Type II Collagen

1.1. Sources and Composition

Type II collagen is a particular type of collagen prevalent in humans, and is synonymous with the dietary supplements such as shark gelatin^[1] and gelatin^[2] while in the process of making the collagen more water soluble it can be referred to as solubilized collagen; collagen type II is commonly appreviated as CII, and will be the phrasing used throughout the course of this article for consistency.

CII's main supplemental purpose is for the treatment of joint pain and arthritic conditions, as for a dietary protein source (collagen or gelatin protein) despite good absorption from the intestinal tract^[2][3] it has lower concentration of essential amino acids and is devoid of L-cysteine.^[4][5]

CII primarily refers to:

• Various CII peptides ranging between 1,000-30,000 Da in weight,^[6] and these peptides have been noted to be absorbed intact from the intestinal tract of rats when orally administered^[2] with one peptide in particular being designated 250-270 as that appears to be the weight range where the most active peptides are located in^[7]

• Various tripeptides containing Glycine such as Gly-Pro-Hyp (Glycine-Proline-Hydroxyproline, which can reach up to 8% weight after enzymatic treatment of collagen^[8]), Gly-Pro-Glu (Glycine-Proline-Glutamine^[9]) and Pro-Gly-Pro (Proline-Glycine-Proline^[9])

There are various peptides contained within CII with the active ones appearing to be fairly low molecular weight and are likely small peptides (like tripeptides) comprised of a high amounts of glycine and proline, two amino acids that are prominent in collagen protein

With sources of CII being:

• Shark-derived CII appears to be 12kDa in weight and effective for similar purposes^[10]

• Porcine CII ha shown efficacy at similar doses to other types^[11]

• Chicken sternum has shown to be a source of CII and a brand name product from this source is UC-II®^[12][13]

CII may also be derived from bovine articular cartilage, but due to bovine spongiform encephalopathy (Mad Cow Disease) alternate sources are sought after since some authors believe that there is a risk of infection associated with bovine sources;^[10] a similar concern led to modern phosphatidylserine supplements being derived from soy rather than bovine cortex.

Various mammals and fish have been shown to be possible sources of CII, and each source appears to be quite similar in potency when supplemented in rodents and humans

1.2. Physicochemical Properties

Denaturation refers to the irreversible process of unwinding peptide chains into their constituent amino acids, and is used in the processing of gelatin protein supplements via heating;^[14] this process when carried to completion is known to degrade CII into its constituent amino acids and ablate its benefits hence why CII supplements that are active in arthritic conditions are sometimes touted to be undenatured (ie. UC-II®^[15]).

Hydrolyzed collagen refers to the process of hydrolysis on collagen, which has a heat and acid component.^[16] However, as hydrolysis of collagen tends to break down peptides from larger forms into smaller ones which are in the weight range for known bioactive peptides^[17] supplemental hydrolyzed collagen may have a similar supplemental role; it has shown joint pain reducing properties in humans although at a much higher dose of 10g.^[18]

The process of hydrolysis and denaturation break down peptides into smaller peptide chains and ultimately into their constituent amino acids, and upon reaching the final solution of amino acids benefits associated with the CII peptide will not occur (as CII is destroyed). Hydrolyzed collagen shows some benefits to joint pain despite this process, but requires a significantly higher oral dose

1.3. Biological Relevance

There are numerous types of collagen in mammals including collagen type II, III, VI, IX, X, XI, XII, and XIV which contribute to the mature matrix while collagens II, IX, and XI contribute to a developing matrix;^[19] CII is the most prominent, with over 80% of total collagen being comprised of this type in youth possibly increasing to over 90% in mature articular cartilage.^[19]

The synthetic capacity of CII in articular chondrocytes appears to decrease after skeletal tissue stops growing,^[19] and its synthesis has been noted to be increased after joint injury.^[20]

1.4. Formulations and Variants

Undenatured CII (UC-II®) is a patented form of collagen derived from chicken sternum that is glycosylated^[21] and has been used in a few rodent^[15][21][22] and industry-funded human studies.^[12][13] and appears to be well tolerated in rats with a very high LD₅₀ value (greater than 5,000mg/kg)^[15] and at the active dose of 40mg in human subjects.^[12]

UC-II is a glycosylated form of CII derived from chicken cartilage and appears to be an effective supplemental form in humans and rodents

2.1. Tolerogenic Dendritic Cells

The first site of activity for oral ingestion of CII in instances of autoimmune inflammation appears to be at the level of dendritic (antigen presenting) immune cells in the intestines, areas known as peyer's patches which have a high population of dendritic cells. Tolerogenic refers to the capacity to form a tolerance to an antigen via influencing other immune cell populations such as T cells.

Ingestion of low doses of CII appears to cause a change in these dendritic cells which promote T cells to differentiate into a more antiinflammatory form known as a T regulatory (Treg) cell, observed via the receptors they express; Treg cells that are relevant to CII ingestion are CD4⁺CD25⁺Foxp3⁺ Treg cells which reduce differentiation of T cells into CD4⁺.

CD4⁺CD25⁺Foxp3⁺ Treg cells produce more of a cytokine known as interleukin 10 (IL-10) and this interleukin suppresses the actions of another type known as IL-17; as IL-17 exacerbates rheumatism, increasing IL-10 concentrations reduces its actions and the subsequent joint pain.

Continuing production of CD4⁺CD25⁺Foxp3⁺ Treg cells from CII is known as 'tolerance', which is achieved within a month of CII supplementation and can be continually held until supplementation is stopped where T cell populations and the two interleukins produced from them normalize about a month later resulting in symptoms recurring.

Via a dendritic cell -> Treg cell -> IL-10 pathway oral ingestion of CII can produce a state of transient tolerance, similar in concept to providing an antigen prior to an infection, which results in less inflammatory cytokine activity and analgesic effects on joints of those affected with inflammatory joint disorders.

Citations for the above can be found in the Inflammation and Immunology section under Interleukins and T cells

3.1. Absorption

In rats given an oral dose of hydrolyzed collagen (gelatin hydrolysate) at 10g/kg bodyweight, the overall bioavailability of the protein supplement over the course of the next 12 hours was 95% reaching peak plasma concentrations after six hours;^[2] when testing the serum, peptides ranging from 500 daltons to 15 kilodaltons in weight from collagen appear to be absorbed intact.^[2]

The role of type II collagen in immunology may not require absorption, as intestinal goblet cells are involved in deliverying antigens to tolerogenic dendritic cells (DCs)^[23] which are located in intestinal Peyer's patches.^[24][25]

3.2. Distribution

Oral ingestion of 10g/kg hydrolyzed collagen in rats, despite being 85% eliminated from plasma within 24 hours, appeared to accumulate in the skin as after peak levels are seen in the skin (after 12 hours) there is still 58% of the dose detectable in the skin after 192 hours;^[2] other organs measured (liver, kidney, spleen, and skeletal muscle) did not appear to accumulate hydrolyzed collagen relative to amino acid control.^[2]

The aforementioned ingestion of hydrolyzed collagen did appear to accumulate in rat cartilage after 12 hours, maintaining concentrations for the entire 192 hour observation period without significant decline.^[2]

3.3. Elimination

With oral ingestion of 10g/kg hydrolyzed collagen in rats, despite good absorption 85% of the radioactivity of the labelled amino acids disappeared from plasma within 24 hours.^[2]

4.1. Neurogenesis

In mice fed collagen peptides either of low molecular weight (2,000 and 8% Gly-Pro-Hyp) or high molecular weight (30,000) as control, both in the drinking water for four weeks, noted that the low molecular weight peptides appeared to enhance neurogenesis in the dentate gyrus (of the hippocampus) about 20% more than control and these effects occurred alongside mild anxiolytic effects.^[8] It was hypothesized that the peptides worked centrally,^[8] as a similar tripeptide (Gly-Pro-Glu) has shown blood brain barrier penetrative capacities^[26] and peptides (like Noopept) have the potential to mimick larger neurotrophic proteins like BDNF and NGF.^[27]

While the mechanisms are largely hypothetical at this point, it is suggested that peptides found in low molecular weight collagen supplements may increase neurogenesis after oral administration

5.1. Osteoblasts

The de novo synthesis of type I collagen appeaers to have a role in promoting osteoblastic differentiation.^[28][29]

5.2. Collagen and Joints

Supplementation of CII is thought to confer some benefits to otherwise healthy subjects in part because when assessing serum antibodies towards the body's own structural CII it is detectable to a similar degree in both normal subjects as well as rheumatic subjects.^[30]

Oral supplementation of undenatured CII at 40mg a day (for four months) in subjects reporting joint pain but with no history of arthritis noted that supplementation was effective in improving knee range of motion (from 73.2º to 81º with no change in placebo) and a longer time for joint pain to occur during exercise and faster recovery, maximal benefits occurring three months after supplementation and maintaining;^[12] this study, however, failed to notice any influence on daily joint pain as assessed by KOOS.^[12]

Low dose collagen appears to be effective for joint pain experienced during exercise despite not affecting whole-day joint pain scores relative to placebo

5.3. Bone Density

Oral ingestion of collagen peptide in rats at three doses of 0.166g/kg, 1.66g/kg, and 16.6g/kg (the lowest being equivalent to 10g daily in a 60kg human) for four weeks noted that only the highest dose mildly increased femur density with no influence on tibial density in growing rats^[6] while in aged calcium-deficient rats it had a growth effect on the femur at 1.66g/kg (no influence on the tibia).^[6]

In male rats given hydrolyzed collagen in their diets at either 20% (normal) or 40% (high) caloric intake, or a combination of 20% collagen with 20% casein (compared to controls with casein), it was noted that after 11 weeks of exercise training both groups experienced increases in bone density, mass, and strength with exercise.^[31] A high protein intake per se is known to exert a bone growth effect when paired with exercise^[32] and when comparing casein against collagen they did not appear to significantly differ in benefits.^[31]

Oral administration of shark gelatin to protein-deficient ovarectomized rats at 100-400mg/kg for two weeks noted some increase in density of the proximal metaphysis of the femur peaking at 200mg/kg, but failed to increase overall bone mass relative to albumin control;^[1] this was associated with an increase in glycosaminoglycan content of the epiphysis,^[1] however gelatin ingestion having been noted to have effects on bone mineral density in instances of protein undernutrition that are not replicated in instances of higher protein intake.^[33]

As a general statement, the immunological effects of CII do not appear to apply much to bone health and the role of CII supplementation in bone density pertain to it simply being a protein source. It might be able to increase bone mass mildly when high doses (estimated 10g in a human) paired with a low protein diet, but when the overall protein intake is increased this benefit is lost and it performs equally to other protein sources

Currently the only human study using collagen and investigating bone mass noted that supplementation of 10g collagen protein alongside intramuscular injections of calcitonin noted an increase in bone mineral density over the course of 24 weeks in osteoporotic women.^[34]

5.4. Osteoarthritis

In osteoarthritic (induced by monoiodoacetate, which promotes cartilage erosion^[35]) rats fed low dose type II collagen (1-10mg/kg) for two weeks appeared to exert analgesic effects with highest efficacy at 1mg/kg associated with an attenuation of plasma CTX-II (40-53%; no effect without osteoarthritis) with no influence on CPII, suggesting a protective mechanism^[11] as CTX-II is produced by action of metalloproteinases acting upon type II collagen^[36] and is a relevant biomarker for osteoarthritis.^[37]

Oral supplementation of UC-II (undenatured CII from chicken sternum) in osteoarthritic subjects at 40mg once daily over 90 days noted that it was effective (relative to baseline values) in improving WOMAC, VAS, and Lequesne scores of osteoarthritic pain and mobility in a somewhat time dependent manner.^[13] This study compared UC-II against a combination of glucosamine hydrochloride (1,500mg) with chondroitin sulphate (1,200mg) and found either equipotency or (in the case of WOMAC) more efficacy with UC-II.^[13]

Oral supplementation of 10g hydrolyzed collagen daily for six months in subjects with osteoarthritis was noted to reduce pain assessed by VAS and WOMAC but failed to affect other parameters of WOMAC such as stiffness and function relative to placebo.^[18]

5.5. Rheumatoid Arthritis

T regulatory (Treg) cells play a role in arthritis as they appear to accumulate at sites of inflammation relative to peripheral blood in arthritic patients despite no alterations in count overall relative to healthy controls,^[38][39] and these Treg cells appear to be more effective at suppressing effector T cells^[40][41] and have numerous surface receptors (Foxp3 mRNA, CTLA-4, and GITR as examples) overexpressed relative to other Treg cell types^[42] with higher serum inflammatory factors being produced in these tissue (ex. IL-6 and TNF-α) relative to synovial fluid from normal controls.^[43][44]

Administration of the active peptide fragment (250-270) to the serum of rheumatic patients is able to increase CD3⁺ T cell populations and particularly both CD3⁺CD25⁺ and CD3⁺CD69⁺ subsets.^[7]

Rheumatism is a disease state associated with abnormal and elevated T regulatory cell function in synovial fluid of those affected

In rodent models of rheumatism such as collagen-induced arthritis (high concentrations of collagen injections can cause autoimmune rheumatism^[45]) oral ingestion of the CII component known as peptide 250-270 in mice (0.1-0.5mg) for one week before induction of arthritis is able to reduce the severity of subsequent arthritis^[7][46] or prevent it from occurring in a few samples.^[7] Other models of rheumatism such as ovalbumin-induced^[47] and Complete Freund’s Adjuvant-induced^[48][10] also see benefit with oral CII administration by similar mechanisms.

The benefits seen in rodent studies are associated with less immunoreactivity from splenocytes^[7] and less CII-specific IgG being produced^[7] specifically IgG_2A as IgG₁ is increased.^[46] T cells seem to take a CD3⁺CD25⁺ phenotype producing more IL-10^[46][47] (which suppresses the pro-rheumatic functions of IL-17^[49]) and TGF-β while T cells overall proliferate less when subsequently exposed to CII in vitro;^[46]

IFN-γ can be stimulated with higher concentrations in vitro (40μg/mL) but are unaltered during oral tolerance^[46] and the T cells which produce it have been noted to be lessened in content after tolerance.^[47]

The oral administration of CII prior to induction of collagen-induced rheumatoid arthritis functions similar to an oral vaccination, allowing immune cells to become tolerant to CII and preventing or reducing the expected adverse autoimmune responses to induction of rheumatism

Oral ingestion of solubilized type II collagen (0.1mg for one month, 1mg for two months) in subjects with severe active rheumatoid arthritis for 90 days appeared to improve symptoms on joints (swelling, pain, and tenderness) and preserved 15m walking time relative to placebo which benefitted to a lesser degree^[50] with four subjects in the collagen group (14% of the sample) reporting resolution of rheumatism.^[50]

One human study noting benefit with supplementation also noted that cessation of supplementation for three months was associated with symptoms returning, supplementation after this period reintroduces benefits.^[50] The benefits seen with supplementation correlate with the reduction in CII-specific antibodies detectable in serum,^[51][30] and while microgram doses seem to outperform low milligrams doses (1-2.5mg^[51]) 10mg has also been noted to outperform lower (1mg) doses.^[30]

6.1. Interferons and Immunglobulins

Oral ingestion of a subactive dose of CII to mice (15µg) prior to induction of arthritis has been noted to become more potent when coadministered alongside TGFβ1 due to more T cell (CD8⁺) induction,^[52] a property known to apply to TGFβ inherently.^[53]

Some rheumatic patients have serum IgA and IgG antibodies to CII at baseline, and despite not changing throughout the course of oral CII therapy (20-2,500µg for 24 weeks) their presence may predict responsiveness to therapy.^[51] Overall antibody titres do not appear to correlate with disease status nor with improvement seen with oral supplementation of CII.^[30]

6.2. Interleukins

Oral ingestion of CII (0.1-1mg/kg) to mice later give arthritis via collagen injections (an animal model of rheumatism) has been noted to alter T cell populations in a manner that promotes IL-10 secretion, by encouraging differentiation into CD4⁺CD25⁺ T cells;^[46] the cytokine itself is known to be antiinflammatory via suppressing the inflammatory cytokine IL-17 (required in the pathology of autoimmune rheumatism^[54]) and is itself therapeutic in instances of rheumatism^[55] by this suppressive mechanism.^[49][56]

Studies assessing concentrations of IL-17 itself do not appear to note any changes with ingestion of CII peptides relative to control groups.^[57]

The activity of IL-10 appears to be increased secondary to higher concentrations of this interleukin, which suppress the activity of IL-17 despite not changing concentrations of the latter; the change of signalling in this interleukin axis, which is due to T cell population changes, mediated the antiinflammatory effects of supplementation

In mice given rheumatism oral supplementation of CII appears to reduce circulating concentrations of IL-6 relative to arthritic control while increasing IL-2 concentrations, also thought to be related to the alteration in T cell differentiation seen during oral tolerance.^[48]

6.3. T Cells

It seems in mice given type II collagen orally (0.1mg) before induction of arthritis (by collagen injection with Freund's adjuvant) can reduce the severity of arthritis^[24][25] which has been noted to be related to a higher intestinal concentration of indoleamine 2,3-dioxygenase (IDO) expressing (aka. CD11c⁺) dendritic cells.^[25] Dendritic cells have a role in stimulating naive T cells^[58] and CD11c⁺ cells in these immunized mice hinder collagen-stimulated CD4⁺ T cell proliferation secondary to increasing activity of CD4⁺CD25⁺Foxp3⁺ regulatory T cells dependent on IDO,^[25] as the latter T cell phenotype can independently increase IDO expression in dendritic cells.^[59][60]

Orally supplemented type II collagen can promote a particular type of T regulatory cell (CD4⁺CD25⁺Foxp3⁺) to prevent overactivity of another type of T cell (CD4⁺) in response to collagen, which reduces the potential autoimmune reaction to collagen exacerbating symptoms of osteoarthritis

When assessing T cell proliferation overall (assessed via thymidine uptake), in rheumatic patients given 1mg or 10mg of CII for twelve weeks there appeared to be suppression of proliferation in most subjects.^[30]

7.1. Skin

Type I collagen comprises approximately 80% of the total collagen of the adult human dermis^[61] and is known to be responsive to serum factors such as glucocorticoid therapy (decreasing type I collagen content^[62]) and UV irradiation (reduces procollagen I content^[63][64]) thought to contribute to the effects of stress and sun exposure (respectively) on photoaging; preventing decreases of procollagen I actually underlies the protective effects of low concentrations of Vitamin A when topically applied.^[64][65]

Type II collagen is not a major constituent of skin like it is in articular cartilage, as types I and III have more of a presence in skin,^[61] although CII can still accumulate in the skin following oral ingestion of hydrolyzed collagen supplements in the rat.^[2]

In skin under normal conditions, the CII content is much less than is found in articular cartilage and instead type I and type II cartilage predominate. Alterations in the production of type I collagen underlie visual changes in the skin in response to environmental factors

Hydrolyzed collagen is thought to be a supplement for skin health since oral ingestion increases serum levels of some dipeptides and tripeptides^[66] of which one of them, proline-hydroxyproline (Pro-Hyp), can stimulate fibroblasts to produce the molecule known as hyaluronic acid (200µM^[67])^[68][69] which is in and of itself a skin health supplement;^[70] this effect is also seen in chondrocytes, thought to underlie some joint health properties.^[68]

Fibroblasts incubated with Pro-Hyp also see an increase in cellular proliferation^[67][69] associated with hyaluron synthase 2 (HAS2) induction, dependent on PKA activation.^[69]

Ingestion of CII is thought to increase synthesis of type I collagen and hyaluronic acid in the skin secondary to being a good source of the dipeptide known Pro-Hyp

In an open-label study with females with some signs of skin aging then given hydrolyzed collagen (Biocell®; comprised of peptides ranging from 1,000-2,500kDa in weight and some hyaluronic acid with chondroitin) at 1g a day for 12 weeks noted that supplementation was associated with transient increases in wrinkling and crow's feet at six weeks while at the end of the study crow's feets normalized while wrinkles were reduced relative to baseline (13.2%);^[71] hydration showed a similar trend over time, ultimately being improved after 12 weeks.^[71]

An increase in hemoglobin content of the skin has been noted with supplementation of the Biocell formulation at both 6 and 12 weeks, thought to be reflective of increased dermal microcirculation.^[71] Dermal collagen content was noted to be increased at both time points while melanin was not affected.^[71]

8.1. General

Undenatured CII (UC-II®) appears to be safe in rodents with an acute oral LD₅₀ of greater than 5,000mg/kg and acute dermal LD₅₀ of over 2,000mg/kg with 90 days continuous ingestion finding no abnormalities^[15] and other studies also using chicken sternum CII finding a similar degree of safety in rats and dogs.^[72]