Collagen: The Biomaterial

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. 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.

The main applications of collagen are for burn/wound cover dressings, osteogenic and bone filling
materials, anti-thrombogenic 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.

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 favourable
environment for periodontal and peri-implant regeneration.

There is a great need in medicine for biomaterials that are versatile and compatible with human
tissues. This need continues despite recent activity in developing and testing a variety of synthetic
polymers. Clinical needs include implantable devices, such as blood vessels or heart valves, and
extracorporeal ones, including artificial kidneys and heart-lung machines. Table I indicates the
general categories of materials that have been used as artificial structures of various kinds. This
review is concerned with a naturally occurring biopolymer, collagen, and its use as a biomaterial.
Collagen is the major connective tissue protein in animals, accounting for about 25% of the total
body protein of vertebrates. An essential feature of this type of biomaterial is that it degrades in
biological environments, eventually disappears, and leaves no permanent foreign residue. A very
practical aspect of the use of collagen is its ease of preparation and its widespread occurrence in
nature. One of the underlying hypotheses in collagen research, as related to biomaterials, is that
evolutionary bioengineering has produced a material that has ideal properties for biologic
applications. Knowledge of the structure and interactions of this material could lead to the
preparation of a unique class of biomaterials. These biomaterials should gradually be metabolized
and replaced by host tissues.
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 fibres. It was
Ramachandran's group from Madras, India, who first postulated a triple helical structure for
collagen.

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.

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 fibres leads to different diseases associated with connective tissues, such
as Ehlers-Danlos syndrome, osteogenesis imperfecta and some types of osteoporosis and arthritis.

The basic molecular subunit of collagen-tropocollagen-is a rigid rod with a molecular weight of
300,000, a length of 2800 A, and a width of 15 A. Collagen molecules are organized in tissues in a
precise way, with each molecule aligncd with its neighbor along about one-fourth of its length.
Native collagen stained with phosphotungstic acid reveals a characteristic band pattern when
studied by electronmicroscopy with a repeat of 690 A -approximately one-fourth the length of the
entire molecule. This type of packing undoubtedly adds strength and resiliency to collagenous
tissues such as skin, tendon, and bone. Actually, the repeat is not quite one-fourth the length of
tropocollagen, indicating the presence of repeating "hole" areas, or areas of decreased molecular
density. These may be sites for enucleation of bone or for large prosthetic groups such as
glycoproteins. The greater part of native collagen is insoluble and only 2-3% can be solubilized in
dilute acid solutions. This acid-soluble collagen is the material used in most studies pertaining to
collagen structure. Most of the insoluble collagen can, however, be solubilized with proteolytic
enzymes, a critical factor in the use of collagetlas a biomaterial. The asymmetry of tropocollagen
leads to high viscosities of dilute collagen solutions and a highly positive flow birefringence. A strong
negative rotation of polarized light indicates that tropocollagen has a high content of helical areas.
The subunit structure of tropocollagen consists of three polypeptide chains, each with a molecular
weight of 95,000-100,000, wound together in a triple helix of the polyproline type II. Each chain is
wound in a left-handed helix with a pitch of 9.5 A, and the three subunit chains coil around each
other in a right-handed superhelix with a pitch of 104A. This tertiary structure is stabilized primarily
by hydrogen bonding between adjacent chains. These weak bonds can be broken, and the molecule
denatured, by heating in dilute acid solution to 38-40°C, or by reagents, such as KSCN, that disrupt
hydrogen bonds. Denatured collagen polypeptides assume a random coil configuration, and this
material is gelatin. By chromatographic or ultracentrifugal methods, however, it can be shown that
two or all three of the subunit polypeptide chains may be covalently bound. Collagen with no
interchain crosslinks is known as collagen, with two chains covalently bound, f3 collagen, and with all
three covalently linked together, y collagen.

Applications
Film
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. 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. 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.
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 atelo collagen matrix
added on the surface of the polyurethane films, enhanced attachment and proliferation of
fibroblasts and supported growth of cells. 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. 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. 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.
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. 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. 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.

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. 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. 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. 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.

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. 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
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, 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.

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.

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