Proc. btdian Acad. Sci. (Chem. Sci.), Vol. 111, No. 2, April 1999, pp.

319-329
© Indian Academyof Sciences

Biomedical and industrial applications of collagen

C MEENA*, S A MENGI and S G DESHPANDE

CU Shah College of Pharmacy, SNDT Women's University, Santacruz (W),
Mumbai 400 049, India

Abstract. Collagen is the principal structural protein present in the skin, tendon and
bone of the vertebrate body. Ten different vertebrate collagens have been identified, of
which the dominant collagen is type I. Interest in collagen as a biomaterial is due to its
low immunogenicity and high biocompatibility. Collagen has diverse general and
biomedical applications and is also a common constituent of many cosmetic and food
products in the form of gelatin. This paper discusses the use of collagen in the fields
of medicine and biotechnology, as well as in the cosmetic and pharmaceutical
industry.

Keywords. Collagen; gelatin; composites; templates; dressings; shields.

1. Introduction
Collagen is the principal structural protein in the vertebrate body. The extracellular
proteins of the main connective tissues consist of 90% or more collagen in tendon and
bone, and more than 50% in the skin. This protein is absent in single-celled animals and in
plants where cellulose and related polysaccharides serve a similar role. Collagen is also
found in invertebrates in the body walls and cuticles. It is an important natural biological
material and has been extensively studied as a polymer for use in manufactured materials.
Technology involving collagen in the form of leather and gelatin goes back several
millennia. Leather is chemically treated animal skin, while gelatin is animal connective
tissue that is denatured and degraded by heat and chemicals. Both consist largely of
collagen but are very different in chemistry and form. The term 'collagen' usually implies
the collagen present in skin, tendon and bone. At present, ten different vertebrate
collagens have been identified. All collagens contain a unique triple helix. However, the
length of the helix and the nature and size of non-helical portions of the molecule vary
from type to type. The dominant collagen of skin, tendon and bone is type-I collagen.
Type II collagen is essentially unique to cartilage, and type-HI collagen occurs in adult
skin (5-10%) in association with type I and may be a minor contaminant of type I
collagen prepared from this source. The other types occur in small amounts and are
usually associated with specific biological structures i. The safety of collagen for human
use is evidenced by its diverse general and biomedical applications. It is a common
constituent of soaps, shampoos, facial creams, body lotions and other cosmetics and of
food-grade gelatin. In medicine, collagen has been used in cardiovascular surgery, plastic
surgery, orthopedics, urology, neurology and ophthalmology. The major medical

*For correspondence
This paper was part of the collagen symposium organized on the occasion of the Golden Jubilee of
the Central Leather Research Institute, Chennai in January 1999.
319
320 C Meena et al

application of collagen has been as catgut suture, which is derived from intestinal collagen
used to promote wound healing. The attractiveness of collagen as a biomaterial rests
largely on the fact that it is a natural material of low immunogenicity and is therefore
considered as a normal constituent rather than as foreign matter by the body.
Immunogenicity relates to the capacity of the material to elicit the production of
antibodies in animals or humans. Nearly all studies on collagen have shown that it has
very low or no immunogenicity. Of the 10 collagen types that have been characterized,
types I, III and V are the most desirable for biomedical applications because of their high
biocompatibility and low immunogenicity 2 Antibodies to collagen and other proteins are
powerful tools used in the laboratory for highly specific and sensitive assays. The
immunology of collagen is important because animal collagens are sometimes used in
humans and it is necessary to understand the potential immunological consequences. The
term antigenicity refers to the ability of a protein or other substance to elicit production of
antibodies. In general, proteins show two types of antigenic determinants, sequential and
conformational. Sequential determinants depend only on the recognition of an amino acid
sequence of typically 5-10 amino acids. Conformational determinants depend on the
presence of a particular three-dimensional structure. Conformational antigenicity is lost
when the protein is denatured, presumably because more than one region of the
polypeptide chains(s) contributes to the antigenic site. In the case of type-I collagen,
although the triple helix is a poor immunogen, some denaturation may take place in vivo,
which reveals sequential determinants 3"4. However collagen is a weak antigen.
Advantages of its use include - (i) abundant sources of highly purified (medical grade)
collagen, (ii) the ability to be reconstituted into high strength forms useful in surgery, (iii)
the wealth of research literature on the characterization of collagen, (iv) improved
processing techniques, (v) introduction of several commercial collagen products and (vi)
recent advances in the use of collagen as a delivery system.

2. Uses 6f collagen

Collagen has both biomedical and industrial applications. It is mainly t/sed for sutures, as
hemostatic agents and tissue replacements and for sausage casings which are made of
dispersed insoluble collagen extruded as a tube and formed or cut into links as it is filled.
However, the industrial uses of collagen in the form of gelatin and leather are very large.
Collagen is also an ingredient in cosmetics, dental composites, skin regeneration
templates, biodegradable matrices and collagen shields in the field of ophthalmology. It is
also used as solid-support microcarrier in the production of enzymes.
Gelatin, another form of collagen, is produced from the collagen contained in bones
and animal hides and is used as the major component in bioreactors. It is found in two
forms - type A, which is prepared by acid treatment of collagen, and type B, obtained by
alkaline treatment. The alkaline processing of hides as the collagen source requires them
to be soaked for 5-12 weeks in a solution of 5-15% lime. There is also a method now in
which lime is replaced by an alkaline protease and the process time is reduced to 24 h,
followed by the conditioning of the gelatin, which is then extracted into hot water and
dried normally 5.
Collagen conditioning is carried out by a process where skin is first cut into small
pieces and washed, after which 200% by weight of enzymatic conditioners is added and
the pH is adjusted to 9. The mixture is agitated for 6-24 h at 25°C and then decanted and
 Biomedical and industrial applications of collagen 321

washed twice with acidified water at pH 2 to activate the enzyme (30 min contact time).
Finally gelatin (pH 6-7) is extracted at 70°C.
The cosmetic industry uses hydrolysed gelatin as a component of shampoos and
ointments. It is found that use of proteins in cosmetic emulsions results in stable and safe
products 6'8. Stable cosmetic lotions containing collagens have been prepared. These
lotions contain stabilizers 0-5-10%, H20 75-95%, and soluble natural collagen (mol. wt.
1 x 106-3 x 10 6) 0.1-5%, and are stable and non-irritating to skin. Collagens are effective
in penetrating aging skin. A lotion containing soluble natural collagens 0.5, glucose 5,
NaC1 (stablizer) 0.8, Na2CO3 0.02, preservative 0.1 and H20 93-58%, and various
perfumes, is stable at room temperature for up to 6 months 9.

3. Dental composites

In the field of dentistry, advantage is taken of the rapidly expanding family of polymeric
materials. Because of desirable properties that can often be engineered to fit specific
requirements, many materials and devices containing polymeric ingredients are employed
in all aspects of dental practice 7. These requirements include adequate strength,
resilience, abrasion resistance, dimensional stability during processing and subsequent
use, translucency or transparency to enable the appliance to match the appearance of the
mouth tissue that is replaced, good colour stability, resistance to mouth fluid, food or
other substances with which the device may come in contact, tissue tolerance, low toxicity
and ease of fabrication into a dental appliance.
Dental composites consist of a monomer system that provides the matrix, which is a
continuous phase, upon polymerization and reinforcing fillers, which constitute the
dispersed phase, a polymerization-initiator system effective under oral conditions and
various stabilizers for optimizing the storage stability of the ingredients and the chemical
stability of the curved composites. Dental composites are used as two-component
formulations, viz. powder-liquid, paste-liquid, paste-paste, or as uniform paste l°'ll. A
typical paste prepared for dental root canal filling contains collagen 8.2-29.2%,
methyluracil 0.45-0.55%, and Bi subnitrate 1.25-2.25%, in addition to eugenol 28.1-
29-1 wt%, the balance being zinc oxide ~2.

4. Skin regeneration templates

Regeneration templates are polymers of natural origin that induce synthesis of

physiological tissue when brought in close contact with the surface of a surgical lesion
(wound) in living, adult mammalian tissue. Polymers that have shown this unusual
biological activity are copolymers formed by grafting chains from chondroitin 6-sulphate
on collagen. Chondroitin 6-sulphate is a glycosaminoglycan (GAG), whereas collagen is a
fibrous protein 13,14
Polymers of natural origin that function as regeneration templates have so far been
synthesized as graft co-polymers of collagen and chondroitin 6-sulphate. Collagen is a
highly crystalline fibrous protein that reinforces the connective tissues of vertebrates
mechanically. The collagen preparation used to synthesize polymers is a fibrillar
(insoluble) collagen extracted from cowhide using lime treatment, followed by mechanical
fragmentation. Because collagen molecules occur naturally in tissues as covalently
crosslinked networks, the protein is "solubilized" by agents that cause degradation of
chain backbone bonds and crosslinks. This process leads to a non-uniform (polydisperse)
322 C Meena et al

population of macromolecular species. As mentioned earlier, collagen derived from skin,

tendon, and bone is predominantly type I collagen. A template measuring - 400 cm 2 in
area with a thickness of - l mm and a pore volume reaction of ~ 0.99 can be prepared.
Collagen (moisture content - 10 wt%) is pulverized in a mill and 1.65 g is dispersed in
600 ml of 0.05 M acetic acid by stirring in a refrigerated homogenizer for 60 rain. During
this period, collagen fibres swell in the acid solvent along the direction lateral to the fibre
axis and, in the process, the lateral packing order is almost completely erased, while the
triple helical structure remains intact. At the end of the swelling stem, 0.13 g GAG in
120 ml of 0-05 M acetic acid is metered into the collagen suspension and a white collagen
GAG coprecipitate forms. The coprecipitate is an ionic complex that can be resuspended
readily and separated reversibly, either by increasing the ionic strength of the aqueous
component or by increasing the pH from acidic to neutral15 The composition of
collagen-GAG coprecipitates varies with the amount of GAG added to the collagen
dispersion and with the pH. Collagen-GAG coprecipitates are converted into highly
porous forms by freeze-drying (lyophilization). Certain collagen-graft glycosamino-
glycans can induce synthesis of dermis within a wound bed in guinea pigs in which the
epidermis and the complete dermis have been removed by surgical excision. These co-
polymers are also capable of significantly delaying or arresting wound contraction.

5. Stra Cor membranes

Stra Cot membranes are a series of three semiocclusive biodegradable hydrogels formed
by the spontaneous co-precipitation of at least two oppositely charged polyelectrolytes,
i.e. solubilized polymers containing charged backbone or side groups. They are derived
from a group of three bipolymers: keratin, collagen and chitin.
Stra Cor I are obtained from ammonium keratinate and collagen acetate, while Stra Cor
II are obtained from ammonium keratinate and chitosan acetate, and Stra Cor III from
collagen acetate and chitosan acetate crosslinked through ammonium keratinate. These
membranes are truly formulated biplastics.
Collagen forms excellent skin-substitute dressings such as collagen (crosslinked)
sponge. These characteristics account for its salient use for burn dressings as it absorbs
wound exudate, provides cell migratory scaffolding, promotes adherence and helps reduce
bacterial count.
Biodegradable collagen-based matrix in sponge or sheet form, in which carrier
compound (fibronectin, laninin, hyaluronate, antibiotic, etc) is incorporated, has been
obtained by a process, which includes isolation of type I, II, III collagens, mixing them
with liquid medium containing a dispersing agent, and then freeze drying. A crosslinking
agent (carbodimer or a succinimidyl), an active ester, is added either prior to or after
freeze drying ~6.
Bandages containing freeze-dried anthcrocollagens which protect wounds and acce-
lerate healing have also been prepared. Collagens isolated from calf skin are treated with
proteinase to give antherocollagens. An antherocollagen solution of strength 0-5-50% is
applied to a gauze until the thickness of the gel becomes 0.5-1-0 mm. The anthero-
collagens are then neutralized, washed with H20 and freeze-dried to obtain a bandage 17

6. Wound dressings
Wounds to the skin are encountered very often. Devices such as "Band Aid" are used to
assist in wound healing. For deeper wounds a variety of wound dressings have been
 Biomedical and industrial applications of collagen 323

developed including cell-cultured artificial skin. These materials are intended to promote
healing of skin which has been damaged or removed as a result of skin grafting,
ulceration, burns, cancer, excision or mechanical trauma. Synthetic materials in the form
of dressings are used primarily for treatment of superficial wounds. These devices prevent
moisture and heat loss from the skin and also prevent bacterial infiltration from the
environment. Artificial skin is used not only to limit entrance of foreign matter into the
skin and prevent mass and heat transfer out of the skin, but also to provide a continuous
cellular layer over the skin. The objective of the use of any implant material is to restore
the normal skin, structure and physiology.
Collagen fibres provide shape to various parts of the body as well as prevent premature
mechanical failure. Among the various layers of the skin, the papillary dermis consists of
an abundant number of fibroblasts and other cell types, as well as wavy fibres, consisting
of types I and III collagens 18. Skin is a multicomponent composite of cells and
macromolecules. The fibroblast is the cell type which is responsible for synthesizing and
depositing collagen fibres in a continuous network that forms the structural scaffold. A
certain amount of collagen also resides in the dermis along with elastic fibres and small
amounts of proteoglycans. Type I-V collagens are found in the form of fibres. Collagen in
the dermis is known to interact with various other macromolecules including heparin
sulphate proteoglycan, vironectin and fibronectin. The proteoglycans are found attached
to and surrounding collagen fibres in the dermis and are involved in the viscoelastic
behaviour of skin. The collagen molecule by virtue of its inherent stiffness dictates the
upper bounds of the modulus or stiffness of the skin. Observations suggest that the
stiffness of collageneous tissues is a consequence of the triple helical structure and
approaches that of an individual collagen molecule in the dry state.
A wide variety of materials have been used as dressings for burns, skin donor sites,
skin ulcers and excisional sites. These include synthetic barrier dressings, animal and
human tissues, reconstituted collagen and tissue culture-produced "artificial skin".
Microfibrous and microcrystalline collagen I is used in medicine and pharmacy.
Collagen is obtained from animal tissues' such as skin, tendon, and bone in solution form
after acid treatment. The soluble collagen obtained is then precipitated or reaggregated by
dialysis. During reaggregation a net of highly birefringent crystalline fibres are tbrmed.
This can be shaped into 0.5-2 cm sheets, aerated to form sponges or dispersed in creams.
Collagen sheets are sterilized by gamma rays or gas and thus made suitable for use in
surgery or burn treatment. The efficacy of a new biological dressing is studied on
experimental skin excisions and burns in rabbits by comparing it with lyophilised porcine
skin (LPS), gauze dressing and collagen wound dressing (CWS). CWS is found to be
better than gauze in healing 0.02 inches thick skin excision, deep excisions and burns.
CWS adhered well to the wound surface allowing the exudate to be absorbed by the gauze
on top and taken away from the wound surface. It also broke down continuously with time
to form pseudocrest and new tissue to help heal the wound. Thus CWS had good efficacy
for skin excisions and burns 19
We studied the transport of radioactivity from 34-CWS, sterilised by gamma radiation,
which is applied to experimental skin excisions. Patches of skin, 2-5 cm square and 0.02
inches deep, were excised with dermatone on bilateral sites of the back of six rabbits.
Then 34-CWS sheets 3.5 cm square, were applied to each excision with gauze and elastic
bandage. Each animal was kept separately in a metabolic cage where its urine and feces
were collected every 24 h. After the animals are sacrificed on the eighth-day, 34-CWS
gauge, elastic bandage and skin-wear treatment area are collected and the radioactivity is
324 C Meena et al

determined by liquid scintillation after combustion in a sample oxidizer. More than 98.2%
of radioactive substance recovered from 34-CWS after eight days by exudate is negligible
and percutaneous transport of collagen is observed. No radioactivity is detected in rabbit
urine, feces and serum z0.
A dressing for the treatment of leg ulcers and skin sores, which is widely used, consists
of undenatured collagen 98.2-99.6, gentian violet 0.1-0.8, xilina 0.1-0-6 and merthiolate
0.2-0.4 parts by weight. The sponge-like product is packed in polythene bags 21
Biodegradable dermal substitutes as biological dressings are temporary dressings that
are replaced or removed prior to rejection by the recipient. They are chiefly composed of
collagen. A number of research groups isolated collagen from mammalian tissues and
formulated it into cell-free renografts. These materials, after cross-linking to
glucosaminoglycans, mask the antigenic determinants and are not rejected but are
biodegraded and replaced by host tissue. Some of the researchers describe the
physicochemical, biochemical and mechanical considerations that form the basis for the
two-stage design of a bilayer membrane which is useful as an experimental burn dressing.
In stage 1, the basic design parameters chosen are physicochemical and mechanical,
including optimization of surface energy, modulus of elasticity, tear strength and moisture
flux rate. In stage 2, the biochemical constraints, including a very low level of antigenicity
and optimum biodegradation rate without release of toxic products, are the critical
parameters. They chose collagen as a starting material because it is hydrophilic and is
capable of biodegradation at controlled rates by varying the cross-link density. Many
studies on glycosaminoglycans (GAG~)-collagen membranes are being carried out.
Coating the collagen-GAG membrane with a layer of silicone rubber resulted in a bilayer
membrane that in an excised guinea-pig model delayed the onset of contraction without
affecting the final wound area reduction. Work on synthetic wound dressings, especially
the affect of adding GAGs and chemical cross-linking agents are being carried out.
Cross-linking with glutaealdehyde results in internal cyclopolymerization and forms
variable length polymers producing polymeric units that cross-link collagen. Ongoing
studies on collagen-GAG matrices include optimization of average pore diameter,
optimization of conditions for growth of human dermal keratinocytes and maximization
of cell proliferation. Collagen is also used as a biodegradable polymeric scaffolding
material.
Biologically active macromolecular collagen, of mol. wt. of 383,000-460,000, having
no antigenic properties and with improved mechanical properties, is prepared by a process
including saponification and degreasing of natural collagen. One kilogram of raw cowhide
is treated for 48 h with a solution containing Ca(OH)z (150 gm), KOH (50 gm), NaOH
(100 gm), NazSOn (144 gin), NaCI (100 gm), KCI (200 gm) and CaSO4 (100 gin). NazSO4
(144 gm), NaCI (100 gm), KCI (100 gm), CaSO4 (100 gm) and H3BO3 (40 gm) in three
litres of water are dissolved in 1% ascorbic acid solution poured into a mould, frozen at
-60°C and lyophilized. Compounds such as medroxyprogesterone and chloromycetin may
be incorporated into reconstituted collagen 22

7. Surgical dressing
Surgical sutures are sterile filaments used to hold tissues together until they heal
adequately for self support or to join tissues with implanted prosthetic devices. They are
normally attached to needles for coaptation of the edges of wounds or surgical incisions.
As ligatures, they are generally used without a needle, to tie off ends of severed tubular
 Biomedical and industrial applications of collagen 325

structures such as blood vessels and ducts to prevent bleeding or other fluid leakage.
Sutures are characterized according to type of material, physical form, biodegradability,
size, surgical use and other criteria. The materials utilized are natural products such as
surgical catgut or silk, many common and synthetic fibres and some fibres from polymers
which have been synthesized specifically for use as sutures 23.
Properties of surgical suture material are improved by treating the fibre with a solution
containing isopropyl alcohol, water, glycerol and at least one carbohydrate such as
sorbitol or xylitol. A carboxylic acid is added to the solution and the pH is maintained
around 7. Raw catgut fibre of length 75 cm is bound to a needle and packed and sealed in
a PVC film with 5 ml alcoholic solution containing glycerol 3%, sorbitol 6% isopropyl
alcohol 79%, water 11% and succinic acid 0.2% (with diethanolamine for p H 7) and
sterilized with ethylene oxide. On storing for 7 days in a container and then storing it for
3 h at room temperature, these sutures had the following properties, viz, swelling 17,
water content 20% and rigidity 50 mN/cm 224
Collagen fibres that do not form knots or nodes, which are easily processed for surgical
use, are prepared by decomposition of Achilles tendons or cattle hides in alkaline media
and mechanical agitation after acid treatment, followed by deswelling, cross-linking and
drying. Achilles tendons are decomposed in alkaline medium to an amino-N-content of
0.15-0.3 ~tmol/kg and after HCI treatment and washing to pH 2-4, the swollen collagen is
tied in thickness and pressure forming a 2-dimensional network. The individual
longitudinal fibres are then exposed by backling. After deswelling, the fibres are cross-
linked with hexamethylene diisocynate in the presence of nonionic or cationic emulsifier
and are dehydrated and further processed on textile meshes 25.
A sterile collagen product with fleece-like structure is separated from pig skin by
homogenizing and extracting at 40°C with NaCI and a surfactant containing NaN3
preservative, followed by washing and treatment with proteolytic enzymes. The pigskin is
frozen, homogenized and then suspended in 10% NaCI and 10% polyxyethylene-nonyl
phenyl ether containing NaN3 for 2 h and centrifuged. The fibres are washed with 0-1 M
NazHPO4 and then 3% HOAc before suspending it overnight in 3% HOAc containing
pepsin and CHCI3 and finally filtering. The filtrate is adjusted to 5% in NaCI to
precipitate collagen and other residues of pepsin are inactivated with NaOH. The collagen
is washed, desalted and filtered to give disks of fleecy collagen, which are packed in
polythene and sterilized by radiation 26.
Collagen fibres for use as sutures, fleeces and other hemostatics and bandages are also
prepared from bovine Achilles tendons by alkaline swelling, acid treatment, pressing or
rolling to reduce thickness, combing to give individual long fibres, treating with
hexamethylene diisocyanate in the presence of an emulsifier, and finally drying. Bovine
Achilles tendons are stripped of fat and washed with H20, soaked for 8 days at 20 ° in a
solution of Na2SO4 20%, NaOH 2%, and H20 150%, based on weight of tendons, and
pressed between fluted rollers on days 2, 4 and 6. After washing, the tendons obtained
contained IJ-23 mol amide N/gin. The tendons are treated with dilute HCt for 4 h, washed
to pH 2.7-3.3, treated with excess water and put in a hammer press to remove solution
and denatured proteins. The product is combed and the fibres are swollen in NaCI
solution mixed with a nonionic emulsifier and stirred with hexamethylene diisocynate.
The cross-linked material is dehydrated with MezCO and air dried. The fibres can be
processed further to fleeces or sutures 27.
326 C Meena et al

8. Ophthalmic collagen shields

Corneal collagen shields for ocular delivery have also been reported. Bandage soft contact
lenses are made of hydrophilic plastics which are widely used to protect eyes with various
problems, including nonhealing epithelial defects after corneal transplantation or
refractive surgery and recurrent epithelial erosions after ocular herpes infection. Although
these soft contact lenses may enhance healing while allowing the eye to remain open, their
cost is relatively high. The hydrophilic lens is placed on the cornea and the drug is
administered topically onto the surface of the lens. The contact lens is considered as
acting a carrier vehicle, binding the drug and releasing it slowly, thereby increasing
retention of the therapeutic agent in the tear film.
Bloomfield et a128 were the first to suggest that collagen might provide a suitable
carrier for sustained ocular drug delivery. The study showed that wafer-shaped collagen
inserts impregnated with gentamycin produced the highest levels of drug in the tear film
and tissue in the rabbit eye, compared to drops, ointments or subconjuctival infection.
In the manufacture of collagen shields, the ability to control the amount of cross-
linking in the collagen subunits by exposure to UV light is an important physicochemical
property because the amount of cross-linking is related to the dissolution time of the
shield on the cornea. The collagen shield is designed to be a disposable, short-term
therapeutic bandage lens for the cornea. It conforms to the shape of the eye, protects the
corneal surface, and provides lubrication as it dissolves. Commercially available shields
are made of porcine scleral tissue and also bovine corium tissue.
Thus it is confirmed that collagen is a protein that can be safely applied to the body for
a variety of medical and cosmetic purposes. The simplicity of use and the convenience
afforded by shields make them an attractive delivery device.

9. Porous collagen microcarriers

Besides these medical and cosmetic uses, collagen is also used in the field of
biotechnology. Highly purified vesicular plant parenchyma material covalently coated
with native collagen can be used as a new type of surface-carrier in animal cell culture.
The carrier material consists of void cellular aggregates with polydisperse diameter
distribution. The highly negative charge of the genuine plant cells surface remains
unchanged unless the charge modifies the effect of the overlaying collagen material.
Different permanent and primary cell lines are grown on this new carrier material. The
basic carrier material (to be collagenated) is derived from freshly harvested cell cultures
from a parenchymatic chenopodium alba cell culture. The material, purified without
alteration of its mechanical and chemical structure, has been used for the first time as
vesicular packing material in chromatography 29.
The chemical modification of collagenated material is a two-step procedure. The
collagenated material is carefully washed, suspended in 0-9% w/w NaCI solution and
finally gamma-sterilised.
The increased use of large-scale animal cell cultures for the production of biologicals
has focussed attention on the economies inherent in high cell density systems. To maintain
high cell concentrations it is necessary to continuously feed the cells by perfusion of
medium. A variety of methods have been developed to retain cells in bioreactors. Ideally
the cells should be maintained in an environment appropriate for growth and product
formation and the system should be readily scaled up. Suspension cultures satisfy these
 Biomedical and industrial applications of collagen 327

criteria but shear effects limit aeration at high cell concentrations. Porous microcarriers
provide an attractive solution to the problem of cell retention for large-scale cell culture.
Porous microcarrier systems can be readily scaled-up in stirred or airlift bioreactors.
Cultispher-G, void volume 50 is made of gelatin and is widely used 30.
Fluidized-bed bioreactor perfusion culture has been used for the production of
therapeutic proteins. Porous collagen microspheres have been developed which are
suitable for both attachment-dependent cells and suspension cells. Advantages of this
macrosporous microsphere cell culture in a fluidized bed reactor are the increased matrix
surface area and open volume resulting in high cell density and efficient mass transfer of
nutrients and transport of secreted products into the harvest.
Culture productivity of a culture process is the result of the interplay between cells,
culture medium, cell support matrix and the bioreactor and process parameters.
Considering these factors that govern culture performance, an innovative high cell density
culture is developed for the mass culture of both suspension cells and anchorage
dependent cells. Employing low-protein and serum-free media, perfusion cultures of
rodent and human hybridaux cells and recombinant mammalian cells have been carried
out. Porous microsphere cell cultures in fluidized bed bioreactors provide increased
matrix surface area of open volume for cell attachment and proliferation to very high cell
densities, thus optimizing cell-cell and cell-matrix interactions. It allows efficient mass
transfer of nutrients through microsphere fluidization resulting in an enhanced diffusive
microenvironment and transport of secreted products and culture metabolites to the
outside of the microsphere and protection of cells from shear and mechanical damage.
The production of high quality antibodies and recombinant proteins in this culture
process is the combined result of the use of collagen microspheres enabling the use of
low-protein media, short residence time of product in the culture system after its secretion
from the cells, very high cell density in the matrix environment, high mass transfer and
efficient substrate utilization for the synthesis of the secreted product 3~.

10. Sustained release preparations

Prolonged sustained release preparations in the shape of bars or needles are useful for
injection or for implantation in the body. They consist of an active ingredient admixed
with 1 or more biodegradable carriers which can be either adsorbed or be subjected to
enzymolysis in the body. Thus, a needle-shaped preparation which contained
indomethacin 4 g is admixed with 1% aqueous solution of antherocollagen (200 g),
concentrated to = 200 ml. The mixture is then poured into a silicone tube, the tube frozen
and cut and the product cross-linked with glutaraidehyde in a gaseous phase for 4 days. In
a release test by the USP basket method, the needle shaped-preparation released the drug
for 10 days.
Sustained-release formulations containing neoplasm inhibitors and polymeric carvers
are formulated. Absorbable polymers are formulated with neoplasm inhibitors the release
to drug slowly in patients. Besides collagen, polyvinyl alcohol, gelatine, starch,
poly(lactic acid) and poly(p-propiolactone) are also employed in the formulations 3z.

11. Conclusion

Besides the leather industry, collagen has a wide array of uses in the field of cosmetics,
surgery, dentistry, ophthalmic drug delivery and sustained drug delivery, as well as in the
328 C Meena et al

Table 1. Biomedical and industrial uses of collagen.

Application Form of collagen

Hemostatic agents Powder, sponge, fleece

Blood vessels Processed human/animal blood vessels,
reconstituted fibrillar collagen
Heart valves Processed porcine heart valve
Tendons and ligaments Processed animal tendon, collagen-carbon
fibre composite
Burn dressings Processed animal skin sponge, composites
Intradermal augmentation Reconstituted fibrillar collagen
Drug delivery systems Various biodegradable forms

field of biotechnology. With chemical modification it has the potential of that o f any other
synthetic polymeric material. Being an endogeneous body protein with low or absolutely
no immunogenecity, collagen seems to be a very promising material for a wide range o f
applications. The biomedical and the industrial uses of collagen can be summarised as in
table 1.

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