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Tecnical Information

About Collagen

Collagen: The Body's Cellular Fabric

Collagen is a common protein that makes up a significant part of the living body, whether human or animal. As a structural protein, collagen is essential to creating the body's physical structure, and as an extracellular matrix it acts as a supporting framework over which our cells are arranged.
The collagen molecule is composed of three intertwined peptide chains as shown in the following figure. It is a rigid protein 300 nm in length, 1.5 nm in diameter, and approximately 300 kilodaltons (kDa) in molecular weight.
The entire molecule consists of about 3,000 amino acids.

 

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Structural protein: Proteins that form the body's anatomical structure and sustain tissue morphology (the shapes and forms of tissue within the body).
Extracellular matrix: Collagen forms a scaffold-like structure between cells that is composed of proteins and polysaccharides.

 

The Role of Collagen in the Body

Collagen is not scattered as individual molecules throughout the body, but is directed to form parts of higher-ordered structures. These structures differ according to their location in the body, however it is believed that each is composed in the same manner from several stages of increasing complexity, as shown in the following figure.
For example, the cornea is composed of collagen, and its transparency is sustained by its unique higher-order structure.
In addition, intermolecular cross-linking occurs between collagen molecules in such higher-ordered structures, and these links serve to increase the strength of tissue and to ensure temperature stability.

fig2

 

Extraction of Collagen

Collagen is widely used in the medical, cosmetic, and research fields. Currently, collagen is obtained from animal tissues, but it is difficult to do so because collagen in the body is completely bound up due to its intermolecular cross-linking. The solution to this problem lies in cleaving these crosslinks, or solubilizing the collagen. One method of collagen solubilization utilizes proteases, enzymes that break the crosslinks between collagen molecules, as indicated in the following figure. Collagen obtained through solubilization is called Atelocollagen.

fig3

 

Characteristics of Atelocollagen

Atelocollagen is a collagen solubilized by protease, but its physical properties are virtually identical to those of natural, unsolubilized collagen. Furthermore, atelocollagen additionally has superior characteristics not found in the natural substance.
Collagen is known for its very low antigenicity (or reactivity, i.e., it causes little immune response) because most of the collagen molecule is composed of a G-X-Y amino acid sequence that differs little even among different animal species. The slight amount of antigenicity that is seen in collagen is thought to be due to the telopeptides attached to each end of the collagen molecule, which do not contain the G-X-Y sequence. Since the telopeptides are not present in atelocollagen, the antigenicity of atelocollagen is even lower than that of collagen.

 

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More Features of Atelocollagen

In addition to its low antigenicity, atelocollagen is generally obtainable with a high degree of purity. This feature is due to the protease treatment, which when used to extract atelocollagen breaks down other protein contaminants.

 

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Atelocollagen can be Processed into Various Configurations.

Another feature of atelocollagen is that it is soluble, so in liquid form it can be engineered into a wide array of physical shapes.
By simply allowing the solution to dry, atelocollagen films can be produced, sponge-like structure can be achieved by freeze-drying, and string-like configurations are possible through extrusion. Atelocollagen beads, powders, and gels are also possible.
In this manner, it is possible to leverage the special properties of atelocollagen to produce the most appropriate configuration for any application.

 

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The Mutable Properties of Atelocollagen

By chemically modifying atelocollagen, its physical properties can be altered. For example, atelocollagen is normally insoluble in water with neutral pH, but this characteristic can be altered to make it soluble in such an environment. It is also possible to use atelocollagen equally as a coagulant or as an anticoagulant, and it is also possible to control the rate at which it is absorbed by the body.

 

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The Mutable Properties of Atelocollagen

By chemically modifying atelocollagen, its physical properties can be altered. For example, atelocollagen is normally insoluble in water with neutral pH, but this characteristic can be altered to make it soluble in such an environment. It is also possible to use atelocollagen equally as a coagulant or as an anticoagulant, and it is also possible to control the rate at which it is absorbed by the body.

 

KOKEN Atelocollagen is absolutely safe.

KOKEN Atelocollagen is obtained from the dermis of Australia bred calves. There is no cause for concern regarding bovine spongiform encephalopathy (BSE) affecting atelocollagen because KOKEN atelocollagen is obtained under the strictest conditions of safety.
BSE is caused by a very stable protein, or prion. It is difficult to remove prions without modifying atelocollagen, so KOKEN ensures the tissue is not contaminated with prions in the first place.
Calf dermis is known to be a tissue free from infection by BSE prions. The source calves are strictly monitored from birth up to the point that the dermis is obtained, to ensure that the KOKEN atelocollagen is produced only from materials where the possibility of BSE has been eliminated.

 

Products

  1. Clinical Medicine
    1. Blood coagulating cotton fabric
    2. Injections to remedy abscesses in soft tissue
    3. Dental bone filling material
    4. Absorptive membranes for periodontal regeneration
  2. Tissue Culture Research
    1. Atelocollagen solution for tissue culture
    2. Atelocollagen films
    3. Atelocollagen sponges
    4. Atelocollagen porous honeycomb sponge
    5. Atelocollagen microspheres
  3. Cosmetics
    1. Atelocollagen
    2. Succinylated atelocollagen
    3. Myristoylated atelocollagen
    4. Succinylated-myristoylated atelocollagen
    5. Collagen derived from tuna
 

KOKEN Special-Purpose Atelocollagen Design

KOKEN has developed techniques for chemical and physical modification, as well as molding into various shapes.
Combining these techniques, KOKEN can supply collagen materials customized to your specifications.
For example, cellular growth matrices play an important role in regenerative treatment and atelocollagen is a material well suited for this purpose. However, atelocollagen must be formed into the right structure to optimally utilize its superior properties. KOKEN has the technical know-how to produce atelocollagen configurations to order.

 

REFERENCES

  1. A Honeycomb Collagen Carrier for Cell Culture as a Tissue Engineering Scaffold, Artificial Org., 25 , 213-217 (2001)
  2. Current Concepts in Tissue Engineering Technique for Repair of Cartilage Defect, Artificial Org., 25 , 172-179 (2001)
  3. Tissue-Engineered Product: Allogeneic Cultured Dermal Substitute Composed of Spongy Collagen with Fibroblasts, Artificial Org., 25 , 180-186 (2001)
  4. Biomaterials for Gene Delivery: Atelocollagen-mediated Controlled Release of Molecular Medicines, Current Gene Therapy, 1, 31-52 (2001)
  5. New Delivery System for Plasmid DNA in vivo Using Atelocollagen as a Carrier Material:the Minipellet, Nature Medicine, 5, 707-710 (1999)
  6. Antisense Oligodeoxynucleotide Targeted to Midkine, a Heparin-binding Growth Factor, Suppresses Tumorigenicity of Mouse Rectal Carcinoma Cells, Cancer Res., 61, 8486-8491 (2001)
  7. Atelocollagen-Based Gene Transfer in Cells Allows High-Throughput Screening of Gene Functions, Biochem. Biophys. Res. Comun., 289, 1075-1081 (2002)
  8. Collagen Engineering for Biomaterial Use, Cinical Materials, 9, 139-148 (1992)
 

KOKEN History

fig8

More than 40 years ago, Dr. Teruo Miyata (KOKEN CEO) developed the technique for solubilizing collagen with pepsin while simultaneously removing its main antigenic structures, the telopeptides. He named this solubilized collagen メAtelocollagenモ. Since then, atelocollagen and this production technique have been used around the world. It has been used as an enabling material in medical fields for more than 20 years, during which widespread use has proven its safety.
KOKEN has been the leader in collagen solubilizing techniques, producing high quality atelocollagen and conducting practical research into its various uses.

 

If Atelocollagen is applied...

There are already medical treatment products using Atelocollagen, some of which have already been in use for more than 20 years. Also, there are products that are being used by millions of people all over the world, so its safety has been thoroughly demonstrated.
Research is also being conducted into new applications of atelocollagen. For example, it is nearing use as a carrier for DDS, as a carrier for gene delivery and as a carrier for Tissue Engineering, and these practical applications are heading for commercial use in the near future.
In this way, Atelocollagen applications will increasingly extend across a wide range in the future.

fig9 Serum IFN concentrations in mice after administration of IFN as an aqueous solution (○), 1×105IU IFN, and Minipellet (●) 5×105IU IFN

fig10 Collagen Minipellet for DDS

Description of the above figure: This graph shows the blood concentration and duration of dosage in animals to which Interferon (IFN) is administered using two different methods. The ○ symbol indicates dosage via injection, and ● indicates dosed via mixing with collagen. Mixing with collagen allows the effect of a single dose of IFN to be sustained for a long period of time.

 

Atelocollagen applied to gene profiling

fig11

The human genome project is complete, and the functions of many available genes remain unknown. It is therefore necessary to elucidate the function of a large number of genes in a short period of time, and to achieve this goal, materials are needed that condense or package DNA into nanoparticles that can be easily taken up by cells and those in which DNA can be kept without degradation. Atelocollagen is a reliable carrier for gene delivery because it is considered safe and appropriate for practical use.
As a complementary approach to gene expression profiling on cDNA microarrays, we developed a basic technique for high-throughput gene transfer and expression screening by pre-coating a microplate with an Atelocollagen/cDNA complex to which cells are then seeded. Further antisense ODNs, siRNAs can be also transferred into in vivo or in vitro cells.

 

Atelocollagen applied to Tissue Engineering

Tissue engineering is an emerging field that aims to regenerate natural tissues and create new tissues using biological cells, biomaterials, biotechnology, and clinical medicine. The current scope of tissue engineering is primarily experimental but is rapidly expanding as developments occur daily. The term 'tissue engineering' was officially coined at a National Science Foundation workshop in 1988 to mean the application of principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues, and the development of biological substitutes to restore, maintain or improve tissue function. Collagen is the best material for tissue engineering scaffold, and can be converted into fiber, sponge, film, gel and various other shapes.

 

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Collagen engineering for biomaterial use
Miyata T, Taira T, Noishiki Y.
Clin Mater. 1992;9(3-4):139-48.

A honeycomb collagen carrier for cell culture as a tissue engineering scaffold
Itoh H, Aso Y, Furuse M, Noishiki Y, Miyata T.
Artif Organs. 2001 Mar;25(3):213-7.

Biomaterials for gene delivery: atelocollagen-mediated controlled release of molecular medicines.
Ochiya T, Nagahara S, Sano A, Itoh H, Terada M.
Curr Gene Ther. 2001 May;1(1):31-52.

New delivery system for plasmid DNA in vivo using atelocollagen as a carrier material: the Minipellet.
Ochiya T, Takahama Y, Nagahara S, Sumita Y, Hisada A, Itoh H, Nagai Y, Terada M.
Nat Med. 1999 Jun;5(6):707-10.

Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions.
Honma K, Ochiya T, Nagahara S, Sano A, Yamamoto H, Hirai K, Aso Y, Terada M.
Biochem Biophys Res Commun. 2001 Dec 21;289(5):1075-81.

Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions.
Honma K, Ochiya T, Nagahara S, Sano A, Yamamoto H, Hirai K, Aso Y, Terada M.
Biochem Biophys Res Commun. 2001 Dec 21;289(5):1075-81.

Antisense oligodeoxynucleotide targeted to Midkine, a heparin-binding growth factor, suppresses tumorigenicity of mouse rectal carcinoma cells.
Takei Y, Kadomatsu K, Matsuo S, Itoh H, Nakazawa K, Kubota S, Muramatsu T.
Cancer Res. 2001 Dec 1;61(23):8486-91.

5'-,3'-inverted thymidine-modified antisense oligodeoxynucleotide targeting midkine. Its design and application for cancer therapy.
Takei Y, Kadomatsu K, Itoh H, Sato W, Nakazawa K, Kubota S, Muramatsu T.
J Biol Chem. 2002 Jun 28;277(26):23800-6.

A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics.
Takei Y, Kadomatsu K, Yuzawa Y, Matsuo S, Muramatsu T.
Cancer Res. 2004 May 15;64(10):3365-70

Antisense oligodeoxynucleotide against HST-1/FGF-4 suppresses tumorigenicity of an orthotopic model for human germ cell tumor in nude mice.
Hirai K, Sasaki H, Sakamoto H, Takeshita F, Asano K, Kubota Y, Ochiya T, Terada M.
J Gene Med. 2003 Nov;5(11):951-7.

Atelocollagen for protein and gene delivery.
Sano A, Maeda M, Nagahara S, Ochiya T, Honma K, Itoh H, Miyata T, Fujioka K.
Adv Drug Deliv Rev. 2003 Nov 28;55(12):1651-77.

Targeted conversion of the transthyretin gene in vitro and in vivo.
Nakamura M, Ando Y, Nagahara S, Sano A, Ochiya T, Maeda S, Kawaji T, Ogawa M, Hirata A, Terazaki H, Haraoka K, Tanihara H, Ueda M, Uchino M, Yamamura K.
Gene Ther. 2004 May;11(10):838-46.

Potential of atelocollagen-mediated systemic antisense therapeutics for inflammatory disease.
Hanai K, Kurokawa T, Minakuchi Y, Maeda M, Nagahara S, Miyata T, Ochiya T, Sano A.
Hum Gene Ther. 2004 Mar;15(3):263-72.

Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo.
Minakuchi Y, Takeshita F, Kosaka N, Sasaki H, Yamamoto Y, Kouno M, Honma K, Nagahara S, Hanai K, Sano A, Kato T, Terada M, Ochiya T.
Nucleic Acids Res. 2004 Jul 22;32(13):e109

Gene expression profiling of cerebellar development with high-throughput functional analysis.
Saito S, Honma K, Kita-Matsuo H, Ochiya T, Kato K.
Physiol Genomics. 2005 Mar 29; [Epub ahead of print]