collagen hydrolysate sports collagen... · collagen hydrolysate is classified as a food, not a drug...

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Collagen Hydrolysate

Its role in reducing joint pain, promoting healthy

joints and treating connective tissue injury

Revitalise Sports Collagen Report © March 2019, Xenca Ltd.

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Contents

What is Collagen?

Why Would I Need a Collagen Supplement?

How Does Collagen Hydrolysate Work and What Are Its Benefits

How Safe Are Collagen Supplements?

Clinical Studies

About Revitalise Sports Collagen

Summary

Appendices

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What is Collagen?

Collagen constitutes between 25-30% of all the protein in the human body. It is a major structural protein which forms molecular chains that strengthen the tendons and large strong sheets which support the skin and internal organs. It is also a major element of our hair and nails, whilst mineral crystals added to collagen make bones and teeth.

Structurally, collagen is composed of three chains which are wound together in a tight triple helix. Each chain is over 1,400 amino acids long and a repeated sequence of three amino acids, proline, glycine and lysine make up this sturdy structure. Vitamin C is vital for the crosslinking of the three chains and this is what gives the structure its strength.

Collagen constitutes 1-2% of muscle tissue and accounts for 6% of the weight of strong, tendinous muscles. Known as ‘the glue that holds the body together’, it provides an essential structure to our bodies, protecting and supporting the softer tissues and connecting them with the skeleton. Collagen in healthy tissue is very strong and, on weight basis, is almost as strong as steel.

Over 20 different types of collagen have been identified but the three most common types are:

Type 1: Found in skin, hair, tendons, vascular ligatures, organs and bones.

Type 2: The main component of cartilage.

Type 3: The main component of reticular fibres, commonly found alongside Type I in connective tissue.




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Why Would I Need a Collagen Supplement?

Wear and Tear and Ageing

Sufficient collagen levels in the body are essential for the maintenance of healthy joints, connective tissue, skin, nails and hair. Our bodies produce collagen naturally but they also break it down constantly too. Unfortunately, as we get older our bodies lose the ability to produce sufficient amounts of collagen and our reserves therefore start to deplete. From their mid-20s onwards the average person loses 1.5% of their collagen reserves annually, a process that accounts for many of the symptoms of wear and tear and the ageing process. Skin loses elasticity, causing fine lines and wrinkles to appear, aches and pains may develop and hair and nails may lose their strength and shine.

Injury

Healthy levels of collagen are also essential for the prevention of injury and sound recovery thereafter. For example, in scar tissue collagen fibres are generally smaller and weaker than those of the normal, healthy, surrounding tissue, attaining only about 80% of their strength. That scar tissue is, therefore, more prone to further injury. Higher levels of collagen by supplementation may, therefore, reduce the risk of repeat injury, as they might have reduced the risk of actual injury if taken before.




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Osteoarthritis

Osteoarthritis is the main cause of physical impairment in people over the age of 65 in the UK, with over 9 million sufferers, but it is by no means exclusive to that age group. A common treatment for osteoarthritis is the use of non-steroidal, anti- inflammatory drugs (NSAIDs). Concern has been expressed that NSAIDs may be toxic to articular cartilage and may even advance the timeline of osteoarthritis. Non-drug alternatives including collagen are therefore certainly worthy of consideration.

Increasingly more people are deriving benefits from Collagen Hydrolysate in terms of pain relief and increased mobility in joint function. (See Paragraph 1. Clinical Studies - Hans-Konrad Selbmann, MD of the University of Tuebingen Germany).

General

Clearly a wide range of people could benefit from supplementation by Collagen Hydrolysate: sportspeople, those recovering from injuries and accidents, those with osteoarthritis, those who wish to lessen the impact of the ageing process and many more.

This short video is an excellent illustrative summary of some of the benefits of Collagen Hydrolysate: https://youtu.be/U5kXexH44oc


https://youtu.be/U5kXexH44oc



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How Does Collagen Hydrolysate Work and What Are Its Benefits?

How it works

When collagen-bearing material undergoes enzymatic hydrolysis, becoming Collagen Hydrolysate, the collagen molecule breaks down into smaller components. These components are then small enough to be digested and become speedily absorbed into the body. When taken orally, the Collagen Hydrolysate passes through the stomach into the gastrointestinal tract. Here it is broken down further into single amino acids and di-peptides, which then pass into the bloodstream from the small intestine and are delivered to the body where it is needed.

90-95% of the Collagen Hydrolysate absorbed reaches the bloodstream (this is known as bioavailability). 90-95% is, therefore, a very high percentage, given that liquids administered intravenously are 100% bioavailable.

The absorption by the intestine of Collagen Hydrolysate can be measured by a widely accepted experimental model.




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It was determined during MRI trials (McAlindon TE, 2011), with a delayed Gandolinium Enhanced Magnetic Resonance Image (dGEMRIC), that the actual take- up of Collagen Hydrolysate can be seen as a positive change in 'Proteoglycan' content (i.e. the filler substance between cells, giving it a gel-like nature) in the knee cartilage after 24 weeks (see Figure 4, above).

In tests a significant amount of collagen has been found in cartilage and skin tissue even after one single administration, also indicating an accumulation of these peptides in the connective tissue.

(S. Oesser, M. Adam, W. Babel, J. Seifert : Oral administration of 14C-labeled gelatine hydrolysate leads to an accumulation of radioactivity in cartilage of mice (C57/Bl).J Nutr, 129 (1999) 1891-1895

(See Appendices 4, 5 and 6)

The Benefits of Collagen Hydrolysate

We are all different and our individual chemistry and metabolisms are unique to us. However, if our choice of supplement is based on reliable, scientifically-derived data, the likelihood of deriving benefit from a supplement is certainly much higher than relying on manufacturers’ (sometimes over-exaggerated) claims.

Bearing this in mind and referring to the reports, studies and data in the Appendix, the following benefits of Collagen Hydrolysate can be reasonably identified:-

Speedier recovery from injury, wounds and soft tissue damage.

Pain relief and increased mobility in arthritic and stiff joints.

Ligament, tendon and cartilage regeneration.

Reduction of fine lines and wrinkles.

Improved texture, strength, and appearance, of skin, nails and hair.




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How Safe Are Collagen Supplements?

There have been many trials in many countries over the years and these have shown Collagen Hydrolysate to be a benign supplement, as it is based on a natural element in our bodies. In fact Xenca Revitalise Sports Collagen has been certified as safe for use by elite athletes by the world renowned Informed-Sport , Informed Choice and LGC Sports Science Laboratory (See Appendix 7 and 8).

Informed-Sport and Informed Choice are global quality assurance programmes for sports nutrition products, suppliers to the sports nutrition industry and supplement manufacturing facilities. The programmes certify that every batch of a supplement product that bears the Informed-Sport / Informed Choice logo has been tested for banned substances by LGC's world-class sports anti-doping laboratory. Supplements users should use the search function on the Informed-Sport / Informed Choice site to find products that have been through this rigorous certification process, while athletes are strongly advised to cross reference the tested batches listed on the product pages with the batches they are consuming. The programmes also aim to provide athletes with an effective risk management service so that they can choose products that have undergone additional quality control testing, as required in elite sport. In monitored clinical trials of over 2,000 patients given Collagen Hydrolysate, no undesirable side effects were reported.

It has a long history of being produced as a food product since the 1800s and has been consumed in Europe since the Napoleonic wars.

Collagen Hydrolysate is classified as a food, not a drug and is therefore safe for regular and long term consumption.

Athletes susceptible to joint impact injuries have been successfully treated in the USA with large doses of 10g a day over a period of three months.

It should be noted, however, that Collagen Hydrolysate should not be stated to cure or prevent any disease.




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Clinical Studies

The effects of Collagen Hydrolysate supplementation in the treatment of degenerative joint disease have been investigated for over thirty years. According to Dr Hans-Konrad Selbmann of the University of Tübingen in Germany, 16 studies involving a combined total of 2,000 subjects have been published on its therapeutic use so far. The general conclusion is that administering five grams of Collagen Hydrolysate daily for the duration of at least three months has a beneficial impact on pain symptoms and joint function in patients with osteoarthritis. During the start of 2019 a clinical study into the reduction of pain in Achilles tendinopathy patients when taking an oral supplement of Collagen Hydrolysate (combined with calf-strengthening exercises) was published. The conclusion made by the Australian Institute of Sport and University of Canberra was that oral supplementation of Collagen Hydrolysate may accelerate the clinical benefits of a well-structured calf-strengthening and return-to-running program in patients with chronic Achilles tendinopathy symptoms. (See Appendix 1) Penn State University in University Park, Pennsylvania conducted a placebo-controlled, double blind 24-week study on the use of Collagen Hydrolysate as a dietary supplement in athletes with activity-related joint pain. They concluded that, despite the study’s size and limitations, the results suggest that athletes consuming Collagen Hydrolysate can reduce parameters (such as pain) that have a negative impact on athletic performance. (See Appendix 2) Recently ‘The Times’ featured an article promoting the importance of a collagen supplement for athletes. An extract from the article quotes Professor Graeme Close, “Collagen is getting a lot of attention in elite sport and that message is now filtering down to the fitness masses. There used to be not much you could do about knee pain or tendon problems in sport, but there’s now convincing evidence that collagen taken with vitamin C can be really helpful both in terms of injury prevention and recovery from intense activity.” (See Appendix 3)




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A randomised, double-blind study by Dr James Rippe in Massachusetts, USA is one of many studies to support these findings. The randomised, double-blind study investigated knee pain, stiffness, joint mobility, flexibility and development of strength in 190 patients with mild osteoarthritis of the knee. Over a period of 14 weeks those affected were given a daily dose of 10 grams of Collagen Hydrolysate (plus 300 milligrams of calcium and 60 milligrams of vitamin C) or placebo. Isometric and isokinetic measurements were then taken which showed a significant improvement in physical joint function in those patients who were given the Collagen Hydrolysate. (See Appendices 2 and 4)

Dr Steffen Oesser of the Collagen Research Institute in Kiel, Germany is one of the foremost researchers in this field. As early as 1999 a research group led by Dr Oesser produced evidence of intestinal absorption of Collagen Hydrolysate at an average peptide size of 3.5 kDa. After absorption collagen fragments accumulated significantly and exclusively in the joint cartilage. In in-vitro experiments Dr Oesser was able to show a significant, dose-related stimulation of type II collagen synthesis in chondrocytes (the cells that make up the cellular matrix of cartilage) when Collagen Hydrolysate was added. (See Appendices 1 and 2)




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After addition of the Hydrolysate there was also a significant increase in pericellular proteoglycans (the material between cells that provides structural support to connective tissue). In contrast, no increase in protease activity was observed. This led Dr Oesser to conclude: "These results indicate an increase in synthesis activity in the whole extracellular matrix of the cartilage. Therefore, Collagen Hydrolysate could become increasingly important, as far as nutrition for individuals with degenerative disease of cartilage tissue is concerned.”

Numerous other clinical and anecdotal findings also suggest that hydrolysed collagen may be helpful in the following: promotion of lean muscle mass through the burning of fat rather than carbohydrates and proteins, toning and thickening skin, joint rebuilding, arterial strengthening, increased energy, organ rebuilding, alleviating osteoporosis, reducing high blood pressure, bladder weakness, chronic fatigue, shallow breathing, autoimmune, skin problems and splitting nails.




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About Revitalise Sports Collagen

It’s always wiser to look at the full picture when understanding quality of life and people’s desire for a healthier and happier self. At Revitalise Sports Collagen we recognise that, as human beings, we aren’t really looking for pills, lotions, creams or bottles. Our souls aren’t aching for a quick fix, our bodies deserve more than mass-produced, poor-quality products, our individuality is paramount, from our unique biology to our innermost desires. By working their magic from within, our exceptional products embody the key mission of Revitalise Sports Collagen, enhancing quality of life in a sustainable, holistic and lasting manner. We stay clear of chemicals and shortcuts, instead harnessing the very best of scientific expertise and natural processes. We believe that our bodies know best how to embrace their full potential and we design our customized products to simply provide a gentle nudge in the right direction by providing our systems with the best blend of nutrients to counteract ageing, pollution, injury and stress.

Revitalise Sports Collagen has been specifically launched to benefit sportspeople and athletes. This is because the peptide process specifically targets strengthening and injury recovery of connective tissue, which forms the vast majority of injuries in sport.

A maintenance dose of 5 g per day will help an athlete’s body stay strong and supple and facilitate more effective recovery from training regimes than unaided natural recovery alone. Where a sportsperson has injured connective tissues (e.g. torn ligaments, tendons and muscles) doses of a minimum of 15g per day (based on body mass and weight) will significantly speed recovery.

Doses of 15g per day combined with resistance training will significantly increase the building of fat-free muscle.

It is safe for elite sportspeople and athletes because it carries full product certification by Informed-Sport and Informed-Choice, which are globally respected testing programs.

Here is a link to the Xenca Ltd YouTube channel, where various athletes talk about their experiences using Xenca Revitalise Sports Collagen: http://www.youtube.com/playlist?list=PL3OemL3-SPZo-vnRQfgK8TiNQ19xN-zT-


http://www.youtube.com/playlist?list=PL3OemL3-SPZo-vnRQfgK8TiNQ19xN-zT-



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Tom Kelly, European Powerlifting Champion

Tom has overcome several injuries and setbacks over the years, including 3 surgeries which most recently involved an arthroscopy of his left knee to clean up some damage to lateral meniscus, underside the patella and articular cartilage at the upper outside aspect of the knee.

On returning from surgery Tom immediately included Xenca’s Collagen Revitalise supplement in his rehab programme and, in the words of his surgeon, has made remarkably good progress considering the extensive nature of the operation. With such a clear endorsement from a specialist, Tom will continue his rehab, aided by this great supplement.




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Dan Smith, World Champion IFBB Wheelchair Pro Bodybuilder

“Being in a wheelchair means that for the rest of my life my elbows and shoulders are going to take a massive amount of wear and tear, the collagen powder has made a huge difference in reducing the amount of pain I was getting in my joints and also helps with faster recovery post training. Due to the manufacturing process this Hydrolysate Collagen powder is cutting edge and the best currently available for sports athletes. Mainly it focuses on elasticity and strength, reduces soft tissue injury/damage, skin rejuvenation, protects from wear and tear, encourages healthy joints and aids in repairing and growing muscle tissue.... Supplements nowadays are not always allowed in sports and some professions, for example pre-workouts, intro workouts and some protein powders etc.... so this amazing product which is tested and supported by informed-sport is 100% safe to use in any sport or occupation that may test for banned substances. So get your hands on some. Each bag has 3 months’ supply at a maintenance dose but with elbow tendon pain I double the dose and really feel the benefits.”




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Here is a link to a series of interviews with the England Hockey Great Grand Masters Team – Gold medallists and Hockey World Cup winners 2018: http://www.youtube.com/playlist?list=PL3OemL3-SPZqJQ7dnXOM4eQYz0sUlcILn

Also, see this blog post by Jason Redding, Physiotherapist and Sports Therapist for the team, which describes how the product has made a difference: http://www.xenca.com/blog/detail/hockey-masters-world-cup-report-from-jason- redding-and-michelle-ravell---sports-therapists-to-the-england-hockey-masters

Since our inception in 2012, we have developed a much valued reputation from within the industry for the way we operate and our business ethics. We believe our customers deserve to enjoy an outstanding experience in their dealings with us and we have committed ourselves to making this experience a refreshingly different kind of partnership. When we say “together we’re better” we really do mean it.


http://www.youtube.com/playlist?list=PL3OemL3-SPZqJQ7dnXOM4eQYz0sUlcILn

http://www.xenca.com/blog/detail/hockey-masters-world-cup-report-from-jason-redding-and-michelle-ravell---sports-therapists-to-the-england-hockey-masters





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Summary

As a key component in our connective tissue (hair, skin, and nails), sufficient collagen levels in the body are essential to our general wellbeing – and at times when the body needs repair. Our bodies produce collagen naturally but they also break it down constantly too so as we get older our bodies lose the ability to produce sufficient amounts of collagen and our reserves start to deplete.

Many of these symptoms of ageing and other physical challenges arising from injury, wounds and soft tissue damage can be lessened, improved and recovered from more speedily by taking hydrolysed collagen supplement on a regular basis.

Collagen is a vitally important protein. Fibrous in nature, it connects and supports the tissues of the body: skin, bone, tendons, muscles and cartilage. It also supports the internal organs and is present in eyes and teeth. In fact collagen makes up some 50% of the entire protein content of the human body.

Collagen can be thought of as the glue that holds the body together. Without it the body would, quite literally, fall apart. From our mid-twenties onwards we lose collagen at an average rate of 1.5% annually, resulting eventually in lines, wrinkles, aches and pains and many other symptoms attributed to the ageing process.

Collagen Hydrolysate (i.e. collagen protein that has been enzymatically or chemically processed to make it more digestible) taken orally has been scientifically proven to safely accumulate in bone joints and cartilage, thus offering a way of combatting the natural depletion process.

Clinical research in Germany, UK and the USA into Collagen Hydrolysate has shown that taking a daily supplement of collagen can significantly help sufferers of arthritis and ease joint pain.

Athletes susceptible to joint impact injuries have been successfully treated in the USA with larger doses of 10g of Collagen Hydrolysate a day over a period of three months.

Following treatment with Collagen Hydrolysate, study results have specifically determined the following benefits: reduced joint pain at rest, reduced joint pain when walking and standing, reduced joint pain when carrying objects and reduced joint pain when lifting weights.




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Japanese clinical research monitored the hydration status of female skin following Collagen Hydrolysate supplementation and noted an improvement of water absorption capacity. Anecdotal evidence from large numbers of users of collagen supplements supports their findings that taking collagen daily helps smooth the appearance of skin, reducing fine lines and wrinkles.

Increased collagen intake may also improve your eyes, strengthen nails and improve the condition of your hair.

In monitored clinical trials of over 2,000 patients given Collagen Hydrolysate no undesirable side effects were reported.

It is recommended that you read all the Appendices to this document, although specific references are made to particular Appendices throughout.








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Appendices

1. Oral Supplementation of Specific Collagen Peptides 2. 24-Week Study on the Use of Collagen Hydrolysate 3. Extract from The Times February 12th 2019 4. Prevention and Treatment of Osteoarthritis 5. Role of Collagen Hydrolysate in Cartilage Metabolism & Regeneration 6. Collagen Bioavailability & Dosage

7. Certificate of Analysis Revitalise Sports Collagen 8. Informed-Choice & Informed-Sport Certificates for Revitalise Sports Collagen 9. Revitalise Sports Collagen Amino Acid Composition






x 1 Oral Supplementation of

Specific Collagen Peptides

Article

Oral Supplementation of Specific Collagen Peptides Combined with Calf-Strengthening Exercises Enhances Function and Reduces Pain in Achilles Tendinopathy Patients Stephan F.E. Praet 1,2,*,Craig R. Purdam 3,Marijke Welvaert 1,2,Nicole Vlahovich 1,Gregg Lovell 1,Louise M. Burke 4,Jamie E. Gaida 2,5,Silvia Manzanero 1,David Hughes 1 andGordon Waddington 1,2 1

Department of Sport Medicine, Australian Institute of Sport, Leverrier St, Bruce ACT

2617, Australia 2

University of Canberra Research Institute for Sport and Exercise (UCRISE), Cnr

Allawoona St & Ginninderra Drive Bruce, ACT 2617, Australia 3

Department of Physiotherapy, Australian Institute of Sport, Leverrier St, Bruce ACT

2617, Australia 4

Department of Sports Nutrition, Australian Institute of Sport, Leverrier St, Bruce ACT

2617, Australia 5

Discipline of Physiotherapy, University of Canberra, Building 1/11 Kirinari St, Bruce

ACT 2617, Australia *

Author to whom correspondence should be addressed. Received: 3 December 2018 / Accepted: 24 December 2018 / Published: 2 January 2019

Abstract : The current pilot study investigates whether oral supplementation of specific collagen peptides improves symptoms and tendon vascularisation in patients with chronic mid-portion Achilles tendinopathy in combination with structured exercise. Participants were given a placebo or specific collagen peptides (TENDOFORTE®) in combination with a bi-daily calf-strengthening program for 6 months. Group AB received specific collagen peptides for the first 3 months before crossing over to placebo. Group BA received placebo first before crossing over to specific collagen peptides. At baseline (T1), 3 (T2) and 6 (T3) months, Victorian Institute of Sports Assessment–Achilles (VISA-A) questionnaires and microvascularity measurements through contrast-enhanced ultrasound were obtained in 20 patients. Linear mixed modeling statistics showed that after 3 months, VISA-A increased significantly for group AB with 12.6 (9.7; 15.5), while in group BA VISA-A increased only by 5.3 (2.3;

https://www.mdpi.com/search?authors=Stephan%20%20F.E.%20Praet&orcid=0000-0001-6714-9027

https://www.mdpi.com/search?authors=Craig%20%20R.%20Purdam&orcid=

https://www.mdpi.com/search?authors=Marijke%20Welvaert&orcid=

https://www.mdpi.com/search?authors=Nicole%20Vlahovich&orcid=

https://www.mdpi.com/search?authors=Nicole%20Vlahovich&orcid=

https://www.mdpi.com/search?authors=Gregg%20Lovell&orcid=

https://www.mdpi.com/search?authors=Louise%20%20M.%20Burke&orcid=

https://www.mdpi.com/search?authors=Jamie%20%20E.%20Gaida&orcid=

https://www.mdpi.com/search?authors=Silvia%20Manzanero&orcid=0000-0002-5294-7082

https://www.mdpi.com/search?authors=Silvia%20Manzanero&orcid=0000-0002-5294-7082

https://www.mdpi.com/search?authors=David%20Hughes&orcid=

https://www.mdpi.com/search?authors=Gordon%20Waddington&orcid=

8.3) points. After crossing over group AB and BA showed subsequently a significant increase in VISA-A of, respectively, 5.9 (2.8; 9.0) and 17.7 (14.6; 20.7). No adverse advents were reported. Microvascularity decreased in both groups to a similar extent and was moderately associated with VISA-A (Rc

2:0.68). We conclude that oral supplementation of specific collagen peptides may accelerate the clinical benefits of a well-structured calf-strengthening and return-to-running program in Achilles tendinopathy patients. Keywords: achilles tendon; microvessels; contrast-enhanced ultrasound; hydrolysed collagen supplementation

1. Introduction The treatment of chronic Achilles tendinopathy remains challenging with a

relatively high percentage of non-responders. There are many proposed treatment options, of which an eccentric exercise program is currently the first treatment of choice. Despite the good results on pain scores after eccentric exercises, a 5-year follow-up study showed that only ~40% of the patients were completely pain free and 48% had received one or more alternative treatments [1]. In accordance with this, there is still a need for treatments that would improve the benefits of a structured eccentric exercise program. Recent studies indicate that both cardiometabolic [2,3], as well as nutritional factors [4,5] can modulate local tendon healing. Although clinical studies are still scarce, a recent International Olympic Committee (IOC) consensus statement on dietary supplements in high-performance athletes [6] proposes that dietary supplementation containing gelatin or hydrolysed collagen could potentially be useful for athletic populations as increased intake of collagen-derived peptides has been shown to modulate collagen synthesis [7] and reduce tendon- [8] and joint-related pain [9,10]. Others have shown that glycine, as the most abundant component of collagen hydrolysates [11], has disease-modifying properties in both animal [12,13] and in vitro [14] models of tendinopathy. There is also first evidence, that collagen peptides might improve functional ankle properties in chronic ankle instability [15]. Therefore, we aimed to study the potential clinical benefits of a specific hydrolysed collagen supplement as add-on therapy to 6 months of an eccentric calf-strengthening and well-structured return-to-running program.

It has been well established that increased (micro) vascularity is associated with chronic (painful) tendon lesions [16]. As a change in (micro)vascularity might be a hinge for improved tendon architecture, our aim was to check whether the oral intake of collagen peptides influence microvascular changes in tendinopathic areas of the Achilles tendon using real-time contrast-enhanced ultrasonography (CEUS) [17]. In a recently published study [18], it was shown that CEUS provides a more objective and reproducible assessment of tendon microvascularity in comparison with power Doppler ultrasound. Furthermore, CEUS was also shown to be moderately associated with increased Achilles tendon symptoms. We hypothesized that improvements in tendon pain and function throughout the course of the intervention would be reflected by a reduction in CEUS-based tendon microvascularity. A previous ultrasonographic tissue characterisation study has shown that structural integrity is decreased in both Achilles tendons in people with unilateral Achilles tendinopathy [19]. As dietary supplementation of hydrolysed collagen appears to have systemic effects on collagen-dense tissue [20,21,22], we aimed to study both tendons of each individual patient, independent of whether participants had uni- or bilateral symptoms.

https://www.mdpi.com/2072-6643/11/1/76/htm#B1-nutrients-11-00076






















2. Methods The current study is a prospective double-blinded placebo-controlled clinical trial

with a cross-over design. Through balanced within block-randomization (computer-based, 5 individuals per block), eligible participants were allocated to each study arm by an independent staff member to either 3 months of bi-daily 2.5 g hydrolysed specific collagen peptides (sCP) (TENDOFORTE®) or a placebo product to be dissolved in water, both pre-packed in identical sachets (see also study flow diagram Figure 1). The bi-daily intake of sCPs was based on the pharmacokinetic profile; the placebo sachets contained 2.5 g of maltodextrin (CARGILL) obtained by enzymatic conversion of starch. The sachets containing sCP or placebo were absolutely identical in appearance and the products were equal in flavor and texture. After 3 months, participants who received the sCPs were crossed-over to the placebo product (group AB) and vice versa (group BA). Allocation concealment was further ascertained by allocating an individual to group AB or group BA. No patient, research nurse, assessor or any other medical or research staff was aware of the treatment assignments for the duration of the study. All data and statistical analyses were also completed before the randomization code was broken at the end of the completed trial [23]. Based on lack of previous data sets that allowed for a power analysis and sample size calculation, we designed a small-scale pilot study with multiple outcome measures that involved 2 groups of 10 participants. Data collection of this single-center trial was performed in an outpatient sports medicine clinic of a national high-performance sports institute. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local ethics committee of the Australian Institute of Sport (Project ID: 20141201V2) and prospectively registered (ANZCTR#12615001035516). Patients were not involved in our study design. Participants were recruited by social media and advertisements. In- and exclusion criteria are summarized in Appendix A Table A1.

Figure 1. CONSORT study flow diagram.

https://www.mdpi.com/2072-6643/11/1/76/htm#fig_body_display_nutrients-11-00076-f001


https://www.mdpi.com/2072-6643/11/1/76/htm#app1-nutrients-11-00076

https://www.mdpi.com/2072-6643/11/1/76/htm#table_body_display_nutrients-11-00076-t0A1

2.1. Exercise Intervention In addition to the nutritional intervention, all participants followed a well-

structured eccentric calf-strengthening program and milestone-based return-to-running program for a total duration of 6 months. The eccentric exercise started with a calf raise using the unaffected limb (or both limbs if the symptoms were bilateral), followed by an eccentric drop using the injured leg. The exercise was performed with both the knee straight and the knee bent to target the gastrocnemius and soleus muscles respectively. Over a 6-month period, participants were instructed to perform 2 × 90 repetitions daily, despite the presence of pain [24]. All participants were instructed to avoid weight-bearing sporting activities for the first four weeks. When participants reported less than 2 out of 10 pain on single leg hopping, they were allowed to start with low-intensity running exercises. Details and milestones used within this program can be found in Appendix A Table A2. Half an hour before the calf-strengthening exercises, participants were instructed to ingest 2.5 g of sCP or placebo product dissolved in a glass of cold water. This provides sufficient time for orally ingested sCPs to be absorbed in the gut and appear in the blood stream [25]. Participants kept a diary of their calf-strengthening exercises and were asked to return any leftover sachets at both their 3 and 6 months follow-up appointments. 2.2. Outcome Measurements.

All participants underwent a series of measurements and evaluations at baseline, 3 and 6 months to evaluate their response to the combined exercise and nutritional intervention. All measurements and questionnaires were scheduled within a time frame of 3 days and performed by the same team of health practitioners and researchers who were blinded for the nutritional intervention and trained in the standardized operating procedures for the different measurements. Both at 3 and 6 months, our participants were asked whether they thought they had been taking the sachets with sCPs or placebo. At the end of the trial and after deblinding this information was matched with true allocation order. 2.3. Victorian Institute of Sports Assessment–Achilles (VISA-A) Questionnaire, Patient Satisfaction and Return-to-Running

One day prior to the ultrasound examinations, all patients were asked to rate their subjective Achilles tendon pain and functional limitations for both the right and left Achilles tendon separately using a Victorian Institute of Sports Assessment–Achilles (VISA-A) questionnaire [26]. After 3 and 6 months patients also received a questionnaire to rate their satisfaction and to what level they had been able to resume their running sports activities (Appendix A Table A3). 2.4. Real-Time Harmonic Contrast Enhanced Ultrasound (CEUS) Measurements

Contrast-enhanced ultrasound (CEUS) measurements scans were performed as described in previous studies [17,18]. Patients were prone with fully extended knees and both ankles immobilized with an ankle angle between 5–10 degrees plantar flexion whilst ensuring minimal tendon tension. Both Achilles tendons were examined in the longitudinal plane with a 58 mm long high-frequency (5–14 MHz) linear array transducer (model 14L5/PLT-1005BT) on an Aplio™500 (TUS-A500, Toshiba/CANON Medical Systems, Tochigi Otawara, Japan). The transducer was longitudinally positioned in the Achilles tendon midline using a retort clamp attached to the bed. A 12G copper wire was placed between transducer, gel and skin to locate and mark tendon insertion in a reproducible manner. Tendon insertion was defined as the attachment point of most ventral tendon fibres to the calcaneal bone. A total of seven skin marks at 1-cm intervals were used to position the ultrasound probe over the tendon mid-portion (2–6 cm). Probe pressure was kept to a minimum to avoid










blood vessel obliteration. Scanning parameters were optimised for 3 cm depth and kept constant for all participants. Real-time harmonic CEUS of the Achilles tendon mid-portion was performed through the injection of 10uL.kg-1 body weight bolus of agitated contrast agent (DEFINITY™, Lantheus Medical Imaging, Billerica North Billerica, MA, USA) in a forearm vein, followed by 10 ml 0.9% saline solution. CEUS is based on second harmonic imaging of intravascular circulating microbubbles with a diameter of ~1–3 microns. These microbubbles act as echo-enhancers and are used with the aim to identify small vessels (down to 40 microns) [18]. Participants were instructed not to talk or change body position during recordings. Blood pressure and heart rate were measured every 5 min throughout the procedure with a digital sphygmamometer and automatically inflating arm cuff (OMRON HEM-7130, Wujia Medical Equipment Co., Ltd., Fengshan District, Kaohsiung City, Taiwan). As peak flow of CEUS signal was achieved within 2 minutes following the bolus injection, only first 120 s of digital CEUS data were used for further analysis. To allow for complete washout of perflutren gas through the lungs, a minimum of 15 min was allowed between consecutive CEUS measurements of right and left Achilles tendon. Dedicated contrast harmonic image quantification software (2B771-005EN*V, Toshiba/CANON Medical Systems, Tochigi Otawara, Japan) was used for time-curve analysis (TCA). In the present study, it involved the examination of a 25 × 13 mm region of interest (ROI) that included at least 2 mm of the anterior fat pad. Within this ROI, the relative microvascularity (MV) values were determined as peak log-scaled signal intensity in decibels (dB) of a normalised smoothed curve during the first 2 min after subtraction of baseline signal intensity 20–25 s after bolus injection. Inter-observer reliability for CEUS-based MV-values was previously shown to be excellent with an intraclass correlation (ICC) of 0.97 [17]. 2.5. Blood Sampling

At baseline, 3 and 6 months, non-fasting blood samples were obtained from venipuncture to determine total cholesterol, triglycerides and uric acid and exclude (familial) hypercholesterolaemia [27] and gout [28] as a contributing factor to tendinopathy. In addition, at each time point a 500 µL serum sample was stored at −80 °C for further analyses in case of a serious adverse event related to the intake of the investigational product. 2.6. Product Safety and Monitoring of Adverse Events

Specific collagen peptides are characterized by a considerably high safety profile. So far no clinical indications of allergies have been observed. No incompatibilities with other diets or medications have ever been described in the medical literature. Collagen peptides received the GRAS status from the Food and Drug Administration. “GRAS” is an official abbreviation which stands for “generally recognized as safe [29]. Nevertheless, participants were instructed to report any gastro-intestinal or other side effects that they thought were potentially associated with the intake of the investigational product or placebo. 2.7. Statistical Analyses

All data are presented as means ± standard deviation (SD), unless indicated otherwise. Baseline values of all parameters were tested for uniformity via discriminant analysis. All data processing and statistical analyses were performed before de-blinding the randomization order. Linear mixed model (LMM) analysis using R statistical software package was used to determine the association between CEUS-based microvascularity values and VISA-A scores at the 3 different time points. For statistically significant interactions, we determined Rc

2, which represents the variance explained by the combination of fixed effects and random effects. Mixed






analysis of variance (ANOVA) was used for all other comparisons. LMM analysis was also performed to test for changes in our main outcome variables between examination at baseline and following the 3- and 6-month intervention periods between and within groups in a cross-over design. The LMM analysis satisfies all conditions of an intention-to-treat analysis and has also been shown to be the most powerful statistical method under a variety of conditions that are common to randomized controlled trials [30]. Carry-over effects within our cross-over design were accounted for in our LMM-analyses by looking at interaction of the order and treatment effect across time. A p value < 0.05 was considered statistically significant.

3. Results 3.1. Study Population

From September 2015 until April 2016, a total of 60 patients (32 men/28 women, age 43 ± 10 years) with subjective Achilles tendon symptoms showed interest in our study. Following initial screening, 40 potential candidates (18 men/22 women, age 43 ± 11 years) were excluded or withdrew for the reasons mentioned in Figure 1. A total of 20 patients (13 men/7 women, age: 44 ± 8 years, body mass index (BMI): 24.4 ± 3.3 kg∙m−2) with clinical symptoms (mean duration 54 ± 90 months) of uni- (n = 7) or bilateral (n = 13) mid-portion Achilles tendinopathy were formally included in the study following a detailed medical history and physical examination by a sport and exercise physician (Figure 1). Demographic characteristics and blood test results of participants at baseline are presented in Table 1. To evaluate homogeneity of the data at baseline between the 2 groups a discriminant analysis was carried out. The data revealed no statistically significant differences between both study groups. All participants wished to return to pain-free running and had either uni- or bilateral Achilles tendon tenderness on palpation. The clinical diagnosis was based on the presence of tenderness and/or tendon thickening 2 to 6 cm proximal to the distal insertion. One asymptomatic tendon that previously had undergone surgery for a complete Achilles tendon rupture was excluded from our analysis. Table 1. Baseline-characteristics study population (n = 20).




https://www.mdpi.com/2072-6643/11/1/76/htm#table_body_display_nutrients-11-00076-t001

3.2. Change in VISA-A Scores Changes in Achilles tendon pain and function scores during the treatment were

evaluated through the VISA-A questionnaire [26]. Baseline VISA-A scores from right and left limb were not significantly correlated (Pearson’s R: 0.39 (95% confidence interval (CI): −0.08; 0.72), p = 0.097). VISA-A score for the group AB at T1 was on average 60.8 (95%CI: (52.0–69.6)), similar to the group BA at T1 (62.8, 95%CI: (54.0–71.6)). Linear mixed modeling statistics showed that after 3 months, estimated mean difference in VISA-A score for group AB was 12.6 (95%CI: (9.7–15.5)) points, while in group BA this was 5.3 (95%CI (2.3; 8.3)) points (Figure 2). After crossing over the AB and BA group showed an estimated mean difference in VISA-A score of respectively 5.9 (95%CI (2.8–9.0) and 17.7 (95%CI (14.6–20.7) between T2 and T3. Both group AB and BA achieved the same estimated mean improvement in VISA-A score between T1 and T3 of respectively 18.5 (95%CI: (15.5–21.6)) and 23.0 (95%CI: (19.9–26.1)) (Figure 2). Importantly, our LMM analyses showed that there was a statistically significant interaction from our sCP randomization order ((LMM, F (2,762) = 20.3, p < 0.0001)) on change in VISA-A scores indicating a significant difference between the groups in evolution of the VISA-A scores over time in relation to sCP versus placebo supplementation.

Figure 2. Progression of the mean VISA-A scores during the course of the study Data represent mean ± SD. Statistical analysis of changes in VISA-A scores at baseline (T1), 3 months (T2) and 6 months (T3) via linear mixed modeling. p values

≤ 0.05 were considered statistically significant and marked with an asterisk.

3.3. Patient Satisfaction and Return-to-Running Sports After 3 months respectively 6 out of 10 participants in group AB and 7 out of 10

participants in group BA reported their treatment as ‘good or excellent’. After 6 months 7 out of 9 participants in group AB and 7 out of 9 in group BA reported being satisfied with their treatment. The success of the intervention is also reflected in Figure 3, summarizing the number of participants that were able to return to their desired (running) sport, although none of them was able to return to their pre-injury level within the duration of the study. None of the participants in group AB reported that they stopped running sport activities after switching over from sCPs to placebo.





Figure 3. Number of participants able to return to their running sport at T2 and T3. Due to low numbers Chi-square statistics were not possible on these data.

3.4. Change in Achilles Tendon Microvascularity and Blood Markers Both group AB and BA showed a significant (LMM, F (2,202) = 12.8, p < 0.001)

decrease in tendon microvascularity over the course of the intervention as assessed by CEUS. As is illustrated in Figure 4, there is no difference in effect between the 2 interventions and no evidence for a benefit of sCP supplementation on tendon vascularisation. The difference at T2 for the 2 different randomisation groups is only marginally significant (LMM, F (2,202) = 2.81, p = 0.062), due to the greater variability in group AB compared to the BA group, in particular on that time point. Our LMM analyses indicate a significant interaction between microvascularity and VISA-A symptom score over the course of the study (Rc

2: 0.68; LMM, F (2,196) = 3.32, p = 0.038). Non-fasting serum levels of mean total cholesterol, triglycerides and uric acid were unchanged at T2 and T3 (Table 2, ANOVA, p > 0.05)


https://www.mdpi.com/2072-6643/11/1/76/htm#table_body_display_nutrients-11-00076-t002

Figure 4. Changes in microvascularity of the Achilles tendon mid-portion (2–4.5 cm from insertion) over time (both symptomatic and asymptomatic tendons combined).

Table 2. Serum blood makers at baseline (T1), 3 (T2) and 6 (T3) months.

3.5. Compliance with Interventions and Adverse Events In group AB, no compliance data on the daily calf strengthening exercise were

available for 1 participant at both T1-T2 and T2-T3. In group BA, compliance data on the daily calf strengthening exercise were missing for 1 participant (T1-T2), 1 participant (T2-T3), and 1 participant (T1-T2 and T2-T3). Based on the available exercise diaries, both groups AB and BA showed and achieved a similar compliance of respectively 84 ± 11% and 78 ± 14% (p = 0.326) to the twice-daily calf-strengthening exercises over the 6 months intervention period.

In group AB, no compliance data regarding intake of investigational product were available for 1 participant at both T1-T2 and T2-T3. In group BA, compliance data were missing for 3 participants (T1-T2) and 1 participant (T2-T3). Compliance to intake of sCPs and/or placebo was respectively 89 ± 15% and 91 ± 9% (p = 0.906) for group AB and BA over the 6-month intervention period. At T2, 6 out of 10 in group AB and 4 out of 10 in group BA correctly guessed their allocation to active or placebo supplement. At T3, 4 out of 9 in group AB and 5 out of 9 in group BA correctly guessed their allocation to active or placebo supplement. A total of 1 participant in group BA reported minor gastro-intestinal discomfort after ingestion of the investigational product during the first week of the treatment whilst using the placebo. Following a medical check-up with an independent physician who was deblinded for the randomization order of this participant, it was decided that there was no clear association with the dietary intervention and this participant was advised to continue with the trial. After 1 week, a medical follow-up learned that all gastro-intestinal symptoms had disappeared. Except for mild delayed onset muscle soreness of the calf muscles in response to the eccentric exercise, no other adverse events were reported during the course of the intervention.

4. Discussion The main finding of this small-scale but well-controlled pilot study is that oral

supplementation of sCPs with high glycine content may accelerate the clinical benefits of a well-structured calf-strengthening and return-to-running program in

patients with uni- or bilateral chronic Achilles tendinopathy symptoms. Although the study was not powered to detect differences in VISA-A scores or return-to-running sport activities between the 2 groups, it is interesting to note that, independent of randomization order, both clinical outcome variables appear to improve more during the sCPs supplementation period. Although the differences between the 2 groups at 3 months may appear small, the mean change in VISA-A score in group AB of 12.6 (9.7; 15.5) points is well above the minimum clinically important difference (MCID) of 6.5 for Achilles tendinopathy patients [31]. In our placebo group BA, VISA-A increased only by 5.3 (2.3; 8.3) points after 3 months, which is below the MCID of 6.5. After crossing over from sCP to placebo and vice versa, group AB and BA showed an opposite response with, subsequently, a significant increase in VISA-A of respectively 5.9 (2.8; 9.0) and 17.7 (14.6; 20.7). These clinical findings extend on two earlier studies [32,33], that reported respectively a pain modulating and in vitro anti-inflammatory effect of a nutraceutical containing collagen, mucopolysaccharides and vitamin C. Furthermore, in a rodent model of collagenase-induced Achilles tendinopathy, a 5% glycine-rich diet for 3 weeks improved hydroxyproline, glyosaminoglycans and non-collagenous protein content of the Achilles tendon [12]. Glycine has also been shown to improve collagen matrix organisation strength and tenocyte remodelling, most likely by modulating both TNF-alpha, matrix metalloproteases and the availability of collagen precursors [14]. As the sCP supplement in the present study contains 22% of glycine [11], it could be postulated that the increased intake of 1.1 g of glycine per day may have contributed to observed clinical improvements in VISA-A scores during sCP supplementation. Due to nature of the co-intervention, a true washout period to bring participants back to their baseline condition is in Achilles tendinopathy patients not possible. From a pharmacokinetic perspective, the serum hydroxyproline content following the ingestion of hydrolysed collagen peptides returns to baseline within 12 h [25]. Nevertheless, any potential carry-over effects have been accounted for in our LMM-analyses by looking at interaction of the order and treatment effect across time.

Given the small scale of the study and unequal distribution of men and women in our 2 study groups, there is a potential of selection bias. Although the adaptability of tendon to loading differs in men and women [34], we are not aware of any intervention study that specifically showed clinically relevant gender difference in VISA-A scores following a calf-strengthening program. To evaluate homogeneity of the data at baseline between the 2 groups a discriminant analysis was carried out. The data revealed no statistically significant differences between both study groups. Unfortunately, the relatively low number of female participants does not allow for a gender-based inference. As women may be more prone to develop Achilles tendinopathy [35,36], future random-controlled trials (RCTs) should aim to include an equal number of men and women in both study arms.

In order to maximize any potential synergistic effect of orally ingested sCPs with bi-daily calf-strengthening exercises (as per Alfredson’s protocol) on tendon collagen synthesis, our participants were instructed to ingest the sCPs 30 min before each exercise session. About 15–30 min after oral ingestion of collagen hydrolysates, free and peptide forms of serum hydroxyproline levels significantly increase and reach a maximum after 30–60 min and are almost back to baseline level after 7–12 h [25,37]. Although this was not investigated as a separate outcome measure, the small time window between the bi-daily intake of a sachet with 2.5 g of sCPs and the calf-strengthening exercise may have contributed to the high compliance rate observed in our trial. A recent proof-of-concept study by Shaw et al. (2017) showed a dose-













dependent improvement of collagen metabolism in ligaments by the oral administration of gelatine 60 min before jumping exercises [7]. This specific time window was based on the fact that peak availability of free and peptide forms of serum hydroxyproline is respectively 1 and 2 h following the oral ingestion of gelatin [38]. Based on the new insight that the stimulation of exercise-induced anabolic processes might be influenced positively by the oral administration of sCPs immediately before an exercise program [7], we recommend that future study designs should take absorption kinetics of the available forms of gelatin or sCPs into consideration.

A strength of the present study is that there were only 2 dropouts after T2, 1 in each group, which is a negligible number. Given that our LMM approach operates on a missing at-random-assumption, we deemed the reasons for dropout not violating the assumptions of the model. Furthermore, attrition is low and no individuals were excluded from our LMM so the possibility of selection bias is considered low in the present study. Nevertheless, as the clinical improvements in VISA-A scores after 3 months are in the same range as previously published studies on eccentric calf-strengthening programs [39], no firm conclusions can be drawn regarding the added benefits of sCP supplementation in chronic Achilles tendinopathy. So, although the present randomized controlled and double-blinded crossover study found a significant interaction of sCP supplementation on change in VISA-A score during a 6-month calf-strengthening plus structured return-to-running program, our findings require duplication in a larger and adequately powered clinical trial.

From a clinical perspective, it is important to note that the majority of our participants had a relatively long history of mid-portion Achilles tendinopathy not responding to rest or other physiotherapy regimes. The latter is also reflected by the fact that placebo group BA only showed a clinically relevant improvement in VISA-A score after they were switched over to sCPs supplementation. This cross-over effect indicates that sCPs supplementation could be a useful adjunct therapy in Achilles tendinopathy patients whom are not responding to a well-standardized eccentric calf strengthening exercise program.

To improve our understanding of the in vivo working mechanism of specific collagen peptides, we also investigated the microvascular changes of the Achilles tendon using a novel medical imaging technique based on contrast-enhanced ultrasonography. The present study is the first of its kind showing that objectively quantified Achilles tendon microvascularity is inversely and moderately associated with Achilles tendon symptoms throughout the course of a therapeutic intervention. The latter indicates that CEUS of tendons may be a useful and more objective medical imaging technique to monitor the clinical response to a therapeutic intervention. Although a previous publication showed that CEUS has a better sensitivity in detecting microvascular abnormalities as compared to power Doppler ultrasound (PDU) [18], the observed change in CEUS-based microvascularity during the course of the intervention was not associated with the intake of specific collagen peptides. Our rather large inter-subject variability in Achilles tendon microvascularity at baseline may have precluded detecting any statistically significant differences between the 2 groups. Nevertheless, our results are well in line with several other PDU-based imaging studies that reported rather equivocal responses in microvascularity following conservative treatment of Achilles tendinopathy [40,41,42,43,44]. Despite the longitudinally observed association between CEUS-based microvascularity and Achilles tendon symptoms, it is unlikely that CEUS will be useful in determining the potential in vivo working mechanism of specific collagen











peptides as an adjunct treatment to a well-structured eccentric calf strengthening exercise program.

Eccentric tendon loading has shown to be a safe and effective method to reduce pain and to improve tendon structure [45]. Although PDU-based studies have been equivocal [40,41,42,43,44], eccentric tendon loading has been shown to reduce the number of neovessels in the tendinopathic area of the tendon matrix. The latter is considered to be an important etiological mechanism for the beneficial outcome of a calf strengthening program as also applied in the current study [46]. Cell-line studies have shown that tenocytes produce the antiangiogenic factor endostatin, a proteolytic fragment of Collagen XVIII, in response to physiological mechanical load, thus limiting neo-angiogenesis [47]. Although endostatin response to eccentric loading has not been investigated in diseased tendons, several mechanistic studies [47,48,49] indicate that only the right amount of tissue loading increases endostatin expression and, as such, may reduce microvascularity as observed in the present study. As the collagen matrix of tendon is a highly mechanosensitive tissue, cytokine homeostasis and cell survival underlie an intimate balance between adequate biomechanical stimuli and disturbance through load deprivation and overload. This delicate balance between tendon blood flow and loading pattern was recently highlighted in a study by showing that the risk of developing Achilles tendinopathy increased if blood flow increase in the tendon after running was reduced [36]. Based on these new insights, future studies on novel therapeutic strategies for tendinopathy should preferably assess tendon blood flow response both at rest as well as following a standardized tendon-loading protocol.

Although from a pathophysiological perspective return-to-running status and tendon microvascularity are likely to interact, the limited statistical power of the present pilot study does not allow us to exclude such a significant interaction using linear mixed modelling. The decision to return to running was a function of pain reduction during single leg hopping as well as the successful and pain-free achievement of pre-defined milestones during the course of our rehabilitation program. The latter can be justified by the well-established fact that progressive (and sports-specific) tendon loading is required for successful tendon healing and return to sport [38]. Furthermore, the milestones described in Appendix A Table A2 entailed significantly lower Achilles tendon loading than present during pre-injury sports participation. Although the VISA-A questionnaires are designed to quantify both activities-of-daily-living (ADL) and running-associated Achilles tendon pain, we cannot exclude that running with incompletely healed tendon pathology may have introduced a certain level of bias towards the overall VISA-A score. As such, for future RCTs we would recommend assessing both ADL-dependent and running sport-dependent Achilles tendon pain on a visual analog scale as secondary outcome measures.

Previous in vitro work on fibroblasts indicates that biosynthesis of ligament and tendon matrix molecules, as well as elastin content can be stimulated by exposure to sCPs [50]. A recently published shear wave elastography study indicates that, compared to controls, participants with Achilles tendinopathy display lower Achilles tendon elastic modulus [51]. Accordingly, we recommend that future randomized clinical trials in Achilles tendinopathy patients should include shear wave elastography to monitor changes in tendon elasticity following the intake of sCPs.

5. Conclusions


















Although this pilot study has limited statistical power and requires duplication in a larger clinical trial, oral supplementation of sCPs may accelerate the clinical benefits of a well-structured calf-strengthening and return-to-running program in patients with chronic Achilles tendinopathy symptoms. Further imaging studies are required to understand the potential in vivo therapeutic working mechanism of sCPs within tendons.

Author Contributions S.F.E.P. conceptualised the study, data collection and its design, implemented

the study design (conducted the study), conducted analysis of the data, wrote the initial drafts of the paper and subsequently revised the paper after feedback from the team. C.P. conceptualised data collection and study design, designed the structured exercise and return-to-running program and revised the paper. M.W. wrote the statistical plan, conducted analysis of the data, and drafted and revised the paper. N.V. implemented the study design, monitored data collection and revised the paper. G.L. designed data collection tools, monitored data collection, conducted cleaning and analysis of the data and revised the paper. SM monitored data collection, conducted analysis of the data and revised the paper. D.H., L.B., J.G. and G.W. conceptualised and designed the study and data collection tools, monitored data collection and revised the paper.

Funding This investigator-initiated study was financially supported by GELITA AG,

Germany.

Acknowledgments The research team would like to thank Jason Cotter, Claire Bushell, Marisabel

Gonzalez and Jacqui Hislop from TOSHIBA/CANON Medical systems Australia Pty Ltd for their technical support and advice on the different ultrasound protocols. We would like to acknowledge Prof. Keith Baar for proposing the original research idea to study hydrolysed collagen supplementation in tendinopathy. Dr. Steffen Oesser (CRI) and Dr. Jutta Hugenberg are acknowledged for their support in the preparation of the trial. We also would like to thank Dr. Joo-Haw Ong, Mr. John Anglim, Ms. Rebekah Alcock and our nursing staff Ruth Fazakerley, Robynn Broadbent and Helen Browning for their assistance with the data collection. Our sports physiotherapist, Ms. Lauren Dixon, is acknowledged for supervising and tailoring the calf-strengthening and return-to-running program. The study sponsor had no role in the collection, analysis and interpretation of data, in the writing of the report and in the decision to submit the article for publication.

Conflicts of Interest The authors declare no conflicts of interest

Appendix A Table A1. Inclusion and exclusion criteria.

Table A2. Exercise and return-to-running program.

Table A3. Questionnaire on subjective patient satisfaction and returning to sports level.

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http://creativecommons.org/licenses/by/4.0/

x 2 24-Week Study on the Use of

Collagen Hydrolysate

24-Week study on the use of collagen hydrolysate

as a dietary supplement in athletes with activity-

related joint pain

Kristine L. Clark,Wayne Sebastianelli,Klaus R. Flechsenhar,Douglas F.

Aukermann,Felix Meza,Roberta L. Millard, show all

Pages 1485-1496 | Accepted 29 Feb 2008, Published online: 15 Apr 2008

https://doi.org/10.1185/030079908X291967

ABSTRACT

Background: Collagen hydrolysate is a nutritional supplement that has been

shown to exert an anabolic effect on cartilage tissue. Its administration appears

beneficial in patients with osteoarthritis.

Objective: To investigate the effect of collagen hydrolysate on activity-related

joint pain in athletes who are physically active and have no evidence of joint

disease.

Design and setting: A prospective, randomized, placebo-controlled, double-blind

study was conducted at Penn State University in University Park, Pennsylvania.

Parameters including joint pain, mobility, and inflammation were evaluated with

the use of a visual analogue scale during a 24-week study phase.

Study participants: Between September 2005 and June 2006, 147 subjects who

competed on a varsity team or a club sport were recruited. Data from 97 of 147

subjects could be statistically evaluated.

Intervention: One hundred and forty-seven subjects (72 male, 75 female) were

randomly assigned to two groups: a group (n = 73) receiving 25 mL of a liquid

formulation that contained 10 g of collagen hydrolysate (CH-Alpha)* and a group

(n = 74) receiving a placebo, which consisted of 25 mL of liquid that contained

xanthan.

Main outcome measures: The primary efficacy parameter was the change in the

visual analogue scales from baseline during the study phase in relation to the

parameters referring to pain, mobility, and inflammation.

Results: When data from all subjects (n = 97) were evaluated, six parameters

showed statistically significant changes with the dietary supplement collagen

hydrolysate (CH) compared with placebo: joint pain at rest, assessed by the

physician (CH vs. placebo (–1.37 ± 1.78 vs. –0.90 ± 1.74 ( p = 0.025)) and five

https://www.tandfonline.com/author/Clark%2C+Kristine+L

https://www.tandfonline.com/author/Sebastianelli%2C+Wayne

https://www.tandfonline.com/author/Flechsenhar%2C+Klaus+R

https://www.tandfonline.com/author/Aukermann%2C+Douglas+F

https://www.tandfonline.com/author/Aukermann%2C+Douglas+F

https://www.tandfonline.com/author/Meza%2C+Felix

https://www.tandfonline.com/author/Millard%2C+Roberta+L

https://www.tandfonline.com/doi/pdf/10.1185/030079908X291967?needAccess=true

https://doi.org/10.1185/030079908X291967

https://www.tandfonline.com/doi/pdf/10.1185/030079908X291967?needAccess=true#FN0001

parameters assessed by study participants: joint pain when walking (–1.11 ± 1.98

vs. –0.46 ± 1.63, p = 0.007), joint pain when standing (–0.97 ± 1.92 vs. –

0.43 ± 1.74, p = 0.011), joint pain at rest (–0.81 ± 1.77 vs. –0.39 ± 1.56, p = 0.039),

joint pain when carrying objects (–1.45 ± 2.11 vs. –0.83 ± 1.71, p = 0.014) and joint

pain when lifting (–1.79 ± 2.11 vs. –1.26 ± 2.09, p = 0.018). When a subgroup

analysis of subjects with knee arthralgia (n = 63) was performed, the difference

between the effect of collagen hydrolysate vs. placebo was more pronounced.

The parameter joint pain at rest, assessed by the physician, had a statistical

significance level of p = 0.001 (–1.67 ± 1.89 vs. –0.86 ± 1.77), while the other five

parameters based on the participants’ assessments were also statistically

significant: joint pain when walking ( p = 0.003 (– 1.38 ± 2.12 vs. – 0.54 ± 1.65)),

joint pain when standing ( p = 0.015 (–1.17 ± 2.06 vs. –0.50 ± 1.68)), joint pain at

rest with ( p = 0.021 (–1.01 ±1.92 vs. –0.47 ± 1.63)), joint pain when running a

straight line ( p = 0.027 (–1.50 ± 1.97 vs. –0.80 ± 1.66)) and joint pain when

changing direction ( p = 0.026 (–1.87 ± 2.18 vs. –1.20 ± 2.10)).

Conclusion: This was the first clinical trial of 24-weeks duration to show

improvement of joint pain in athletes who were treated with the dietary

supplement collagen hydrolysate. The results of this study have implications for

the use of collagen hydrolysate to support joint health and possibly reduce the

risk of joint deterioration in a high-risk group. Despite the study's size and

limitations, the results suggest that athletes consuming collagen hydrolysate can

reduce parameters (such as pain) that have a negative impact on athletic

performance. Future studies are needed to support these findings.

x 3 Extract from The Times

February 12th 2019

Why collagen shots are a must-have for the gym set

It’s already used in beauty products and by elite sportsmen to prevent injuries — and now the masses are catching on

February 12th 2019, The Times

In the world of the impeccably beautiful, there is a well-established truth: you can never have enough collagen. Scientifically, collagen is our body’s structural protein, a sort of scaffolding that glues together tissues such as bones, tendons, ligaments, cartilage and skin. Cosmetically, it bestows radiant, plump skin and bouncy hair, healthy nails and a dewy youthfulness, which is why products promising to replenish supplies when collagen production slows due to stress, illness and, of course, age are the latest nutritional nirvana.

We are buying into the claims that we can boost our stores of it in any way we can. Celebrities — Yasmin Le Bon, Sienna Miller and Christy Turlington are all said to be fans — swallow it for its purported anti-ageing effects, and the idea that we can reboot our skin’s elasticity simply by ingesting more of what our bodies produce naturally is a tempting one. And while evidence is scarce for some of the claims made by manufacturers, for others there is emerging proof that it might actually work.

In a study published in the journal Nutrients this month, scientists from the Australian Institute of Sport (AIS) became the latest to serve up evidence that consuming collagen can help our tired, overworked bodies to recover after exercise and to suggest it may even prevent injury. Collagen forms an important part of the body’s extracellular matrix, a mix of water and proteins and carbohydrates that enables the cartilage, tendons and bones to absorb energy and force during any sort of physical activity. In the AIS paper, taking supplements of it was shown to speed up a return to running in people with achilles tendon problems, but others have shown that it can boost the body’s collagen synthesis, helping to strengthen and protect the body against joint and back problems. Cue the gym set joining its tribe of loyal followers and, inevitably, the launch of a new wave of products targeting those who want to stay running and Spinning into their dotage.

Graeme Close, professor of human physiology at Liverpool John Moores University, uses it with the sports people he advises, including professional football and rugby clubs such as Queens Park Rangers and London Irish, and says it is formulated to protect and repair tendons and tissues. “Collagen is getting a lot of attention in elite sport and that message is now filtering down to the fitness masses,” Close says. “There used to be not much you could do about knee pain or tendon problems in sport, but there’s now convincing evidence that collagen taken with vitamin C — which is critical in terms of collagen production — can be really helpful both in terms of injury prevention and recovery from intense activity.”

Much of the research that looks at how collagen might help those in need of such a fitness aid has been carried out by Keith Baar, a professor of molecular exercise physiology at the

University of California. In one of his trials, published in The American Journal of Clinical Nutrition in 2017, Baar showed how a regimen of taking a collagen and vitamin C combo an hour before a five-minute protective “prehab” session in which you stimulate the tendons and ligaments you will be working — a few shoulder exercises if you intend to throw or perform handstands or some box jumps or squats if you intend to run around or jump — and at least six hours before or after your workout proper will maximise the benefits for those prone to injury. “The idea is that by doing specific exercises to target an area this will increase collagen synthesis and that this could accelerate recovery of tendon, ligament, cartilage and bone injuries,” he says.

For those who are not slaves to the gym, there are signs that taking collagen could be useful. A study of 200 people by University of Liège researchers in 2012 showed it reduced pain in the joints and back. And in a 2017 review of clinical trials published in the Journal of Arthritis, a team from Cardiff University school of medicine and the South Wales Orthopaedics Research Network found “some evidence that suggests oral collagen is effective for osteoarthritis and has been shown to be tolerable and safe”. Close says that tendons and joints “act like sponges to suck in the nutrients” and that “scientists are beginning to recognise collagen’s wider uses and its potential to prevent different types of pain”.

Even more encouragingly, you don’t need to spend a fortune on flashy products. Most supplements, in whatever form, are made from the kind of collagen-rich animal tissues that are usually discarded by food manufacturers, including the bones and skin of pigs, chickens and cows, and fish skin and scales. Usually, the collagen is “hydrolysed”, meaning its proteins are broken down into a smaller molecular size, forming gelatin, then further broken down before being added to capsules, drinks and protein bars.

Baar is also an advocate of the DIY approach. “I make my own drinks by putting six strawberries and a little milk into a blender and, once it is smooth, I add hydrolysed collagen powder and it has everything I need,” he says. “I take it, and so do my daughter and wife, an hour before we work out.” Baar says there’s no such thing as vegan or plant-based collagen, despite a boom in products that claim to contain it. “Collagen is only made in animals,” he says. “Vegan ‘collagens’ are actually sugars like the agar used to make some gummy sweets.”

Looking after our joints is one thing, but what more of us want to know is if a collagen intake will improve our skin. Creams containing collagen apparently have the drawback that its molecule is too big to be absorbed through the skin. “There is one company that is making collagen in bacteria for cosmetic manufacturers, but these are still early days,” Baar says. “At the moment there is some limited evidence that collagen taken orally helps skin.”

A small study from the University of Kiel in Germany in 2014 found that collagen supplementation for several months did improve skin elasticity and moisture; another last year found that 24 weeks of supplementation increased nail growth and strength.

x 4 Prevention and Treatment of

Osteoarthritis

The role of Collagen Hydrolysate in cartilage metabolism: Satellite Symposium at the World Congress on Osteoarthritis Sponsored by the GELITA Health Initiative A potential causative therapeutic approach: Interview with Steffen Oesser, PhD, Director of the Collagen Research Institute, Kiel, Germany

GHI NEWS ARSI World Congress is the global forum for intern

Satellite Symposium at the World Congress on Osteoarthritis Sponsored by the GELITA Health Initiative

P R EVE NTION AN D TR EATME NT OF OSTEOARTH R ITIS WITH COLL AGE N HYDROLYSATE

Osteoarthritis is the main cause of physical impairment in people over the age of 65. Currently

there is no causal therapy for this degenerative joint disease, only symptomatic treatment options.

Therefore more research in this field is important. The World Congress of the Osteoarthritis

Research Society International (OARSI) was dedicated to the presentation and discussion of scientific

research related to osteoarthritis.

Ideally treatment must target chondrocyte metabolism to counteract the catabolic processes taking place

in the joint cartilage. Two therapeutic approaches are conceivable: inhibiting the degradation of the

extracellular matrix or stimulating the biosynthesis of cartilage cells. There is increasing evidence that

collagen hydrolysate stimulates the collagen synthesis of the chondrocytes, thus alleviating the pain of

osteoarthritis patients. At the OARSI World Congress in Berlin (October 12 – 15, 2003), experts

presented research on the potential benefits of collagen hydrolysate on joint health at a Satellite

Symposium sponsored by GELITA.

GHINEWS | May 2004 | Issue 1

Steffen Oesser, PhD, of the University of Kiel,Germany, in his investigations examined whethercollagen hydrolysate can influence the metabo-lism of chondrocytes. [2,3]

Mature bovine chondrocytes were harvestedfrom the joints and prepared as dense monolayercultures under low-oxygen conditions for 11 days.After pre-incubation for three days the basalmedium was enriched with collagen hydrolysate.Cultures with no collagen hydrolysate or withcollagen-free protein hydrolysate were used ascontrols. ELISA tests showed that type II colla-gen production in chondrocytes was stimulated significantly by collagen hydrolysate. (Fig.2)

There was a dose-related increase up to 2.5-foldhigher. Immunocytochemical studies confirmedthat, in the chondrocyte cultures treated withcollagen hydrolysate, significantly more newtype II collagen had been produced than in the control cultures. (Fig. 3) In addition, in the cultures with collagen hydrolysate, signifi-cantly more pericellular proteoglycan was identified. This can be interpreted that the stimu-lated chondrocytes synthesize a complete extra-cellular matrix.

In another study, collagen biosynthesis inchondrocyte cultures was investigated by also

Latest findings from the cell laboratory

STIMULATION OF

COLLAGEN PRODUCTION

PROVEN

NUTRITIONAL FACTORS IMPACTING JOINT CARTILAGE

Time (days)

Type

II c

olla

gen

(µg/

106

chon

droc

ytes

)

Fig. 2: Stimulation of collagen secretion in chondrocyteculture mediums by collagen hydrolysate.[3]

00

1

2

3

2 4 6 8 10 12

collagen hydrolysate

basal medium

To achieve therapeutic effects in the joints, anactive substance administered orally must beabsorbed by the intestine before reaching the joints.

In an animal experiment, Jürgen Seifert,MD, from the University of Kiel, Germany and

colleagues followed the passage of radioactivelylabeled collagen hydrolysate through the body.The control group received radioactivelylabeled proline. There were no differencesbetween the two study groups regarding theradioactivity measured in the gastrointestinaltract and in plasma. However collagenhydrolysate accumulated significantly more inbone and cartilage than did the proline.(Fig.1) Inthe animals treated with collagen hydrolysate,radioactivity measured in the cartilage wastwice as high as in the control animals. Furtherstudies on the quality of the collagen peptideshowed that collagen hydrolysate moleculeshaving relatively high molecular weight (up to ~10 kDa) were absorbed. This means that collagen hydrolysate macromolecules areavailable to body tissues in addition to the individual peptides that comprise collagenhydrolysate. Absorption did not alter the mole-cular structure, Seifert emphasized. In additionthe researchers were able to show that collagenhydrolysate is absorbed primarily in thejejunum and proximal ileum and in a maximum of 30 minutes.

Results of laboratory experiments

COLLAGEN HYDROLYSATE ACCUMULATES IN THE JOINTS

BoneCartilage

Radi

oact

ivity

in ti

ssue

(Bq/

g)

Fig. 1: Radioactivity in cartilage and bone after oral administration of labeled collagen hydrolysate and proline

0

10

20

30

40

50

60

p < 0,01

Mean±SD, n=3

p < 0,01

14C-collagen hydrolysate14C-proline

Mean±SD, n=6

Genetic factors, stress, metabolic andendocrine factors, loss of proteoglycans andapoptosis of chondrocytes underlie the progressof osteo-arthritis. Joints in overweight people, inparticular, are subject to added stress. In theUS, two thirds of all adults and six out of 10children are overweight or obese.

“Being overweight is a dangerous factor in almost all chronic diseases,” said KristineClark, PhD, RD, of the Pennsylvania StateUniversity. Weight is one of the most impor-tant direct factors that has an impact on jointhealth. Other factors are adequate levels ofvitamins C and D, calcium, proteins, phospho-rus and zinc, which contribute to the normal formation of the extracellular matrix and cartilage. Modern dietary trends, including fastfood and vegetarian diets, however mean thata large part of the population has inadequateintakes of vitamin C, calcium and phosphorus.How this affects cartilage metabolism has so far, in contrast to the influences of nutritional

factors on bone metabolism, not been the subjectof much research.

Managing health through diet and supple-mentation is prevalent in the population.According to government data, about half ofall women and 40 percent of men over the ageof 50 regularly consume nutritional supplements.

A number of nutritional supplements have shown positive effects on stiffness, pain and inflammation, symptoms of joint disease.These include ginger, omega-3 fatty acids,gamma-linolenic acid, glucosamine and chondroitin sulfate. Dr. Clark noted collagenhydrolysate in particular as a very promisingnutrient for osteoarthritis. Studies on collagenhydrolysate support a beneficial effect onsymptoms and may impact cartilage regenera-tion. These types of nutritional supplements arebeneficial as part of a multifactorial approach,which includes weight reduction, appropriatephysical exercise and a balanced and healthydiet, concluded Dr. Clark.

incorporating 14C-proline. Neither the additionof high molecular weight collagen I nor collagenII to the culture medium induced any significantincrease in cell-associated radioactivity. Onlycollagen hydrolysate with collagen fragments (< 10 kDa) was able to significantly stimulate theaccumulation of radioactively labeled proline in the chondrocytes and as a consequence ofcollagen biosynthesis. (Fig.4) “The size of thepeptide fragments seems to be a decisive factor –not whether it originates from type I or type II collagen,” commented Dr. Oesser.

As Dr. Oesser explained, this study demon-strates a physiological regulation process: Smallcollagen fragments, produced in the joint whencollagen is degraded by proteases, stimulate thesynthesis of collagen.

Collagen hydrolysate introduced exogenously,which is absorbed by the intestine and accumu-lates in the joint cartilage, therefore has thepotential to stimulate collagen production in the remaining chondrocytes in patients withosteoarthritis. This counteracts the degenerativeprocesses and may prevent the development ofosteoarthritis.

“Collagen hydrolysate is a food, not a phar-maceutical product. It has no undesirable sideeffects and therefore long-term regular use issafe,” Dr. Oesser concluded.Ra

dioa

ctiv

ity (d

pm/1

06ch

ondr

ozyt

es)

Fig.4 : Stimulation of collagen synthesis by collagenhydrolysate (0.5mg/ml). [3]

Fig. 3: Immunocytochemical evidence of type II collagen (brown staining) after stimulation with collagen hydrolysate. [3]

Collagen hydrolysate

Control

0

200

400

800

600

BM CollI CollII PLA CH CH-F1 CH II

BM Basal medium

Coll I native type I collagen

Coll II native type II collagen

PLA collagen-free wheat protein hydrolysate

CH collagen hydrolysate

CH-F1 collagen hydrolysate fraction

CH II type II collagen hydrolysate

Collagen hydrolysate

Control

Literature:

1 Dickinson A.: The Benefits of Nutritional Supplements.Council for Responsible Nutrition 2002

2 Oesser et al.: Journal of Nutrition 129, 1999, 1891-1895

3 Oesser et al.: Cell & Tissue Research 311, 2003, 393-399

4 Adam et al.: Therapiewoche 41, 1991, 2456-2461

5 Moskowitz et al.: Semin Arthritis Rheum 30, 2000, 87-99

Hans-Konrad Selbmann, MD, University of Tuebingen, Germany, presented the results of 16 published clinical studies using collagenhydrolysate. He focused in particular on twostudies in which patients with osteoarthritis weregiven collagen hydrolysate as a monotherapy.

The first study compared four treatments in across-over design in 52 patients: [4]

• Collagen hydrolysate• Gelatine• Gelatine plus glycin plus

calcium hydrogen-phosphate• Egg albuminThe study lasted for 16 months and, in each

case, treatment was for two months with a two-month washout phase between treatments.

Collagen hydrolysate and the gelatine preparations decreased the pain score by 81 to 85 percent. The effect was significantly less common from the egg albumin (23 percent). Infact, symptoms were significantly more frequentthan for the other treatments, (40 percent versus 0 to 2 percent). The analgesic intake was also

reduced by more than 50 percent in up to three-quarters of patients taking collagen hydrolysateor gelatine. This was significantly lower for eggalbumin at 35 percent.

In the second multi-center study 389patients with osteoarthritis of the knee wererecruited in three countries (England, Germanyand the United States).[5] The patients ingestedeither 10 grams of collagen hydrolysate per dayor placebo for 24 weeks. Paracetamol was alsoallowed as needed. In the entire populationgroup, no significant effects from the collagenhydrolysate could be noted. However, in theGerman subgroup, which included 112 patients,significant improvement was seen for collagenhydrolysate in all primary end points (WOMACpain and WOMAC function score and also theoverall evaluation by the patient).

When comparing the German and the US group, which had 173 patients, there weredifferences: In the US group, protocol violationswere counted in 48 percent of cases versus 17 percent in the German group. In addition

the withdrawal rates in the US were consider-ably higher, which led to a shorter period of treatment. Furthermore, a comparatively highconsumption of paracetamol in the US groupcould have masked the effects of collagenhydrolysate. The English group was not includedbecause of the small number of patients (29 total).

Overall it must be assumed that different cultural conditions, (e.g. use of medications forpain symptoms) impacted the results.

Conclusion:There is evidence that administeringcollagen hydrolysate (10 grams per day for atleast 3 months, based on certain studies) canhave a positive impact on pain symptoms andjoint function in patients with osteoarthritis. Of the nearly 2000 patients given collagenhydrolysate in various studies, no undesirableside effects were reported.

Results of clinical studies

IMPROVEMENT IN PAIN AND JOINT FUNCTION

GELITA Health Initiative drivesresearch with collagen hydrolysate

Collagen hydrolysate is a special type ofgelatine obtained by enzymatic hydrolysis of collagen. It contains 95 percent fragmentsof type I collagen. The peptide fragmentsrange between 0.5 and 13 kDa in size, onaverage 3.5 kDa.

In 1975, collagen hydrolysate obtainedGRAS status (Generally Recognized as Safe)from the Federal Drug Administration,which was re-affirmed in 2003. Varioustypes of collagen hydrolysate or gelatineproducts are available on the market, such ascold water-soluble powdered gelatine orgelatine drinks in various flavors. Based oncurrent research, the daily dose associatedwith benefit is 10 grams of collagenhydrolysate.

To promote research of collagen hydrolysatein degenerative joint disease, the GELITAGroup, the world's leading manufacturer of gelatine, established the GELITA HealthInitiative. The findings from research aredisseminated to physicians, dietitians andconsumers. Scientific material and researchon collagen hydrolysate and its role inosteoarthritis are available on the GELITAHealth Initiative Web site.

www.gelita-health-initiative.com

Interview with Dr. Steffen Oesser

A POTENTIAL CAUSATIVE THERAPEUTIC APPROACH

What leads you to believe that collagenhydrolysate after oral administration stimulates chondrocytes in vivo inhuman joints? Oesser: The question of how experimental research applies to humanpatients frequently arises. We were able to show that our cell cultureexperiments are transferable to human cells. In addition we demonstratedthat collagen hydrolysate is absorbed, reaches the joint and stimulates thechondrocytes. In view of this and the positive clinical effects of collagenhydrolysate, it is reasonable therefore to conclude that the experimentalresults are transferable to human patients.

What are the most important clinical benefits of collagen hydrolysate?Oesser: The clinical benefits to a patient, as suggested by current research,are a significant reduction in pain, a considerably lower need for analgesicsand, associated with this, an overall improvement in mobility. In a newstudy conducted in the US, it has been possible for the first time to objectifythese effects, generally expressed by scores, by means of isometric or isokinetic measurements.

Is collagen hydrolysate a possible leading option for secondary prevention?Oesser: To date, the treatments available for osteoarthritis address the pain,not the underlying progressive loss of cartilage. There are currently no indi-cations that collagen hydrolysate has any direct analgesic effect; the clinicalimprovement in symptoms seen to date in laboratory experiments may beexplained by the direct impact collagen hydrolysate has on the joint cartilage.Collagen hydrolysate has the potential to rebuild some of the cartilage thatmay be lost during the osteoarthritic process by the stimulation of chondro-cytes and the increased synthesis of extracellular matrix. Therefore collagenhydrolysate could be a significant and unique treatment option.

The GELITA Group markets a special-

ized form of collagen hydrolysate. The

use of collegen hydrolysate is patented

for the treatment of osteoarthritis

(EP0254 289 B1).

Can collagen hydrolysate slow down the degenerative process or is it evenpossible to prevent osteoarthritis?Oesser: The current body of research certainly suggests that taking collagenhydrolysate at a very early stage has the potential to slow down the degen-erative process, and thus effect the occurrence of symptoms. It will beextremely difficult to achieve a complete standstill in cartilage degeneration.However, more research is needed to confirm this effect and to further elucidate the role of collagen hydrolysate in prevention.

Who would you recommend to take collagen hydrolysate? Oesser: Any patient diagnosed with osteoarthritis should consider takingcollagen hydrolysate. People who demand a lot of their joints – includingthose who are overweight or those who apply mechanical physical stressbecause of occupational demands or through intensive sporting activities – may also benefit from taking collagen hydrolysate.

How long should the treatment period last?Oesser: The first clinical effects occurred in studies after six to eight weeks.Patients should maintain this regime in order to experience a positive effect.Patients who discontinued the use of collagen hydrolysate experienced arecurrence of symptoms. This observation fits the postulated mechanism ofaction. In principle, this is a long-term treatment, which involves, accordingto current research, taking a daily dose of 10 grams. Best results seem to beobtained when it is taken continuously. Since collagen hydrolysate is a food,no significant undesirable side effects have been reported.

At the OARSI World Congress, Dr. Steffen Oesser presented the latest researchfindings on a possiblemechanism of actionfor collagen hydrolysate.

x 5 Role of Collagen Hydrolysate

in Cartilage Metabolism & Regeneration

ROLE OF COLLAGEN HYDROLYSATE IN CARTILAGE METABOLISM & REGENERATION

Satellite Symposium at the World Congress on Osteoarthritis (OARSI)

October 14, 2003 Palais am Funkturm Berlin

AB

STR

AC

TS

CONTENT

Kristine Clark, Pennsylvania State University, USA »Role of Nutrition in Joint Health«

Jürgen Seifert, University of Kiel, Germany »The Absorption and Distribution of Collagen Hydrolysate after Oral Application«

Steffen Oesser, University of Kiel, Germany»Impact of Collagen Fragments on the Extracellular Matrix Metabolism«

Hans-Konrad Selbmann, University of Tübingen, Germany »Clinical Experience with Collagen Hydrolysate in Osteoarthritis«


CURRICULUM VITAE

Dr. Kristine Clark is the Director of Sports Nutrition for Penn State

University's Athletic Dept. where she counsels more than 800 varsity athletes

from 29 teams. In addition, she advises head coaches, team physicians, athle-

tic trainers, strength and conditioning coaches, and athletic administration on

policies regarding eating disorders, weight management, and supplement use

among athletes. While most of Dr. Clark's time is devoted to athletics, she also

holds a position of assistant professor in the Dept. of Nutrition at Penn State

teaching a course titled, "Nutrition for Exercise and Health throughout the

Lifecycle."

Dr. Clark was appointed to the Sports Medicine Advisory Board of the United

States Olympic Committee in 1999. She began working as the nutritionist for

the United States Women's Soccer Team in 1995 and continues as their nutri-

tion consultant. In addition, she serves as the nutritionist for the United States

Soccer Federation. Clark is the nutrition columnist for the Women's United

Soccer Association website.

Dr. Clark holds a Ph.D. in Nutrition Science from Penn State University, a

Masters degree in Health Education from the University of Wisconsin-

LaCrosse, and a B.S. degree in Nutrition and Dietetics from Viterbo College,

LaCrosse, WI. She is a registered dietitian, a Fellow in the American College of

Sports Medicine, a Board of Trustees member of the ACSM, and is active in the

Am. Dietetic Association.

KRISTINE CLARK,

Ph.D., R.D., FACSM


Kristine Clark, Ph.D., R.D., FACSMDirector of Sports Nutrition, Penn State OrthopedicsThe Pennsylvania State University, USA

// Currently, there is no medical cure for OA, only treat-ments to alleviate pain and symptoms. Accumulating evidenceindicates that promising preventive measures such as nutritionmay help slow down the onset of disease, improve symptoms anddelay disease progression.

Information of joints due to aging, excess stress from physical acti-vity or obesity may decrease mobility, effecting energy balanceand further escalating weights.

The diet of an individual can therefore play a critical role in theprevention of disorders that can influence joint disease. Foodchoices that offer optimal levels of calcium, vitamin C, protein,phosphorus, and vitamin D contribute to normal formation of theextracellular matrix and articular cartilage required for healthyjoint movement. In addition, weight management would helpreduce any negative impact on the joints. In the United Statesalone, 64 percent of all adults are either overweight or obese. Bythe year 2008 obesity alone is predicted to reach 39 percent of allU.S. adults and 26 percent of children.

Though more clinical research is needed to determine the level ofefficacy of various dietary supplements on improving measures ofjoint disease, research results on many have also shown promise.Ingredients such as omega-3 and omega-6 fatty acids, ginger andglucosamine with chondroitin sulfate are shown to relieve somedegree of discomfort with joint stiffness, pain and inflammation.In addition, studies suggest that collagen hydrolysate, safe for usein food, may positively influence articular cartilage regenerationand support OA therapy. //


THE ROLE OF NUTRITION IN JOINT HEALTH

CURRICULUM VITAE

Professor Seifert studied medicine at the University of Munich, Germany, and,

having qualified as a Medical Assistant, worked in the Institute for Surgical Rese-

arch at the university from 1967-1981. In 1974 he qualified as a university lectu-

rer with his work on the enteral resorption of large molecular proteins and was

awarded his professorship in 1979. In 1981 he was appointed Professor of Expe-

rimental Surgery at the Christian-Albrechts University in Kiel, Germany, and is

currently Head of Surgical Research at the Hospital for General and Thoracic

Surgery at the Kiel campus of the University Hospital of Schleswig-Holstein.

Apart from numerous studies on circulatory regulation and other clinical pro-

blems, Professor Seifert did much research work on the enteral resorption of

foodstuffs, particularly proteins. He was able to show that proteins, even in large

molecular form, could be resorbed whilst remaining biologically active. He fol-

lowed up this work with studies where special emphasis was placed on the immu-

nological aspects of the resorption-dependent influence of humoral defense

mechanisms.

Professor Seifert has been awarded a number of prizes for his scientific work. He

is a member of numerous national and international specialist societies and asso-

ciations.

PROF JÜRGEN SEIFERT,

M.D.



Prof Jürgen Seifert, M.D.Head of Surgical Research, Department of General Surgery andThoracic Surgery of the University of Kiel, Germany

// Over the past several decades interest has expanded inthe role of nutritional supplements as agents which may have aspecific effect on disease pathophysiology. Collagen hydrolysate(CH), an enzymatically degraded collagen that received GRASstatus by the FDA, has been used in the treatment of degenerativediseases of the musculo-skeletal system. In recent years a numberof clinical studies have shown the benefits of orally administeredCH on joint health. The therapeutic mechanisms, however, and inparticular the intestinal absorption of CH, remain essentiallyunclear.

Therefore, the time course of CH absorption and its subsequentdistribution in various tissues was investigated in a well-definedmouse (C57BL) model. Absorption of 14C-labeled CH was compa-red to control mice administered 14C-labeled proline followingintragastric application. Plasma and tissue radioactivity was mea-sured over 192 hours. Moreover, additional experiments wereconducted to quantify the molecular weight distribution of theabsorbed collagen fragments using SDS-electrophoresis andHPLC.

Experimental investigations have demonstrated that subsequentto rapid intestinal absorption of CH, measured radioactivity incartilage was more than doubled compared to the control group,indicating a preferential and long lasting accumulation of CHderived fragments in cartilage tissue (FIG1).

Furthermore absorption of CH in its high molecular form, with pep-tides up to 10 kDa, was detected following intestinal passage(FIG2).

THE ABSORPTION AND DISTRIBUTION OF COLLAGENHYDROLYSATE AFTER ORAL APPLICATION

Radioactivity (dpm/100mg cartilage)

Time course of radioactivity in cartilage of mice subsequent to absorption of oral-ly administered 14C-labeled collagen hydrolysate and 14C-labeled proline in thecontrol group. The mice received a standard dose of radioactivity of 580 Bq/g bodyweight and 10 mg collagen hydrolysate / g body weight. The results are presentedas mean ± SD, n = 6. Asterisks indicate p < 0.05 significantly different from the con-trol group.

UV-absorption (%)

GPC-HPLC chromatograms of the absorption medium from ”mice gut sac“ experi-ments 30 min subsequent to absorption of collagen hydrolysate and 9g/L NaClsolution in the control experiment.


300

250

200

150

100

50

00 12 24 36 48 60 72 84 96 192

* * **

Time (h)

Time (min)14 16 18 20 22 24 26

25.0

kD

13.5

kD

3.5

kD

< =

0 .5

kD

100

80

60

40

20

0

▲ 14C-proline + collagen hydrolysate

control (absorption of 9g/L NaCl)

absorption of collagen hydrolysate

■ 14C-collagen hydrolysate

Based on these results the observed beneficial effect of orallyadministered CH on osteoarthritis might be explained by thedemonstrated accumulation of collagen fragments in cartilage tis-sue and the impact of these peptides on cartilage turnover. //


CURRICULUM VITAE

As a Scientific Assistant at the Institute for Physiology of the University of Kiel,

he initially concentrated on the areas of cell physiology and protein chemistry.

Since 1993, Dr. Oesser has been active in medical research at the Schleswig-

Holstein Hospital. Subsequent to his doctoral thesis on the influencing of

chondrocyte metabolism, he has been principally involved in researching the

pathophysiology of osteoarthritis and the development of new therapy possi-

bilities for the treatment of degenerative disease of joint cartilage.


STEFFEN OESSER,

Ph.D.

Satellite Symposium at the World Congress on Osteoarthritis (OARSI)Satellite Symposium at the World Congress on Osteoarthritis (OARSI)

Steffen Oesser, Ph.D.Surgical Research, Department of General Surgery andThoracic Surgery of the University of Kiel, Germany

// Consensus exists that the therapeutic goal of causal treat-ment of osteoarthritis can only occur by targeting chondrocytemetabolism to counteract the catabolic processes taking place inthe joint cartilage. In principle, two therapeutic concepts are con-ceivable: inhibiting the degradation of the structural macromole-cules in the extracellular matrix (ECM) or stimulating the biosyn-thesis of cartilage cells to compensate for pathologically causeddegradation of the ECM.

Experimental investigations have demonstrated intestinal absorp-tion of collagen hydrolysate (CH) in its high molecular form withpeptides up to 10 kDa as well as a preferential accumulation ofthese CH derived fragments in cartilage tissue (Oesser et al. 1999).

In recent studies the influence of CH on the metabolism of matu-re chondrocytes has been investigated in a primary cell culturemodel (Oesser and Seifert 2003). It was shown that the presenceof CH in the culture medium led to a dose-dependent increase intype II collagen biosynthesis, whereas native collagen as well ascollagen-free hydrolysates failed to stimulate the production oftype II collagen in chondrocytes. These results clearly indicate astimulatory effect of CH on the type II collagen biosynthesis ofchondrocytes and suggest a possible mechanism for the regula-tion of collagen turnover in cartilage tissue.

IMPACT OF COLLAGEN FRAGMENTS ON THE

EXTRACELLULAR MATRIX METABOLISM


Type II collagen (μg/106 chondrocytes)

Type II collagen secretion measured in the supernatants of 11-day-old bovinechondrocyte cultures after treatment with collagen hydrolysate. Date representmean ± SD of 6 chondrocyte preparations performed in duplicate.

Type II collagen (μg/106 chondrocytes)

Time course of type II collagen secretion into the supernatants of bovinechondrocytes cultured in basal medium (BM) or in medium supplementedwith 0.5 mg/ml collagen hydrolysate (CH). Data represent mean ± SD of 4chondrocyte preparations performed in triplicate.

0

1

2

3

Collagen hydrolysate (mg/ml)0 0,05 0,1 0,5 1 5 10

Time (days)00

1

2

3

2 4 6 8 10 12

* p < 0.01 compared with untreated controls

** p < 0.01 compared to treatment with 0.1 mg/ml CH

* p < 0.01 compared with untreated controls

Satellite Symposium at the World Congress on Osteoarthritis (OARSI)Satellite Symposium at the World Congress on Osteoarthritis (OARSI)

Moreover, utilizing immunocytochemical methods, it wasdemonstrated that in addition to an enhanced synthesis of type IIcollagen in chondrocytes treated with CH, the amount of pericel-lular aggrecan was significantly increased as well, indicating thatthe stimulated cells synthesize a complete extracellular matrix.

Based on these results CH might be of particular importance forthe nutrition of cartilage tissue and might help to reduce degene-rative alterations in the ECM. //

LITERATURE

Oesser et al. (1999): Oral administration of 14C labeled gelatin hydrolysate leads

to an accumulation of radioactivity in cartilage of mice;

Journal of Nutrition 129: 1891-1895

Oesser and Seifert (2003): Stimulation of type II collagen biosynthesis and secre-

tion in bovine chondrocytes cultured with degraded collagen;

Cell & Tissue Research 311: 393-399


CURRICULUM VITAE

In 1967 Prof Selbmann graduated in mathematics at the University of

Stuttgart, Germany. After three years of serving as Head of the Scientific

Computing Centre of the Du Pont Company at Neu-Isenburg he re-entered

university and completed with a PhD graduation in Human Biology at the

University of Ulm. In 1976, he took his Habitational Degree (postdoctoral lec-

ture qualification) in Medical Statistics and Data Processing and worked as

Professor and Deputy Director of the Institute of Medical Information

Processing, Statistics and Biomathematics at the University of Munich from

1980 till 1984. Since 1984 he is Full Professor and Head of the Department of

Medical Information Processing at the University of Tübingen. 1993 to 1995

he served as Dean of the Medical Faculty at the University of Tübingen and

subsequently became for three years Speaker of the German University

Professors for Medical Informatics, Biometry and Epidemiology.

Prof Selbmann was appointed to the Expert Council of the Concerted Action

in Health Care of the Federal Republic of Germany (1988-1991) and to the

Expert Council of Health Research of the Federal Minister of Education and

Research (1990-2000). From 1992 on he was Chairman of this scientific com-

mittee. Since 1999 he officiates as Chairman of the Committee of Health

Reporting of the Federal Republic of Germany.

In addition Prof Selbmann held office in several scientific societies and asso-

ciations: he was President of the German Association of Medical Informatics,

Biometry and Epidemiology (GMDS) (1985-1987) and Founding Chairman of

the German Association of Quality Management in Health Care (GQMG)

(1993-1997). Since 1993 he serves as Chairman of the Jury of the Golden

Helix Award for Quality Management in Health Care.

PROF H.-K. SELBMANN,

Dr. rer. biol. hum.,

Dipl.-Math.


Prof Hans-Konrad Selbmann, Dr. rer. biol. hum., Dipl.-Math.Institute for Medical Information Processing University of Tuebingen, Germany

// In the age of evidence-based medicine, observationsfrom experienced clinicians do not count, only good clinical stu-dies do. A search for studies on the use of collagen hydrolysate inman has found 16 published and assessable studies. In 7 studiescollagen hydrolysate was applied as monotherapy to patients suf-fering from osteoarthritis. These studies include about 850patients. There were no serious adverse events reported in any ofthese studies.

The first of these 7 studies was conducted in the mid-eighties. Themost recent, largest and methodically most extensive was a mul-tinational, study dated from the years 1996-1998. In the last 20years the requirements of good clinical studies, above all in thenon-pharmaceutical field, have constantly increased.

Four studies were designed as double-blind, placebo controlledrepresenting the state of the art in therapeutic research. They wereexamined more closely within the scope of a systematical review:

Study Country Number Outcome Characteristicsof Patients

Adam 1991 and CSSR 52 Positive effect of Cross over with 4 therapyevaluation report PCH very likely groups over 16 months,

reanalysis not possible.Beuker and Rosenfeld D 92 Positive outcome Inadequate evaluation,1996 is reported. reanalysis not possible.Moskowitz 1999 D, UK, 314 Effects significant Cultural differences can

USA only in D. have an influence.Adam 2001 and CSSR 44 Only Lequesne-index Unpublished, study aban-biometric report shows improvement. doned because of

recruitment problems.

Two of these studies – Adam 1991 and Moskowitz 1999 – showstatistically proven influences of collagen hydrolysate comparetoplacebo.


CLINICAL EXPERIENCE WITH COLLAGEN HYDROLYSATE

IN OSTEOARTHRITIS – RESULTS OF A SYSTEMATIC REVIEW


The study by Adam (1991) compared 4 treatments (collagenhydrolysate, non-hydrolyzed gelatin, non-hydrolyzed gelatin+glycine+CaHPO4*2H2O, egg albumin) in a cross-over design (2-month washout, 2-month treatment). All three gelatin prepara-tions were significantly superior to egg albumin, though no signi-ficant difference between them, in reducing pain and theconsumption of analgesics.

Unfortunately, collagen hydrolysate was not analyzed separatelyin the evaluation report and a reanalysis was not possible.

The multinational study (Moskowitz 1999) had a two arm (PCH10g daily and placebo for 24 weeks) double-blind design. The 19sites of the study were used as strata for the randomization per-mitting a separate analysis for all three countries. Paracetamolwas allowed as an escape medication, the dosage was left to thepatients. Differences in mean scores for the three primary efficacyvariables between baseline and week 24 are presented in the fol-lowing table:

Total* USA UK D**Number of PatientsPCH 161 86 18 57Placebo 153 87 11 55MEAN DIFFERENCES WEEK 24 AND BASELINE:WOMAC Pain Score PCH 63.9 64.4 36.1 71.9Placebo 58.9 78.2 38.5 32.4WOMAC Physical Function ScorePCH 191.1 206.7 93.3 198.3Placebo 167.1 240.9 127.3 58.3Patient Global EvaluationPCH 0.4 0.4 -0.2 0.5Placebo 0.3 0.3 0.4 0.2

* no significance; ** all variables significant at adjusted 5%

There were no statistically significant differences for the total stu-dy group in the three primary efficacy variables. However, all vari-ables showed a significant improvement in the German part of thestudy. The different results between Germany and the other coun-


tries point to influences that could not be standardized in the mul-tinational study, in spite of a common study protocol and only oneinvolved CRO. Thus, between USA and Germany (in UK the num-ber of patients was too small for a separate analysis) differencesbecome apparent in:

USA D

Protocol violations 48% 17%

Drop out rate after 24 weeks 43% 18%

Paracetamol consumption>30 tab/week 45% 3%

Baseline values (WOMAC Pain Score) 253,3 217,9

Physician Rheumatologists Orthopedics

All these impact factors – especially the drop out rate with itsunderlying shortening of therapy duration and the consumptionof analgesics with its impact on all three primary efficacy varia-bles - are good reasons not to combine the results of the threecountries in one analysis.

The two studies reported in more detail illustrate a distinct clinicalimpact of the collagen hydrolysate in patients suffering from oste-oarthritis and confirm the results of recent experimental investi-gations. The other – methodically less extensive – studies point atleast to the same direction although they do not have the samestrength of evidence. //

LITERATURE

Adam M (1991): Welche Wirkung haben Gelatinepräparate?

Therapiewoche 41: 2456-2461

Moskowitz RW (2000): Role of Collagen Hydrolysate in Bone and Joint Disease;

Semin Arthritis Rheum 30: 87-99

x 6 Collagen Bioavailability &

Dosage

Nutritional Information © Sept 2012 Xenca Ltd. All rights reserved V01

™

Collagen Bioavailability & Dosage This document covers the use, take-up and suggested dosage for orally-ingested, pure Type I & III Collagen Hydrolysate (also known as Collagen Peptide) and Type II biosynthesis. It has no chemical additives, is widely considered by food agencies to be a safe food product for human consumption and no maximum consumptive amount is recorded. Suggested dosages of collagen hydrolysate are based on available public domain clinical trials and published research undertaken by universities and refining companies. However, science can be somewhat imprecise and the suggestions in this document should be taken in the spirit with which it has been compiled: a suggestion based on currently available resources. This document should be read in conjuction with our Health and Medical Policy.

The Collagen Molecule

Briefly, collagen comprises about a quarter of all the protein in the human body. It is a major 'structural' protein, forming what can be thought of as molecular cables, or biological ropes that strengthen tendons and vast resilient sheets that support the skin and internal organs. Currently, some 28/29 types of collagen have been identified and described (for additional information see the 'Collagen Types' document). However, over 90% of the collagen in the body is Type I, II or III (Sabiston, Textbook of Surgery 18th Ed):

The five most common types of collagen and where they are found:

Type I; skin, tendon, vascular ligature, organs, scar tissue & repair, artery walls, bone & teeth

Type II; Hyaline cartilage, 50% of all cartilage protein, vitreous humour of the eyeballs

Type III; Reticular fibre, artery walls, skin, intestines, uterus (found alongside type I), blood vessels

Type IV; cell basement membrane

Type V; cell surfaces, hair, placenta

Gelse K, et al, Collagens-structure, function, & biosynthesis, Adv Drug Deliv Rev, 2003

Collagen is composed of three molecular chains, wound together in a tight triple helix (see figure 1, below). It mostly exists in the form of elongated 'fibrils' (i.e. a fine fibre) and is mainly found in fibrous tissues such as tendon, ligament, cartilage, cornea, bone, blood vessels, interverteral discs and the gut.


™

Figure 1; The Collagen Triple Helix Molecule

The fibroblast is the most common cell which creates collagen (i.e. a fibroblast is a type of cell that synthesizes, or produces the extracellular matrix and collagen for tissues). The 'Tropocollagen' or collagen molecule is a sub-unit of larger collagen aggregates such as fibrils. It measures approximately 300 nanometers (nm) long and 1.5 nm in diameter, (note: a water molecule measures only 0.278 nm in diameter) and is made up of three polypeptide strands. Hence, collagen molecules are too large to penetrate the human skin’s outer layers (note: skin is composed of about 75% collagen) from a lotion, for example and are similarly too large to be broken down by the stomach.

Figure 2, Example Showing Tendon Structure

If collagen-rich materials (e.g. bovine hides) undergo enzymatic hydrolysis, the collagen molecule will break down into smaller components. This process typically reduces collagen proteins in molecular size/weight from a value of around 300,000 Daltons (Da), down to between 2,000 and 5,000 Da (see Ricard-Blum et al. 2005 & http://en.wikipedia.org). This low molecular size/weight makes hydrolysed collagen easier to digest and permits relatively quick intestinal absorption by the body. However, some detailed testing has revealed that larger molecules up to 10,000 Da can be absorbed straight into the bloodstream (Oesser at al 2003).

http://en.wikipedia.org/wiki/Hydrolyzed_collagen


™

Basic Pharmacology – Absorption

After the collagen hydrolysate is orally ingested, it passes through the stomach into the gastrointestinal tract. Here, several enzymes from the pancreatic juice and the lining of the intestine break it down to single amino acids and di-peptides. These smaller molecules, now in the hollow of the small intestine (note: 95% of absoption of nutrients occurs in the small intestine), exist in a greater concentration than they do in the adjacent bloodstream. Hence, what is referred to as passive diffusion can occur, as the higher concentration gradient allows the collagen hydrolysate to pass through the cell membrane into adjacent blood vessels. The absorbed collagen hydrolysate is then transported elsewhere in the body. (http://www.nurse-prescriber.co.uk/education/modules)

Figure 3a. Human Gastrointestinal Tract, Wikipedia

Figure 3b. Small Intestine Showing Nutrient Absorption http://jmgreer71.wordpress.com

Bioavailability of Collagen

Bioavailability refers to the proportion of, in this case, collagen hydrolysate that has reached the circulation system and that is available to have an effect. Usually, liquids given intravenously may be considered to be 100% bioavailable, as they are administered directly into the circulation and all of the liquid may potentially cause an effect. Administration by other routes means that some of the molecules will be lost during absorption and distribution, thus bioavailability would be reduced.

http://www.nurse-prescriber.co.uk/education/modules/pharmacology/pharmacy3.htm

http://www.nurse-prescriber.co.uk/education/modules/pharmacology/pharmacy3.htm


™

Two Japanese studies (Iwai K, et al in J Ag Food Chem 2005, and Watanabe-Kamiyama M, et al in J Agric Food Chem. 2010) undertook field and clinical laboratory tests on healthy subjects and ensured fasting prior to the tests. After oral ingestion of collagen hydrolysate, testing of blood serum and plasma showed how peptides were found in high hydroxyproline concentrations in the blood stream. These high peptide levels persisted for about 4 hours following oral ingestion but had achieved a maximum level after 1-2 hours.

When hydrolysed collagen was consumed the amount that was absorbed by the body was found to be high. In fact Oesser et al, in the Journal of Nutrition (2003), demonstrated that orally-administered, hydrolysed collagen was digested and absorbed by more than 90% within 6 hours and 95% within 12 hours. This was determined during clinical laboratory tests when the passage of radioactively-labelled collagen hydrolysate was monitored through the body. As part of this trial, a clear pattern of tissue accumulation was seen in the joint region comprising cartilage and bone.

In experimental laboratory studies i.e. cell cultures, high accumulations/concentrations of Type I collagen hydrolysate in soft tissue (e.g. cartilage) were found to stimulate the chondrocyte metabolism to produce Type II collagen biosynthesis (note: chondrocytes are the only cells found in healthy cartilage and they produce and maintain the cartilaginous matrix comprising mainly collagen and proteoglycans). Hence, it is considered essential that good quality, pure collagen hydrolysate be utilised to ensure proportionately higher bioavailability and absorption (Gelita Internal Research White Paper, 2011).

Figure 4:Top row; Placebo example with higher 'red' content showing a cartilage decrease. Bottom row; Collagen Hydrolysate example showing increased yellow/green clouration indiacting a thickening of the catilage. Delayed Gandolinium Enhanced

Magnetic Resonance Imaging (dGEMRIC) of Knee Cartilage after 24 weeks. McAlindon TE, et al Osteoarthritis & Cartilage (19 (4), 2011 ).


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It was recently determined during MRI trials (McAlindon TE, 2011), with a delayed Gandolinium Enhanced Magnetic Resonance Image (dGEMRIC), that the actual take-up of collagen hydrolysate can be seen as a positive change in 'Proteoglycan' content (i.e. the filler substance between cells, giving it a gel-like nature) in the knee cartilage after 24 weeks (see Figure 4, above).

Collagen Hydrolysate Dosage

No formal, specific, recommended dosages exist for oral collagen hydrolysate (and no industry standard exists at this stage). However, a large number of clinical and pre-clinical studies have taken place over the last thirty years. Of these studies, many are in the public domain and provide evidence of the methods utilised and dosage employed to achieve positive results (some of the key research documents are set out in the table below). These documents, by definition, provide an indication of suitable suggested dosage range and optimum criteria. Two of the most important research studies are briefly set out below:

− In 2012 Dr Olivier Bruyere (et al. Comp. Ther Med;20, 2012) undertook a comparative, randomised, placebo-controlled, double-blind, multi-centre study of 200 patients who were at least 50 years old and experiencing joint pain at lower or upper limbs or at the lumber spine. This evaluation was to investigate the effects of 1,200mg/day of collagen hydrolysate when compared to the placebo group. These tests showed little difference after 3 months, however, clear differences between the groups could be seen at 6 months. Hence, it is recommended that collagen hydrolysate at this dosage be taken orally for at least 6 months.

− In 2008 an important first attempt at 'primary prevention of joint tissue injury' commenced with subjects who were young, healthy and active. Dr Kristine Clark (et al. Curr Med Res Opin; Vol 24, 2008) at Penn. State University undertook a randomised, placebo-controlled, double-blind study of 72 male and 75 female athletes. This evaluation was to investigate the effect of 10g/day collagen hydrolysate on activity-related joint pain caused by strenuous effort and physical exercise over a 24-week period. These trials showed a positive result for the use of collagen hydrolysate.


™

Collagen Hydrolysate Key Clinical & Pre-Clinical Research Documents

1979 Krug undertook an open study administering collagen hydrolysate to 193 subjects and six months later recorded that 75% reported improved symptoms.

Gotz (1982) undertook a non-randomised study of 60 subjects and noted after 1 month 56% of subjects had improved considerably and after 3 months that 45% were symptom free and a further 30% had clearly improved symptoms..

1985 Oberschelp studied comparatively 154 subjects with Osteoarthiritis of the hip, knee or lower spine with positive results.

1989 Seeligmuller, undertook an open study of 356 patients

Adam in 1991 undertook a randomised, double-blind crossover study over 16 months with collegen hydrolysate and reported that 81 test subjects noticed an improvement in their symptoms.

1993 Seeligmuller, undertook an open study of 519 patients

1996 Beuker & Eck undertook an open study of 40 patients

1996 Beuker & Rosenfield undertook a double-blind study of 100 patients

Between 1996-1998 Moskowitz undertook a double-blind study of 389 patients at 20 test centres in 3 countries, with pain levels monitored according to the WOMAC score (see below).

1998 Fernandez undertook a comparative study of 16 members of a mountain bike team and 10 members of a Spanish basketball team, each given 10g of Collagen Hydrolysate. This test showed a demonstrable thickening of cartilage by between 5%-27%.

Zuckley in 2004 recorded significant improvement in a test group of 250 patients.

Rippe in 2004 undertook a double-blind study on 250 patients with mild osteoarthritis and showed improved function of the knee joints.

Rippe in 2005 undertook a randomised study on 102 patients with severe osteoarthritis and again showed improvements in knee joint function.

2005 Flechsenhar undertook an open study on 100 athletes with joint symptoms and some pain. Over 12 weeks 10g of collagen hydrolysate was administered and improvements in clinical parameters occurred.

2006 Adam undertook a comparative, double-blind study of 81 patients over 16 months and found a clear improvement in test parameters.

2008 Clark, undertook a 'primary prevention', randomised, double-blind study of 147 fit active athletes over a 24 week period, again showing positive results.

2011 McAlindon, undertook a single centre prospective randomised, placebo controlled, double-blind, study of 30 subjects over 24 weeks. It detected positive change in knee cartilage proteoglycan.

2012 Bruyere, undertook a comparative, randomised, double-blind study of 200 patients with joint pain at lower or upper limbs and lumber spine, successfully administering 1200mg/day.

Most of the clinical and pre-clinical studies in the public domain use a variety of monitoring methods, depending on the aim of the research and this can make cross-comparison difficult. However, use of the WOMAC Index (see below), or a derivative of this, allows for some commonality between research and subsequent practical use. The WOMAC Index can be completed by a medical or sports specialist, or indeed by the patient themselves, on a regular frequency to allow monitoring of a recovery.


™ Note: The Western Ontario and McMaster Universities Arthritis Index (WOMAC)

A set of standardised questionnaires developed in 1982 used initially by health professionals to evaluate the condition of patients with osteoarthritis of the knee and hip. This includes pain, stiffness and physical functioning of joints.

The WOMAC has also now been used to assess back pain, rheumatoid arthritis, lupus and fibromyalgia (see for examples http://www.orthopaedicscores.com/).

The WOMAC measures five items for pain (on a score range of 0-20), two for stiffness (score range 0–8), and 17 for functional limitation (score range 0–68). Physical functioning questions cover everyday activities such as stair use, standing up from a sitting or lying position, standing, bending, walking, getting in and out of a car, shopping, putting on or taking off socks, lying in bed, getting in or out of a bath, sitting, and heavy and light household duties.

http://en.wikipedia.org/wiki/WOMAC

On the basis of the various known industry and university research currently in the public domain, dosages can be suggested for orally-consumed, pure collagen hydrolysate;

The lowest maintenance dosage to assist in general pain management would be in line with Bruyere's (2012) research, which recommends 1,200mg/day of pure collagen hydrolysate (approx 3x 400mg capsules or just under 1 level teaspoon of powder per day). This is thought likely to be suitable for general aches and pains, anyone with a long term chronic joint injury or for those some 3 months on from a recovering soft tissue injury.

The upper end of the spectrum would be in line with Clark's (2008) research on competing professional athletes, where as much as 10g/day collagen hydrolysate (approx 25 x 400mg capsules or 5 teaspoons of powder per day) could be used. This large amount of collagen hydrolysate would only really be required/suitable for professional sports men/women with a very severe injury and an extremely large height/body mass.

Although beyond the remit of this document, some correlation can be made between the baseline maintenance dose (i.e. 1,200mg/day) and that of the needs of a person with a slight injury and a small frame, right through to a person with a large frame and physique requiring a dose estimated to be somewhere in the region of 5g – 10g/day.

The reality for most people struggling with a soft tissue, joint-based injury, whether competing in sport, undertaking physical work, struggling with arthiritis and general aches and pain associated with joints, is likely to be in the order of 4g/day and this is likely to be needed for some 6 weeks to 3 months, in turn followed by a maintenance dose.

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x 9 Revitalise Sports Collagen Amino Acid Composition

Technical Information

Amino acid composition of gelatin and collagen peptides

Amino Acid g Aa/100g crude protein Aa/100g Aa Alanine 8.6 10.7 Arginine 7.3 4.5 Aspartic Acid 5.8 4.8 Glutamic Acid 10.2 7.7 Glycine 22.2 32.6 Histidine 1.0 0.6 Hydroxyproline 11.9 10.0 Isoleucine 1.4 1.1 Leucine 2.7 2.3 Methionine 0.9 0.7 Phenylalanine 2.1 1.4

Proline 12.7 12.1 Serine 3.2 3.4 Threonine 1.8 1.7 Lysine 3.6 2.6 Hydroxylysine 1.6 1.1 Tyrosine 0.8 0.4 Valine 2.4 2.3

Analytical Method The amino acid composition was determined by amino acid analysis as described in Pharm. Eu. 2.2.56 (Version 8).The proteins were hydrolyzed for 24 h to their individual amino acid constituents in the presence of 6 n HCl and 0.1 % phenol at 110 °C. The amide links in the side chains of glutamine and asparagine are hydrolyzed to form glutamic acid and aspartic acid. Following the hydrolysis, the amino acids are covalently labeled with 6 – aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ-Flour reagent, Waters Inc.) using a pre-column derivatisation technique. L-2 Aminobutyric acid (AAbA) with a final concentration of 10pmol/μl was used as internal standard. The derivatives are separated by C18 reversed-phase HPLC and quantified by fluorescence detection (Determination of data PROTAGEN AG, Dortmund, Germany).

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