the Taylor cone, thus creating the fibers by stretching the cone, whipping it. This process is generally preferred to create nano fibers, because it is cost effective, and can produce nano collagen scaffolds for various purposes including tissues engineering, tissue repair and regeneration [47], and matrices that mimic the native ECM. The fibers produced through electrospinning are dry, and devoid of any solvent molecules, which are then collected in a metallic collector, which also determines the shape [51]. Over a period of time, electrospun collagen nanofibers have been endowed with certain ‘smart’ abilities, to improve their applications. Some smart abilities include response to external stimuli such as change in pH, exposure to light, and magnetic fields, etc., retaining a shape memory, self-cleaning, and some more [46].

4.2 Electrospray deposition

As the name suggests, electrospray deposition is a process which involves the spraying of nano collagen solution as a fine mist onto a specific target. This method is mostly used for the applications of nanoparticles in the biomedical field for pharmaceutical application. This is mostly because, in this technique, collagen is used in its particle form. It is then sprayed through a nozzle onto a target with a high negative voltage, in the form of a fine mist. The solvent of the collagen particles generally evaporates on deposition onto the target surface, leaving an even spread of nano collagen particles, making it ideal for drug delivery purposes. This evaporation prevents the aggregation of molecules, and thus reduces the risk of contamination [52].

4.3 Milling

Milling is a process in which nano collagen is produced by the application of great amounts of mechanical stress onto a polymeric solution of collagen, to form particles of the nano scale range. This process is one of the most inexpensive methods for the large-scale production of nano collagen [53]. The mechanical energy along with the kinetic energy in the milling container also produces large amounts of heat, which can lead to the denaturation of collagen [54]. Therefore, this generation of heat is contained by performing this process at cryogenic temperatures, with the use of liquid nitrogen, thereby preserving the integrity of collagen.

4.4 Nanoemulsion

Nanoemulsion is a method used to integrate collagen with nanoparticles in a droplet form. Two immiscible liquids in different phases, i.e., oil-in-water-phase (oil is dispersed in water) and water-in-oil phase (water is dispersed in oil) when combined, form a concoction called an emulsion. Nanoemulsions differ from emulsions in their size ranges. The size of a nanoemulsion droplet ranges from 20 to 200 nm, while a normal emulsion droplet size is around 1 μm [55]. An aqueous phase with collagen, and a hydrophilic surfactant in water, is mixed with an organic phase with a lipophilic surfactant in a solvent that is immiscible in water and is continuously agitated under room temperature conditions to produce a uniform emulsion system. Nano collagen emulsion particles are then obtained by combining this emulsion system with a heated oil in a drop-by-drop manner [56]. Nanoemulsions naturally tend to penetrate deep into the tissue to deposit active compounds. This property has been exploited for purposes such as drug delivery in pharmaceutical, food and cosmetic industries. The same properties can be attributed to the collagen Nanoemulsion droplets to enhance the wound healing mechanism and speed up the process. The production of collagen nanoemulsions has increased greatly along with their application mainly in the field of cosmetics and drug delivery due to the technological advantages it offers for the manufacturers [57].

5. Applications

5.1 Bone grafting

Collagen is a major component of the bone matrix. Bone formation is facilitated by the osteoblasts, which are involved in the production of collagen type I protein. The ECM supports the collagen fibers (50–500 nm) synthesized by the osteoblasts. The hydroxyapatite crystals are then deposited on these collagen fibers, leading to the hardening and maturation of the bone [58]. This mechanism can be exploited for the purposes of bone remodeling, in the case of a grave bone injury such as a compound fracture. A collagen scaffold can be grafted onto the damaged tissue area, to provide a solid support onto which the apatite crystals can be deposited, to increase the speed and efficiency of new bone formation. It is thus prudent that the collagen scaffold mimics native collagen fibers to achieve successful bone grafting and promote optimal bone regrowth.

It is well known that bone related tissue trauma is difficult to treat and is a time-consuming process, due to the complexity of the bone healing process itself, and the loss of bone from non-sterile wounds, creating a high risk and susceptibility for infections. Cardoso et al., proposed the use of silver nanoparticles stabilized with type I collagen to form nano collagen biomaterials (AgNPcol) for the collagen scaffold to support rapid bone remodeling. This was an optimal solution for the problem of infections caused due to the non-sterility of the bone wounds. The silver nanoparticles in the collagen also showed anti-microbial activity against a number of microorganisms. Thereby proving to be effective in wound healing. The developed cells also showed no signs of cell toxicity [59]. In another study by Sun et al., collagen scaffolds were infused with AgNPs along with BMP2, a bone morphogenic protein to improve the bone healing process effectively. The role of silver nanoparticles in antibacterial property was already established. However, the incorporation of the bone morphogenic protein induced an increase in the expression of runt related transcription factor 2, osteopontin and osteonectin, which are known to accelerate the differentiation of the bone marrow derived mesenchymal stromal cells, thereby proving the therapeutic potential of nano collagen in bone grafting, and healing [60].

Poor development of alveolar ridge after tooth extraction is an issue faced by most dental patients due to the lack of oral hygiene or knowledge about it. Wang et al., in their research, proposed the usage of artificial nano collagen bone implants. This was done to support the alveolar ridge post extraction of tooth. The implantation was followed by a CT scan to track the bone mineral density progressively. It was found that the implanted nano collagen bone has successfully fused with the native alveolar bridge. It also showed an increase in the overall bone mineral density [61].

5.2 Nerve tissue

Treatment of damaged nerve tissues has been a topic of interest for many researchers. This can be attributed to the inability of terminally differentiated neurons to undergo further cell division and also the fact that the nervous system controls and coordinates most of our body’s processes. Damage or injury caused to the nerve tissue can seriously impair many functions of the body. Autografts of the nerve tissue has been performed in some cases. However, this has proven to be more challenging, due to the shortage of the donor sites, or occurrence of deformities. This has fuelled the search for alternative methods or materials to treat nerve damages effectively. The extensive study on collagen and nano collagen has tested the ability of collagen to act as an effective scaffold and promote cell attachment and growth [62]. Collagen has been used in the manufacture of nerve guidance conduits to aid the nerve regeneration in small nerve gaps of 2–3 cm across the peripheral nerve tissue. The use of collagen hydrogels for the treatment of lesions in the central nervous system effectively has been demonstrated by Orive et al. [63]. Further degradation of the nerve tissues can be prevented on injection of collagen nanospheres, which have the potential to deliver therapeutic drugs, and other stem cells for structural support as well [64]. Zhang et al., illustrated the application of collagen—nano size β tricalcium phosphate, together with growth factors of nerves and some collagen fibers, for the treatment of facial nerve repair and regeneration. Improved action potential was seen in the muscles, along with the formation of thicker myelin sheath, making it a highly promising avenue for further innovation and studies in nerve regeneration [65].

5.3 Articular cartilage

Articular cartilage covers the edge of a bone, and it is a connective tissue which forms a synovial joint that provides low frictional surface and enables the smooth movement of the joint. So, any damage to the articular cartilage results in acute pain during the movement of the joint. However, unlike most tissues in the body, articular cartilage lacks the potential to heal itself by replacing damaged areas in the tissue with new cells. This is mainly due to its avascular nature, i.e., there is no direct blood supply to the cartilage, thereby making it a difficult to heal by targeting therapeutic drugs. Treatment for articular cartilage necessitates surgical intervention techniques such as chondrocytes implantation and osteochondral transplant. However, the high cost and numerous other risk factors of patients has given rise to much needed research in the field of cartilage tissue engineering [66].

Cartilage tissue engineering employs the use of 3D bioprinting for the creation of collagen 3D scaffolds, which are then treated in vitro to make them suitable for implantation. The application of 3D bioprinting techniques along with the nano collagen scaffolds effectively reduces the requirement of a cartilage transplantation from a donor, along with the need for other less effective surgical options. A collagen-hydroxyapatite hydrogel nanocomposite was developed and effectively used in an investigation which showed promising results. Hydrogel composite was found to be suitable to facilitate fluid transport, and also thermally stable up to a temperature of 90°C [67]. Jiang et al., illustrated a different approach to stimulate the differentiation of the chondrocytes in the articular cartilage in order to initiate the repair mechanisms. The inhibition of chondrocyte dedifferentiation was achieved by the use of nano hydroxyapatite collagen scaffolds [68].

5.4 Skin wound healing

The process of wound healing involves four steps viz., hemostasis, inflammation, proliferation, and remodeling which occur in a sequential order [69]. Disruption of any of these steps will make the process lengthy. The main issue involved in wound healing is infection by pathogens that results in inflammation, interrupting the healing process [45]. Schimek et al., developed full-thickness skin equivalents (ftSEs) to hold the 96-well cell culture [70]. Collagen powder can be used as the dermal substitute as they are part of the ECM that shows slow biodegradation and accelerates wound healing [45]. Collagen with nanoparticles is widely used in therapy. Munish et al., used collagen granules for the diabetic foot ulcer treatment and the results were compared with the saline dressing. The study demonstrated that the wound, when treated with collagen showed a speedy recovery [71]. In another study, Akturk et al., developed gold nanoparticles (AuNPs) based collagen scaffold, and they were incorporated into the cross- linked collagen scaffolds. It was found that it helps in enhancing the stability against enzymatic degradation and increases the tensile strength [72]. The main advantage includes the absence of rejection and the fact that they can reduce the inflammation in and around the wound. Apart from gold nanoparticles, the use of silver as an antimicrobial agent has also been of great interest recently. Silver nanoparticles (AgNPs) are usually used in the treatment of burns and infection as they are known to demonstrate antibacterial property. There is sufficient evidence to prove that the bacterial resistance against AgNPs may not be a matter of concern, for AgNPs are known to hinder quorum sensing mechanisms in bacteria [45].

Collagen-based dermal scaffolds are coated with silver nanoparticles that act as antimicrobial dressing without having any toxic side effects. Nano silver reacts with gram-negative and gram-positive bacteria, causing damage to the intracellular structure. The positively charged silver nanoparticles react with negatively charged bacterial surfaces leading to the disruption of the inner membrane. During electrospinning, the synthesized silver nano particles are incorporated into the collagen nano fibers. The in vitro results prove that the AuNPs and AgNPs can provide the antimicrobial conditions for wound healing. The rate of wound healing in case of collagen composite nanofiber mat was significantly higher compared to the regular nanofiber collagen [44].

5.5 Drug delivery

Collagen nanoparticles have shown promise as treatment carriers [73]. The recent trends in nanotechnology research and development aims to create collagen scaffolds that deliver the drug to the specific site and are released in a controlled manner [74]. Gold nanoparticles with different concentrations of gold (Au) was synthesized and coated onto collagen to form an amalgamation of nanoparticles and collagen (Au-Hp-Col). This amalgamation was found to be effective in the delivery of the drug Doxorubicin [70]. Poloxamer 407 (PM) is a polymer soluble in water used in the delivery of ophthalmic drugs like Ketorolac Tromethamine (KT). The PM is incorporated into the cellulose nano collagen particles that showed controlled release of the drug in vitro [73]. One of the case studies demonstrated the use of collagen nanoparticles in drug delivery to treat tumors. Collagen is a major component of the tumor microenvironment. The study involved the development of tumor spheroids based on collagen that are optimized using cell lines like 95-D, U87, HCT116 [75]. It was observed that the conjugated nanoparticles showed greater penetration into the gel matrix and were able to gain access to the tumor cells [76].

Preparation method	Principle	Advantage	Limitation	References
Electrospraying	Uses electrostatic field to create nano collagen fibers from a polymeric solution of collagen	Can be upscaled for industrial purposes; ease of particle synthesis due to single step process; formation of dry particles	Reduced flow; can degrade some macromolecules	[46]
Electrospinning	Uses a high voltage difference to generate dispersible nanoparticles from collagen solute	Can produce fine fibers of collagen; Emulates ECM closely; cost effective	Time consuming	[47]
Milling	Uses mechanical energy to break down a polymeric material of collagen to nanoparticle sizes	Economical; easy experimentation; controllable nanoparticle size	Chamber has to be cooled due to heat release; cannot control nanoparticle shape	[48]
Nanoemulsion	Uses mechanical agitation to form nanoemulsions by the combination of two immiscible liquids in different phases.	Simple process; easy recovery; high flexibility and selectivity	Requires appropriate surfactant due to unstable thermodynamic nature; the organic solvent needs to be removed, for the residues may be toxic	[49, 50]

Table 1.

Collagen nanoparticle preparation methods, their principles, advantages and limitations.

5.6 Vascular grafting

Cardiovascular disease is the major cause of death worldwide [77]. These disorders are caused by reduced blood flow by blockage of blood vessels [78, 79]. Presently, the saphenous vein, the internal thoracic artery, and autologous vessels are used as grafts which are known to perform better than the synthetic alternative [80]. However, their limited availability and invasive harvest make them unsuitable for use. Tissue-engineered vascular grafts (TEVG) are currently used in order to overcome these limitations [48]. TEVG makes use of modern technology for the construction of vascular medical implants. The collagen along with the other components are used as a scaffold in the preparation of the TEVGs. In a previous study, Park et al., described a poly-epsilon-caprolactone (PCL) vascular graft, and its suitability for healing process. It was observed that the graft undergoes gradual degradation replaced by natural blood vessels. Collagen is also incorporated on to the inner layer and silica (sol-gel-derived ceramic) into the outer layer of PCL to improve the vascular response [49].

6. Conclusion

This chapter conclusively describes the importance and role of nanoparticles-based collagen biomaterials in the treatment of various wounds. The ECM is mainly comprised of collagen, which provides support and elasticity against mechanical stress. While collagen in itself is useful in the form of various biomaterials like scaffold s and hydrogels, the introduction of nanotechnology to it comes with its own set of challenges as well as advantages. The reduction of collagen to the nano particle’s sizes, giving it a large surface-to-volume ratio, is known to increase its efficiency of dealing with mechanical stress, thereby making it a viable option for treatment of wounds. Multiple research studies are conducted on wound healing using various materials and methods to reduce risk infection and aid in speedy recovery of the patient. The antimicrobial properties of nanoparticles of various elements such as gold and silver has already been proven, which can be further exploited in the effective treatment of wounds and injuries, in combination with collagen. The current challenge lies in the effective incorporation of nanoparticles and collagen in the production of nano collagen biomaterials, upscaling the production of nano collagen and making it affordable to the general public.

Acknowledgments

NM thank Global Innovation and Technology Alliance (GITA), Department of Science and Technology (DST), India [Project Number-GITA/DST/TWN/P-95/2021], and Indian Council of Medical Research (ICMR), (Project Number-ITR/Ad-hoc/43/2020-21, ID No. 2020-3286) Government of India, India for financial support.

Conflict of interest

The authors declare no conflict of interest.