Biomedical Applications of Collagen

International Journal of Pharmaceutics 221 (2001) 1–22

Review

Biomedical applications of collagen

Chi H. Lee a,*, Anuj Singla a, Yugyung Lee b

a Department of Pharmaceutics, College of Pharmacy, The Uni�ersity of Missouri-Kansas City, 5005 Rockhill Rd, Katz Bdg c108,Kansas City, MO 64110, USA

b School of Interdisciplinary Computing and Engineering, The Uni�ersity of Missouri-Kansas City, Kansas City, MO 64110, USA

Received 5 January 2001; received in revised form 26 March 2001; accepted 3 April 2001

Abstract

Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and safety due to itsbiological characteristics, such as biodegradability and weak antigenecity, made collagen the primary resource inmedical applications. The main applications of collagen as drug delivery systems are collagen shields in ophthalmol-ogy, sponges for burns/wounds, mini-pellets and tablets for protein delivery, gel formulation in combination withliposomes for sustained drug delivery, as controlling material for transdermal delivery, and nanoparticles for genedelivery and basic matrices for cell culture systems. It was also used for tissue engineering including skin replacement,bone substitutes, and artificial blood vessels and valves. This article reviews biomedical applications of collagenincluding the collagen film, which we have developed as a matrix system for evaluation of tissue calcification and forthe embedding of a single cell suspension for tumorigenic study. The advantages and disadvantages of each systemare also discussed. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Collagen; Biomaterial; Drug/protein/gene delivery; Tissue engineering

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1. Introduction

Collagen, a well-known protein, has beenwidely used in medical applications. Many naturalpolymers and their synthetic analogues are usedas biomaterials, but the characteristics of collagenas a biomaterial are distinct from those of syn-

thetic polymers mainly in its mode of interactionin the body (McPherson et al., 1986). Collagenplays an important role in the formation of tissuesand organs, and is involved in various functionalexpressions of cells. Collagen is a good surface-ac-tive agent and demonstrates its ability to pene-trate a lipid-free interface (Fonseca et al., 1996).Collagen exhibits biodegradability, weak anti-genecity (Maeda et al., 1999) and superior bio-compatibility compared with other naturalpolymers, such as albumin and gelatin.

* Corresponding author. Tel.: +1-816-2352408; fax: +1-816-2355190.

E-mail address: [email protected] (C.H. Lee).

0378-5173/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S0378-5173(01)00691-3

C.H. Lee et al. / International Journal of Pharmaceutics 221 (2001) 1–222

The primary reason for the usefulness of collagenin biomedical application is that collagen can formfibers with extra strength and stability through itsself-aggregation and cross-linking. In most of drugdelivery systems made of collagen, in vivo absorp-tion of collagen is controlled by the use of crosslink-ing agents, such as glutaraldehyde (Barbani et al.,1995), chromium tanning (Bradley and Wilkes,1977), formaldehyde (Ruderman et al., 1973),polyepoxy compounds (Tu et al., 1993), acyl azide(Petite et al., 1990), carbodiimides (Nimni et al.,1988), and hexamethylenediisocyanate (Chvapil etal., 1993). Physical treatment, such as ultra-violet/gamma-ray irradiation and dehydrothermal treat-ment, have been efficiently used for the introduc-tion of crosslinks to collagen matrix (Harkness1966; Stenzel et al., 1969; Miyata et al., 1971;Gorham et al., 1992). The thiolation of denaturedcollagen, which allows precise amounts of SHgroups to be attached onto the protein backbone,was also investigated (Nicholas and Gagnieu,1997). It was reported that, under optimized condi-tions, oxidized and denatured thiolated collagenfilms are more resistant and rigid than glutaralde-hyde treated ones.

The use of collagen as a drug delivery system isvery comprehensive and diverse. Collagen can beextracted into an aqueous solution and molded intovarious forms of delivery systems. The main appli-cations of collagen as drug delivery systems arecollagen shields in ophthalmology (Kaufman et al.,1994), sponges for burns/wounds (Rao, 1995),mini-pellets and tablets for protein delivery (Lucaset al., 1989), gel formulation in combination withliposomes for sustained drug delivery (Fonseca etal., 1996), as controlling material for transdermaldelivery (Thacharodi and Rao, 1996), andnanoparticles for gene delivery (Rossler et al.,1995). In addition, its uses as surgical suture (Milleret al., 1964), hemostatic agents (Cameron, 1978;Browder and Litwin, 1986), and tissue engineeringincluding basic matrices for cell culture systems(Kemp, 2000) and replacement/substitutes for ar-tificial blood vessels and valves (Chvapil et al.,1993; Auger et al., 1998; Huynh et al., 1999) werereported earlier.

Due to its excellent biocompatibility and safety,the use of collagen in biomedical application has

been rapidly growing and widely expanding tobioengineering areas. However, some disadvan-tages of collagen-based systems arose from thedifficulty of assuring adequate supplies, their poormechanical strength, and ineffectiveness in themanagement of infected sites (Friess, 1998). Im-provement of the physical, chemical and biologicalproperties will be necessary to address some ofdrawbacks in collagen based applications. Thebetter collagen delivery systems having an accuraterelease control can be achieved by adjusting thestructure of the collagen matrix or adding otherproteins, such as elastin, fibronectin or gly-cosaminoglycans (Doillon and Silver, 1986; Lefeb-vre et al., 1992, 1996). A combination of collagenwith other polymers, such as collagen/liposome(Kaufman et al., 1994; Fonseca et al., 1996) andcollagen/silicone (Suzuki et al., 2000), has beenproposed to achieve the stability of a system andthe controlled release profiles of incorporated com-pounds.

The purpose of this article is to review biomedicalapplications of collagen including the collagen film,which we have developed as a matrix system forevaluation of tissue calcification and controlleddelivery of cardiovascular drugs. This collagenmatrix system further serves as the substrate for theembedding of a single cell suspension or a smalltumor specimen in the evaluation process of cancertherapy. The advantages and disadvantages of eachsystem are also discussed.

2. Characterization of collagen as a biomaterial

Collagen is the primary structural material ofvertebrates and is the most abundant mammalianprotein accounting for about 20–30% of total bodyproteins (Harkness, 1961). It is present in tissues ofprimarily mechanical function. About one half ofthe total body collagen is in the skin and about 70%of the material other than water present in dermisof skin and tendon is collagen. Collagen made itsappearance at the early stage of evolution in suchprimitive animals as jellyfish, coral and seaanemones (Bergeon, 1967). Collagen is synthesizedby fibroblasts, which usually originate frompluripotential adventitial cells or reticulum cells.

C.H. Lee et al. / International Journal of Pharmaceutics 221 (2001) 1–22 3

The molecular structure of collagen has beenfirmly established on the evidence from earlierstudies, such as amino acid composition analysis,X-ray diffraction analysis, electron microscopyand physicochemical examination of solutions(Gross, 1963; Ramachandran and Sasisekharan,1965; Burge, 1964). Collagen has a uniquestructure, size and amino acid sequence (Miyataet al., 1992; Rao 1995). The collagen moleculeconsists of three polypeptide chains twinedaround one another as in a three-stranded rope.Each chain has an individual twist in the oppositedirections. The principal feature that affects ahelix formation is a high content of glycine andamino acid residues (Piez, 1984). The strands areheld together primarily by hydrogen bonds be-tween adjacent �CO and �NH groups, but also bycovalent bonds (Harkness, 1966). The basic colla-gen molecule is rod-shaped with a length and awidth of about 3000 and 15 A� , respectively, andhas an approximate molecular weight of 300 kDa(Traub and Piez, 1971; Nimni and Harkness,1988).

At least 19 types of collagen have been re-ported. Types I, II and III collagen as well astypes V and XI are built up of three chains and allare composed of the continuous triple-helicalstructure. Types I, II, III, and V are called as fibrilforming collagens and have large sections of ho-mologous sequences independent of species(Timpl, 1984). In type IV collagen (basementmembrane), the regions with the triple-helical conformation are interrupted with largenon-helical domains as well as with the shortnon-helical peptide interruption. Fibril associatedcollagens (Type IX, XI, XII and XIV) havesmall chains, which contain some non-helical do-mains. Type VI is microfibrilla collagen and typeVII is anchoring fibril collagens (Samuel et al.,1998).

An investigation on the native collagen has ledto a better understanding of a structure functionrelationship between target drugs and collagen. Athree-dimensional (3-D) model of fibril-forminghuman type II collagen was proposed for thedevelopment of synthetic collagen tissues and thestudy of the structural and functional aspects ofcollagen (Chen et al., 1995). This system also

allows the studies of the stereochemistry of all theside chain groups and specific atomic interactions,and further evaluation of its therapeutic effects oncollagen related diseases. The orderly arrange-ment of triple helix tropocollagen molecules re-sults in a formation of fibrils having a distinctperiodicity. Nonhelical telopeptides are attachedto both ends of the molecule and serve as themajor source of antigenecity. Atelocollagen,which is produced by elimination of the telopep-tide moieties using pepsin, has demonstrated itspotential as a drug carrier, especially for genedelivery (Kohmura et al., 1999; Ochiya et al.,1999).

Table 1 summarizes the major characteristics ofcollagen, which are suitable for medical applica-tions (Jerome and Ramshaw, 1992; Rao, 1995;Friess, 1998; Fujioka et al., 1998; Maeda et al.,

Table 1Advantages and disadvantages of collagen as a biomateriala

Ad�antagesAvailable in abundance and easily purified from living

organisms (constitutes more than 30% of vertebratetissues);

Non-antigenic;Biodegradable and bioreabsorbable;Non-toxic and biocompatible;Synergic with bioactive components;Biological plastic due to high tensile strength and minimal

expressibility;Hemostatic — promotes blood coagulation;Formulated in a number of different forms;Biodegradability can be regulated by cross-linking;Easily modifiable to produce materials as desired by

utilizing its functional groups;Compatible with synthetic polymers;

Disad�antagesHigh cost of pure type I collagen;Variability of isolated collagen (e.g. crosslink density, fiber

size, trace impurities, etc.);Hydrophilicity which leads to swelling and more rapid

release;Variability in enzymatic degradation rate as compared with

hydrolytic degradation;Complex handling properties;Side effects, such as bovine spongeform encephalopathy

(BSF) and mineralization

a Sources; Jerome and Ramshaw, 1992; Rao, 1995; Friess,1998; Fujioka et al., 1998.


1999). It is easily absorbable in the body and hasvery low antigenicity. It has high tensile strengthand high affinity with water. Moreover, it is non-toxic, biocompatible and biodegradable (Friess,1998; Maeda et al., 1999). It can be prepared in anumber of different forms including strips, sheets,sponges and beads. Collagen can be solubilizedinto an aqueous solution, particularly in acidicaqueous media, and can be engineered to exhibittailor-made properties. Collagen is relatively sta-ble due to its function as the primary structuralprotein in the body, but it is still liable to col-lagenolytic degradation by enzymes, such as colla-genase and telopeptide-cleaving enzymes(Woolley, 1984). Collagenase binds tightly totriple helices, and degrades collagen starting fromthe surface. The melting profile of the pepsin-solu-bilized collagen showed a biphasic transition, in-dicating that age-related decrease in thermalstability has implications for the mechanicalstrength and turnover of the bone collagen(Danielsen, 1990).

Reports of adverse reactions to collagen havebeen restricted to localized redness and swellingfollowing plastic surgery using collagen implantsand wound breakdown with the use of catgutsuture material (Webster et al., 1984; Carrol,1989). Clinical reactions to collagen were rare, buttwo cases of allergic (IgE-mediated) reactions tobovine collagen were reported (Mullins et al.,1996). Patients in both cases developed conjunc-tive edema in response to the topical applicationof highly purified bovine collagen to eye duringophthalmic surgery. When irritant effects and cy-totoxicity of various products developed fromcollagen were evaluated, cell response to exoge-nous collagen starts shortly after the material iskept in contact with tissues, evoking a local andfast inflammatory response, whose intensity de-pends on the pharmaceutical formulation in use(Trasciatti et al., 1998). Even though, unknownreverse effects may be discovered in the futureapplication of collagen for gene delivery or tissueengineering, since collagen can help to avoid sideeffects originated from incorporated drugs, thera-peutic peptides and proteins, the need for thecontinuous effort in development and evaluationof collagen based systems is manifest.

3. Collagen-based drug delivery systems

3.1. Film/sheet/disc

The main application of collagen films is asbarrier membrane. Films with the thickness of0.01–0.5 mm and made of biodegradable materi-als, such as prepared from telopeptide-free recon-stituted collagen, demonstrated a slow releaseprofile of incorporated drugs (Rubin et al., 1973).The drugs can be loaded into collagen membranesby hydrogen bonding, covalent bonding or simpleentrapment. They can be sterilized and becomepliable upon hydrolyzation, while retaining ade-quate strength to resist manipulation. When colla-gen film was applied to eye, it was completelyhydrolyzed after 5–6 h (Bloomfield et al., 1978).This finding adds to evidence that collagen-basedsystems are suitable for resembling current liquidand ointment vehicles.

Collagen film/sheet/disc have been used for thetreatment of tissue infection, such as infectedcorneal tissue or liver cancer. Soluble ophthalmicinsert in the form of a wafer or a film wasintroduced as a drug delivery system for the treat-ment of infected corneal tissue using a high doseof antibiotic agents, such as gentamicin(Bloomfield et al., 1978) and tetracycline (Minabeet al., 1989a). The wafer route of administrationgave the highest tissue concentration of incorpo-rated drugs. The duration of therapeutic effectafter administration of the collagen film contain-ing tetracycline demonstrated that tetracyclinecould be detected in the plasma for more than 7days after implantation into rabbits (Minabe etal., 1989a,b). The microfibrous collagen sheets asa local delivery carrier for the treatment of cancerwas evaluated (Sato et al., 1996). The local appli-cation of collagen sheets loaded with anticanceragent, ectopocide (VP-16), resulted in a relativelylong maintenance of drug concentrations at thetarget site, which, in this case, is the liver.

Some modifications on collagen film/sheet/dischave been made to control the release rate ofincorporated drugs from delivery systems. Colla-gen films crosslinked by chromium tanning, form-aldehyde or a combination of both have beensuccessfully used as implantable delivery systems


in achieving the sustained release of medrox-yprogesterone acetate (Bradley and Wilkes, 1977).A modification by attaching another membrane tocollagen based film/sheet/disc was attempted toachieve the controlled release rate of incorporateddrugs. A transdermal delivery device containingnifedipine, whose release rate was well controlledby an attached membrane made of chitosan,showed the highest therapeutic efficacy in thetreatment of tissue infection (Thacharodi andRao, 1996).

Collagen film and matrix were used as genedelivery carriers for promoting bone formation. Acomposite of recombinant human bone morpho-genetic protein 2 (rhBMP-2) and collagen wasdeveloped to monitor bone development and ab-sorbent change of carrier collagen (Murata et al.,1999, 2000). The rhBMP-2/collagen onlay implantresulted in active bone formation, whereas thecollagen alone resulted in no bone formation.Collagen provides an anchorage for cell differenti-ation and remains as an artificial matrix in wovenbone. In a similar study, collagen matrix loadedwith BMP and placed in a close contact withosteogenic cells achieved direct osteoinductionwithout causing a cartilage formation (Nakagawaand Tagawa, 2000). These results indicate that acollagen based gene delivery system is very effi-cient as a biological onlay implant.

Collagen film and disc as gene delivery systemshave many advantages. Systems that isolate trans-planted cells from the host immune system mightbe beneficial and economically attractive, becausethey would allow the use of allogenic or evenxenogenic cells in many patients (Liu et al., 1993;Al-Hendy et al., 1996; Tani et al., 1989; Braukeret al., 1995). The use of genetically modified cellsfor a long-term delivery of a therapeutic transgeneproduct has been an attractive option for thetreatment of monogenetic hereditary as well asmany multifactorial nongenetic diseases (Barr andLeiden, 1991; Scharfmann, et al., 1991; Heartlein,et al., 1994). Biodegradable collagen films or ma-trices have served as scaffolds for a survival oftransfected fibroblasts (Rosenthal and Kohler,1997).

While these methods seem to allow an adequatecell survival, the concerns about long-term bio-

compatibility of non-degradable materials alsoarose. In several animal models, a long-term ex-pression of a foreign gene after implantation oftransfected cells has not been achieved (Aebischeret al., 1996). A combination of collagen and otherpolymers, such as atelocollagen matrix added onthe surface of polyurethane films, enhanced at-tachment and proliferation of fibroblasts and sup-ported growth of cells (Park et al., 2000).Transplantation of cells embedded in a lattice ofpolytetrafluoroethylene fibers coated with rat col-lagen followed by a mixing with matrigel as wellas basic fibroblast growth factor showed that along-term expression of human �-glucuronidaseby retroviral-transduced murine or canine fibrob-lasts was achievable using a collagen-based matrixsystem. (Moullier et al., 1993a,b, 1995). There-fore, collagen-based film/disc systems seem toneed to contain extra matrices that improve con-ditions for a long-term cell survival.

3.2. Collagen film as a calcifiable matrix system

One of the problems with implantable biomate-rials is their calcification, which is influenced bystructure of the implantable system, and deter-mines its in vivo therapeutic efficiency and clinicalfate (Schoen et al., 1992). Calcification of tissue orsystems depends on chemical factors that operateat the cellular level around various tissues orbiomaterials (Wada et al., 1999). Both collagenand elastin are major components of connectivetissues, which possess a structure that compro-mises collagen fibers intimately associated with aremarkably stable elastin network. The suitabilityof collagen and elastin in many potential medicalapplications in reconstructive and plastic surgeryincluding controlled delivery of bone morpho-genetic protein has been reported (Vardaxis et al.,1996).

In our laboratory, the matrix films, which arecomposed of various combinations of collagenand elastin, have been developed and evaluatedfor its uses in tissue calcification and as a con-trolled delivery device for cardiovascular drugs.Collagen films, which we have developed, wereused to simulate the calcification process of im-plantable biomaterials, such as bioprosthetic heart


valve (BHV). In BHV, the aortic wall and leaflethave mainly served as calcifiable matrix. Recently,the calcification of the bioprosthetic aortic wallhas been extensively studied due to increasing useof the stentless BHV. The stentless BHV has arelatively large segment of exposed aortic wallcomparing to the stented valve, in which virtuallyno aortic wall is exposed outside the stent. Thus,bioprosthetic aortic wall calcification has a poten-tial to cause the clinical failure of the stentlessBHV. Aortic wall is composed of 90% collagenand 10% of elastin, while leaflet is mainly com-posed of collagen (99%). Due to their differencesin composition, the calcification rates of aorticwall and leaflet and their response to anti-calcifi-cation agents are different. For example, ethanolpretreatment completely inhibited calcification ofporcine leaflet, while only partially (about 50%)inhibited calcification of porcine aortic wall (Leeet al., 1998). This may indicate that elastin has amore critical role in tissue calcification than colla-gen. This result has led to investigation of theelastin concentration effects on the calcificationrate of implantable biomaterials.

Collagen films made of various combinations ofcollagen and elastin were evaluated for their suit-ability as drug delivery systems. Biomaterialsshould possess mechanical properties capable ofwithstanding the forces and motions experiencedby the normal tissues and have sufficient fatiguestrength to ensure a long life of the implant invivo (Meaney, 1995). Tensile strength can be usedfor evaluation of mechanical strength, resilientactivity, endurance and biocompatibility of thesystems. Collagen films, we have developed, has asize of 6×10 mm and a thickness of 1 mm. Itstensile strength was determined using the follow-ing equation,

Tensile strength (�)=Force or load (F)

MA

where F is the maximum load (in Newton) andMA is the minimum cross-sectional area of thefilm specimen (in square meters) (Fell and New-ton, 1970). Chatillon DFM-10 Gauge equippedwith LTC Manual Test Stand and GF-1 Grips(Ametek Test & Calibration Instruments Divi-sion, Largo, FL) was used. The tensile strength

Table 2The tensile strength of films containing various combinationsof collagen and elastina

FILM Composition Tensile Strength(Collagen:Elastin) (MPa�S.D.)

2.9�0.290:1050:50 3.0�0.3

3.1�0.420:80

a N=6 each. Values are not significantly different.

was almost constant irrespective of a loading ratioof collagen and elastin and within a durable rangeas shown in Table 2. There was no sign ofswelling of the system, which is a sensitive markerof collagen deterioration (Bank et al., 2000). Thissystem was well endurable after subcutaneous im-plantation in the rat for up to three weeks and noside effect or infection was detected. As shown inFig. 1, as the concentration of elastin in a systemincreased from 10 to 90%, the total amount ofcalcium accumulation in a system implanted inthe rat subcutaneous model also increased. Thisresult indicates that elastin has a more critical rolein tissue calcification than collagen and that thissystem can be used as a calcifiable matrix simulat-ing calcification process of implantable tissues,such as BHV. This system can be further applica-ble to local and systemic delivery of various anti-calcification agents, cardiovascular drugs andantibiotics for the treatment of heart diseases.

The collagen-based matrix, we have developed,can be used as a lattice for tissue culture systems.In the collagen based culture system, a film pre-pared from collagen fiber provides the substratefor the embedding of a single cell suspension or asmall tumor specimen. Three-dimensional aggre-gates can be kept viable and proliferating forweeks, demonstrating very good degrees of resem-blance compared with the in vivo tumors fromwhich they were derived. The matrix remainedstable in shape and size during the cell culture.The three-dimensional models show more resis-tance to treatment with anticancer drugs thancells growing as monolayers. They can serve asbetter models for the biological and biochemicalcharacteristics of human solid tumors than mono-layer subconfluent cell cultures. Collagen-based


matrix cultures offer unique opportunities for thestudy of the mechanisms behind human tumorprogress.

3.3. Collagen shields

The collagen shield was originally designed forbandage contact lenses, which are gradually dis-solved in cornea (Wedge and Rootman, 1992).The idea of using a shield or a hydrogel lens as adelivery device has led to the development ofvarious drug delivery systems for ophthalmic ap-plications. One of the merits of the collagen-baseddrug delivery systems is the ease with which theformulation can be applied to the ocular surfaceand its potential for self administration (Friedberget al., 1991; Lee, 1990). The collagen cornealshield is fabricated from porcine sclera tissue thatclosely resembles collagen molecules of the humaneye (Harrison, 1989). The mechanical propertiesof the shield protect the healing corneal epithe-lium from the blinking action of the eyelids(Mondino, 1991). The collagen corneal shieldwould promote epithelial healing after cornealtransplantation and radial keratomy (Robin et al.,1990; Shaker et al., 1989; Marmer 1988; Poland

and Kaufman, 1988; Waltman and Kaufman,1970; Unterman et al., 1988).

Drug delivery by collagen shields depends onloading and a subsequent release of medication bythe shield (Leaders et al., 1973). The collagenmatrix acts as a reservoir and the drugs are en-trapped in the interstices of the collagen matrix ina solution for water-soluble drugs or incorporatedinto the shield for water-insoluble drugs. As tearsflush through the shield and the shield dissolves, itprovides a layer of biologically compatible colla-gen solution that seems to lubricate the surface ofthe eye, minimize rubbing of the lids on thecornea, increase the contact time between thedrug and the cornea, and foster epithelial healing(Kaufman, 1988; Kaufman et al., 1994). A bolusrelease of drug from the lenses was attributable tothe enhanced drug effect (Waltman and Kaufman,1970; Podos et al., 1972). Therefore, this systemallows the higher corneal concentrations of drug,and the more sustained drug delivery into thecornea and the aqueous humor.

As shown in the Table 3, collagen shields wereused as delivery devices for the treatment of vari-ous local infections and their therapeutic effectswere compared with the conventional formula-

Fig. 1. The Effect of ethanol or gramicidin on the calcification rates of porcine aortic wall implanted in rat subcutaneous models(N=6).


Table 3The application of collagen shields for various topical agents

ReferenceDrugs/Agents

AntibioticsBloomfield et al., 1978; Baziuk etGentamicinal., 1992; Liang et al., 1992; Milanet al., 1993

Vancomycin Phinney et al., 1988Tobramycin O’Brien et al., 1988; Poland and

Kaufman, 1988; Unterman et al.,1988; Aquavella et al. 1988; Hobdenet al., 1988; Sawusch et al., 1988a,b;Assil et al., 1992Dorigo et al., 1995NetilimycinPalmer and McDonald, 1995Polymyxin B SulfatePalmer and McDonald, 1995Trimethoprim

AmphotericinB Schwartz et al., 1990; Mendicute etal., 1995

Trifluorothymidine Gussler et al., 1990Willey et al., 1991AcyclovirTaravella et al., 1999Ofloxacin

SteroidsAquavella et al., 1988; Sawusch etal., 1988a,b; Hwang et al., 1989;Milan et al., 1993; Palmer andMcDonald, 1995

Cholinergic:Aquavella et al., 1988Pilocarpine

Antineoplastic:Finkelstein et al., 19915-fluorouracil

Anticoagulant:Murray et al., 1990Heparin

ImmunodepressantCyclosporin Chen et al., 1990; Reidy et al., 1990;

Sato et al., 1996; Gebhardt andKaufman, 1995

Gene therapyPlasmid DNA Angella et al., 2000

et al., 1990), and ofloxacin (Taravella et al., 1999)to the anterior segment. Delivery of drugsthrough the impregnated collagen shield was morecomfortable and reliable than frequent applica-tion of other conventional treatments, such asdrops, ointment or daily subconjunctive injection(Friedberg et al., 1991).

An application of collagen shield was extendedto gene delivery area. Delivery of plasmid DNAinto the bleb through a collagen shield increasedchloramphenicol acetyltransferase, the reportergene, 30-fold over injection of plasmid DNAthrough saline vehicle (Angella et al., 2000). Genetherapy using naked plasmid DNA and a simplecollagen shield delivery was very useful for regu-lating wound healing after glaucoma surgery.

Modifications of collagen were made to sim-plify the application, to meet the highest compli-ance, to reduce blurring of vision, and to enhancethe drug concentration and bioavailability ofdrugs in the cornea and aqueous humor (Kuwanoet al., 1997). The cross-linked collagen using glu-taraldehyde or chromium tanning can serve as adrug reservoir and provide more desirable drugdelivery than non cross-linked collagen shields byincreasing the contact time between the drug andthe cornea. Collasomes, in the form of collagenpieces, were developed by adding long hydrocar-bon side chains to the collagen (Kaufman et al.,1994). This modification increases not only thehydrophobicity of the collagen but also the totalsurface area, which effectively decreases the diffu-sion rate of hydrophilic drug molecules from thecollagen matrix. Collasomes can be formulatedwith various constituents and chemically alter-nated with the addition of lipid (calledLacrisomes) for the treatment of dry eyes (Kauf-man et al., 1994). In this work, a delivery systemin which collagen pieces were suspended in aviscous vehicle was instilled into the lower for-niceal space. Collasomes hydrated in a solution ofsodium fluorescein and suspended in a methylcel-lulose vehicle as a model for delivery of water-sol-uble drugs produced fluorescein concentrationsmuch higher in the cornea and aqueous humor,comparing with fluorescein-containing vehiclealone.

tions. The use of antibiotic collagen shields sup-plemented by frequent topical applications hasbeen clinically useful for preoperative and postop-erative antibiotic prophylaxis, the initial treatmentof bacterial keratitis, and the treatment of cornealabrasions (Poland and Kaufman, 1988; Friedberget al., 1991). Most of these studies showed thatshields provided equal or better drug delivery offluorescein (Reidy et al., 1990), prednisolone ace-tate (Sawusch et al., 1988a,b), cyclosporine (Reidy


Collagen shields as a drug carrier for topicalagents have many advantages. Experimental andclinical studies showed that the speed of epithelialhealing is faster and more complete with the useof the collagen shield than conventional formula-tions (Marmer, 1988). There was less stromaledema at the wound sites in collagen-treatedcorneas. The collagen shield seemed to protectkeratocytes adjacent to the wound sites and di-minish inflammatory reaction of keratocyte.Moreover, the surface epithelial bonding ap-peared to be normal with the use of the collagenshield. However, the application of collagenshields for drug delivery is limited by severaldisadvantages, such as reducing visual activity,causing slight discomfort, and a short duration atthe inserted site (Friess, 1998). Some side effectsof collagen shields were also reported. Shieldswere implanted into rabbit and guinea pig eyesand a possible toxic response was tested to deter-mine whether collagen shields produce histologi-cal evidence of inflammation when implantedsubconjunctively (Finkelstein et al., 1991). Theinflammatory response in rabbit was noticeableafter 7-day implantation and was much severethan in guinea pig. This study may indicate thatthe inflammatory response in rabbits is speciesspecific and that collagen shields are useful as adrug delivery system for antifibroblast drugs inother species.

3.4. Collagen sponges

Collagen sponges have been very useful in thetreatment of severe burns and as a dressing formany types of wounds, such as pressure sores,donor sites, leg ulcers and decubitus ulcers as wellas for in vitro test systems (Geesin et al., 1996).Human collagen membrane has been a majorresource of collagen sponge used as a biologicaldressing since 1930s (Rao, 1995). Collagensponges have the ability to easily absorb largequantities of tissue exudate, smooth adherence tothe wet wound bed with preservation of low moistclimate as well as its shielding against mechanicalharm and secondary bacterial infection (Pachenceet al., 1987; Yannas, 1990). Experiments usingsponge implantation demonstrated a rapid recov-

ery of skin from burn wounds by an intenseinfiltration of neutrophils into the sponge (Boyceet al., 1988). Coating of a collagen sponge withgrowth factor further facilitates dermal and epi-dermal wound healing (Marks et al., 1991; Royceet al., 1995).

The sponges made from pure collagen isolatedfrom bovine skin, were swollen at pH 3.0 andstabilized into the physical form of a sponge layer.In order to achieve highly resilient activity andfluid building capacity, collagen sponges havebeen combined with other materials like elastin,fibronectin or glycosaminoglycans (Lefebvre etal., 1992, 1996; Doillon and Silver, 1986). Thestarting material can be crosslinked with glu-taraldehyde and subsequently graft-copolymerizedwith other polymers, such as polyhydroxyethylmethacrylate (PHEMA) (Sastry and Rao, 1991).The grafted PHEMA chains, which are hy-drophilic, keep the membranes wet and increasetheir tensile strength. This further affects the effi-ciency in the management of infected wounds andburns. The importance of matrix compliance tomechanical response was also reported (Prajapatiet al., 2000). The physiological loading of fibrob-lasts in three-dimensional collagen lattices elicitedcomplex and substantial changes in protease ac-tivities, which were subjected to matrixmodification.

Collagen sponges were found suitable for short-term delivery (3–7 days) of antibiotics, such asgentamicin (Wachol-Drewek et al., 1996). Gen-tamicin containing collagen sponges placed on aseptic focus in the abdomen reduce local infection(Vaneerdeweg et al., 1998, 2000). This therapyachieved high concentration of gentamicin at thelocal site, while low concentration in serum. Col-lagen sponges containing antibiotics did not showany side effects and the collagen is reabsorbedafter a few days (Stemberger et al., 1997).

Like collagen film, which was used as genedelivery carriers for promoting bone formation,collagen sponges were also used for osteoinduc-tion. An absorbable collagen sponge containingbone morphogenetic protein 2 (rhBMP-2) wastested in the rat model for the evaluation of theefficacy of rhBMP-2 produced in Escherichia colion promoting bone healing (Kimura et al., 2000).


Bacterially expressed rhBMP-2 loaded in collagensponge was osteogenic in vivo. An absorbablecollagen sponge containing bone morphogeneticprotein 2 (rhBMP-2) stabilize endosseous dentalimplants in bony areas and normal bone forma-tion was restored without complication (Cochranet al., 2000).

Collagen sponges were also used for delivery ofsteroids through topical applications, such as in-travaginal delivery of lipophilic compounds in-cluding retinoic acid (Dorr et al., 1982; Chvapil etal., 1985). Collagen-based sponge was insertedinto a cervical cap made of hydrogel hypan,which, when in contact with wet tissue surfaces,adheres to them by the force of differential os-motic pressure (Peng et al., 1986). This novelsystem is associated with high local concentra-tions of drugs without producing any systemicsymptoms, and has been very useful for local drugdelivery.

Collagen sponge as a drug carrier or a vaginalcontraceptive barrier showed many advantages,such as the controlled release of spermicidalagents and reducing the tissue-irritation activity,over the rubber diaphragm. The main drawbacksof sponges appear to be the difficulty of assuringadequate supplies and their preservation. Otherproblems arose from their poor mechanicalstrength and ineffectiveness in the management ofinfected wounds and burns.

3.5. Gel, hydrogel, liposomes-collagen

Hydrogels have been widely used as a drugcarrier due to its ease in manufacturing and self-application. The production of a large and con-stant surface area is one of the major merits forthem to be widely used for clinical and fundamen-tal applications. Various combinations of poly-mers were made into hydrogel formulations toinvestigate their potential as a drug delivery sys-tem. The combination of natural and syntheticpolymers may provide mechanical stability andbiological acceptability, acquiring from synergisticproperties of both materials.

An attempt of combining collagen and polyhy-droxyethyl methacrylate (PHEMA) into hydrogelswas made to develop a delivery system for anti-

cancer drugs, such as 5-FU (Jeyanthi and Rao,1990). The hydrogels were found stable and re-silient and did not show any adverse effects, suchas calcification, after 6 month of subcutaneousimplantation in rats. This system was also veryefficient in therapeutic application, which, in thiscase, was anticancer therapy (Jeyanthi et al.,1991). Hybrid copolymers of collagen withpolyethylene glycol-6000 and polyvinylpyrrolidone were prepared for the controlled de-livery of contraceptive steroids (Shantha and Rao,1993). Two synthetic polymers, poly(vinyl alco-hol) (PVA) and poly(acrylic acid) (PAA), wereblended with two biological polymers, collagenand hyaluronic acid (HA), to enhance the me-chanical strength of natural polymers and to over-come the biological drawbacks of syntheticpolymers. These blends were formulated into hy-drogels, films and sponges, and subsequentlyloaded with growth hormone (GH) (Cascone etal., 1995). The results of this work showed thatgrowth hormone could be released from collagen-PVA hydrogels in a controlled release pattern andthe rate and quantity of growth hormone releasedwere mainly dependent on collagen content in thesystem.

One of the successful applications of collagenfor the controlled delivery systems is collagen-based gel as an injectable aqueous formulation.An injectable gel formulation in a combination ofcollagen and epinephrine for delivery of 5-FU wasdeveloped for cancer treatment (Sahai et al.,1995). The disappearance of 5-FU by diffusionafter intratumoral injection in mice was sustainedand its therapeutic effect was improved. The sub-cutaneous injection of soluble collagen efficientlydemonstrated its potential in the repair of derma-tological defects, such as vocal fold immobility(Remacle et al., 1995) and urinary incontinence(Shortliffe et al., 1989).

Collagen hydrogel was also used as gene deliv-ery carriers. Plasmids noncovalently bound to theFab fragment of antibody can be efficiently intro-duced into cells that express the polymeric im-munoglobin receptor (pIgR). Human trachealepithelial cells grown on plastic, a condition thatdown regulates the expression of the receptor,failed to express the reporter gene, whereas cells


from the same trachea maintained on collagengels were transfected (Ferkol, et al., 1993). G-CSFtransfected clonal murine fibroblast lines can sur-vive with grafts and continue to synthesize thetransgene as well as collagen in vivo (Rosenthaland Kohler, 1997). Due to their low antigenecity,the bovine and equine collagen matrices were welltolerated in clinical applications. Gel made ofatelocollagen, which is produced by elimination ofthe telopeptide moieties using pepsin, has beenused as a carrier for chondrocytes to repair carti-lage defects (Uchio et al., 2000; Katsube et al.,2000). Chondrocytes embedded in the atelocolla-gen gel gradually proliferated and maintain thechondrocyte phenotype. The grafted type I atelo-collagen provided a favorable matrix for cell mi-gration in relation with collagenase expressionand cell behavior was modulated by graft collagen(Suh et al., 2001).

To achieve both formulation stability and con-trolled release rates of entrapped materials fromcollagen based hydrogels, a system consisted ofliposome and collagen has been developed (Raoand Alamelu, 1992). Liposomes are widely used asa drug carrier due to their biodegradability andremovable versatility in terms of composition andsize. It was found that a coupling of liposomes tothe gel-matrices enhanced the stability of the sys-tem due to the antioxidant effect of collagenmolecules when they were immobilized (Weiner etal., 1985; Pajean and Herbage, 1993). Cross-link-ing the functional liposomes to a collagen gelmatrix further sustained the release rate of theentrapped marker (Rao, 1995). Thus, the processof cross-linking of the liposomes to the gel-matrixcan significantly manipulate the release rates ofentrapped drugs.

A novel drug delivery system comprising lipo-somes sequestered in a collagen gel has demon-strated controlled release profiles of insulin andgrowth hormone into the circulation (Weiner etal., 1985). Moreover, since coated vesicles weremore stable than control liposomes, the perme-ation rates of incorporated drugs from small unil-amellar liposomes coated by collagen intosystemic circulation were much greater (Fonsecaet al., 1996). Blood elimination and liver uptakeof collagen containing vesicles were about 2-fold

faster and 1.5–2-fold higher than those of controlliposomes, respectively. Collagen-based systemssoaked in liposomes were found to deliver signifi-cantly higher levels of immunosuppressive agentcyclosporin A to the cornea, anterior sclera,aqueous humor and vitreous in rabbit eyes thancollagen shields without liposomes (Pleyer et al.,1994). Inclusion of antimicrobials or cell growthfactors within the liposomes could facilitate en-hanced cell growth and prevention of infection,while a base collagen provided a substrate for cellattachment and proliferation (Weiner et al., 1985).

The formulation, in which liposome was se-questered in collagen gel, appears to have severaladvantages over other liposome formulations orgel formulations. This technology seems to have apotential for topical application in the treatmentof surgical or nonsurgical wounds and burns.Since the release kinetics of hydrophilic andlipophilic substance encapsulated in liposomewere similar to those of a non-encapsulated drug,the combination of liposomes with collagen isvery useful for drugs which do not penetrate theocular surface as well as systems in need of pro-longed corneal contact time (Grammer et al.,1996). Liposome encapsulated formulations didnot show any toxicity associated with a variety ofpotentially beneficial drugs.

3.6. Pellet/tablet

Minipellets made of collagen have been devel-oped for various candidate compounds (Take-naka et al., 1986; Yamahira et al., 1991;Matsuoka et al., 1988; Fujioka et al., 1995;Maeda et al., 1999). A rod with a diameter and alength of 1 mm and 1 cm, respectively, is a usefulshape as a drug delivery device, because this rod(minipellet) is small enough to be injected into thesubcutaneous space through a syringe needle andstill spacious enough to contain large molecularweight protein drugs, such as interferon (Miyataet al., 1992) and interleukin-2 (Matsuoka et al.,1988). A single subcutaneous injection of a minipellet caused a prolonged retention of interleukin-2 and decreased its maximal concentration in theserum. This pellet-type carrier was used for localdelivery of minocycline and lysozyme for the


treatment of periodontitis symptoms. An attemptto produce a pellet type controlled-release deliveryvehicle made of purified type I collagen for water-soluble osteogenic proteins was described (Lucaset al., 1989). Water-soluble proteins from a colla-gen-based system induced cartilage and bone witha success rate of 76%, demonstrating the feasibil-ity to formulate controlled-release delivery sys-tems for soluble bioactive factors, which interactwith local responsive cells.

Collagen-based pellet as a gene delivery carrierhas been extensively studied. The effect of atecol-lagen-based mini-pellet on the mRNA expressionand functional status of facial nerve in the ratmodel was investigated (Kohmura et al., 1999).The facial nerve transaction and immediate repairwas accelerated by this system and the facial nerveregeneration was ultimately achieved. A minipelletwith a cylindrical shape (0.6 mm in diameter and10 mm in length) containing 50 �g of plasmidDNA and human HST-1/FGF-4 cDNA was eval-uated as a controlled delivery system for plasmidDNA (Ochiya et al., 1999). This gene transfermethod allowed a sustained release and expres-sion of plasmid DNA in normal adult animalsthrough the use of atelocollagen, a biocompatiblepolymer, as a carrier.

The solid nature of atelocollagen in vivo seemsto have a great potential for site- or tissue-specifictransportation of target genes. The controlledgene transfer using atelocollagen in a form ofpellet has allowed a prolonged systemic circula-tion of target products and has facilitated a long-term use of naked plasmid vectors for somaticgene therapy (Ochiya et al., 1999). The mecha-nism of direct bone formation by BMPs–collagencomplex was ultrastructurally investigated (Naka-gawa and Tagawa, 2000). This study proved thatdirect bone formation is ectopically induced bybone morphogenetic proteins (BMPs) withoutcartilage formation when atelocollagen type I col-lagen pellet is used as a carrier. Further studiesare needed to assess the applicability of atelocol-lage-based pellet systems for the augmentation ofthe bioavailability of low molecular weight mate-rials, such as antisense oligonucleotides and bio-logically active oligopeptides, or virus vectors.

3.7. Nanoparticles/nanospheres

A property, in which the crystallites in the gelaggregates appear as multiple chain segments inthe collagen-fold configuration, has been used toprepare aggregates as colloidal drug delivery car-riers (Muller et al., 2000). Nanosphere formationis driven by a combination of electrostatic andelectropic forces with sodium sulfate employed asa dissolving reagent to facilitate greater charge–charge interactions between plasmid DNA andcollagen (Marty et al., 1978). The molecularweight of collagen or gelatin has a decisive influ-ence on the stability of the manufactured gelatinnanoparticles (Coester et al., 2000). The molecularweight profile of the collagen solution was af-fected by pH and temperature, both of whichfurther influenced the noncovalent interactions re-sponsible for the molecular structure of collagen(Farrugia and Groves, 1999). The relationshipbetween electropic forces and gene factors wasalso evaluated. Polyion complexation between ba-sic fibroblast growth factor and gelatin was stud-ied by turbidity change of a mixed solution andisoelectric electrophoresis (Muniruzzaman et al.,1998). It was found that an electrostatic interac-tion was the main driving force for the complexa-tion between acidic gelatin and basic fibroblastgrowth factor.

The biodegradable collagen based nanoparticlesor nanospheres are thermally stable, readilyachieving their sterilization (Rossler et al., 1995).Moreover, nanoparticles can be taken up by thereticuloendothelial system (Marty et al., 1978),and enable an enhanced uptake of exogenouscompounds, such as anti-HIV drugs, into a num-ber of cells, especially macrophages (Bender et al.,1996), which may be an additional advantage ofcollagen based nanoparticles as a systemic deliv-ery carrier. Thus, nanoparticles were used as aparenteral carrier for cytoxic agents and othertherapeutic compounds, such as campthocin(Yang et al., 1999) and hydrocortisone (Bertholdet al., 1998). Gelatin microspheres were used as adrug carrier for parenteral delivery of cancerdrugs, such as methotrexate (Narayani and Rao,1994). The microspheres showed zero order kinet-ics in the release profiles of incorporated drugs,


demonstrating prolonged action against ratfibrosarcoma and improved antitumor activity.

Due to a small size, a large surface area, highadsorptive capacity, and ability to disperse inwater to form a clear colloidal solution, collagen-based nanoparticles have demonstrated their po-tential to be used as a sustained releaseformulation for anti-microbial agents or steroids(El-Samaligy and Rohdewald, 1983). Delivery ofhydrocortisone, one of lipophilic steroids, was notaffected by the pH of the receptor medium or itsbinding affinity to the particles. Collagennanoparticles were used to enhance dermal deliv-ery of retinol (Rossler et al., 1994). The retinol inthe system was very stable and facilitated a fasterand higher transportation of the incorporateddrug through the skin than the freshly precipi-tated drug.

4. Collagen-based systems for tissue engineering

4.1. Collagen as skin replacement

Collagen based implants have been widely usedas vehicles for transportation of cultured skin cellsor drug carriers for skin replacement and burnwounds (Bell et al., 1983; Leipziger et al., 1985;McPherson et al., 1986; Deatherage and Miller,1987; Harriger et al., 1997; Boyce, 1998). Sincesponge implant was originally developed for re-covery of skin and was very efficient in thatpurpose, various types of artificial skin were de-veloped as a form of sponge. Cultured skin substi-tutes developed on collagen lattice were also usedfor skin replacement and skin wounds. Reconsti-tuted type I collagen is suitable for skin replace-ment and burn wounds due to their mechanicalstrength and biocompatibility (Rao, 1995).Chronic wounds resulting from diabetes havebeen successfully cured with allogenic culturedskin substitutes prepared from cryopreserved skincells (Boyce, 1998). In the cultured skin substi-tutes, the contracted collagen lattice was used as asupport for epithelial growth and differentiationto replace pathological skin (Yannas et al., 1989).Allogenic cultured dermal substitute prepared byplating fibroblasts on to a collagen sponge matrix

and subsequently freeze dried from a 1% aqueoussolution of atelocollagen provided a good envi-ronment for epithelialization (Yamada et al.,1999). The effectiveness of collagen sponges as asubstrate for human corneal cells was demon-strated and corneal cells exhibited normal cellphenotype when cultured individually on an engi-neered collagen sponge matrix (Orwin and Hubel,2000). Addition of selected antimicrobial drugslike amikacin to the bovine skin implantable col-lagen managed to control microbial contamina-tion and increased healing of skin wounds (Boyceet al., 1993).

The modified sponge for artificial skin was de-veloped by combining fibrillar collagen withgelatin (Koide et al., 1993). Dehydrothermalcrosslinks were used to stabilize collagen-basedsponge physically and metabolically. Spongesmade of gelatin by itself in a resorbable gelfoamwere also used as a carrier matrix for humanmesenchymal stem cells in cartilage regenerationtherapy (Ponticiello et al., 2000). When thisgelatin sponge was implanted in an osteochondraldefect in the rabbit femoral condyle, gelfoamcylinders were observed to be very biocompatible,with no evidence of immune response orlymphatic infiltration at the site.

Some limitations inherent to cultured skin sub-stitutes, such as deficient barrier function in vitroand delayed keratinization after grafting in com-parison to native skin autografts, were reported(Supp et al., 1999). To address those limitations,modifications of collagen-based systems by thecombination of collagen with other proteins, suchas glycosaminoglycan, fibrin, and biotin, wereproposed. The role of glycosaminoglycan and dif-ference in its concentrations between pathologicaland normal tissues were reported earlier(Sobolewski et al., 1995). Dermal skin substitutes(membranes) made of collagen and glycosamino-glycan were found to be suitable substrates for theculture of human epidermal keratinocytes (Boyceet al., 1988). Cultured skin substitutes consisted ofhuman keratocytes and fibroblasts attached tocollagen-glycosaminoglycan substrates, whichwere subsequently crosslinked, decreased the rateof biodegradation and further reduced the en-graftment of skin substitutes (Boyce et al., 1995;


Harriger et al., 1997). Restoration of functionalepidermis by cultured skin substitutes developedfrom collagen was stimulated by incubation inreduced humidity in vitro (Supp et al., 1999). Inthe case of collagen and fibrin combination, cul-tured cells were best grafted directly onto thewound bed or in combination with either a thinlayer of collagen or fibrin but not both (Lam etal., 1999). Biotinylation of bovine skin collagen bycovalent addition of biotin has been used to at-tach peptide growth factors with avidin as abridge. This technique retained the activity ofpeptide growth factors and demonstrated a poten-tial to be used as a modulator of the response inwound treatment (Boyce et al., 1992; Stompro etal., 1989).

Sponges in a combination of silicone and colla-gen were also used to address the limitationsinherent to cultured skin substitutes. Acellularbilayer artificial skin composed of outer siliconelayer and inner collagen sponge was used for athin split thickness skin graft and achieved goodperformance in the long term postoperative ap-pearance of the split thickness skin graft site(Suzuki et al., 2000).

4.2. Collagen as bone substitutes

Among the many tissues in the human body,bone has been considered as a powerful markerfor regeneration and its formation serves as aprototype model for tissue engineering based onmorphogenesis. Collagen has been used as im-plantable carriers for bone inducing proteins, suchas bone morphogenetic protein 2 (rhBMP-2)(Reddi, 2000). Recently, collagen itself was usedas bone substitutes due to its osteoinductive activ-ity (Murata et al., 1999). The uses of collagen filmas gene delivery carriers for osteoinduction andcollagen sponge for bone related protein carrierswere described earlier in Sections 3.1 and 3.4,respectively, and this section mainly focused on itsuses as a bone substitute and a bone marker.

Type I collagen crosslinked N-telopeptide wasused as a marker of bone resorption and clinicallyused as a marker of bone metastasis of prostatecancer and breast cancer (Kobayashi et al., 2000;Ulrich et al., 2001). The polymorphisms of colla-

gen type Ialpha1 and vitamin D receptor as ge-netic markers for osteoporotic fracture in womenwas also reported (Uitterlinden et al., 2001). Thisresult added to evidence that interlocus interac-tion is an important component of osteoporoticfracture risk.

Collagen in combination with other polymersor chemicals was also used for orthopaedic de-fects. Demineralized bone collagen was used as abone graft material for the treatment of acquiredand congenital orthopaedic defects either by itselfor in combination with hydroxyapatite (Takaokaet al., 1988). The result of this study showed thatgrafted demineralized bone collagen in combina-tion with hydroxyapatite was an excellent osteoin-ductive material and could be used as a bonesubstitute. A recent study showed that addition of500 IU of retinoic acid to collagen at a site of abone defect enhanced regeneration of new bone,achieving union across the defect and leading toits complete repair (Sela et al., 2000).

4.3. Collagen as bioengineered tissues

Collagen gel as human skin substitutes havedemonstrated its usefulness in tissue engineeringand led to the development of bioengineered tis-sues, such as blood vessels, heart valves and liga-ments (Auger et al., 1998). Collagen showshemostatic properties that promote blood coagu-lation and play an important role in tissue repairprocess. Collagen sponge or gel initiates adhesionand aggregation of platelets that lead to a throm-bus formation (Miyata et al., 1992). Monomericcollagen does not activate platelet aggregation,while polymeric collagen having a regular ar-rangement of the molecules with a length ofaround 1 �m does activate it. Arginine side chainsof collagen seemed to be responsible for its inter-action with platelets (Wang et al., 1978).

A provisional extracellular support was devel-oped using type I collagen lattice to organize thecells into a three-dimensional structure in vitro(Kemp, 2000). A small-diameter (4 mm) graftconstructed from type I bovine collagen was ear-lier used to integrate into the host tissue andprovide a scaffold for remodeling into a func-tional blood vessel (Huynh et al., 1999). Three-di-


mensional collagen scaffolds, that are biodegrad-able in vivo and have a large surface area for cellattachment, can support vascularization processesand can be used as artificial blood vessels, heartvalves or cell transplant devices (Kuzuya andKinsell, 1994; Chevallay and Herbage, 2000). Theuse of collagen as a coated material for perme-ation filters made of culture endothelial cellmonolayers was also tested to evaluate in vitrovascular permeability of a drug to contrast media(Matin-Chouly et al., 1999).

A method for generating a cellular layer ofintestinal collagen from the porcine submucosawithout compromising the native collagen struc-ture further facilitated the use of collagen in tissueengineering (Abraham et al., 2000). Biologicaltissue grafts in the form of collagen-based matrixhave been derived from bladder, ureter or smallintestine (Clarke et al., 1996; Desgrandchamps,2000). These collagen constructs were designed tobe similar to synthetic polymer prostheses interms of their ability to persist. The structure-me-chanical behavior relationship of biomaterials ac-quired from intestine submucosa demonstratedmechanical anisotropy and stiffer direction pre-ferred in biomaterials (Gloeckner et al., 2000).Using a phenomenological constitutive model, itwas demonstrated that glycan increased the tensilestiffness and ultimate tensile strength of collagen-based matrix and further increased their resistanceto collagen degradation (Girton et al., 2000).

Natural collagenous materials were used forsurgical repair and abdominal wall repair by tak-ing advantage of their inherent low antigenecityand their ability to integrate with surroundingtissues (Van der Laan et al., 1991). Moreover,new generations of collagen-based biological tis-sue are practical and remodelable due to its sim-ple membranous configuration, relativeuniformity and abundant availability. These char-acteristics are employed in a new type of surgicaladhesive made from porcine collagen and polyglu-tamic acid, developing for sealing air leaking fromthe lung, which takes a relatively long period forrecovery (Sekine et al., 2001). The absorption rateof collagen-based adhesive can be controlled bycollagen concentration in the system. Recent pro-gress in tissue engineering may lead to well-char-

acterized and reproducible biomaterials fromnatural collagenous materials.

5. Concluding remarks

Collagen has various advantages as a biomate-rial and is widely used as carrier systems fordelivery of drug, protein and gene. The examplesdescribed in this paper represent selected applica-tions of collagen in the biomedical field. Thesuccessful demonstration of usefulness of humanskin substitutes made of collagen has lead to thedevelopment of bioengineering tissues, such asblood vessels and ligaments. Autologous tissueengineering provides an alternative for allogenictissue transplantation.

The study of native collagen for drug deliverysystems and tissue engineering may lead to abetter understanding of pathological diseases. Theconcepts of high binding affinity and specificityplay a critical role in targeting delivery of drugs.By understanding the nature of drug deliverysystems and their durability in the body, theessential parameters for designing effective lig-ands, which can interact with the systems, can beidentified. It can further provide a new guide fortissue growth and organization and leading tobioactive signals for tissue-specific gene expres-sion. Collagen-based biomaterials are expected tobecome a useful matrix substance for variousbiomedical applications in the future.

Acknowledgements

This work is supported in part by grant fromthe University of Missouri Research Board.

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Biomedical Applications of Collagen