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The following table lists some major antibodies used in the study of collagen type II and gives the details the species they were derived from, their working concentrations in different methods and an example of a research paper they were used in. An example of a secondary antibody has also been included.
 
The following table lists some major antibodies used in the study of collagen type II and gives the details the species they were derived from, their working concentrations in different methods and an example of a research paper they were used in. An example of a secondary antibody has also been included.
  
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Revision as of 07:26, 3 June 2015

Extracellular Matrix 2015 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7

Projects are now locked for final Assessment.

Type II Collagen

Introduction

Collagens are polymeric proteins that have a important role in the structure of the cells. These tripe helical molecules occur in the extracellular matrix and in the cell-extracellular matrix interface. [1] There are 28 known isoforms of collagen in humans, where this protein family is present in a variety of structures and in a wide range of tissues. [2] In this page, the main topics about the type II collagen are discussed. This subcategory consist on fibrils that are the major components of cartilage, intervertebral discs, and the vitreous humor of the eye, being also vital to the normal development of bones and teeth. [3]

History

Collagen was first discovered to have a molecular structure in the mid-1930s [4]. During the 1950s, many well-known scientists including Crick, Rich, and Ramachandran were researching the structure of collagen.

In 1955, Ramachandran developed the “Madras” model, which depicted the triple helical structure of collagen [4][5]. Ramachandran also proposed the structure of collagen to contain two interchain hydrogen bonds. However, Crick and Rich proposed an alternative model showing collagen possessing only a single interchain hydrogen bond [5]. However, after much speculation, Ramachandran’s model provided the correct quaternary structure for the molecule, although it did require some minor changes [4]. He used collagen samples from the tendons of kangaroo tails and produced X-ray diffraction patterns from collagen fibres [5].

Collagen is also additionally packed into ‘sheet-like’ fibrillar collagen types with hexagonal shapes. In 2006, the microfibrillar structure of the adult tendon was confirmed by Frase, Miller and Wess [4]. They discovered the D-periodic pentameric arrangement and defined it as the microfibril.

In 1969, Collagen type II was discovered by Miller and Matukas [6].

Structure

The mainly characteristic of a collagen molecule is its helical tape rigid and long structure . In this helical tape, three polypeptide chains (alpha chains) are wound on each other forming a kind of super twist rope. To enable that, collagen has plenty of prolines and glycines on its structure, the first one stabilizes the alpha chain helice conformation and the second is regularly spaced throughout the central region of the alpha chain. Since glycine is the smallest amino acid, it also allows the three alpha chain helices to cluster and form final collagen super-helice. [7]

The type II collagen is fibrillar, which means being composed by fine structures (10 to 300nm diameter) with hundreds micrometers lengh visible using eletronic mycroscopy in mature tissues. [8]

Collagen triple-helix


Biosynthesis

The collagen precursor, procollagen, is the first molecule to be synthesized. The procollagen is mainly composed of glycine and proline amino acids and it is modified by an addition of hydroxyl groups onto the amino acids proline and lysine. This modification has no important significance to step of the protein biosynthesis that happens in the sequence: the glycosylation of hydroxylysine, that consists on the addition of either glucose or galactose onto the lysines’ hydroxyl group. These hydroxyl groups are important once they form interchain hydrogen bonds that helps the triple-stranded helix to stabilizes. After the glycosylation, when the triple-helix is formed, the new molecule, now called procollagen, presents two different structural parts: a twist central portion and two loose ends in both terminations. This molecular conformation is packed and transported to the Golgi apparatus to be modified with the addition of oligosaccharides onto its structure. Ended those modifications, the procollagen is sended to the extracellular space where membrane bound enzymes called “collagen peptides” will remove the loose ends of the molecule and transform it into tropocollagen. The final step of the collagen biosynthesis is coordinated by other extracellular enzymes known as “lysyl oxidases" that act on hydroxylysine and also lysine and produce the aldehyde groups that will eventually undergo covalent bonding with another tropocollagen molecule. The polymers formed by tropocollagen chains are what we know as collagen fibrils.

Developmental Stages

Studies have shown that in human embryos, over weeks 4-12 gestation, there is a temporal and spatial distribution of Collagen Type II mRNA in the vertebral column development. [9] Collagen is first observed in the vertebrate embryo at about the time of gastrulation (see Green et al., 1968; Cohen and Hay, 1971; Manasek, 1975). Gastrulation is the time the cleaving embryo begins to form the primary embryonic tissues - ectoderm, mesoderm, endoderm. [10]

The skeleton of vertebrates forms via two processes: intramembranous and endochondral ossification [11]. Throughout intramembranous ossification, mesenchymal cells differentiate into osteoblasts where bone is directly produced. During endochondrial ossification (EO), the mesenchymal cells differentiate into chondrocytes, which undergo proliferation, maturation and hypertrophy. When chondrocytes reach hypertrophy there is matrix calcification, degradation of the cartilage matrix, and bone deposition. It is during EO, the growth plate is established. The growth plate is another tissue that experiences compressive loads, and hence 80% of the total collagen in this tissue comprises of collagen II [12].

Function

Fibrillar collagens play a structural role by contributing to the shape, and mechanical properties of tissues such as the tensile strength in skin and the resistance to traction in ligaments [13]. In cartilage, the extracellular matrix is mainly composed to collagen II. The small yet strong fibrils of collagen II [12] offer the matrix strength and compressibility, which is vital for synovial joints to maintain their shock absorbing properties properties [14].

Articular Cartilage

Synovial joints are covered by a highly specialised form of connective tissue known as articular cartilage [15]. It provides a load-bearing and lubricating surface with low friction, and facilitates painless skeletal movement in areas such as the ends of long bones [16]. Articular cartilage is limited in its capacity for healing and repair because it lacks blood vessels, nerves and lymphatics [15].

Structure of Healthy Articular Cartilage

In articular cartilage, collagen II is cross-linked to proteoglycans by collagen XI to form a complex 3D heterotypic fibrillar network, which has a vital function in maintaining the tensile stiffness and strength of cartilage [14] [17].

The structure of the cartilage can be broken down into three zones [15]:

  • The articular surface contains densely aligned collagen type II and IX fibers, as well as an increased number of flat chondrocytes. The integrity of this layer is vial for the protection of the deeper layers. This layer is in contact with synovial fluid, and accounts for most of the tensile properties of cartilage – resistant to sheer and compressive forces imposed by articulation.[15]
  • The transitional zone contains thicker collagen fibrils and proteoglycans. The chondrocytes in this layer are almost spherical. This zone is also involved in the resistance against compressive forces. This zone represents 40% to 60% of the total cartilage volume, whereas the articular surface only makes up 10% to 20% of articular cartilage thickness. [15].
  • The deep zone offers the highest resistance against compressive forces due to the perpendicular arrangement of the collagen fibrils. The chondrocytes are structured in a columnar orientation (parallel to the fibrils). There is a high proteoglycan content and the largest collagen fibrils are located in this region. This zone makes up 30% of articular cartilage volume. A calcified layer sits underneath the deep zone to ensure the cartilage is attached to the bone through the anchoring of collagen fibrils of the deep zone to the subchondral bone.[15]
Callus formation at fracture sites in mice femurs after different treatments

Skeletal Bone Development

Studies [11] have shown that the loss of the two abundant cartilage components, collagen II and perlecan, can have severe effects on skeletal development. This is because the strong heterotypic fibrils of collagen II have an essential role in maintaining the correct structure and development of bones [11]. Perlecan maintains the collagen network by regulating the activity and binding of matrix metalloproteinases (MMPs), which are known to degrade collagen, in particular MMP-13, which is linked to the degradation of collagen II [18].

Fracture Healing

Collagen II is also known to play an essential role in maintaining the process of bone fracture healing[19]. It is involved in the osteogenic differentiation of bone marrow derived mesenchymal stem cells (BMMSCs) to increase the success of bone formation, and even marrow organisation. In recent studies [19] it was found that there was a greater bone mass and callus formation in the femur of rats treated with collagen II as opposed to collagen I. The histological data showed more densely woven bone tissue and marrow formation with a cartilaginous external callus. This implies that the new bone is forming by a process similar to EO.

Abnormalities

Collagen Type II gene, COL2A1, when mutated or defects are present, can significantly affect the phenotypes of cartliage and bone [20]. The focus of this section will be on Stickler Syndrome, Kniest dysplasia, Achondrogenesis type 2 and Hypochondrogenesis.

Where is the COL2A1 gene located?

Location of COL2A1 gene on chromosome 12.jpeg
  • on the long (q) arm of chromosome 12 at position 13.11. = 12q13.11 [21]
  • the COL2A1 gene is located from base pair 47,972,964 to base pair 48,025,285 on chromosome 12 [21]

The mutations are expressed in the heterozygous state, and inheritance of type II collagenopathies is autosomal dominant.[22]

When discussing collagen type II disorders, there is a phenotypic spectrum that is often referred to [23]. The lethal achondrogenesis type 2 and hypochondrogenesis are at the severe end, Kniest dysplasia in the middle of the spectrum, and Stickler Syndrome and familial osteoarthritis, at the milder end [23].

When the gene, COL2A1, is mutated, the disorders and defects, mentioned above, are commonly known as Type II Collagenopathies. Below is a table listing the Type II Collagenopathies that we have focused on and we have compared their phenotypes and pathogenesis to one another.

Abnormality/Disorder # of mutations in COL2A1 gene that cause this mutation Phenotype Pathogenesis Image
Stickler Syndrome Type I 200 A clinically variable mutation: characterized by ocular, auditory, skeletal, and orofacial abnormalities [24].

Common ocular abnormalities include: high myopia, vitreoretinal degeneration, retinal detachment, and cataracts [24].

Mutations in the COL2A1 gene that result in Stickler Syndrome include an abnormally short pro-alpha1(II) chain produced that cannot be incorporated into a type II collagen fiber or there’s a premature stop signal in the instructions for making the pro-alpha1(II) chain [21].

As a result, cells only produce half the normal amount of this collagen chain, which therefore affects the amount of type II collagen in cartilage and other tissues [21].

Kniest Dysplasia 20 Characterized by short trunk and limbs, kyphoscoliosis, midface hypoplasia, severe myopia, and hearing loss [25]. The deletion of one or more DNA building blocks (nucleotides) in the COL2A1 gene result in the production of abnormally short pro-alpha1(II) chains, which then join to normal-length chains. This mismatch of normal and short pro-alpha1(II) chains brings about abnormal type II collagen molecules that are shorter than usual [21].

This shorter, atypical type II collagen molecule collagen prevents bones and other connective tissues from developing properly [21]. Overall, in this disorder, an abnormality of collagen fibril organization is responsible for the symptoms of Kniest Dysplasia [25].

Achondrogenesis Type II or the Langer-Saldino type 18 Characterized by severe micromelic dwarfism with small chest and prominent abdomen, incomplete ossification of the vertebral bodies, sacrum and pubic bones, disorganization of the costochondral junction [26] and underdeveloped lungs [21]. Mutations in the COL2A1 gene that cause Achondrogenesis type II alter the amino acid glycine by replacing it with a different amino acid along the pro-alpha1(II) chain.

This mutation consequently prevents the normal production of mature triple-stranded type II collagen molecules, which results in the lethal skeletal abnormalities seen in this disorder [21].

Hypochondrogenesis 18 A lethal disorder of bone growth characterized by a small body, short limbs, and abnormal bone formation in the spine and pelvis [21]. Some mutations delete part of the COL2A1 gene and result in a pro-alpha1(II) chain that is missing critical segments or the mutations, like in Achondrogenesis Type II, alter the amino acid glycine by replacing it with a different amino acid along the pro-alpha1(II) chain [21].

All of these mutations interfere with the formation of mature triple-stranded type II collagen molecules, which results in the features of hypochondrogenesis by affecting tissues that are rich in type II collagen [21].


FACTS

  • In both, Achondrogenesis, type II and Hypochondrogenesis, death occurs in utero or the early neonatal period (1)
  • Achondrogenesis, type II/Langer-Saldino type and Hypochondrogenesis are often grouped together clinically due to their similarities in phenotype and pathogenesis.
  • Spranger et al. (1974) distinguished 2 forms of achondrogenesis, which they called types I and II. Type I was subdivided into type IA (200600) and IB (600972). (1)

Current Understanding and Areas of Research

The fact that collagen type Ii makes up the bulk of mammalian cartilage means that current research is centred around the cure and treatment of diseases involving collagen type II such as Rhuematoid Arthritis. The following is a breakdown of recent articles:

The effect of estrogen on the expression of cartilage-specific genes in the chondrogenesis process of adipose-derived stem cells.[27]

The aim of this study was to determine the effect of oestrogen on genes that control chondrogenesis. Adipose-derived stem cells (ADSCs) were differentiated into cartilage and then treated with estrogen in order to determine the genetic markers associated with the expression of type II collagen. The study found that type II collagen was found in the control group but not in the experimental group. Aggrecan was detected in both groups but there was an significant decrease in aggrecan expression in the experimental group. The results of the study demonstrate the inhibitory effect of oestrogen on the expression of collagen type II. The implication is that oestrogen is not suitable for use in the chondrogenesis of type II cartilage from ADSCs. This information can be used to optimise methods in developing cartilage from ADSCs in order to produce effective treatments for conditions pertaining to joint degeneration such as rheumatoid arthritis (RA).

Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis.[27]

This aim if this study was to assess the potential of collagen type II regulatory T cells (Col-Treg) for treatment of RA. The results of the study indicated that introduction of Col-Treg cells reduced the incidence and clinical symptoms of arthritis in both preventive and curative settings. Significant impacts were seen on

Macroscopic Comparison of Rat Knee Articular Cartilages After Treatment with BMMSCs.[28]

collagen type II antibodies as well as noticeable decreases in antigen-specific effector T cells. This means that collagen type II T regulatory cells could be an effective treatment from patients suffering from RA.

Remission of Collagen-Induced Arthritis through Combination Therapy of Microfracture and Transplantation of Thermogel-Encapsulated Bone Marrow Mesenchymal Stem Cells.[27]

A new therapeutic strategy for autoimmune inflammatory diseases such as RA was suggested in this study focusing particularly on the effect on collagen type II. The results indicated that the majority of SD rats developed some form of irreversible bone or cartilage degradation with the exception of the bone marrow mesenchymal stem cell (BMMCs) treated group. The BMMCs group displayed significantly less symptoms of arthritis onset than the non-treatment groups but were still possessed higher degrees of disease than normal rats. This means that BMMSC therapy could reverse synovial hyperplasia to an extent and could be effective in treating joint degradation in patients suffering from RA.

TGF-β1 conjugated chitosan collagen hydrogels induce chondrogenic differentiation of human synovium-derived stem cells. [27]

This study tested the effectiveness of a biofunctional hydrogel consisting of collagen type II nanofibers and transforming growth factor β1 (TGF-β1) in the regeneration of cartilage. Results indicated that collagen type II impregnation and TGF-β1 delivery significantly promoted chondrogenesis in human synovium-derived mesenchymal stem cells(hSMSCs). This hydrogel system could be an effective treatment for cartilage defects in patients and the results also provide evidence to support the hypothesis that collagen type II impregnation in conjunction with TGF-β1, promotes chondrogenesis in hSMSCs.

The following link presents all the articles on pubmed related to the search term "collagen+type+II": Collagen Type II

Antibodies

The following table lists some major antibodies used in the study of collagen type II and gives the details the species they were derived from, their working concentrations in different methods and an example of a research paper they were used in. An example of a secondary antibody has also been included.

Antigen Species Working Concentrations Secondary Antibody Example of Use
MAB8887 Anti-Collagen Type II Antibody, clone 6B3 MAB8887 Mouse Western Blot: Assay Dependent, Immunofluorescence: Assay Dependent, Immunohistochemistry: 1-2 µg/ml, ELISA: Assay Dependent Type II collagen expression is regulated by tissue-specific miR-675 in human articular chondrocytes.[29]
Collagen II Antibody MA5-37493 MA5-37493 Mouse Western Blot: 1-2 µg/ml, Immunofluorescence: Assay Dependent, Immunohistochemistry: 1-2 µg/ml, Flow Cytometry: Assay Dependent Biotinylated secondary antibody to rabbit IgG[29] Articular cartilage increases transition zone regeneration in bone-tendon junction healing.[29]
Collagen II Antibody (MA5-12789) MA5-12789 Mouse Western Blot: 1-2 µg/ml, Immunofluorescence: Assay Dependent, Immunohistochemistry: 1-2 µg/ml, Flow Cytometry: Assay Dependent Mouse anti-Human IgA (Alpha heavy chain) GA01 A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers.[30]
Collagen II Antibody (CIIC1) CIIC1 Mouse ELISA: 1-2 µg/ml. Immunohistochemistry: Assay Dependent, Functional assay: Assay Dependent Collagen II Antibody (M2139) Arthritogenic anti-type II collagen antibodies are pathogenic for cartilage-derived chondrocytes independent of inflammatory cells.[31]

Models

Methods (IHC, WB)

Glossary

Ossification: The process of bone formation

Mesenchymal cells: Unspecialised cells that are capable of differentiation into all supporting tissue cell types (e.g. bone and cartilage).

Osteoblasts: Synthesises osteiod, ultimately giving rise to bone formation.

Osteocytes: Phagocytic cells which are capable of eroding bone.

Hypertrophy: The enlargement of an organ or tissue due to the increased size of constituent cells.

Chondroblasts

Chondrocytes

References

  1. Weis, M., Hudson, D., Kim, L., Scott, M., Wu, J. and Eyre, D. (2009). Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly. Journal of Biological Chemistry, 285(4), pp.2580-2590.
  2. Weis, M., Hudson, D., Kim, L., Scott, M., Wu, J. and Eyre, D. (2009). Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly. Journal of Biological Chemistry, 285(4), pp.2580-2590.
  3. Weis, M., Hudson, D., Kim, L., Scott, M., Wu, J. and Eyre, D. (2009). Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly. Journal of Biological Chemistry, 285(4), pp.2580-2590.
  4. 4.0 4.1 4.2 4.3 Mandal, A. (2014). What is Collagen? Viewed May 21, 2015. Available from: http://www.news-medical.net/health/What-is-Collagen.aspx Cite error: Invalid <ref> tag; name "newsmedical" defined multiple times with different content Cite error: Invalid <ref> tag; name "newsmedical" defined multiple times with different content Cite error: Invalid <ref> tag; name "newsmedical" defined multiple times with different content
  5. 5.0 5.1 5.2 Subramanian. E., (2001). ‘G.N. Ramachandran.’ Nature Structural Biology, vol. 8, pp. 489-491. Cite error: Invalid <ref> tag; name "ramachandran1" defined multiple times with different content Cite error: Invalid <ref> tag; name "ramachandran1" defined multiple times with different content
  6. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003457/
  7. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, et al. Biologia molecular da celula. 5ª edição. São Paulo: Artmed; 2010. P (1184). "Os colágenos são as principais proteínas da matriz extracelular".
  8. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, et al. Biologia molecular da celula. 5ª edição. São Paulo: Artmed; 2010. P (1185-1186). Tabela 19-7. "Alguns tipos de colágeno e suas propriedades".
  9. <pubmed>8838495</pubmed>
  10. Hay, ED. (1981). Cell Biology of Extracellular Matrix, "Collagen and Embryonic Development" pp379-409. Viewed on 20 May, 2015. doi:10.1007/978-1-4613-0881-2_13 http://link.springer.com/chapter/10.1007/978-1-4613-0881-2_13
  11. 11.0 11.1 11.2 <pubmed> 12814946 </pubmed>
  12. 12.0 12.1 <pubmed> 24088220 </pubmed>
  13. Ricard-Blum, S. (2011). The Collagen Family. Cold Spring Harbor Perspectives in Biology, 3(1), a004978. doi:10.1101/cshperspect.a004978
  14. 14.0 14.1 Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 22.3, Collagen: The Fibrous Proteins of the Matrix. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21582/ Cite error: Invalid <ref> tag; name "lodishmatrix" defined multiple times with different content
  15. 15.0 15.1 15.2 15.3 15.4 15.5 Sophia Fox, Alice J., Asheesh Bedi, and Scott A. Rodeo. “The Basic Science of Articular Cartilage: Structure, Composition, and Function.” Sports Health 1.6 (2009): 461–468. PMC. Web. 26 Apr. 2015. Cite error: Invalid <ref> tag; name "foxcartilage" defined multiple times with different content Cite error: Invalid <ref> tag; name "foxcartilage" defined multiple times with different content Cite error: Invalid <ref> tag; name "foxcartilage" defined multiple times with different content Cite error: Invalid <ref> tag; name "foxcartilage" defined multiple times with different content Cite error: Invalid <ref> tag; name "foxcartilage" defined multiple times with different content
  16. <pubmed> 25786729 </pubmed>
  17. <pubmed> 12658447 </pubmed>
  18. <pubmed> 25044117 </pubmed>
  19. 19.0 19.1 <pubmed> 24411332 </pubmed>
  20. Genetics Home Reference - Kniest Dysplasia. Viewed 21 May, 2015. Available from: http://ghr.nlm.nih.gov/condition/kniest-dysplasia
  21. 21.00 21.01 21.02 21.03 21.04 21.05 21.06 21.07 21.08 21.09 21.10 Genetics Home Reference - COL2A1. Viewed 20 May, 2015. Available from: http://ghr.nlm.nih.gov/gene/COL2A1 Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content Cite error: Invalid <ref> tag; name "COL2A1" defined multiple times with different content
  22. <pubmed>8157027</pubmed>
  23. 23.0 23.1 <pubmed>10406661</pubmed>
  24. 24.0 24.1 Online Mendelian Inheritance in Man - Stickler Syndrome. Viewed 23 May, 2015. Available from: http://www.omim.org/entry/108300 Cite error: Invalid <ref> tag; name "OMIM108300" defined multiple times with different content
  25. 25.0 25.1 Online Mendelian Inheritance in Man - Kniest Dysplasia. Viewed 23 May, 2015. Available from: http://www.omim.org/entry/156550 Cite error: Invalid <ref> tag; name "OMIM156550" defined multiple times with different content
  26. Online Mendelian Inheritance in Man - Achondrogenesis Type II. Viewed 26 May, 2015. Available from:http://www.omim.org/entry/200610
  27. 27.0 27.1 27.2 27.3 <pubmed> 25789269</pubmed> Cite error: Invalid <ref> tag; name "”PMID" defined multiple times with different content Cite error: Invalid <ref> tag; name "”PMID" defined multiple times with different content Cite error: Invalid <ref> tag; name "”PMID" defined multiple times with different content
  28. <pubmed>25774788</pubmed>
  29. 29.0 29.1 29.2 <pubmed>18987921</pubmed> Cite error: Invalid <ref> tag; name "“PMID18987921”" defined multiple times with different content Cite error: Invalid <ref> tag; name "“PMID18987921”" defined multiple times with different content
  30. <pubmed>17205546</pubmed>
  31. <pubmed>15934095</pubmed>

The five most recent articles related to the search term "collagen+type+II" on pubmed:

<pubmed limit=5>Collagen type II</pubmed>