ReengineeRing injectable

hyaluRonic acid filleRs:

the scienceMariano Busso, David Applebaum, Thomas Tzikas, Birgit Lundskov Fuhlendorff, and James Finney explore the use of Bacillus subtilis as a novel bacterial source of

hyaluronic acid for injectable soft tissue fillers used in aesthetic medicine

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ABSTRACT Aesthetic medicine is dedicated to satisfying the aesthetic goals of patients, while optimising outcomes and minimising adverse events. Soft tissue fillers are now the second most commonly performed minimally-invasive procedure in aesthetic medicine. Hyaluronic acid (HA) is the most abundant glycosaminoglycan in the human dermis, and it is the injectable biomaterial of choice for this use. Procedures using HA fillers are predicted to increase in frequency by 8–12% per year in North America alone1. A primary challenge for manufacturers of soft tissue fillers has been to obtain HA of high quality and purity. HA is currently derived from three sources: the rooster comb of male chickens, the bacterium Streptococcus equi subsp zooepidemicus, and — most recently — the bacterium Bacillus subtilis, first available in 2011. The B. subtilis-derived HA process allows for a high level of purity and a homogeneous end-product because it does not require the use of powerful organic solvents to extract it from the bacterial capsule, in contrast to the process required for Streptococcus-derived HA. Adverse events are generally injection-related and not serious or systemic. B. subtilis-derived HA is well placed in the market to complement existing sources of HA used in soft tissue fillers.

KeywoRdS aesthetic medicine, Bacillus subtilis, hyaluronic acid, Bacillus subtilis-derived fillers, soft tissue fillers, fillers, facial injectables

MARiAno BuSSo, Md, FAAd, Dermatologist, Coconut Grove, FL; dAvid AppleBAuM, Md, FACS, Plastic Surgeon, Boca Raton, FL; ThoMAS TziKAS, Md, Facial Plastic Surgeon, Delray Beach, Fl; BiRgiT lundSKov FuhlendoRFF, Head of Technical Service, Novozymes Biopharma DK A/S, Bagsvaerd, Denmark; JAMeS Finney, MSc, PGCert, Project Manager, Novozymes Biopharma UK Ltd, Nottingham, UK

email: drbusso@aol.com; biopharma@novozymes.com

outcomes. Health professionals practicing aesthetic medicine (dermatologists, plastics surgeons, and other aesthetic medicine providers) are trained in both invasive and non-invasive treatment modalities, and typically use a combination of both to meet the needs of the patient.

Biomaterials and their aesthetic applicationthe international Union of Pure and applied chemistry (iUPac) defines biomaterial as ‘material exploited in contact with living tissues, organisms or microorganisms’2. However, a clearer definition may be ‘any synthetic material that is used to replace or restore function to a body tissue and is continuously or intermittently in contact with body fluids’3.

there are many different types of materials (metals and alloys; ceramics and glasses; polymers; composites) that can be used in a variety of medical or dental settings2. biomaterials used within the field of minimally-invasive aesthetic medicine are typically injectable.

an ideal injectable biomaterial used as a facial soft tissue filler should be non-allergenic, non-carcinogenic, non-immunogenic, non-migratory, and non-teratogenic4, 5. an ideal injectable biomaterial should also be reversible, long-lasting but not permanent, and versatile in its application, and it should possess reproducible safety outcomes4, 5. Hyaluronic acid (Ha)

Aesthetic medicine is focused on satisfying the aesthetic desires and goals of patients. procedures in this area are generally elective and are dedicated to the dual goals of optimising patient outcomes and

minimising adverse effects.this rapidly emerging specialty is primarily focused

on the pathophysiology and mechanobiology of facial ageing and adheres to science and evidence-based

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The expanding use of soft tissue fillers in aesthetic medicinesoft tissue fillers are now the second most commonly performed minimally-invasive procedure behind botulinum toxin injections9, 10. according to the american society of Plastic surgery (asPs), approximately 2 million procedures were performed using soft tissue fillers in 2012, and the facial injectable market is estimated to be worth $1 billion in 20131, 11. Procedures using Ha soft tissue fillers are predicted to increase in frequency by 8–12% per year in north america alone1, 11. Drivers for this growth include greater awareness and acceptance of aesthetic medicine, improved accessibility to practitioners in the field, an ageing population, and the opportunity for individuals to increase their general wellbeing.

Sources of HA soft tissue fillers Ha soft tissue fillers are currently derived from three sources. the first source, the rooster comb of male chickens, is now largely an historic source; Ha derived from this method is currently used in only a small number of Us proprietary products designed for non-aesthetic use6, 12.

most Ha used in aesthetic medicine today is derived from the bacterium Streptococcus equi subsp zooepidemicus (of various strains)5, 12. Streptococcus-derived Ha is well established in the worldwide aesthetic market, having been used for over two decades with catalogued efficacy and safety data. even so, S. equi is considered a

pathogen in horses, and there are a number of potential disadvantages of this source, such as

trace residual endotoxin and lack of uniformity in Ha molecular weight and strand length12, 13.

For example, restylane® (manufactured by Q-med, a Galderma division, and

distributed in the Us by medicis, a division of Valeant Pharmaceuticals) is a Streptococcus-derived Ha product, which was initially associated with a hypersensitivity reaction frequency of 0.8% pre-200014. However, after improvements in the manufacturing

process that led to a raw product containing less protein, this incidence

decreased to 0.6% post-200014. to that end, alternative production sources have been

explored to negate these potential disadvantages12.

the newest source of Ha is from the bacterium Bacillus subtilis, first available in 2011 as Hyasis®

(manufactured by novozymes biopharma DK a/s., bagsvaerd, Denmark)5, 12. (currently, enhancement

medical, llc, Wauwatosa, Wi is manufacturing B. subtilis-derived Ha injectable gel under the trade name expression. as of january 2014, expression has a Food and Drug administration (FDa) indication for use as an intranasal splint, but it is used off-label in aesthetic medicine with no official aesthetic indication.)

demonstrates all of these beneficial attributes; therefore, it is currently the biomaterial of choice in aesthetic medicine.

The link: HA and aesthetic medicineHa, a complex sugar, is the most abundant glycosaminoglycan in the human dermis5. approximately half of the 14–16 g of Ha in the human body is found in the cell surface and extracellular matrix of the skin6. it is the major component of the vitreous humour in the eye (0.1 mg/ml), and large concentrations are found in the cartilage and synovial fluid of the joints.

Ha can be considered to have both biological and mechanical functions in the human body6. biologically, it regulates cell proliferation and migration, as well as angiogenesis5. in a mechanical capacity, Ha maintains volume by drawing water into the skin and other structures; it also cushions, protects, and supports by binding collagen and elastin fibres5. aesthetic medicine uses the mechanical properties of Ha with the aim of restoring this function that may degenerate over time. Ha-based soft tissue fillers are typically used in the face in such areas outlined in Table 1. Other, less common areas of treatment include reshaping the nose, recontouring the forehead, and revolumising earlobes. in addition, Ha fillers have been used to rejuvenate the hands and the décolletage8.

Table 1 Typical areas of use for HA fillers in aesthetic medicine5, 7

SeCTion of fACe AreA

Upper face n Eyebrows n Forehead lines n Forehead recontouring n Glabellar lines n Periocular areas n Temple lipoatrophy

Mid face n Earlobes n Medial/lateral malar cheek augmentation n Nasojugal groove augmentation n Nasolabial folds n Tear trough

Lower face n Lip and perioral area (restoration or augmentation) n Oral commissures n Marionette lines (corners of the mouth) n Mentalis crease of chin n Jawline or prejowl sulcus areas

Other areas n Décolletage n Hands

figure 1 Bacillus subtilis morphology

the Bacillus subtilis-derived hA offers a number of

bioprocessing advantages over Streptococcus-derived hA.

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Figure 2 Diagram comparing Streptococcus and Bacillus hyaluronic acid fermentation

the B. subtilis-derived Ha offers a number of bioprocessing advantages over Streptococcus-derived Ha. in the Streptococcus-based process, Ha is produced by the cells and surrounds the bacterial capsule. in order to liberate Ha, the cells have to be physically disrupted: sonicated or homogenised, depending on the methodology. this results in a lack of uniformity in the resulting Ha, so there is a wider range of molecular weights and chain lengths. When compared with the Bacillus process, which secretes Ha, there is a narrower range of molecular weights (~850 mDa) and chain length, hence more uniformity in the final product.

Furthermore, in contrast to Streptococcus, which requires use of organic solvents to separate or extract Ha from the bacterium cell surface, B. subtilis-derived Ha is secreted directly into the medium from the host bacterium5. as such, the Ha from B. subtilis can be separated from the host cell without the use of organic solvents. in order to physically separate the Ha in the Streptococcus-derived process, organic solvents have to be used. in most of the methodologies reviewed, this requires a large volume of solvent. in the Bacillus-derived Ha process, the elimination of the organic solvents reduced the costs and also increased the sustainability of the process because it is a 100% water-based process. the organic solvents are recovered in the Streptococcus process, but the solvents can affect the structure of the Ha molecules. this can affect the bioprocessing of the final end product and steps must be taken to adjust for the presence of solvent, which could affect cross-linking and other properties. Whether this has a true effect in clinical translation is not known and will have to be further elucidated. therefore, by eliminating organic solvents in the Bacillus-process, a ‘cleaner’ end product is the result.

there are a number of other source organisms (e.g. Agrobacterium sp, Escherichia coli, and Lactococcus lactis) from which Ha can be derived, but many of these are restricted to laboratory-based primary research12. currently, B. subtilis and S. equi are the only two organisms in use on a commercial scale12.

Bacillus subtilis: a microbial mini factory?B. subtilis, first identified in 1835, was one of the first bacteria ever studied in microbiology (Figure 1). it is found naturally in soil, but also resides in the digestive tract of some animals15, 16. it is one of the most well characterised bacterial organisms in nature from a biotechnological perspective. the literature has evaluated its probiotic activity against the common digestive pathogens Helicobacter pylori and enterobacteriaceae, which illustrate the varying dynamics of this organism16. Widely used in science and technology and having been granted ‘Generally recognized as safe’ (Gras) status by the FDa, it is seen as an ideal host for production of Ha6,  17.

the fermentation process using B. subtilis offers inherent bioprocessing advantages over Streptococcus-derived Ha (Figure 2). B. subtilis-derived Ha is expressed from the cell into the fermentation environment; it is then separated without the use of organic solvents and spray-dried, making it a completely 100% water-based process. in contrast, in the Streptococcus processing model, the Ha grows and surrounds the bacterial capsule and must be extracted using organic solvents (2-propanol and sodium acetate) to disrupt the cells to liberate Ha6. this creates a number of processing difficulties, including a risk for the inclusion of trace bacterial endotoxins, cellular debris, and solvents when the Ha is extracted, which limits its application in the biomedical field12. in contrast, B. subtilis-derived Ha does not produce endotoxins6. in addition, the Bacillus model produces more homogenous strands of Ha as compared with Streptococcus-derived Ha.

Bioengineering injectable biomaterials for soft tissue fillers most of the currently available Ha injectable biomaterials used in soft tissue fillers consist of particles (a solid phase) suspended in a fluid phase18. the physicochemical structure of this soft tissue filler is established during the manufacturing process by the adjustment of a number of variables including, but not limited to:

n concentration of the solid-phase particles n method and percentage of cross-linking of the

solid-phase particles n type and technology of cross-linker used n Proportion of gel in the fluid phase (gel-to-fluid ratio). Ha soft tissue fillers with different physicochemical

properties produce different clinical outcomes with regard to their rheology — elasticity and viscosity18. One important rheological property of a soft tissue filler gel

B. subtilis-derived hA is

expressed from the cell into the

fermentation environment; it is

then separated without the use of

organic solvents and spray-dried,

making it a completely 100%

water-based process.e

Bacillus fermentation 100% water based process

Bacillus subtilis

hA secreted into medium

gentle physical separation

Steptococcus fermentation Organic solvent based process

Streptococcus equi subspecies

hA produced and surrounds the bacterial capsule

Cellular disruption to liberate hA

Organic solvent precipitation to recover HA

Risk of residual endotoxin

less homogenous hA strands More homogenous hA strands

No use of organic solvents to recover HA

B. subtilis strain does not produce endotoxin

HAproduction

recovery and

purification

resulting HA macromolecules

Microbialstrain

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that can be quantified is its elastic modulus (G’). a high G’ in a Ha soft tissue filler appears to be a predictor of a better resistance to skin tension forces; therefore, it deforms less under pressure18.

the rheological properties can be used as a scientific rationale in choosing a soft tissue filler — a strategy known as rheological tailoring18. this can be individualised for each patient and facial area to achieve the desired goals and outcomes. to that end, viscosity and elasticity should be part of the selection process when choosing an appropriate soft tissue filler. Other clinical considerations, such as injection technique and implantation depth of soft tissue filler, are also important18. the addition of B. subtilis-derived Ha to aesthetic medicine practitioners’ armamentarium can increase their ability to achieve customised, evidence-based outcomes from a rheological perspective.

Potential complications associated with injectable HA biomaterialsas with every medical procedure, there is a degree of risk associated with the use of injectable Ha soft tissue fillers, although serious complications arising from their use are infrequent19. Unwanted adverse events do occur with all soft tissue filling compounds (including Ha biomaterials); however, their true prevalence is unknown4, 20–22. these adverse events may be injection-related, technique-related, or (rarely) may be owing to localised exposure to Ha itself, potential residual purification solvents, or trace presence of endotoxin. injection-related events are those that are caused by the injection of the soft tissue filler rather than the soft tissue filler itself, while technique-related events are the result of the specific manner in which a physician injects the substance into the patient.

injection-related adverse eventsby far the most common adverse events associated with Ha soft tissue fillers are injection-related19. these events are usually transient, resolving within 4–7 days, and are localised to the site of injection (Table 2). rarely, an inadvertent intravascular injection or adjacent vascular compression may result in a non-localised adverse event (i.e. necrosis)19, 23.

Technique-related adverse eventsOne of the most common technique-related adverse events is inappropriate placement of the soft tissue filler. lumps of visual product or bluish bumps under the skin (the tyndall effect) can result from a too superficial placement of product24, 25. For the most part, such reactions can be prevented by the use of correct injection technique and proper training on the part of the injector19. the occurrence of these events can lead to anxiety, dissatisfaction, and less than optimal results for the patient24, 25. the advantage of Ha-based soft tissue fillers over other classes (e.g. Poly-l-lactic acid, calcium

n Swelling n Hyperaemia/erytheman Local oedema n Pain/tendernessn Contusions (bruising) n Pruritis (itching)

Table 2 Injection-related adverse events associated with HA-based fillers19, 23

As with every medical procedure, there is a degree of risk associated with the use of injectable hA soft tissue fillers, although serious complications arising from their use are infrequent

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references1. Millennium research Group. facial injectable regional Markets. http://tinyurl.com/oontfaf (accessed 25 february 2014)2. Vert M, Doi Y, Hellwich K-H et al. Terminology for biorelated polymers and applications (iUPAC recommendations 2012). Pure Appl Chem 2012; 84(2): 377–4103. Davis Jr. ed, Handbook of Materials for Medical Devices. Materials Park, oH: ASM international, 20034. Alijotas-reig J, fernández-figueras MT, Puig L. Late-onset inflammatory adverse reactions related to soft tissue filler injections. Clin rev Allergy immunol 2013; 45(1): 97–1085. Brandt fS, Cazzaniga A. Hyaluronic acid gel fillers in the management of facial aging. Clin interv Aging 2008; 3(1): 153–96. Schiraldi C, La Gatta A, De rosa M. Biotechnological Production and Application of Hyaluronan. http://tinyurl.com/q3rc34y (accessed 25 february 2014)7. Muhn C, rosen n, Solish n et al. The evolving role of hyaluronic acid fillers for facial volume restoration and contouring: a Canadian overview. Clin Cosmet investig Dermatol 2012; 5: 147–588. Streker M, reuther T, Krueger n, Kerscher M. Stabilized hyaluronic acid-based gel of non-animal origin for skin rejuvenation: face, hand, and décolletage. J Drugs Dermatol 2013; 12(9): 990–49. ozturk Cn, Li Y, Tung r, Parker L, Piliang MP, Zins Je. Complications following injection of soft-tissue fillers. Aethet Surg J 2013; 33(6): 862–77

10. American Society for Aesthetic Plastic Surgery. Statistics 2012. www.surgery.org/media/statistics (accessed 25 february 2014)11. American Society of Plastic Surgeons. 14.6 Million Cosmetic Plastic Surgery Procedures Performed in 2012. http://tinyurl.com/ovcx5v5 (accessed 25 february 2014)12. Liu L, Liu Y, Li J, Du G, Chen J. Microbial production of hyaluronic acid: current state, challenges, and perspectives. Microb Cell fact 2011; 10: 99 13. nCBi. Streptococcus equi. http://tinyurl.com/p4y9ftp (accessed 25 february 2014) 14. André P. evaluation of the safety of a non-animal stabilized hyaluronic acid (nASHA -- Q-Medical, Sweden) in european countries: a retrospective study from 1997 to 2001. J eur Acad Dermatol Venereal 2004; 18(4): 422–515. nCBi. Bacillus subtilis. http://tinyurl.com/pqa8rln (accessed 25 february 2014)16. Pinchuk iV, Bressollier P, Veneuil B et al. in vitro anti-Helicobacter pylori activity of the probiotic strain Bacillus subtilis 3 is due to secretion of antibiotics. Antimicrob Agents Chemother 2001; 45(11): 3156–6117. US food and Drug Administration. Microorganisms & Microbial-Derived ingredients Used in food (Partial List). http://tinyurl.com/nqmu5r7 (accessed 25 february 2014)18. Sundaram H. Going With The flow: An overview of Soft Tissue filler rheology and its Potential Clinical Applications, Part i. http://tinyurl.com/o868zok (accessed 25 february 2014)

19. Cohen JL. Understanding, avoiding, and managing dermal filler complications. Dermatol Surg 2008; 34(Suppl 1): S92–920. Hirsch r, Stier M. Complications of soft tissue augmentation. J Drugs Dermatol 2008; 7(9): 841–521. Descamps V, Landry J, francès C, Marinho e, ratziu V, Chosidow o. facial cosmetic filler injections as possible target for systemic sarcoidosis in patients treated with interferon for chronic hepatitis C: two cases. Dermatology 2008; 217(1): 81–422. Alijotas-reig J, Garcia-Gimenez V. Delayed immune-mediated adverse effects related to hyaluronic acid and acrylic hydrogel dermal fillers: clinical findings, long-term follow-up and review of the literature. J eur Acad Dermatol Venereol 2008; 22(2): 150–6123. Gilbert e, Hui A, Meehan S, Waldorf HA. The basic science of dermal fillers: past and present Part ii: adverse effects. J Drugs Dermatol 2012; 11(9): 1069–7724. narins rS, Jewell M, rubin M, Cohen J, Strobos J. Clinical conference: management of rare events following dermal fillers -- focal necrosis and angry red bumps. Dermatol Surg 2006; 32(3): 426–3425. Brody HJ. Use of hyaluronidase in the treatment of granulomatous hyaluronic acid reactions or unwanted hyaluronic acid misplacement. Dermatol Surg 2005; 31(8 pt 1): 893–726. Dougherty AL, rashid rM, Bangert CA. Angioedema-type swelling and herpes simplex

virus reactivation following hyaluronic acid injection for lip augmentation. J Am Acad Dermatol 2011; 65(1): e21–227. He MS, Sheu MM, Huang ZL, Tsai CH, Tsai rK. Sudden bilateral vision loss and brain infarction following cosmetic hyaluronic acid injection. JAMA ophthalmol 2013; 131(9): 1234–528. Chader H, Bose r, Hersant B et al. infectious cellulitis of the face complicating injection for aesthetic nasolabial sulcus by hyaluronic acid: about seven cases. Ann Chir Plast esthet 2013; 58(6): 680–329. Kwon SG, Hong JW, roh TS, Kim YS, rah DK, Kim SS. ischemic oculomotor nerve palsy and skin necrosis caused by vascular embolization after hyaluronic acid filler injection: a case report. Ann Plast Surg 2013; 71(4): 333–430. Alijotas-reig J. recurrent urticarial vasculitis related to nonanimal hyaluronic acid skin filler injecction. Dermatol Surg 2009; 35(Suppl 1): 395–731. McGuire LK, Hale eK, Godwin LS. Post-filler vascular occlusion: a cautionary tale and emphasis for early intervention. J Drugs Dermatol 2013; 12(10): 1181–332. Glaich AS, Cohen JL, Goldberg LH. injection necrosis of the glabella: protocol for prevention and treatment after use of dermal fillers. Dermatol Surg 2006; 32(2): 276–8133. Alam M, Gladstone H, Kramer eM et al. ASDS guidelines of care: injectable fillers. Dermatol Surg 2008; 34(Suppl 1): S115–48

hydroxylapatite) is their reversibility when treated with hyaluronidase, which can successfully resolve many unwanted adverse events20, 25.

Serious adverse events related to HAinjectable Ha soft tissue fillers are typically well tolerated with few adverse events. serious events are possible, although rare. typically these events have not been associated with aesthetic use of Ha products.

Ha-derived injectable soft tissue fillers may rarely be associated with localised reactions, such as persistent swelling, pain, and nodule formation. these effects may require physical removal and/or enzymatic degradation with hyaluronidase4, 19. Other rare effects include angioedema, arterial occlusion, loss of vision, infection, necrosis, vasculitis and vascular occlusion23, 26–31. Practitioners should be able to recognise these adverse events and apply the appropriate clinical algorithm for treatment should they occur9, 31–33.

Conclusionsa primary challenge for manufacturers of soft tissue fillers has been obtaining Ha of high quality and purity. contrary to Streptococcus-derived Ha, B. subtilis-derived Ha is produced by a host, free of endotoxin and without organic solvents.

the B. subtilis-derived Ha process overcomes the manufacturing and safety challenges associated with Ha of other origins, allowing for a high level of purity and a homogeneous end-product. the bioengineering process associated with production of B. subtilis-derived Ha does not require the use of powerful organic solvents to extract it from the bacterial capsule, in contrast to the process required for Streptococcus-derived Ha. in addition, B. subtilis-derived Ha results in uniform strands and has a homogenous molecular weight with a narrow and well-

Most hyaluronic acid (HA) used in aesthetic medicine today is derived from Streptococcus sp

The newest source of HA is from Bacillus subtilis

A primary challenge for manufacturers of soft tissue fillers has been to obtain HA of high quality and purity

The B. subtilis-derived HA process allows for a high level of purity and a homogeneous end-product

B. subtilis-derived HA is well placed in the market to complement existing sources of HA used in soft tissue fillers

Key points defined weight distribution. in summary, B. subtilis-derived Ha is well placed in the market to complement existing sources of Ha used in soft tissue fillers.

Declarations of interest Dr Mariano Busso is a consultant for Merz Aesthetics; Dr David Applebaum is a consultant and speaker for Valeant Pharmaceuticals; Dr Thomas L. Tzikas is a shareholder of Enhancement Medical; Birgit Lundskov Fuhlendorff is an employee of Novozymes Biopharma DK A/S; and James Finney is an employee of Novozymes Biopharma UK Ltd Figures 1 © Sebastian Kaulitzki; 2 Original chart data courtesy of Novozymes Biopharma UK Ltd, Nottingham, UK, reproduced by Prime Journal

A primary challenge for

manufacturers of soft tissue fillers

has been obtaining hA of high quality

and purity.

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