Bio lubricants: The applications of Hyaluronic Acid in knee joint treatment
Advanced Rheology and Materials H84ARM
In the last few years there have been large improvements in the research and development of replacement biofluids, in particular those used to lubricate bones in knees joints. Knee joints are the largest joint in the human body and must cope with a wide range of varying forces which can be up to four times the body weight (1). Consequently the lubricating synovial fluid inside the joint is a highly complex fluid, able to adapt its properties depending on the forces acting on the knee. Loss of lubrication within the knee is believed to be one of the underlying causes of osteoarthritis (2), a disease characterised by the degeneration of joint cartilage (Figure 1), which affects about 80% of people over 65 and is the highest cause of work loss in the US and Europe (3). With an aging population, a cure for osteoarthritis in the form of a bio-lubricant would be worth millions to the manufacturer. This report sets out to assess the potential for a replacement bio-lubricant within a knee joint, in particular the potential for using Hyaluronic Acid (HA), a naturally occurring component of synovial fluid.
2.1 Synovial Fluid
Synovial joints are the most common type of joint in the human body (Figure 1) with the knee joint being the largest. The knee joint is comprised of two major bones; the tibia and the fibula. Both bones are coated in a layer of articular cartilage which distributes the joint forces over the underlying bone and acts as a bearing surface (2). The bones are enclosed within a fibrous capsule which is where the synovial fluid is contained .
Synovial fluid lubricates the articular cartilage surfaces, which aids the mechanical function of the joint by acting as a shock absorber and reducing friction. It does this by forming a microscopic film between the layers of cartilage, and also acts as a transport medium for delivering nutrients and removing waste products from the cartilage (6). Synovial fluid is composed of water, a lubricant glycoprotein (lubricin) and hyaluronic acid (HA), which maintains the necessary viscosity of the fluid film (7). Synovial fluid composition varies between rest and exercise due to water rapidly entering the joint during inflammation. HA is unable to leave the joint so rapidly due to its macromolecular structure; however it does have a half life in the joint cavity of about 24 hours (7).
Hyaluronic acid is a naturally occurring substance in all vertebrates, with an adult of an average body weight of 70 kg containing around 15g of HA (8). Within a knee joint there is approximately 2mL of synovial fluid with HA concentrations varying from 2.5 to 4.0mg/ml (9), depending on the water content. The typical structure of HA can be seen in Figure 3 below.
HA is a polysaccharide chain consisting of a basic unit of two sugars, D-glucuronic acid and N-acetyl D-glucosamine, polymerised into large macromolecules of over 30,000 repeating units (11). The polymer is a basic constituent of many tissues within the body and has a molecular weight between 104 and 107 Daltons (10).
2.3 Osteoarthritis and available treatments
A reduction in HA viscosity, and hence lubrication, is believed to be one of the underlying causes of osteoarthritis (2). Osteoarthritis is the most common form of arthritis and affects around 8.5 million people in the UK, with 80% of over 65s suffering from some form of it (12), which indicates that there is a sizeable market for drugs and remedies.
Treatments for osteoarthritis can be classified in three main categories: non pharmacologic treatments (mainly exercise and weight loss), surgery, and pharmacologic. The available treatments and their market share are shown in.
Total knee replacement (TKR) surgery is usually advised for advanced osteoarthritis when all conservative measures and minor surgical procedures have failed (13). Surgery is a last resort as there are many potential problems during the procedure; such as nerve damage, formation of blood clots and wound infections. There is no guarantee the surgery will be successful, and complications can occur after the operation when further surgery may be required.
Pharmacologic choices include acetaminophen, non-narcotic analgesics, non steroidal anti-inflammatory drugs (NSAIDs), topical analgesics, intra-articular corticosteroids, and disease modifying osteoarthritis drugs (14). NSAIDs are the most widely used treatment for OA and work by blocking prostaglandins; the substances that dilate blood vessels and cause inflammation and pain. Unfortunately some patients cannot tolerate NSAIDs or suffer serious side effects, such as gastrointestinal bleeding and ulceration (15), so it is not a universal treatment.
Corticosteroid treatment is rarely used for chronic OA as the injections only mask the pain, so the patient must be careful not to overuse the affected joint. They are used for short term pain relief as opposed to a permanent cure. Most clinicians recommend at least three months between injections, out of concern for potential effects on joint structure from the increased use of a less painful, but still diseased, joint (16).
HA treatment is classed as a disease modifying drug, because rather than simply treating the pain and inflammation, it targets the root cause of the problem. HA comprises approximately 25% of the market so any improvements in HA products would result in large financial rewards.
2.4 HA properties
The most important property of HA which makes it such a useful fluid in the body is its viscoelasticity. This is believed to be due to a number of contributing factors, namely the high molecular weight and the molecular interactions of the HA molecule. HA is a polyanion composed of repeating monomer units with evenly spaced, repelling, negative charges on the glucuronic acid molecules (Figure 3) (17). This repulsion helps explain why HA forms an extended polymer chain. The intermolecular and intra-molecular hydrogen bonding of the HA molecule, along with its hydrophobic interactions provides a rigid structure to the chains (18), which enables viscous behaviour. Adjacent carboxyl and N-acetyl groups in HA are therefore able to hydrogen bond with the water contained within the synovial fluid. The hydrated molecule occupies 1000 times the volume of the unhydrated molecule, by forming highly crowded and entangled polyanionic molecular networks (17) which further increase HA viscosity. Up to six litres of water can be bound per gram of HA (19).
HA is a pseudoelastic material; its viscosity is reduced when it is subjected to a shear force (20). Under low shear conditions, hydrogen bonding stiffens the backbone formation, and under high shear forces the hydrogen bonds break leading to shear thinning (21), as can be seen from Figure 5.
The Synovial fluid from osteoarthritic joints is lower in viscosity than that from healthy joints (22) which is due to a reduction in the molecular weight and concentration of HA in the fluid (23). Therefore by adding HA of a high molecular weight into the knee joint, the viscosity of the synovial fluid could theoretically be increased, and the symptoms caused by osteoarthritis could be reduced if not cured.
Viscosupplementation is the injection of HA into joints to manage osteoarthritis, improve synovial fluid viscosity and elasticity, and relieve pain. It was initially suggested as a treatment because the concentration and molecular weight of HA appeared decreased in osteoarthritis joints. This was down to HA de-polymerisation by free radicals and the production of low molecular weight HA (24). It is claimed that viscosupplemented high molecular mass HA increases the joint fluid viscoelasticity, stimulates the production of endogenous HA, inhibits the effects of inflammatory mediators, decreases cartilage degeneration, and promotes cartilage matrix synthesis (8).
The effect of viscosupplementation seems to last longer than intra-articular steroid injections. Combining HA injections with steroids could have some benefits; as the first lasts for up to one year, and the latter begins to have an effect at an earlier stage (4 to 6 weeks) (8).
Current HA products reduce pain but do not control inflammation. Based on the knowledge that methotrexate has anti-inflammatory properties but adverse effects if consumed orally, an HA - MTX conjugate was designed. This product connects MTX with HA through peptides susceptible to cleavage by lysosomal enzymes. Intra-articular injection on this conjugate produced significant reduction of knee swelling in rats (25).
There are currently two main ways of producing hyaluronic acid commercially (26). The conventional method of manufacturing HA is by extraction from rooster comb while the other method involves the fermentation of Streptococcus bacterium. Different rheological properties of the product result from the two methods (27).
3.1 Extraction from rooster comb
The main focus of this method is in the preparation of HA in its pure form, and within an acceptable molecular weight range which is suitable to be absorbed, assimilated and utilized in the human body (28). This conventional method includes steps such as providing the solution of extraction, removing the impurities from the solution, decomposing and separating the HA to a desired molecular weight range, before finally drying the product. The complete process is illustrated in Figure 6.
The raw rooster comb is harvested as part of the chicken slaughtering process. The first step is to dehydrate the rooster comb using acetone before grinding it in to particles. This step is repeated approximately three times, after which the rooster comb is soaked in distilled water to form a solution of extraction. The solution contains both the desired HA and undesired proteins which are partially removed by the addition of chloroform. The next step is to add ethanol to form a precipitation to produce crude HA. The crude HA is then dissolved with a 0.1 mol/l sodium chloride solvent to produce a solution where the remaining undesired proteins are removed using chloroform, and a suspension is produced. The HA is then decomposed to the desired low molecular weight range by enzymatic digestion using the Strptomycia-protease enzyme. This occurs at pH 7.5 and a temperature of 37oC for approximately 24 hours to produce a decomposed solution (29). The temperature and time interval of this digestion step is vital in order to produce HA of the desired molecular weight range.
The solution now contains 1% by volume HA. The solvent is removed to form a precipitate where it is soaked in 0.4 mol/l sodium chloride in water (29). This forms an ionized solution where it is then precipitated again using 95% ethanol to further separate the solvent. Finally, the precipitate is dehydrated by vacuum sterilisation to produce HA in the form of sodium hyaluronate powder. The product can then be packaged for the distribution and consumption of human use. The approximate timescale of the process can be seen in table 1.
Table 1: Time needed for preparation of HA on a lab scale (29)
Steps Time (days) Comb slice / ethanol wash 3 Water extraction 3 Comb removal / suspension / centrifuge / precipitation 1 Dissolve / chloroform extraction / centrifuge / precipitation 6 Dissolve 2 Suspension / chloroform extraction / centrifuge / precipitate 3 CPC / washes / re-dissolve / centrifuge / precipitate 3 Formulate / sodium hyaluronate powder 1 Total 22
There are some disadvantages associated with this method; it is presently impractical to control the molecular weight of the product while it is synthesized in animal tissue, and the extraction and origin determines the purity of the product. The subsequent extraction and purification steps result in an inherent molecular weight reduction (19). It is important to remove the impurities, such as proteins, to avoid side effects after injection into the joints. There is also some risk of cross-species viral infection and the acetone and chloroform used during the purification process are hazardous materials (30). The collection of rooster combs along with the extraction and purification processes are time consuming and labour intensive causing the HA production to be very costly (31). There are also ethical issues involved in slaughtering the chicken. Commercial factory farming operations often involve raising the chicken in small crowded cages, preventing the chickens from engaging in their natural behaviour. Some chickens are subjected to debeaking which might cause severe pain leading to the chicken starving to death. Some standards have to be met such as "Cage Free", "Natural", and "Certified Humane" standards (32).
However, these disadvantages can be avoided by carrying out a microbial production process, using the fermentation of Streptococcus bacterium.
3.2 Batch fermentation using Streptococcus bacterium
The two most important factors in HA production are its subsequent purity and molecular weight. The high value of HA means that that the raw material costs are not a major factor, therefore quality rather than quantity has been the major focus (30).
The main advantage of this process is that the key process parameters can be controlled and optimised to achieve the desired molecular weight and poly-dispersion of the final product unlike the conventional method of production (34).
In this process there are 2 main alternatives, the first is to supply air through the reaction medium and allow the cells to reproduce aerobically and the other is to supply no air to the reactor and allow the cells to grow anaerobically.
Table 2: Comparison between anaerobic and aerobic conditions, adapted from (30)
Anaerobic Aerobic Specific growth rate (h-1) 0.85 1.02 Acetate yield (mg/g) 4.45 4.96 Ethanol yield (mg/g) 68 7 Formulate yield (mg/g) 122 0 Lactate yield (mg/g) 651 662 HA yield (mg/g) 65 88 Biomass yield (mg/g) 191 206 Molecular weight (Mda) 1.1 2.4
The yield of HA as well as the molecular weight of the final product is much enhanced under aerobic conditions. The specific growth achieved is 20% higher in aerobic condition which explains the higher yield.
Another process condition which requires optimisation is agitation of the reaction medium. Results have proved that there is a higher yield from increased agitation rates due to enhanced oxygen transfer (35), but within literature there is also experimental data which contradicts this. These results showed that at higher agitation rates there is an initial increase in the yield of the HA product, but as there is a build up in the concentration of HA in the reactor the viscosity increases. The outcome of higher viscosity within the system means that higher impeller forces are required to achieve the same agitation. At these higher impeller forces, high shear stress causes a decrease in the HA molecular weight and yield. Other experimental data also proves that the HA molecules are resilient to shear forces where there is an increase in yield and molecular weights at 1000rpm (36).
Any deviation from pH 7 was found to reduce the yield and molecular weight as the growth of cells is optimum at this pH (37). Temperature did not have an effect on the polydispersity, but did have a negative effect on the molecular weight at temperatures above and below 37C.
This process is the most common in industry, but has an inherent disadvantage of a large turnaround time which rapidly decreases the volumetric production rate; this results in higher fixed costs per unit product.
One way to rectify this problem is to increase the HA concentration which is achieved by high cell density. It was found that when concentrations of higher than 4g/l were achieved the broth became too viscous to achieve efficient agitation and aeration. As a result, the benefit of high HA concentration is reduced by low efficiency of HA synthesis. Another report suggests that beyond concentrations of 5-10mg/l, due to the high viscosity, the production of hyaluronic acid is not practical (30).
Another way to increase production rate is to skip the turnaround phase, and hence the lag phase, by using a continuous culture. Continuous cultures could also offer two benefits. Firstly, cell growth could be maintained at the exponential phase so that the excretion of cell wall proteins at the stationary phase can be avoided. Secondly, the cells could be controlled to grow at a lower specific growth rate, which might result in HA of higher molecular weight (38). However a continuous culture has its inherent defect; low efficiency of substrate utilization. This is the main reason why continuous cultures are seldom used in a commercial process. Another adverse factor is that the efficiency of HA production would decrease during prolonged operation.
At higher seed volumes it was found that there was a decrease in the specific growth rate of cells and a decrease in the yield of HA. These results suggest that there are inhibitors in the broth that are reducing the synthesis of HA. With an external cartridge filter to retain the cells when draining the broth, the repeated batch culture can be employed successfully for HA production of low yields.
Streptococci can be difficult or expensive to ferment as can be seen from the above and are also challenging to genetically manipulate, and have the potential to produce exotoxins. This therefore justifies finding alternative methods of producing HA.
Product performance of HA, in its orthopedic application, is highly related to its molecular weight. It accounts for the viscoelastic properties and the ability of the polymer to retain large volumes of water, which are important in determining the physiological functions of HA (35). It is therefore critical that the molecular weight distribution, specifically the average molecular weight, be controlled. This can be achieved by batch fermentation but there is a growing demand to produce HA on a larger scale. and to solve the problems of using a continuous culture.
More research is needed in solving the problems of using a continuous culture by finding the origins of the inhibitors in the broth, which become more apparent at larger seed volume. This way it may be possible to manipulate the process conditions in a way which inhibits the production of these inhibitors.
Table 3: Comparison between the Conventional method and the Fermentation method
Source Rooster Comb Fermentation of Streptococcus Molecular Weight Up to 5 million DA 50,000 - 2.5 million DA Synthesis Uncontrolled Made according to requirements Impurities Possible protein content Possible endotoxin content Risks Might cause allergy Immunologic reactions
Few lines comparing both processes?
4.1 Treatment for Knee Osteoarthritis
As mentioned previously, osteoarthritis affects 8.5 million people in the UK with the knee being the most affected joint. The UK population is steadily increasing, and with people living longer the number of people suffering from osteoarthritis will also increase. With recent studies showing that a course of hyaluronic acid injections into the affected area have at least a partial effect on patient's lives, the demand for the treatment will increase, leading to greater commercialisation.
A study in 2008 (39) conducted a trial to determine the effectiveness of hyaluronic acid as a treatment for osteoarthritis in the knee. The patients were given one of two HA products - Hylan G-F 20 or Sodium Hyaluronate. Patients were given a course of three injections and were monitored at 6 weeks, 3, 6 and 12 months. The pain as experienced by the patient was measured using the Visual Analogue Scale (VAS). If a patient were to use Sodium Hyaluronate, secondary injections would have to be conducted in order to stop the pain increasing. Measurements of physical activity were also made, using the WOMAC (Western Ontario and McMaster Universities Osteoarthritis) to see if the products had any effect . As was the case in measurements of VAS, there was an improvement in both Hylan and Sodium Hyaluronate, but Hylan lasted for a longer period of time.
Table 4: WOMAC scores for the two products (reference)
Pre Injection 6 weeks 3 months 6 months 12 months Hylan 36.1 29.3 17.7 14.3 15 Sodium Hyaluronate 34.7 28.6 18.4 27.9 33.3
With other trials displaying similar results (40) patients suffering from the condition will be keen to have access to the treatment to ease their pain. A recent study conducted in Canada compared prices for different brands of hyaluronan products with prices ranging from 100 to 200 (currency conversion correct as November 9th 2009) for a course of three injections (41). These injections contained 2mL of doses and were effective for between six months and one year. If only one tenth of those suffering from the condition are willing to pay for the treatment, it will result in an expenditure of between 85million to 170million and a need for approximately 2,000 L of hyraluronic acid to be produced. These are quite signficant sums and would be valuable to any prospective manufacturers. Furthermore if the treatment were to be made available on the NHS, demand would dramitcally increase, leading to great revenue for manufacturers.
As the population of the country increases - it is expected to grow by ten million in the next 20 years - as well as the number of people living to an older age, more people will be diagnosed with the condition, implying that the future scale of hyaluronic acid as a treatment for osteoarthritis will increase.
Hyaluronic Acid also has the potential as an alternative option to a total knee replacement. While a knee replacement has the option of totally eliminating the pain caused by arthritis, but it is subject to a number of risks. The patient would have to undergo surgery, which could potentially cause damage or even prove fatal. This risk would be greater amongst older people, which constitute a large proportion of arthritis sufferers. There is also the consideration that the pain might not be treated by the replacement, or acquiring an infection during surgery.
A typical knee replacement lasts for between 12 to 15 years and can cost upwards of 8,000 (42). While it may seem an attractive proposition for younger sufferers of osteoarthritis, older patients may not find it such a viable option. There is the risk that they may not live for the full live span of the prosthetic joint or may not be able to afford the surgery. In this case a course of HA injections may be a more attractive option. A report from the US (43) found that patients who underwent surgery spent up to $57,900 including costs after surgery, which was $20,800 more than those who did not. Furthermore, a report from the BBC (44) estimated that knee operations will cost the NHS 1 billion by the year 2010, a significant sum of money for a public healthcare organisation. With such large costs involved, as well as potential injuries incurred during surgery, HA becomes a very viable alternative.
4.2 Future Treatments
A recent study conducted Manfredini et al (45) have shown that pain levels and masticatory efficiency of 76 patients suffering from temporomandibular joint osteoarthritis improved following a course of hyaluronic acid. The patients were injected once a week, for a five week period, followed by 4 supplementary injections spread out over six months. The results obtained indicated that the hyaluronic acid worked, with patients reporting a significant difference.
Table 5: Results from clinical trial reference
Symptom 1st Injection 5th Injection 4th Follow Up Masticatory efficiency 5.702.05 6.622.15 7.782.10 Pain at mastication (max) 5.942.95 4.482.49 2.332.65 Pain at phonation (max) 3.983.31 2.392.74 1.952.73 Pain at rest (max) 3.913.36 2.242.77 1.572.34
The hyaluronic acid used in this test was the same as those mentioned above. Additional studies have been conducted for use of HA on the hip (46) and ankle (47). HA works as effectively in them as it does in the knee, with no changes having to be made to the structure of the molecule. There is potential for HA to expand into these markets and increase its market value, which is currently estimated at $1 billion worldwide (8).
Hyaluronic Acid can be seen to be an effective treatment for sufferers of Osteoarthritis. It is a viable alternative to surgery or other treatments, and is well priced to meet market demand. After further clinical trials targeting other joints, it should be approved for use which will increase consumer demand and hence its market value. Furthermore if the necessary research is conducted to produce a continuous reactor for the production of HA, operating costs will decrease, resulting in a lower price for the treatment, making it more competitive. Thus reduced production costs for HA, combined with an increasing market size would suggest a very worthwhile and lucrative investment for any potential financiers.
1. Walker, P S, et al. Features of the Synovial Fluid Film in Human Joint Lubrication. Nature. 1970, Vol. 225.
2. Techniques for assessment of wear between human cartilage surfaces. Verberne, G, et al. 2009, Wear, Vol. 266, pp. 1216-1223.
3. Grinstaff, M W and Wathier, M. Novel Hydrophilic polymers as medical lubricants and gels. US2009/0208589 A1 USA, 2009.
4. Muscles-and-bones.com. What is osteoarthritis? Muscles-and-bones.com. [Online] [Cited: November 15th, 2009.] http://www.muscles-and-bones.com/articles/about-osteoarthritis/index.php.
5. Tortora, G J and Grabowski, S R. Introduction to the human body: The essentials of anatomy and physiology. 6th Edition. s.l.: John Wiley & Sons, 2004.
6. Synovial Fluid Analysis. Brannan, S and Jerrard, D. 3, 2005, Journal of Emergency Medicine, Vol. 30, pp. 331-339.
7. Edwards, J. Normal joint structure. Notes on Rheumatology. [Online] 2000. [Cited: November 10, 2009.] http://www.ucl.ac.uk/~regfjxe/Studentinfo2.htm.
8. Role, Metabolism, Chemical Modifications and Applications of Hyaluronan. Volpi, N, et al. 2009, Current Medicinal Chemistry, pp. 1718-1745.
9. Rosen, J E, et al. Osteoarthritis pain management update. Institute of clinical care. [Online] 2009. http://www.clincare.org/PDF/BioRestorativeProfilingMonograph.pdf.
10. Anomalous viscosity behaviour in aqueous solutions of hyaluronic acid. Maleki, A, Kjoniksen, A L and Nystrom, B. 2007, Polymer Bulletin, Vol. 59, pp. 217-226.
11. Hyaluronic acid: the scientific and clinical evidence. Price, R D, Berry, M G and Navsaria, H A. 2007, Journal of Plastic, Reconstructive and Aesthetic Surgery , pp. 1110-1119.
12. The National Collaborating Centre for Chronic Conditions. Osteoarthritis: National clinical guideline for care and management in adults. London: Royal College of Physicians, 2008.
13. Mir, N A and Mustafa, A. Osteoarthritis and propects of total knee replacement surgery in Jammu and Kashmir state. MedIND. [Online] http://medind.nic.in/haa/t02/i1/haat02i1p80g.pdf.
14. Updated therapy in elderly patients with knee osteoarthritis. Chen, D Y. 1, 2007, International Journal of Gerontology, Vol. 1, pp. 31-39.
15. The role of viscosupplementation with Hygal G-F 20 (Synvisc) in the treatment of Osteoarthritis of the knee: a canadian multicenter trial comparing Hylan G-F 20 alone, Hylan G-F 20 with non steroidal anti-inflammatory drugs (NSAIDs) and NSAIDs alone. Adams, M E, et al. 1995, Osteoarthritis and Cartilage, Vol. 3, pp. 213-226.
16. Injectable corticosteroids in treatment of arthritis of the knee. Schumacher, H R and Chen, L X. 2005, The American Journal of Medicine, Vol. 118, pp. 1208-1214.
17. Weiss, C. Hyaluronan and hylan in the treatment of osteoarthritis. [book auth.] J F Kennedy, G O Phillips and P A Williams. Hyaluronan. Volume 2: Biomedical, medical and clinical aspects. s.l.: Woodhead Publishing Ltd, 2002.
18. Kuo, J W. Practical aspects of hyaluronan based medical products. s.l.: Taylor & Francis Group, 2006.
19. Nielsen, L K. Bio Engineering. University of Queensland. [Online] 2003. http://www.cheque.uq.edu.au/research/bioengineering/research/Metabolic_Engineering/HA.html.
20. Kwo, J W. Practical Aspects of Hyaluranon Based Medical Products. Boca Raton, FL: CRC Press, 2006.
21. Physical properties of hyaluronic acid and hydroxypropylmethylcellulose in solution: evaluation of coating ability. Silver, F H, et al. 1, 1993, Journal of Applied Biomaterials, Vol. 5, pp. 89-98.
22. Hyaluronic acid in synovial fluid. I. Molecular parameters of hyarulonic acid in normal and arthritic human fluids. Balazs, E A, et al. 4, 1967, Arthritis & Rheumatism, Vol. 10, pp. 357-376.
23. The role of viscosupplementation with hylan G-F 20 (Synvisc) in the treatment of osteoarthritis of the knee: a Canadian multicenter trial comparing hylan G-F 20 alone, hylan G-F 20 with non-steroidal anti-inflammatory drugs (NSAIDs) and NSAIDs alone. Adams, M E, et al. 1995, Osteoarthritis and Cartilage, Vol. 3, pp. 213-226.
24. Factors predicting long term efficacy of Hylan GF-20 viscosupplementation in knee osteoarthritis. Conrozier, T, et al. 70, 2003, Joint bone spine, pp. 128-133.
25. Novel hyaluronic acid-methotrexate conjugates for osteoarthritis treatment. Homma, A, et al. 2009, Bioorganic and Medicinal Chemistry, pp. 4647-4656.
26. Recombinant production of hyaluronic acid. H Brown, Stephen and E Pummill, Philip. 4, s.l.: Current pharmaceutical biotechnology, 2008, Vol. 9. ISSN: 1873-4316.
27. Comparative chemical evaluation of two commercially available derivatives of hyaluronic acid used for soft tissue augmentation. F, Mannaa, et al. 13, s.l.: Journal of the European Academy of Dermatology and Venereology, 1999.
28. Human Body Recon Co, Llc. Frequently Asked Questions about Hyaluronic Acid. Human Body Recon Co, Llc Website. [Online] 2005. [Cited: November 25, 2009.] http://www.humanbodyrecon.com/faqs.htm.
29. Alkayali, Ahmad. Preparation of low molecular weight hyaluronic acid as a food supplement. EP 1 707 578 A2 United States of America, October 4, 2006.
30. Microbial hyaluronic acid production. Chong, Barrie Fong, et al. 66, s.l.: Appl Microbiol Biotechnol, 2005.
31. Microbial Synthesis of Hyaluronan and Chitin: New Approaches. Takashi, Yamada and Kawasaki, Takeru. 6, Hiroshima: Journal of Bioscience and Bioengineering, 2005, Vol. 99.
32. The Humane Society of the United States. Scientists and Experts on Battery Cages and Laying Hen Welfare. Washington: The HSUS.
33. Fermentation process develoupment for hyaluronic acid production by Streptococcus Zooepidemicus. Shu-Jen Chen, Jia-Ling Chen, Wei-Chih Huang, Hsin-Liang Chen. Taiwan: Korean Journal of Chemical Engineers, 2009, Vol. 26.
34. Mechanism for the effect of agitation on the molecular weight of hyaluronic. Xu Zhang, Xu-Jie Duan, Wen-Song Tan. Shanghai: East China University of Science and Technology, 2010, Vol. 119.
35. Culture Conditions Affect the Molecular Weight Properties of Hyaluronic Acid Produced by Streptococcus Zooepidemicus. David C. Armstrong, Michael R. Johns. Queensland: American Society for Microbiology, 1997, Vol. 63.
36. The role of dissolved oxygen and function of agitation in hyaluronic acid fermentation . Wei-Chih Huanga, Shu-Jen Chenb and Teh-Liang Chen. Taiwan: Biochemical Engineering Journal, 2006, Vol. 32.
37. High production of hyaluronic and lactic acids by Streptococcus zooepidemicus in fed-batch culture using commercial and marine peptones from fishing by-products. Jos Antonio Vzqueza, Mara I. Montemayora, Javier Fraguasa, and Miguel Anxo Murado. Galicia: Biochemical Engineering Journal, 2008, Vol. 44.
38. Production of hyaluronic acid by repeated batch fermentation. Wei-Chih Huang, Shu-Jen Chen, Teh Liang Chen. Taiwan: Biochemical Engineering Journal , 2008, Vol. 40.
39. Efficacy of Hylan G-F 20 and Sodium Hyaluronate in the treatment of osteoarthritis of the knee A prospective randomized clinical trial. Raman, R, et al. 4, s.l.: Elsevier, 2008, The Knee, Vol. 15, pp. 318-324.
40. Hyaluronic acid injections for knee osteoarthritis. Aggarwal, A and Sempowski, I.P. 3, s.l.: Canadian Family Physician, 2004, Vol. 50, pp. 249-256.
41. Intra-articular hyaluronic acid products for osteoarthritis. Delbaere, M. 5, Ottawa: s.n., 2006, Pharmacy Practice, Vol. 22, pp. 31-45.
42. Private Healthcare UK. Private knee replacement costs in the UK and abroad. [Online] [Cited: November 20, 2009.] http://www.privatehealth.co.uk/hospitaltreatment/whatdoesitcost/knee-replacement/.
43. Reuters. Knee replacement surgery worth the price. Chicago: Reuters, 2009.
44. BBC. Knee op 'will cut hospital stays'. London: BBC, 2003.
45. Temporomandibular joint osteoarthritis: an open label trial of 76 patients treated with arthrocentesis plus hyaluronic acid injections. Manfredini, D, et al. 8, s.l.: Elsevier Ltd, 2009, International Association of Oral and Maxillofacial Surgeons , Vol. 38, pp. 827-834.
46. Intra-articular treatment of hip osteoarthritis: a randomized trial of hyaluronic acid, corticosteroid, and isotonic saline. Qvistgaard, E, et al. 2, Copenhagen: Elsevier, 2006, Osteoarthritis and Cartilage, Vol. 14.
47. Efficacy of intra-articular hyaluronic acid in patients with osteoarthritis of the ankle: a prospective study. Sun, S, et al. 9, Taipei: Elsevier , 2006, Osteoarthritis and Cartillage, Vol. 14, pp. 867-874.
48. Comparative chemical evaluation of two commercially available derivatives of hyaluronic acid used for soft tissue augmentation. Manna, F, Dentini, M and Desideri, P. 1999, European Academy of Dermatology and Venereology, pp. 183-192.
49. Hyaluronic Acid Production in Bacillus subtilis. Widner, B, et al. 2005, Applied and Environmental Microbiology, pp. 3747-3752.
50. High production of hyaluronic and lactic acids by Streptococcus zooepidemicus in fed-batch culture using commercial and marine peptones from fishing by-products. Vazquez, J A, et al. 2009, Biochemical Engineering Journal, pp. 125-130.
51. Cartilage lubricant synthesis and tissue. Zhu, Y A, et al.
52. Intra-articular hyaluronan treatment for osteoarthritis. George, E. s.l.: Annals of the Rheumatic Diseases, 1998, Vol. 57, pp. 637-640.
53. Microbial hyaluronic acid production. Chong, Barrie Fong, et al. 341-351, s.l.: Appl Microbiol Biotechnol , 2005, Vol. 66.
54. Prescott, Al. Method of purifying high molecular weight hyaluronic acid. US 6660853 B2 United States of America, December 9, 2003.
55. Modeling the microbial production of hyaluronic acid. Wei-Chih Huang, Shu-Jen Chen, Teh-Liang Chen. Taiwan: Journal of the Chinese Insituite of Chemical Engineers, 2007, Vol. 38.