SURGERY

Field of invention

The present invention relates to biomaterials in the form of gel or fibres, comprising gellan or derivatives thereof, alone or in association with hyaluronic acid or derivatives thereof, for use as cellular fillers in the surgical field.

Prior art

A wide range of materials, such as membranes, sponges, gauzes (or meshes) and gels, are used in surgery.

The material to be used is selected on the basis of the type of surgery to be performed.

Different materials with variable characteristics, such as adherence, elasticity, biocompatibility and biodegradability, are therefore necessary.

In some cases, the material must be biocompatible but not biodegradable, in which case a second surgical operation is required to remove the material (for example in urogynaecological and ENT surgery). In other cases, the material must be both biocompatible and biodegradable, in particular in dermocosmetic surgery.

The first filler to be used, in the Seventies, was silicone oil, which is now prohibited because of its serious side effects.

The use of bovine collagen, introduced in the late Seventies, gave very good results but also triggered some allergic reactions.

There are numerous materials currently on the market, in the form of membranes (GORE® Medical Membranes); sponges (Valentine R. et al., Otolaryngol Clin North Am; 2009 Oct; 42(5):813-28); gauzes or meshes (Markar Mrcs S.R. et al., Surg Laparosc Endosc Percutan Tech; 2010 Aug; 20(4):213-9); and gels ( adiesse®; Jacovella P.F., Clin Interv Aging; 2008; 3(1): 161-74), for specific use in dermocosmetic, urogynaecological and ENT surgery, which have variable characteristics depending on the type of compound of which they are made.

Meshes of non-resorbable derivatives based on polyesters

(MERSILENE), polyamides and polypropylene (Dolphin mesh) are used in gynaecological surgery, and in particular as materials for the prevention of urinary incontinence (AMS SPARC™, AMS Monarc™, AMS BioArc™ SP and AMS BioArc™ TO); sponges made of partly resorbable derivatives are used in ENT surgery (Meropack, Nasopore); and gels or membranes based on modified/crosslinked hyaluronic acid, either alone or combined with other polymers, are mainly used in dermocosmetic surgery (Hydrelle™, Juvederm®, Perlane® and Restylene®).

A variety of complications can arise following surgery due to the use of the various known materials, often correlated with the nature of the material used or inflammatory reactions caused by lengthy residence of implanted material in situ (Altman D. et al., Obstet Gynecol Surv. 2005 Nov; 60(l l):753-60).

In particular, complications can arise with materials which are biocompatible but not biodegradable, or during the degradation of bioabsorbable materials that release inflammatory substances, and the formation of such substances is still one of the most serious complications of numerous surgical procedures (Tatakis D.N. and Trombelli L., J. Periodontal.; 1999 May; 70(5):542-7).

Although clinical trials designed to evaluate the efficacy of these materials have given strongly conflicting results, there is no doubt that the said materials are associated with substantial contraindications.

For example, the use of membranes (made of PTFE or polypropylene) as an alternative to gels involves the implant of a synthetic material foreign to the human body, which is not biodegradable and may require a second surgical operation to remove the material, which causes adverse reactions such as inflammation or, as in the case of nasal packing in ENT surgery, often causes pain and bleeding.

WO 2006/037592 describes formulations based on gellan, used alone or in combination with hyaluronic acid derivatives, in particular with sulphated hyaluronic acid, for use in the prevention of surgical adhesions, in particular in the prevention of spinal adhesions.

However, there is still a need to identify new biomaterials which can be used as fillers in the surgical field.

Summary of the invention

The present invention relates to biomaterials comprising gellan or derivatives thereof, either alone or in combination with hyaluronic acid or derivatives thereof, for use as fillers in surgery.

Detailed description of the invention

The present invention relates to the use of biomaterials, comprising gellan or derivatives thereof, either alone or in combination with hyaluronic acid (HA) or derivatives thereof, as fillers in the surgical field.

Said biomaterials are characterised by high biocompatibility and are completely biodegradable; they are therefore suitable for a variety of uses in surgery, and a second surgical operation is not required to remove them from the sites of application.

The biomaterials can preferably be in the form of gels or fibres.

Gellan is a linear anionic heteropolysaccharide with a repeating unit consisting of glucose-glucuronic acid-glucose-rhamnose.

A gellan derivative which can preferably be used is deacetylated gellan.

It has been found that gellan is an inert material which can be advantageously used as a filler.

Gellan does not perform an active biological role, and is characterised by lengthy residence at the site of application and high viscosity.

Gellan presents biocompatibility, prolonged residence at the site of application, a degree of bioadhesion and a consistency which allow it to be used as an injectable gel or fibre in biomedical applications.

Gellan, or derivatives thereof, is preferably pyrogen-free; in other words it is obtained by following a particular purification procedure which leads to a product with endotoxin content limits of less than 24 EU per millilitre of gel.

Gellan or deacetylated gellan is preferably freeze-dried and soluble up to concentrations of 20-30 mg/ml in saline solutions at 85-90°C.

The hyaluronic acid used in the present invention can derive from any source; for example, it can be obtained by extraction from rooster combs (EP 0138572 B l), by fermentation (EP 0716688 B l) or by a technological process.

Hyaluronic acid can have a molecular weight of between 400 and 3x10 ^~6 Da, in particular between 10,000 and lxlO ^"6 Da, and more particularly between 100,000 and 250,000 Da.

According to a preferred embodiment, hyaluronic acid is functionalised with sulphate groups, i.e. is sulphated hyaluronic acid (HA-S).

The process of sulphation of hyaluronic acid and the derivatives thereof can take place as known to the skilled person, but is preferably performed as described in patent EP0702699 B I.

The HA derivatives which can be used to undergo the sulphation process are listed below:

1. Salified HA with organic and/or inorganic bases having a molecular weight of 50-730 KDa (EP0138572 B l) or a high molecular weight of 750- 1230 KDa (EP 535200 Bl); preferably having a molecular weight of between 100 and 250 KDa;

2. Hyaff®: HA esters with alcohols of the aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic series (EP 216453 B l); the percentage esterification of hyaluronic acid subsequently subjected to the sulphation process ranges between 5 and 65%, depending on the type and length of the alcohol used, because the product obtained must be soluble in water;

3. ACP®: inner HA esters (EP 0341745 B l); the percentage esterification of hyaluronic acid subsequently subjected to the sulphation process ranges from 1 to 15%, because the product obtained must be soluble in water;

4. Hyoxx™: percarboxylated derivatives of HA obtained by oxidation of the primary hydroxyl of the N-acetylglucosamine fraction (EP1339753); the percentage percarboxylation of hyaluronic acid subsequently subjected to the sulphation process ranges from 1 to 50%.

All the HA-free carboxyl groups can be salified with organic and/or inorganic bases.

The degree of sulphation of hyaluronic acid and/or of the derivatives thereof listed above, measured as the number of sulphate groups per repeating unit, can range from 0.5 to 3.5, and is preferably 3.

Its solubility in water or saline solutions is directly proportional to the degree of sulphation of hyaluronic acid.

Hyaluronic acid is normally characterised by very rapid resorption times, whereas it is not processable when it is incorporated in the biomaterials according to the invention. The association of hyaluronic acid or derivatives thereof with gellan or derivatives thereof therefore allows it to be administered during surgery.

Biomaterials containing gellan or derivatives thereof and hyaluronic acid or derivatives thereof can be used as fillers, as they also have biological properties conferred by hyaluronic acid or derivatives thereof. Said biomaterials can be used as biorevitalising fillers, for example in the dermocosmetic field.

According to a preferred embodiment, the biomaterial consists of a mixture comprising deacetylated gellan and sulphated hyaluronic acid (HA-S).

Sulphated hyaluronic acid (HA-S) possesses anti-inflammatory and/or antimicrobial properties. These properties are advantageous in a filler used in cosmetic, urogynaecological or ENT surgery, because they moderate the onset of inflammatory stress due to the conditions in the original tissue and the stress induced by surgery.

According to a preferred embodiment, the biomaterials comprise freeze-dried, pyrogen-free gellan or deacetylated gellan and grade 3 sulphated hyaluronic acid.

The biomaterials preferably only consist of gellan or deacetylated gellan at the maximum concentration of 30 mg/ml (3% solution) or of a mixture comprising gellan or deacetylated gellan with a concentration ranging from 18 to 29 mg/ml and sulphated hyaluronic acid with a concentration ranging from 1 to 12 mg/ml; even more preferably, the biomaterial comprises gellan or deacetylated gellan with a concentration of 20 mg/ml and sulphated hyaluronic acid with a concentration of 10 mg/ml.

The weight ratio between gellan or derivatives thereof and hyaluronic acid or derivatives thereof, preferably sulphated hyaluronic acid, can range between the ratios 1.5: 1 , 2: 1 and 2: 1.5; the weight ratio 2: 1 is preferable.

The biomaterials have similar characteristics to native hyaluronic acid, but with residence times suitable to perform the various functions required, according to the type of surgery in which they are used.

The biomaterial based on deacetylated gellan alone or combined with sulphated hyaluronic acid presents a long residence in situ together with a very high tolerability profile, demonstrated by the results of the biocompatibility tests according to ISO 10993 conducted on the central nervous system. During said tests, it was demonstrated that a biomaterial based on deacetylated gellan and sulphated hyaluronic acid according to the present invention does not induce sensitisation reactions after application. The biomaterial also presents a residence time after application exceeding two years.

Extrusion tests conducted on the biomaterials based on deacetylated gellan and sulphated hyaluronic acid according to the present invention confirm their properties of manageability, easy injection and comfortable positioning which meet the needs of the doctor performing the operation.

The biomaterials according to the present invention can be used as acellular fillers in surgery, in particular in dermocosmetic surgery, urogynaecological surgery and ENT surgery, for example to increase the physiological tissue for biofunctional or cosmetic purposes.

As fillers in the urogynaecological field they can be used to solve problems caused by urinary incontinence due to their characteristics of residence time and injectability.

In urogynaecology, the primary indication of the fillers is ISD (intrinsic sphincter deficiency), but they are also used in cases of urinary incontinence caused by hypermobility of the bladder. In this case the action of the filler is to improve the sphincter function. The characteristics of the ideal filler include, as well as the tolerability profile, ease of injection, maintenance of volume over time and persistence at the site of injection, despite constant stresses due to movement, for at least 2 years.

The materials according to the present invention meet these requirements.

In the ENT field, the biomaterials according to the present invention can be used in phonosurgery, in vocal fold surgery, for example to restore the vibratile activities of the vocal folds by medialisation of cords affected by paresis, or in rhinosurgery, when a barrier between the paranasal sinuses is required.

In the case of applications in the field of vocal fold surgery, the ideal filler must be biocompatible, not immunogenic, injectable through needles with dimensions compatible with common surgical practice, have an adequate residence time and possess viscoelastic properties similar to the physiological properties of the vocal folds so as to guarantee biofunctional reinstatement.

In dermocosmetic surgery, for example, biomaterials can be used as fillers, injected into the skin of the face through a very fine needle, to fill wrinkles, folds or depressions or increase the volume of the lips, chin and cheekbones.

The biomaterials according to the present invention, comprising gellan or deacetylated gellan and hyaluronic acid and/or derivatives thereof, can be advantageously obtained, for example, by simple dry mechanical mixing, with no need for the formation of new chemical bonds between the ingredients to obtain derivatives of various kinds, such as crosslinked substances, esters, ethers, starches, ion complexes, etc.

The examples given below further illustrate the invention.

Examples

Preparation example 1

Preparation of the biomaterial in the form of a hydrogel consisting of gellan in combination with sulphated HA in 2:1 weight ratio

The HA is sulphated according to EP 0702699 Bl with a degree of sulphation of 3.

A solution of 20 mg/ml of deacetylated gellan (Gelrite®) is prepared by heating (75-85°C) and dissolving 1 g of deacetylated gellan in 50 ml of NaCl, 0.9%. Once solubilisation is complete, 500 mg of sulphated HA is added and left to dissolve completely. The mixture is then cooled to room temperature until a hydrogel is obtained which can then be steam-sterilised.

Preparation example 2

Preparation of the biomaterial in the form of a hydrogel consisting of gellan in combination with sulphated HA, 1.5:1 weight ratio

Proceed as for Example 1 , dissolving 750 mg of deacetylated gellan and 500 mg of sulphated HA.

Preparation example 3

Preparation of the biomaterial in the form of a hydrogel consisting of gellan in combination with sulphated HA benzyl ester, with 25% esterification, 2:1 weight ratio

The deacetylated gellan solution is prepared as described in Example 1. 500 mg of sulphated HA benzyl ester is then added and left till solubilisation is complete. It is then left to cool to room temperature giving a hydrogel that can then be steam-sterilised.

Preparation example 4

Preparation of a fibre consisting of a mixture of gellan (98%) and HYAFF ^® -11 p75 (2%)

9.8 grams of Kelcogel ^® CG-LA deacetylated gellan is mixed dry with

0.2 grams of powdered HYAFF ^® -1 1 p75.

The mixture of powders is placed in a mixer, and 125 ml of water is added slowly. A mixture with a concentration of 80 mg/ml is obtained.

The mixture is transferred to a screw extruder connected to a 60 μ die. The temperature of the extrusion chamber is set to 54°C, and the material is extruded.

A fibre with an average diameter of 25-30 μ is obtained. Preparation example 5

Preparation of a fibre consisting of a mixture of gellan (95%) and HYAFF ^® -11 p50 (5%)

9.5 grams of Kelcogel ^® CG-LA deacetylated gellan is mixed dry with 0.5 grams of powdered HYAFF ^® -1 1 p50.

The mixture of powders is placed in a mixer, and 1 10 ml of water is added slowly. A mixture with a final concentration of approx. 90 mg/ml is obtained. The mixture is transferred to a screw extruder connected to a 40 μ die. The temperature of the extrusion chamber is set to 54°C, and the material is extruded.

A fibre with an average diameter of 15-25 μ is obtained.

Preparation example 6

Preparation of a fibre consisting of a mixture of gellan (90%) and sulphated HA with sulphation grade 1 (10%)

9 grams of Kelcogel ^® CG-LA deacetylated gellan is mixed dry with 1 gram of freeze-dried sulphated HA with sulphation grade 1.

The mixture of the two polymers is placed in a mixer, and 100 ml of water is added slowly. A mixture with a final concentration of 100 mg/ml is obtained. The mixture is transferred to a screw extruder connected to a 150 μ die. The temperature of the extrusion chamber is set to 54°C, and the material is extruded.

A fibre with an average diameter of 50-70 μ is obtained.

Preparation example 7

Preparation of a fibre consisting of a mixture of gellan (95%) and HYADD™ (5%)

9.5 grams of Kelcogel ^® CG-LA deacetylated gellan is mixed dry with 0.5 grams of powdered HYADD™-4 (hexadecyl amide with 2% amidation).

The mixture of the two polymers is placed in a mixer, and 125 ml of water is added slowly. A mixture with a final concentration of 80 mg/ml is obtained. The mixture is transferred to a screw extruder connected to a 60 μ die. The temperature of the extrusion chamber is set to 54°C, and the material is extruded.

A fibre with an average diameter of 25-30 μ is obtained.

Preparation example 8

Preparation of a fibre consisting of a mixture of gellan (95%) and ACP ^® (5%)

9.5 grams of Kelcogel ^® CG-LA deacetylated gellan is mixed dry with 0.5 grams of powdered ACP ^® (autocrosslinked hyaluronic acid).

The mixture of the two polymers is placed in a mixer, and 1 10 ml of water is added slowly. A mixture with a final concentration of approx. 90 mg/ml is obtained. The mixture is transferred to a screw extruder connected to a 100 μ die. The temperature of the extrusion chamber is set to 50°C, and the material is extruded.

A fibre with an average diameter of 30-50 μ is obtained.

Preparation example 9

Preparation of a fibre consisting of a mixture of gellan (98%) and HYAFF ^® -11 (2%)

9.8 grams of Kelcogel ^® CG-LA deacetylated gellan is mixed dry with 0.2 grams of powdered HYAFF ^® - 1 1.

The mixture of powders is placed in a mixer, and 125 ml of water is added slowly. A mixture with a concentration of 80 mg/ml is obtained. The mixture is transferred to a screw extruder connected to a 60 μ die. The temperature of the extrusion chamber is set to 54°C, and the material is extruded.

A fibre with an average diameter of 25-30 μ is obtained.

Preparation example 10

Stage 1 : Preparation of a solution of gellan TBA (tetrabutylammonium salt) 25 grams of powdered Kelcogel ^® CG-LA deacetylated gellan, sieved and collected between 140 and 38 micron sieves, is added to 300 ml of water and maintained under slow stirring in a thermostated mixer at a temperature of 60°C. After approx. 10 minutes another 100 ml of water is added, and stirring is continued for a further 30 minutes until a homogenous mixture is obtained. Stirring is stopped and 150 ml of activated TBA resin suspended in 100 ml of water is added to the hot gellan mixture. Stirring is resumed, always maintaining the minimum speed. The ion exchange (Na→TBA) takes place after 4 to 6 hours, leading to complete dissolution of the gellan gum. After 6 hours the solution of gellan TBA is separated from the resin by filtration through a steel filter with a porosity of 45 microns. The gellan solution obtained must have a pH of between 7 and 8, and the deacetylated gellan content must be between 45 and 60 mg/ml.

Preparation example 11

Stage 2: Preparation of gellan fibre

The deacetylated gellan solution prepared according to example 10 is filtered through a filter cloth with 20 micron pores and transferred to an extrusion reactor thermostated at 55°C connected to a spinneret for multiple strand wet-extrusion consisting of 3000 80-micron holes. The material is extruded through a first coagulation bath with continuous recirculation of 3 litres of a solution of sodium chloride in ethanol/water. The threads are introduced through transport rollers into a second coagulation tank filled with 0.5 litres of a solution of calcium chloride in ethanol, into 3 successive washing tanks filled with 0.5 litres each of absolute ethanol, and finally into a last washing tank filled with 0.5 litres of acetone. When the extrusion process has been completed, the fibre is continuously dried with hot-air ejectors and wound onto a reel. A deacetylated gellan fibre with a diameter of 10-30 microns is obtained. Example 12

Study of bio degradation, local tolerance and neurotoxicity of filler (gel) based on gellan gum and sulphated hyaluronic acid compared with a dermal filler (gel) based on carboxymethylcellulose and polyethylene oxide (PEO) (tradename Laresse™, marketed as a dermatological filler)

The study evaluated the effects of a gel based on deacetylated gellan and sulphated hyaluronic acid and a gel based on carboxymethylcellulose and polyethylene oxide after subdural application in the rabbit.

The gellan filler and the commercially available filler (control) were applied bilaterally in the subdural space of 21 rabbits.

Each group was observed for 3, 6, 9, 15 or 24 months.

Clinical and neurobehavioural observations were conducted, and food consumption and body weight were monitored.

After the animals were euthanised, the cerebrospinal fluid and some blood values were analysed, and histological analysis of the target organs was conducted. Finally, macroscopic and histological analysis of the implant sites was performed.

The results indicated that the deacetylated gellan and sulphated hyaluronic acid gel under study and the control had comparable safety profiles, with no macroscopic or microscopic signs of intolerance or toxicity. No harmful effect on the cerebrospinal fluid was observed, or signs of local intolerance or systemic toxicity.

The control material was no longer detectable at the follow up (3 months), whereas the deacetylated gellan and sulphated hyaluronic acid gel under study presents a very slow degradation process which had not been completed after 24 months.

In the field of application as dermocosmetic filler, this aspect of lengthy residence at the injection site represents a desirable characteristic for a successful product. The results of the study indicate that the gel based on deacetylated gellan gum and sulphated hyaluronic acid is equally safe and represents a device with a longer residence in situ than the commercial comparator.

Example 13

Measurement of extrusion force of a filler based on a gel consisting of gellan and sulphated hyaluronic acid in relation to aging processes

For the purpose of measuring the extrusion force of the gel based on deacetylated gellan and sulphated hyaluronic acid, a 10 cm long cannula was fitted to the syringe, and tests were conducted on four syringes per sample with an INST ON 4520 instrument.

The deacetylated gellan and sulphated hyaluronic acid gel was tested immediately after preparation (Time 0) and after aging processes at ambient temperature and at 40°C. A test was also conducted after freezing (-20°C) of the material.

Time Temperature Average force Semidispersion Standard

°C out of (∑) deviation

4 measurements

(^max )

(N)

0 ambient 6.9 0.6 0.6

17 h -20 3.6 0.4 0.4

3 months ambient 8.1 0.8 0.8

6 months ambient 6.7 0.3 0.4

9 months ambient 6.6 0.3 0.4

12 months ambient 6.9 0.3 0.5

2 months 40 7.9 0.6 0.6

3 months 40 6.8 0.7 0.5

6 months 40 4.6 0.5 0.3 The study demonstrates that during aging of the deacetylated gellan and sulphated hyaluronic acid gel at ambient temperature, no phenomena liable to influence its extrusion characteristics (i.e. degradation) take place.

When the product is aged at 40°C, the extrusion force gradually declines up to 6 months.

Example 14

Evaluation in a biofunctional canine model of injection of a filler based on a gel consisting of gellan gum and sulphated hyaluronic acid in laryngoplasty procedures

A study was conducted to test the efficacy, longevity and safety of injection of a filler based on a gel consisting of deacetylated gellan and sulphated hyaluronic acid, used in laryngoplasty procedures in a canine model. During the experiment, a commercially available control material called Radiesse® Voice (based on calcium hydroxyapatite, CaHA) was used.

The study was carried out with 30 beagle dogs (all males, average weight of 13.6 kg). After inducing complete unilateral paralysis through resection of the laryngeal nerve of the dogs, Radiesse® Voice and the gel based on deacetylated gellan and sulphated hyaluronic acid were injected.

1 , 3, 6, and 9 months after the injection, clinical outcomes and videostroboscopic findings were evaluated by investigators blind to the injection materials. Histological study was also performed.

In the videostroboscopic examination 1 and 9 months after CaHA injection, it was found that the mucosal wave was normal in one vocal fold, and decreased in the other. 6 months after the injection, the mucosal waves were well detected, but after 3 months there were some decreases in amplitude and periodicity compared to the vocal fold not injected, due to insufficient augmentation and mass effect of CaHA. Histologically, the H-E and masson trichrome stainings of the larynx specimens obtained 1 , 3, 6, and 9 months after injection showed a giant cell reaction indicating phagocytosis by macrophages in some areas, and revealed that the foreign body reaction was predominant in tissue injected with CaHA.

The larynx specimens injected with the filler based on deacetylated gellan and sulphated hyaluronic acid showed a foam cell formation indicating resorption, and inflammation was found in 4 specimens. Signs of fibrosis were found in 2 cases.

Stroboscopically, the mucosal waves of all except one of the vocal folds treated with the gel based on deacetylated gellan and sulphated hyaluronic acid were excellent, and similar between the paralysed and normal vocal folds.

During the study period, protrusion of the material did not occur, and no other systemic or local complications were observed in any group.

The mucosal waves of the vocal folds were preserved in amplitude and periodicity, and were still well detected during the follow-up periods.

In conclusion, the gel based on deacetylated gellan gum and sulphated hyaluronic acid is a safe, lasting filler in the vocal folds, and can be considered to be a suitable material for laryngoplasty injections, on a par with the products currently on the market. Compared with the control material, it has the added value of a better effect on the restoration of the natural characteristics and mucosal waves of paralysed vocal folds due to the superior stroboscopic results and lower inflammatory reactions associated with administration of the gel based on deacetylated gellan gum and sulphated hyaluronic acid. The type of material injected during the laryngoplasty procedure influences the result in terms of quality.