Literature Review 2: Collagen
Literature Review 2: Collagen
1. Introduction
Collagen is the main protein of connective tissue in animals and fish, where it is the most abundant protein in mammals, constituting up to 25-30% of the total protein content of the body. On the other hand, collagen constitutes 1-2% of muscle tissue
besides contributing to the tune of 6% of the weight of strong and tendinous muscles. Even the people of early civilization discovered collagen’s multiple utility value, such as waterproofing, adhesive, and decoration.
Literature Review 2: Collagen
In the modern era collagen has become an inseparable instrument in both bio
medical and non-bio medical industries, with an extended range of usage. For example, it provides structure additive to food besides providing food supplements of collagen;
enables the pharmaceutical industry to produce hard and soft capsules, artificial skins, injections of regeneration of cells, provides cosmetic industry for the beautification and elimination of age-related skin wrinkles, acts as a source of glue in its hydroslated form, helps in films in the photographic industry
The main sources of collagen are pig and bovine skins due to their easy
availability, though fish collagen is also making its mark and the researchers are
contemplating with the potential of chicken skin. The major advantages of marine collage
sources are that they are free from the risk of BSE (bovine spongiform encephalopathy),
culturally more acceptable across the world, more suitable to the human skin than its
counterparts, and are available in abundance, since fish skin is a major by-product of the
fish-processing industry.
2. Background
In the period between 1920-1935 Nageotte (1927a, b, 1928, 1930, 1933),
Nageotte & Guyon (1933), Leplat (1933), and Faure-Fremiet (1933) had studied and
observed the partial dissolution of collagen in dilute solutions of weak acids such as
formic and acetic acid. Renewed interest in this soluble protein was generated in the
period between 1940-1955 with the works of Orekhovich and his colleagues in the
U.S.S.R., who reported the extraction of a soluble collagenous-type protein from the skin
of various animals using dilute citrate buffers, which they suggested as a soluble
precursor of collagen (Plotnikova, 1947; Tustanovskii, 1947; Orekhovich et al. 1948;
Chernikov, 1949; Orekhovich, 1950, 1952). Such findings on soluble collagens were
further reviewed by Harkness et al. (1954), who also reported the presence of a small
amount of a protein of collagen type which was extracted from skin by dilute phosphate,
pH 9-0 (alkali-soluble collagen).
Later Harkness et al. (1954) experimented on the feeding of labeled glycine to
rabbits to arrive at a conclusion that this is a true precursor of collagen, whereas the
metabolic role of the acid-soluble collagen described by Orekhovich is less certain, and it
is not necessarily an intermediate in the formation of all insoluble collagens of the skin.
Harkness et al. (1954) determined the hydroxyproline and tyrosine content of the alkali-
soluble and acid-soluble collagen, and also of the gelatin obtained from the remaining
insoluble collagen.
Both soluble collagens contained less tyrosine and more hydroxyproline than the
insoluble collagen, and the acid-soluble had a higher hydroxyproline and tyrosine content
than the alkali-soluble collagen. Bowes et al. (1953) also observed similar differences
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between the hydroxyproline and tyrosine content of the acid soluble collagen of calf skin and the adult collagen of ox hide and between the acetic acid-soluble and insoluble
fractions of tendon collagen.
3. Methods
It requires solvents like salt, dilute acid, alcohol, detergents and H2O2 for
removing non-collagenous proteins, as well as for removing fat and odors. Alongside it
requires chemicals (EDTA) for deashing. It is after that the solvents like acetic acid,
lactic acid, pepsin enzyme, Bacillus bacteria, and yeast are used to extract collagen under
the temperature range of 0-40C. Altogether three steps are involved in the extraction of
collagen, such as pre-treatment steps, extraction of acid-soluble collagen, and purification
of collagen. Apart from the above, there is also super-critical extraction method where
CO2 is used as solvent, which has some advantages like ease of controlling the dissolving
power of supercritical fluid (SCF) by controlling temperature and pressure. It is also easy
to recover SCF by decompressing pressure, and it is impossible to separate the precipitate
from extracts by centrifugation. SCF is a non-toxic solvent and is applicable to extract the
thermally decomposed compounds. However, this method is costly and requires expertise
to handle the proceedings under high pressure.
3.1. A Typical Process
In a typical extraction process, at least eight steps are taken (Mingyan et al. 2009). For example, in process of squid (Ommastrephes bartrami) collagen extraction, the steps could be like below.
1, Fish skin preparation: After collecting the squid skins they are manually
descaled and are made free from the residual meat, before placing them at -200 C till the
time of use. Alongside, all the reagents are checked to ensure that they are of analytical
grade.
2. Histological observation: At this stage the squid skins are cut into pieces (0.5 cm x 0.5 cm) and fixed in 4% buffered formalin for 24 hours. After that the specimen is dehydrated in a series of graded ethanol solutions (70%, 80%, 90%, and 100%), clarified in xylene and is finally embedded in paraffin. Eight-mocron sections are then cut
perpendicular to the skin surface and the slides with the sections are cleared off paraffin with xylene, rehydrated to water through graded alcohols, now in reverse order (100%, 90%, 80%, and 70%), and then are stained with hematoxylin and eosin and again with the Van Gieson stain (collagen – red color; muscle – yellow color). The stained sections are then observed through light microscopy and using color video camera at an original
magnification of 20x the digital images are then collected.
3. Extraction of collagen: Generally it takes 40C to conduct the extraction process
(Nagai and Suzuki, 2000), where the skin is extracted with 0.1 mol L-1 NaOH to remove
non-collagenous materials and to eliminate the effect of endogenous proteases on
collagen (Sato et al., 1987). After that it is rinsed thoroughly with distilled water to
neutralize the pH of the wash water. Then the samples are defatted with 10% butyl
alcohol at a solid to solvent ratio of 1:10 for 24 hours, after which they are washed with
plenty of distilled water. The further minced skins are gently stirred in 0.5 mol L-1 acetic
acid solution for 48 hours, and the extract is centrifuged at 10 000×g for 30 minutes.
Then the acid-solubilized collagen (ASC) in the supernatant is salted out by adding NaCl
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to a final concentration of 0.9 mol L-1. Then after leaving the solution overnight, the
resultant precipitate is collected by centrifugation at 8000×g for 20 minutes, before
dissolving the same in 0.5 mol L-1 acetic acid. Then it is dialyzed against 0.1 mol L-1
acetic acid for 24 hours and distilled in water for 48 hours, and then it is lyophilized.
After acid extraction, the insoluble fraction is kept suspended in 10 times of 0.5
mol L-1 acetic acid (by volume) and is digested with porcine pepsin (EC 3. 4. 23. 1;
powderized; 750 U mg-1 dry matter) at an enzyme/ substrate ratio of 1:100 for 48 hours
at 40C with gentle stirring. The extract is then centrifuged at 10 000×g for 30 min. Then
the pepsin-solubilized collagen (PSC) in the supernatant is salted out by adding NaCl to a
final concentration of 0.9 mol L-1 and after leaving it the solution overnight, the resultant
precipitate is then collected by centrifugation (8000×g for 20 min) was dissolved in 0.5
mol L-1 acetic acid, dialyzed against 0.02 mol L-1 Na2HPO4 for 1 d to inactivate pepsin.
The precipitate was collected at low speed centrifugation and the same is then dissolved
in 0.5 mol L-1 acetic acid. After that the solution is dialyzed with 0.1 mol L-1 acetic acid
and distilled through water, before putting to lyophilize.
4. Amino acid analysis: At this stage, ASC and PSC samples are hydrolyzed under reduced pressure with 6 mol L-1 HCl at 1100C for 24 hours before analyzing the hydrolysates on an amino acid analyzer (e.g. Hitachi 835-50).
5. Sodium Dodecyl Sulphate Polyacylamide Gel electrophoresis (SDS-PAGE): It
can be performed by following the method of Laemmli (1970), using the discontinuous
Tris-HCl/ glycine buffer system with 7.5% resolving gel and 5% stacking gel. After
electrophoresis, the gel is kept stained for 20 minutes with 0.1% Coomassie Brilliant
Blue R-250 dissolved in distilled water, methanol and acetic acid (9:9:2), and then the
same is destained using a solution of distilled water, methanol and acetic acid (8:1:1).
6. Fourier Transform Infrared Spectroscopy (FTIR): FTIR spectra is obtained
from samples placed on discs containing a mixture of 0.2 mg lyophilized collagen and
about 10 mg potassium bromide (KBr) ground under drying conditions. The spectra are
recorded using infrared spectrophotometer (e.g., Nicolet 200SXV) from 4000 to 500 cm-1
at a data acquisition rate of 2 cm-1 per point. The resulting spectra are analyzed using the
software (e.g. Omnic 6.0).
7. Determination of Denaturation Temperature: The denaturation temperature is
measured from changes in viscosity, where Ubbelohde viscometer can be used (Zhang et
al. 2007). Usually ten milliliters of 0.03% collagen solution in 0.1 mol L-1 acetic acid
with 0.2 mol L-1 sodium acetate buffer (pH 5.0) are used for viscosity measurements.
Thermal determination curve is obtained by measuring the solution viscosity at seven
stepwise-raised temperatures from 16 to 420C, each temperature being maintained for 30
min. Fractional viscosity at a given temperature is calculated by the following equation:
Fractional viscosity=(ηsp(T)−ηsp(420C))/(ηsp(160C)−ηsp(420C)), where ηsp is the specific viscosity.
These fractional viscosities are plotted against the temperature and the denaturation
temperature is determined as the temperature where the fractional viscosity is predicted to
be 0.5.
8. Differential Scanning Calorimetry (DSC): DSC is performed on a calorimeter
(e.g., Netzsch DSC 200PC) fitted with an air-cooling compressor and a liquid nitrogen
cooler at ambient temperature (Cui al., 2007). The temperature is calibrated using indium
as standard. Collagen fiber is weighed (3.00 mg) and sealed in aluminum pans (BO
6.239.2−64.502). Triplicate samples are heated from 20 to 1000C at a scanning rate of 2
3
K min-1, with an empty sealed pan as a reference. The shrinkage temperature is taken at the peak of the plotted thermal transition curve (Minyang et al. 2009: 191-196)
4. Extraction of Collagen
4.1. Acid Solubilized Collagen (ASC) Extraction
4.1.1. Typical Process of Extraction at Earlier Times
For the preparation of proteins from ox-hide collagen, the middle layer of a hide
taken from a 2-year-old bullock immediately after flaying the skin is shaved, cut into
pieces about 1 cm.2 and disintegrated in a Wiley mill. However it is necessary to stop the
mill at intervals to remove the macerate by hand since it would not go through even the
coarsest sieve. The macerated material (1800 g.) is then placed in a cotton bag and
extracted, first with 0 l m-Na2HPO4, pH 8-6, and then with 0-12M sodium citrate buffer,
pH 3-62. After the last phosphate extraction the macerate is suspended for a short period
in two 3-1portions of the citrate buffer in order to remove the phosphate buffer before
going on to the citrate extractions proper. Each extraction takes a period of 24 hr., during
which the contents of the bag are agitated intermittently. All extractions are carried out
between 20 and 4°.
The macerated skin swells considerably during the extractions; as much liquid as
possible is to be squeezed out after each extraction, but relatively large amount remains
behind. After the last extraction special efforts required to squeeze out liquid, and the
relatively high protein content of this extract suggests that some liquid remaining from
the earlier extractions is removed from the interstices of the protein. A small amount of
the macerate (5 g.) was further extracted with successive 200-ml. portions of the citrate
buffer. Samples are taken at various stages, dehydrated with acetone, and total nitrogen
and hexosamine determinations are carried out. (Bowes & Kenten, 1948a, b)
The protein in the citrate extracts is precipitated by the addition of sufficient 30% (w/v) sodium chloride solution to bring the final concentration to 5% (w/v). The next morning the lower clear layer of liquid is withdrawn, and the top layer, containing the protein in a gelatinous form, is centrifuged. The precipitate is then washed with a small mount of water and dehydrated with acetone after ascertaining that there is no alteration in the solubility of the extracted protein. Redissolving in citrate buffer and dialysing against tap water then further purifies the precipitated protein.
The citrate-soluble collagen is converted into gelatin by heating a 2% (w/v)
suspension in slightly acidified water to 400 for 5 min. The protein went into solution during the heating and on cooling the solution set to a gel.
4.1.2. Example of Modern Times
In another instance of extraction and characterization of collagen from chicken
skin, the skins are first ground and then are heated to 40 or 60°C to extract the fat. After
the mechanical separation, the collagen content remaining in the resulting solid phase are
extracted with pepsin or ethylene diamine. Type I and type III collagen are then isolated
and characterized by SDS PAGE, antigen labeling, determination of tyrosine residues,
and transmission electron microscopy. The total collagen content of the skin is recovered
from the solid phase following heat treatment at 40°C. Extraction yields vary with the
solubilization process; for example 38.9% of the collagen content in the solid phase could
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be extracted with pepsin and 25.1% with ethylene diamine (Cliche et al. 2003). The
collagen obtained is characterized by its electrophoretic migration pattern and is then confirmed by antigen labeling. The presence of telopeptides is determined by measuring tyrosine content. Transmission electron micrographs are also obtained. For example, 1% phosphotungstic acid is applied to the collagen samples as a negative stain, and then they are mounted on Formvar grids before being viewed at 105,000× (Piez, 1984), using an EM 420 transmission electron microscope. The results are then compared to those
obtained from analysis of Atelohelogen avian collagen.
4.2. Pepsin Solubilized Collagen (PSC) Extraction
Fujimoto (1968) found that collagen could be solubilized by pepsin treatment as a preliminary to purification from the muscle layer of Ascaris lumbricoides and pig kidney, which prompted other researchers to apply the same technique. For example, Bannister and Burns (1972) applied the same technique to solubilization of avian intramuscular
collagen, where they found that tropocollagen from intramuscular sources was more
highly cross-linked than the material from skin treated in similar fashion. Although
solubilization results from digestion of non-collagenous protein, it is also the result of
cleavage of telopeptides containing covalent cross-links, and precise comparison between collagens from different sources is difficult to make.
4.2.1. Solubilization and Purification of Collagen
In an instance of pepsin treatment of avian skin collagen, Bannister and Burns
(1972: 677-681) selected the skin of a strain of domestic fowl derived from commercial
broilers as their source of material. The birds were all female and belonged to 12-18 week
age group. The plucked skins from freshly killed birds were cleaned of adhering fat and
muscle before mincing with ice in a hand-operated mincer. Next, the material was
defatted by two extractions with chloroform-methanol (2:1, v/v), before thoroughly
washing with tap water and distilled water and extracting twice with 0.2M-Na2HPO4 at
room temperature. However, all subsequent operations were performed at 0-40C, where
the researchers obtained acid-soluble collagen by extracting the defatted minced skin
three times (for 2-3 days in each case) with 3% (v/v) acetic acid. The remaining material
was then resuspended in 3% (v/v) acetic acid and digested for 2-3 days with approx. 5mg
of pepsin/ml.
The collagens solubilized by both procedures were then purified, where the
extracts were clarified by centrifugation for one hour at approx. 33000g and the
supernatant was dialyzed against aq. NaCl so that the final concentration turns 7% (w/v). The precipitated collagen was then collected by centrifugation and was redissolved in 3% (v/v) acetic acid and centrifuged for another hour at approx. 77000g. Entire of the above procedure was then repeated and the protein was precipitated a third time by dialysis against 0.02M-Na2HPO4. After dissolving in, and dialysis against, 3% (v/v) acetic acid the collagen was centrifuged for 1.5 hrs. at 109000g. The supernatant containing the
purified protein was then freeze-dried and stored in a desiccator.
4.2.2. Pepsin Treatment of Soluble Collagens
The researchers adjusted the portions of acid-soluble and pepsin-solubilized
collagens to 1.Omg/mI in 3% (v/v) acetic acid and treated with crystalline pepsin (concn.
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0.1 mg/ml) for a period between 3 and 14 days at 4°C. After digestion, pepsin was
removed by one salt precipitation and one Na2HPO4 precipitation. Thus four types of
collagen were available for study, such as acidsoluble (A), acid-soluble pepsin-digested
(Ap), pepsin-solubilized (P), and pepsin-solubilized pepsindigested (Pp) collagen, out of which collagen Pp was mostly treated for 14 days.
4.2.3. Hexosamine and Aldehyde Contents of Collagen Preparations
The amount of hexosamine present is considered to be a measure of
contamination of collagen preparations by the mucopolysaccharide constituents of
connective tissue. Therefore the researchers took measurements by the Elson-Morgan
procedure as described by Davidson (1966), with glucosamine as standard. The aldehyde
content was assayed by the method of Paz et al. (1965), with acetaldehyde as standard.
4.2.4. Determination of Subunit Composition
The percentage of α-, β-, and γ-chains in denatured collagen solutions was determined by polyacrylamidegel electrophoresis and densitometry.
4.2.5. Rate of Fibril Formation
Since the rate at which native-type fibrils can be formed from collagen solutions may be used as a measure of the aggregation properties of the tropocollagen molecule, the researchers dissolved collagen dissolved in 3% (v/v) acetic acid at 0°C and carefully raised the pH to 7.2 by adding 2M- and 0.01 MnaOH using a Pye model 291 pH-meter. When the required pH was achieved, they adjusted the volume with water to give a
collagen concentration of 0.1 %. Fibrillogenesis was monitored continuously at 400nm in a spectrophotometer (e.g. Unicam SP.800). The process was initiated by transferring
solution from an ice bath to a cuvette maintained at 38°C.
4.2.6. Effects of Pepsin Treatment on Hexosamine Andaldehyde Content
Hexosamine: Contamination by hexosamine was low and similar in all
preparations, which meant pepsin treatment is probably without effect in the further purification of already highly purified collagen.
Aldehyde: Here the findings appeared somewhat different from those of
Deshmukh & Nimni (1971), where chick skin acid-soluble collagen contained about one-
third as much aldehyde as did neutral-salt-soluble collagen from rat skin. However, that
was not surprising in view of the biologically older nature of acid-soluble collagen and
the differing methods employed in purification. Pepsin-solubilized collagen contained
about one-half as much as the acid-soluble material, which the researchers presumed as a
reflection of greater biological maturity and also some loss of telopeptides due to action
of the enzyme. When they treated with pepsin, both preparations (Ap and Pp) sustained
further decreases in aldehyde content, in conformity with the location of this group in the
telopeptide region.
4.2.7. Pepsin Treatment
The subunit composition was expressed as the percentage of α-chains
(monomers), β-chains (dimers) and γ-chains (trimers), although this last group probably
contained higher-molecular-weight subunits as well. The compositions of collagens A
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and P showed that, although there is no great difference in the content of α-chains,
collagen P is very much richer (about four times) in γ- chains. Sampling at 24h intervals
and examining the ureadenatured material by gel electrophoresis enabled the study of the
pepsin-digestion of these two collagens. In this experiment the researchers did not
remove pepsin by repurification because it did not interfere with separation of the subunit
classes. The results of treatment over a 4-day period showed that Collagen A was almost
completely converted into α-chains with little β- and no detectable γ-chains remaining. In
contrast, collagen P gave rise to fewer α-subunits than did collagen A, and significant
quantities of β- and γ-chains survived pepsin treatment. This observation suggested that
there are covalent cross-links present in collagen P that resist pepsin attack, and confirms
a similar conclusion reached by Steven (1966) with bovine and human collagens.
4.2.8. Rate of Fibril Formation
Researchers of 1950s and 1960s (Gross and Kirk,1958; Bensusan, 1960;
Bensusan and Scanu, 1960) studied the effects of a variety of substances on the rate at
which solutions of collagen can form rigid gels containing native-type fibrils.
Subsequently, other researchers (Rubin et al., 1963; Connel & Wood, 1964) also
investigated the results of limited proteolysis on the rate of fibril formation. This
prompted Bannister and Burns (1972) to use a similar turbidimetric technique to test
whether further pepsin treatment of collagens A and P results in a decrease of fibril-
forming capacity. Resultantly they found that collagen A was markedly inhibited whereas
pepsin treatment of collagen P was virtually without effect. The finding with collagen A
was much as expected, but the failure to produce significant retardation of fibrillogenesis
with collagen P was not expected, which in turn suggested that sufficient quantity of a
‘nucleus-forming’ collagen remained despite removal of a large percentage of the covalent
cross-links by pepsin. In spite of the absence of a direct evidence, the researchers found it
tempting to suggest that this is related to the previously demonstrated pepsin-resistant
collagen.
5. Isolation and Characterization
5.1. Western Blot
Western Blot method is used to detect functional proteins after the cell produces
them. Both procollagen and collagen can be detected using this method. Proteins in the
mixture are separated using electrophoresis based on their electrical charge, which
corresponds to the molecules weight or size, and then transferred to a membrane to which
the proteins become affixed. Antibodies that are specific to react or bind with the protein
of interest are applied to the membrane and these complexes are then detected by
chemilumenescence and film development (Cao et al. 1997; Ikenou et al. 2003; Lim et al.
2002).
In an instance of Western Blot analysis (antigen labeling) Cliche et al. (2003: 504)
adopted the method suggested by Timmons and Dunbar (1990), where the collagen was
transferred (10 V for 50 in) with the Trans-Blot Semi-Dry Transfer Cell to an
Immun-Blot polyvinylidene difluoride (PVDF) membrane (0.2 μm). They used rabbit
anti-chicken collagen type I as primary antibody, and alkaline phosphatase rabbit IgG as
the secondary antibody. The primary and secondary antibodies were diluted in the
blocking solution (3% BSA) at 1:3,000. Alongside, they developed the color associated
7
with antigen labeling by using alkaline phosphatase provided with the kit.
5.2. SDS-PAGE
Sodium dodecyl sulphate polyacrylamine gel electrophoresis (SDS-PAGE) is one
of the methods to determinate the forms of collagen (alpha chain, beta chain); SDS-
PAGE to determine peptide mapping. Determination of hydroxyproline content is
required besides the determination of denaturation temperature, viscosity of collagen
solution, effect pH and NaCl on collagen solubility, FT-IR and SEM to determine
structure of collagen. In an instance of extracting collagen from chicken skin (Cliche et
al. 2003: 504) the SDS PAGE was performed using the procedure of Laemmli (1970)
with a Mini-Protean II electrophoresis unit, where the stacking gels contained 3%
polyacrylamide, and the separation gels contained 6.5% polyacrylamide. Ten micrograms
of collagen deposited in the wells, and the migration was induced at 80 V for 5 min, until
the collagen subunits had passed through the stacking gel, and then at 150 V for 55 min,
or until the migration reached the end of the resolving gel. The researchers stained the
gels with Coomassie blue R-2507 for 30 min and destained with a solution of 10% glacial
acetic acid, 40% methanol, and 50% water.
In another instance of SDS-PAGE Mingyan et al. (2009) examined collagen from
squid skins by using a 7.5% resolving gel, where both ASC and PSC had the similar
electrophoretic pattern of typical type I collagen, consisting of α chains with two distinct
types like α1 and α2, varying in their mobility. The electrophoretic positions of α chains
of squid skin collagen were different from those of the walleye polylock (Yan et al.
2008), grass carp, bovine (Zhang et al. 2007), and porcine, (115 kDa for α1 chain and 66
kDa for α2 chain) which suggested the distinctiveness of the squid skin collagen at its
primary structure. Alongside, the inter and intra molecular crosslinked components such
as β (dimers) and γ (trimers) were also found in squid collagen, which resembled with
bigeye snaper (Kittiphattanabawon et al. 2005) and ocellate puffer fish skin (Nagai et al.
2002). The electrophoretic results eventually underpinned type I collagen as the major
collagen in squid skin.
6. Other Important Factors
6.1. Tyrosine Measurement
Tyrosine content requires to be measured in collagen samples, usually hydrolyzed at 1050C in 6 N hydrochloric acid for 24 hours under a nitrogen atmosphere. Amino acids are quantified by liquid phase ion-exchange chromatography using an analyzer (e.g.
Biochrom 20) (Cliche et al. 2003: 504).
6.2. Hydroxyproline Determination.
A colorimetric assay is used to determine the hydroxyproline content following
hydrolysis of collagen at 1050C in 7 N sulfuric acid for 16 hours. Colorimetric reaction is
done with chloramine-T and 4-dimethylbenzaldehyde using the Kolar (1990) method.
Hydroxyproline content is converted to total collagen using a factor of 7.57 (Bonifer and
Froning, 1996).
7. Problems with Extraction and Identification
A major difficulty in the identification of intact tissue antigens is the need to
preserve antigenicity, i.e., the conformational integrity of the epitopes and to achieve
adequate ultrastructural preservation. The methods like aqueous fixation, dehydration,
8
and resin embedding for electron microscopy give rise to considerable changes in tissue
structure, including extraction, precipitation, and collapse of labile molecular constituents
(Hunziker, 1993). Furthermore, conventional chemical fixation in aldehyde solutions can
lead to complete loss of antigenicity owing to the formation of intermolecular cross-links.
Collagen epitopes appear to be particularly sensitive to aldehyde fixation. To overcome
the problems associated with structural artifacts induced by conventional aqueous
fixation and dehydration procedures, alterative methods of tissue preparation adopt
employing low temperature. These methods have been applied to cartilage, with
considerable improvements in the ultrastructural preservation of chondrocytes and matrix (Hunziker, 1993; Keene and McDonald, 1993; Hunziker and Schenk, 1984; Hunziker et al., 1984; Akisaka and Shigenaga, 1983) and superior immunolocalization of matrix
proteoglycan epitopes (Hunziker and Herrmann, 1987). Till 1995, it was not possible to successfully localize specific collagens by those methods, which prompted the
researchers to assume that a particular stage in the cryotechnical process may give rise to conformational changes in collagen epitopes (Hunziker, 1993).
8. Conclusion
The review depicts a promising evolution of the extraction process, where several
researchers have enriched the methods, besides providing valuable insights regarding the
nature of collagens in different sources, where a growing trend of exploiting nonbovine
sources of collagen such as fish or chicken-skin is observed. Such trend is justified since
the usage of collagen has been ramified over the years and the disease and cultural factors
associated with bovine or porcine collagen have become points of concern in
commercially exploiting them.
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