Use of collagen as a biomaterial: An update.

Biomaterial science is an expanding area, which encompasses a wide range of medical knowledge involving arthroplasty, cochlear implants, heart valves designing, lenses, dental fixation and tissue engineering. Within this context, in vitro cell culture on polymer scaffolds is one of the adopted strategies for tissue creation. It consists of a specific cell line that is seeded onto a particular substrate. This scaffold should provide excellent biocompatibility, controllable biodegradability, appropriate mechanical strength, flexibility as well as the ability to absorb body fluids for delivery of nutrients. Collagen certainly fulfils these demands; therefore, it is often chosen as a biomaterial. Moreover, this protein is abundant in the animal kingdom and plays a vital role in biological functions, such as tissue formation, cell attachment and proliferation.

INTRODUCTION

Collagen is a highly versatile material that is extensively used in the medical, dental and pharmacological fields. Collagen is capable of being prepared into cross-linked compacted solids or into lattice-like gels.[1] Use of collagen in the form of tendons as suture material goes back to millennia and can hold its ground with catgut, which is still representing a useful suture material in surgery.[2]

The main applications of collagen are for burn/wound cover dressings, osteogenic and bone filling materials, antithrombogenic surfaces and immobilization of therapeutic enzymes. Recently, use of collagen as a carrier for drug delivery has attracted many researchers throughout the world. Collagen-based drug delivery systems include injectable microspheres based on gelatin (degraded form of collagen), implantable collagen–synthetic polymer hydro gels, interpenetrating networks of collagen and synthetic polymer collagen membranes for ophthalmic delivery.[3]

Resorbable forms of collagen have been used to dress oral wounds, for closure of graft and extraction sites and to promote healing. Collagen-based membranes have also been used in periodontal and implant therapy as barriers to prevent epithelial migration and allow cells with regenerative capacity to repopulate the defect area. It has been hypothesized that membrane regenerative techniques facilitate the natural biological potential by creating a favorable environment for periodontal and peri-implant regeneration.[1]

This present review does not aim to be comprehensive. Rather, it intends to draw attention to some important properties of collagen, how these properties have been used in certain collagen-based biomaterials and how they provide opportunities for regeneration and development of human tissues in medicine and dentistry.

STRUCTURE OF COLLAGEN

Astbury was the first to suggest a structure for collagen in 1938, which consisted of a mixture of Trans and Cis peptide units, and the same feature was incorporated by Pauling and Corey in the model proposed by them in 1951 that had three co-axial helices. However, neither of these structures was in agreement with the observed X-ray diffraction pattern of collagen fibers. It was Ramachandran's group from Madras, India, who first postulated a triple helical structure for collagen.[4]

The collagen molecule consists of three polypeptide chains twined around one another as in a three-stranded rope. Each chain has an individual twist in the opposite direction. The principal feature that affects a helix formation is a high content of glycine and amino acid residues. The strands are held together primarily by hydrogen bonds between adjacent CO and NH groups and also by covalent bonds. The basic collagen molecule is rod shaped with a length and a width of about 3000 and 15 A, respectively, and has an approximate molecular weight of 300 kDa. The triple helix structure contains three basic amino acids: Glycine, proline and hydroxyproline. The pattern is glycine, proline and X, with X being any amino acid. Specific amino acids cause specific functions for the collagen. Hydrogen bonds hold the helix structure together by linking peptide bonds.[2]

The individual triple helices or tropocollagen molecules, as they are sometimes called, are arranged to form fibrils that are of high tensile strength and flexibility and can be further assembled and cross-linked so as to support stress efficiently. Abnormalities in the collagen molecular structure or its organization into mature fibers leads to different diseases associated with connective tissues, such as Ehlers-Danlos syndrome, osteogenesis imperfecta and some types of osteoporosis and arthritis.[4]

COLLAGEN-BASED DRUG DELIVERY SYSTEM IN MEDICINE

Film/sheet/disc

The main biomaterial application of collagen films is as barrier membrane. Films with a thickness of 0.01-0.5 mm are formed by air-drying a casted collagen preparation similar to ophthalmological shields and made of biodegradable materials. The drugs can be loaded into collagen membranes by hydrogen bonding, covalent bonding or simple entrapment. They can be sterilized and become pliable (easily bended) upon hydroxylation while still retaining adequate strength to resist manipulation. Collagen film/sheet/disc is used for the treatment of tissue infection, such as infected corneal tissue or liver cancer, and as a drug carrier for antibiotics such as tetracycline.[5] Collagen film and matrix were used as gene delivery carriers for promoting bone formation. A composite of recombinant human bone morphogenetic protein 2 (rhBMP-2) and collagen was developed to monitor bone development and absorbent change of carrier collagen.[6] The rhBMP-2/collagen onlay implant resulted in active bone formation, whereas the collagen alone resulted in no bone formation. Collagen matrix loaded with bone morphogenetic protein (BMP) and placed in close contact with osteogenic cells achieved direct osteoinduction without causing a cartilage formation.[7] Biodegradable collagen films or matrices have served as scaffolds (give support) for survival of transfected fibroblasts. A combination of collagen and other polymers, such as atelocollagen matrix added on the surface of the polyurethane films, enhanced attachment and proliferation of fibroblasts and supported growth of cells.[8]

The matrix films, which are composed of various combinations of collagen and elastin, are used in tissue calcification and as a controlled delivery device for cardiovascular drugs.

Collagen shields

The collagen shield was designed for bandage contact lenses, which are gradually dissolved in the cornea. The use of collagen-based drug delivery systems is the ease with which the formulation can be applied to the ocular surface and its potential for self-administration.[9] The mechanical properties of the shield protect the healing corneal epithelium from the blinking action of the eyelids. Drug delivery by collagen shields depends on loading and a subsequent release of medication by the shield.[10] The collagen matrix acts as a reservoir and the drugs are entrapped in the interstices of the collagen matrix. As tears flush through the shield and the shield dissolves, it provides a layer of biologically compatible collagen solution that seems to lubricate the surface of the eye, minimize rubbing of the lids on the cornea, increase the contact time between the drug and the cornea and increase the epithelial healing. A bolus release of drug from the lenses leads to the enhanced drug effect.[11]

Collagen sponges

The sponges made from pure collagen isolated from bovine skin are swollen at pH 3.0 and stabilized into the physical form of a sponge layer. In order to achieve highly resilient (elastic) activity and fluid-building capacity, collagen sponges have been combined with other materials like elastin, fibronectin or glycosaminoglycans.[12] The starting material can be cross-linked with glutaraldehyde and subsequently co-polymerized with other polymers, such as polyhydroxyethylmethacrylate (PHEMA). The PHEMA chains, which are hydrophilic, keep the membranes wet and increase their tensile strength. This further affects the efficiency in the management of infected wounds and burns. Collagen sponges have been very useful in the treatment of severe burns and as a dressing for many types of wounds, such as pressure sores, donor sites, leg ulcers and decubitus ulcers, as well as for in vitro test systems. Collagen sponges have the ability to easily absorb large quantities of tissue exudates, smoothly adhere to the wet wound bed with preservation of low moist climate and also shield against mechanical harm and secondary bacterial infection. Coating of a collagen sponge with growth factor further facilitates dermal and epidermal wound healing.[13] Collagen sponges are also used for delivery of steroids through topical applications, such as intravaginal delivery of lipophilic compounds including retinoic acid. Collagen-based sponges are inserted into a cervical cap made of hydrogel hypan to deliver all-trans-retinoic acid to patients with cervical dysplasia.[2]

Gel, hydrogel, liposomes–collagen

Collagen gels are flowable, suggesting the possibility of an easily injectable, biocompatible drug delivery matrix. Collagen gels are primarily used for injectable systems. The most readily available forms of such injectable collagen gels are: (a) injectable suspensions of collagen fibers and (b) non-fibrillar, viscous solutions in aqueous media. For ophthalmic use, formulations are patented, which are initially liquids but turn to gel after administration to the eye. When applied, the gel will remain in place in the cul-de-sac of the eye substantially longer than liquid formulations and will allow for a sustained delivery of non-steroidal anti-inflammatory drugs or antibiotics.[2] Gel made of atelocollagen, which is produced by elimination of the telopeptide moieties using pepsin, has been used as a carrier for chondrocytes to repair cartilage defects.[14] It has been reported that grafted type I atelocollagen provided a favorable matrix for cell migration in relation with collagenase expression and cell behavior was shown to be modulated by graft collagen.[15] An attempt of combining collagen and PHEMA into hydrogels has been made to develop a delivery system for anticancer drugs, such as 5-FU. A novel drug delivery system comprising liposomes sequestered in a collagen gel has demonstrated controlled release profiles of insulin and growth hormone into the circulation.[2]

Pellet/tablet

Minipellets made of collagen have been developed for various candidate compounds. A minipellet is small enough to be injected into the subcutaneous space through a syringe needle and is still spacious enough to contain large molecular weight protein drugs, such as interferon and interleukin-2. A single subcutaneous injection of a mini pellet causes a prolonged retention of interleukin-2 and decreases its maximal concentration in the serum. This pellet-type carrier has been used for local delivery of minocycline and lysozyme. Collagen-based pellet as a gene delivery carrier has been extensively studied. The mechanism of direct bone formation by collagen complex has been ultrastructurally investigated.[15] This study has proved that direct bone formation is ectopically induced by BMPs without cartilage formation when an atelocollagen type I collagen pellet is used as a carrier.

Nanoparticles/nanospheres

Nanosphere formation is driven by a combination of electrostatic and electropic forces, with sodium sulfate employed as a dissolving reagent to facilitate greater charge–charge interactions between plasmid DNA and collagen. Nanoparticles can be taken up by the reticuloendothelial system and enable an enhanced uptake of exogenous compounds, such as anti-HIV drugs, into a number of cells, especially macrophages,[16] which may offer an additional advantage of collagen-based nanoparticles as a systemic delivery carrier. Thus, nanoparticles are used as a parenteral carrier for cytotoxic agents and other therapeutic compounds, such as campthocin and hydrocortisone.

USE OF COLLAGEN IN DENTISTRY

It has been reported that collagen has the following properties: (1) it controls the evaporation of fluid, keeping the wound pliable and flexible, (2) it promotes the development of granulation tissue, (3) it diminishes pain and (4) it provides mechanical protection against physical and bacterial insult.[17]

Clinical researches have reported that collagen powders exhibit excellent adhesion to the wound, hemostatic properties, tissue fluid binding and an adequate stimulation of cell reactivity with the formation of a highly vascularized granulation bed.[13]

Films made from hydrolyzed collagen have been used as a tissue adhesive for suture replacement due to its chemical resemblance to connective tissue and its tissue fluid-binding properties. Moreover, it is biodegradable, non-toxic and readily absorbed; therefore, it does not impose a hindrance to the healing process.[13]

Collagen has the ability of enhancing wound healing following dental therapy by clot formation and stabilization, neovascularization and epithelial cell rejuvenation thus acting as a natural hemostatic agent.[1819] Collagen also serves as a biologic scaffold for ingrowths of endothelial cells and progenitor cells from the periodontal ligament.[20]

For oral applications, homogenized reconstituted collagen mixed with cell culture media has been used for endodontic repair.[21]

Notably, collagen-based membranes have been widely used in periodontal and implant therapy as barriers that prevent the migration of epithelial cells and encourage wound repopulation by cells with regenerative potential.[22]

Several investigators have examined type I collagen as a possible membrane barrier for use in guided tissue regeneration (GTR) procedures. Collagen is absorbable, does not require a second surgical procedure for removal and has some unique properties.[23]

Collagen is used as a membrane due to the following reasons: It is the major extracellular macromolecule of the periodontal connective tissue and is physiologically metabolized by these tissues, it is chemotactic for fibroblasts, it has been reported to act as a barrier for migrating epithelial cells in vitro and it is a weak immunogen that has been used experimentally in animals and humans. Pitaru et al. in a series of experiments concluded that type I collagen has the capacity to support regeneration of periodontal tissues.[23]

Formation of the fibrin clot on the root surface is a critical event for new attachment formation. The use of a membrane stabilizes the wound and protects the root surface-adhering fibrin clot from tensile forces acting on the wound margin. It has been observed that collagen is chemotactic for fibroblasts. The collagen membrane barrier may act to enhance and protect the initial clot formation onto the root surface by acting as a scaffold for cell adhesion and in growth. It may also attract fibroblasts to the area, which may aid in the formation of new attachment and regeneration during GTR procedures.[18]

A combination of dexamethasone and platelet derived growth factors (PDGF) in a collagen carrier matrix (CM) has been tested on local experimental periodontitis lesions in monkeys. It was observed that application of PDGF/dexamethasone/CM produced five-fold more new cementum and ligament and seven-fold more supracrestal bone than the control treatments that had a collagen carrier only. CM produced an environment that favored connective tissue formation and acted as a barrier to epithelial migration.[24]

CONCLUSION

Collagen has various advantages as a biomaterial and is widely used as carrier system for delivery of drug, protein and gene. The examples described in this review represent selected applications of collagen in the biomedical field. The successful demonstration of usefulness of human skin substitutes made of collagen has led to the development of bioengineering tissues, such as blood vessels and ligaments. Autologous tissue engineering provides an alternative for allogenic tissue transplantation. The study of native collagen for drug delivery systems and tissue engineering may lead to a better understanding of pathological diseases. It can further provide a new guide for tissue growth and organization, leading to bioactive signals for tissue-specific gene expression. Collagen-based biomaterials are expected to become a useful matrix substance for various biomedical applications in the future.