Bone cement formulation exhibiting antibacterial effect

Abstract

The present invention describes use of a bone cement formulation comprising polymethyl methacrylate (PMMA), a liquid component and a bone cement softener comprising linoleic acid, wherein the bone cement formulation is loaded with linoleic acid and wherein a concentration of linoleic acid is at least 6 vol.% of the liquid component in the bone cement formulation to provide for an antibacterial effect of the bone cement formulation.

Description

[0001] BONE CEMENT FORMULATION EXHIBITING ANTIBACTERIAL EFFECT Field of the invention

[0002] The present invention relates to a bone cement formulation exhibiting antibacterial effect.

[0003] Technical Background

[0004] Different bone cement formulations are known. For instance, in WO 2014 / 168565 there is described an injectable composition for a bone cement material comprising a dry powder component, a liquid component and a modifier configured to modify a Young's modulus of said bone cement material, wherein said modifier is linoleic acid or a derivative thereof and is present in a concentration of 0.1 to 12 v / v of said liquid component.

[0005] The present invention provides a bone cement formulation with enhanced antibacterial effect.

[0006] Summary of the invention

[0007] The stated purpose above is achieved by use of linoleic acid of at least 6 vol.% of the liquid component in a bone cement formulation, said bone cement formulation comprising polymethyl methacrylate (PMMA), a liquid component and a bone cement softener comprising linoleic acid, optionally in combination with at least one antibiotic agent, for promoting an increased level of at least released linoleic acid and thus antibacterial effect in the bone cement formulation, preferably the concentration of linoleic acid is at least 7 vol.% of the liquid component in the bone cement formulation, more preferably at least 8 vol.%, more preferably at least 9 vol.%, most preferably at least 12 vol.% of the liquid component in the bone cement formulation.

[0008] According to the present invention, as understood from the below, the inventors have found that there is a lower limit of the concentration needed of linoleic acid in a bone cement formulation according to the present invention to ensure for an antibacterial effect of the bone cement formulation.

[0009] In relation to the present invention it should be noted that all concentrations provided of the linoleic acid are calculated on the liquidcomponent and not based on the total volume of the bone cement formulation.

[0010] It is stated in WO 2014 / 168565 that the bone cement formulation described therein comprises linoleic acid or a derivative thereof is present in a concentration of 0.1 to 12 v / vof said liquid component. First of all, it should be noted that the concentration of the linoleic acid shown in WO 2014 / 168565 is preferably from 2 to 6 vol.% of the liquid component, i.e. below the set minimum range according to the present invention. Secondly, it should also be noted that there is no link in WO 2014 / 168565 with reference to providing an antibacterial effect by changing the concentration of linoleic acid. In WO 2014 / 168565 it is stated that antibiotics may be used to reduce any inflammatory side effects or bacterial infections after e.g. a hip replacement procedure. Thus, WO 2014 / 168565 provides a different direction for providing possible anti-bacterial properties, i.e. using antibiotics. According to the present invention, the antibacterial effect of the bone cement formulation is obtained by the needed concentration of linoleic acid in the bone cement formulation. This effect may then be further enhanced or complemented by also using certain antibiotic agents, as will be further explained below.

[0011] Moreover, it should be noted that the concentration of linoleic acid is preferably above 6 vol.% of the liquid component in the bone cement formulation to provide for an enhanced antibacterial effect of the bone cement formulation, more preferably at least 7 vol%, more preferably at least 8 vol%, more preferably at least 9 vol%, and most preferably at least 12 vol% of the liquid component, as supported by the example provided below.

[0012] Brief description of the drawings

[0013] Fig. 1 shows experiments conducted on bone cement formulations with different concentrations of linoleic acid.

[0014] Fig. 2 shows released linoleic acid concentration from bone cement formulations with different concentrations of linoleic acid.Specific embodiments of the invention

[0015] Osteoporosis is a bone disease that develops when bone mineral density and bone mass decrease, or when the structure and strength of bone change. This can lead to a decrease in bone strength that can increase the risk of fractures. When the vertebrae in the spine weaken from osteoporosis, they can become flatter. The vertebra can collapse during normal activity, leading to a spinal fracture. This type of compression fracture can cause a great deal of pain and can permanently alter the shape and strength of the spine. Spinal fractures due to osteoporosis often occur while doing something that causes relatively minor trauma to the spine, such as an insignificant fall, or twisting while lifting. Patients showing a DEXA T-score below -2.5 would be classified as having osteoporosis. Osteoporosis primarily affects the elderly, particularly postmenopausal women as estrogen, which plays a protective role in bone health, decreases significantly aftermenopause. However, men can also develop osteoporosis as they age. Other risk factors include a family history of the condition, certain medications, low body weight, smoking, excessive alcohol consumption, and a sedentary lifestyle.

[0016] A spinal fracture is a compression or fracture of the vertebrae and can occur anywherealong the spine. Most spinal fractures are caused by injury or trauma from car accidents, falls, sports or some sort of high velocity impact. A compression fracture occurs when one or more vertebrae in the spine collapse. This can lead to a decrease in the height of the affected vertebrae. Compression fractures commonly occur in the thoracic and lumbar regions of the spine and are often the resultof conditions that weaken the bones, such as osteoporosis, trauma, or metastatic cancer.

[0017] Moreover, both men and women are diagnosed with vertebral compression fractures. The incidence of clinically diagnosed vertebral compression fractures in women (123 / 100,000 patient years) is comparable to that for hip fractures, with fracture painusually lasting from two weeks to three months, associated with intense, deep, and sometimes intractable pain. Each additional new vertebral compression fracture is associated with a further increase in functional limitation, of a magnitude similar to thatin patients with diabetes, ischemic heart disease and rheumatism.Women with a prevalent vertebral fracture have a significantly lower quality of life than women without a prevalent vertebral fracture.

[0018] Furthermore, women with clinically diagnosed vertebral compression fractures have a 15% higher mortality than women who do not have vertebral compression fractures. Severe kyphosis is strongly predictive of pulmonary deaths, plausibly because those with underlying lung disease and decreased respiratory reserves may not tolerate restrictivechanges in thoracic anatomy resulting from vertebral fractures.

[0019] Vertebral augmentation is a well-established technique that has been reported to provide significant relief to patients with painful vertebral compression fractures related to osteoporosis. Immediate improvement in the quality of life follows the reduction of pain. Moreover, the mobility of elderly patients is crucial to reducing the risk of thromboticevents, pulmonary complications and decubitus ulcers. The immediate pain relief is likely to allow patients to resume their normal activity much quicker than if they are treated conservatively.

[0020] Furthermore, osteopenia is a medical condition characterized by lower than normal bone mineral density, which is not as severe as osteoporosis. It indicates that bones are weaker thannormal and an increased risk of fractures but not to the extent found in osteoporosis. Patients showing a DEXA T-score between -1 and -2.5 would be classified as having osteopenia.

[0021] Moreover, hemangiomas are benign vascular tumors that can occur in the spine. The tumors can grow and cause the affected vertebra to weaken. The weakening can lead to structuralinstability of the spine and eventually vertebral compression fractures. Multiple myeloma is a blood cancer that develops in plasma cells in the bone marrow. Myeloma cells stimulate osteoclasts activation, the cells responsible for bone resorption, which leads to increased bone breakdown. Simultaneously, osteoblasts are inhibited, hence leading to less bone formation. This imbalance leads to osteolytic lesions that weaken the bone. Fractures commonly occur in the spine. There are other kind of cancers, that often cause metastasis in the spine weakening the bone, such as lung cancer, breast cancer or prostate to give a few examples. Also these types of indications are relevant with reference totreatment with bone cement. Moreover, also degenerated discs may be treated with a bone cement according to the present invention.

[0022] Poly(methyl methacrylate) (PMMA), is a bioinert material commonly used to repair andaugment spinal compression fractures and to fixate hip and knee implants. Since PMMA has a Young’s modulus that is much higher than that of human cancellous bone, it may affect the biomechanics negatively, which may promote fractures in thetissues adjacent to the augmented vertebrae.

[0023] The use of PMMA based bone cement has been common in orthopedic surgeries fordecades and is well-established and considered safe. In addition, it is relatively inexpensive compared to alternative materials, making it an attractive material, especially in healthcare systems with limited resources.

[0024] PMMA-based bone cement is generally well-tolerated by the body, and allergic reactions are rare, which makes it suitable for use in orthopedic surgeries. It can be modified with additives such as antibiotics to reduce the risk of infection or with radiocontrast agents to improve visibility during surgery. Since PMMA with the addition of a rad iocontrast is radiopaque, meaning it appears clearly on X-rays and other imaging modalities, it enables surgeons to assess the placement and integrity of implants and bone cement during and after surgery.

[0025] In general, PMMA based bone cement comprising a softener can be obtained by mixing liquid components and powder components, wherein the liquid component comprises a liquid cement component and a liquid softener. The liquid and powder components can be mixed in any sequential order as long as the components are thoroughly mixed to form an extrudable paste. It should also be said that the PMMA of the bone cement initially comprises both monomers of methacrylic acid methyl ester in the liquid component, and partially pre-polymerized PMMA in the powder component. Upon mixing the polymerization process is initiated.

[0026] Below there are provided some embodiments of the present invention. According to one embodiment, the concentration of linoleic acid is at least 7 vol.%, more preferably at least 8 vol.%, more preferably at least 9 vol.% of theliquid component in the bone cement formulation, most preferably at least 12 vol.% of the liquid component in the bone cement formulation. The benefit of an increased concentration of linoleic acid for the antibacterial effect of the bone cement formulation may be understood from the examples presented below.

[0027] According to yet another embodiment, the bone cement formulation also comprises at least one antibiotic agent to further promote the antibacterial effect. According to one embodiment, said at least one antibiotic agent is gentamicin, vancomycin, tobramycin, or clindamycin, or a combination thereof.

[0028] It should further be noted that linoleic acid has another improved effect than “just” providing antibacterial effect. Linoleic acid provides a temperature decrease in the bone cement formulation during the polymerization thereof, which implies that an increased concentration of linoleic acid as suggested according to the present invention, provides a temperature protection. In turn this implies that also temperature sensitive antibiotics may be used according to the present invention. Below there is provided examples of such temperature sensitive antibiotics, and further considerations for such agents.

[0029] Temperature-sensitive antibiotics are those that require specific storage conditions to maintain their potency and effectiveness. Many antibiotics degrade when exposed to high temperatures, while some require refrigeration. Below is a list of commonly used temperature-sensitive antibiotics:

[0030] 1. Amoxicillin-Clavulanate (Augmentin) suspension - Note that clavulanic acid degrades at room temperature.

[0031] 2. Erythromycin Ethylsuccinate suspension - Note that stability decreases at higher temperatures.

[0032] 3. Cefaclor suspension - Note that this loses potency if not refrigerated.

[0033] 4. Cefixime suspension - Note that some formulations require refrigeration.

[0034] 5. Cefpodoxime suspension - Note that this requires refrigeration to maintain stability.6. Cefuroxime Axetil suspension - Note that this needs refrigeration for stability.

[0035] 7. Penicillin V suspension - Note that this maintains potency better when refrigerated.

[0036] 8. Vancomycin Oral solution - Note that this must be refrigerated to prevent degradation.

[0037] For the above it should be noted that refrigeration is required at temperatures of 2-8°C, as these antibiotics must be stored in a refrigerator to prevent degradation.

[0038] Furthermore, often it is important to avoid excessive heat, thus storage should be held below 25°C. Below there are provided antibiotics which should not be exposed to high temperatures, but which do not always require refrigeration:

[0039] 1. Doxycycline - Note that heat exposure can reduce effectiveness. 2. Azithromycin suspension - Note that some formulations do not require refrigeration but should be kept below 30°C.

[0040] 3. Clarithromycin suspension - Note that this should not be refrigerated but must be kept below 25°C.

[0041] 4. Linezolid suspension - Note that this should be stored at controlled room temperature.

[0042] Freezing can inactivate some antibiotics, making them ineffective. Examples are:

[0043] 1. Amoxicillin suspension - Note that freezing can alter the formulation.

[0044] 2. Cefuroxime suspension - Note that this can crystallize when frozen.

[0045] 3. Penicillin G injectable solutions - Note that freezing can cause precipitation of this.

[0046] 4. Vancomycin IV solution - Note that this should not be frozen to maintain efficacy.

[0047] Bone infections, also known as osteomyelitis, require antibiotics that can penetrate bone tissue effectively. The choice of antibiotic depends on thecausative organism, severity, and whether the infection is acute or chronic. Below there is provided is a list of commonly used antibiotics for bone infections:

[0048] Empirical therapy (before culture results)

[0049] • Vancomycin - covers Staphylococcus aureus, including MRSA.

[0050] • Ceftriaxone - has a broad coverage for Gram-negative bacteria.

[0051] • Piperacillin-Tazobactam - covers Pseudomonas aeruginosa and other Gram-negative organisms.

[0052] • Meropenem - is used in severe polymicrobial infections.

[0053] Definitive therapy (based on culture results)

[0054] For Methicillin-sensitive Staphylococcus aureus (MSSA)

[0055] • Nafcillin / Oxacillin

[0056] • Cefazolin

[0057] • Clindamycin (if allergic to beta-lactams)

[0058] For Methicillin-resistant Staphylococcus aureus (MRSA)

[0059] • Vancomycin (IV)

[0060] • Linezolid (Oral & IV)

[0061] • Daptomycin (IV)

[0062] • Clindamycin (if susceptible)

[0063] For Streptococcus Species

[0064] • Penicillin G

[0065] • Ceftriaxone

[0066] • Vancomycin (if resistant)

[0067] For Gram-Negative Bacteria (e.g., Pseudomonas aeruginosa)

[0068] • Cefepime

[0069] • Piperacillin-Tazobactam

[0070] • Meropenem

[0071] • Ciprofloxacin or Levofloxacin (if oral therapy is needed)

[0072] For Anaerobic Infections (Polymicrobial Osteomyelitis)

[0073] • Metronidazole (combined with a broad-spectrum agent)

[0074] • Clindamycin

[0075] Chronic Osteomyelitis (long-term suppressive therapy)

[0076] • Doxycycline• Trimethoprim-Sulfamethoxazole (Bactrim)

[0077] • Rifampin (used in combination, especially for prosthetic joint infections)

[0078] With reference to antibiotics for prosthetic joint infections (PJIs), below there are provided suitable examples. It should be mentioned that prosthetic joint infections (PJIs) are serious complications that require prolonged antibiotic therapy, often in combination with surgical intervention. In relation to prosthetic joint infections (PJIs), it is only the antibiotics mentioned in the passage that should be seen as relevant according to the present invention, i.e. otherwise there is no direct correlation for the bone cement formulation according to the present invention and use in treatments against prosthetic joint infections (PJIs). Treatment depends on the infecting organism, presence of biofilm, and the duration of infection.

[0079] 1. Empirical therapy (before culture results)

[0080] • Vancomycin - covers Staphylococcus aureus (including MRSA) and Streptococcus species.

[0081] • Cefepime or Piperacillin-Tazobactam - covers Gram-negative bacteria, including Pseudomonas aeruginosa.

[0082] • Metronidazole (if anaerobic infection is suspected).

[0083] Note: Empirical therapy should be guided by local antibiograms and adjusted based on culture results.

[0084] 2. Pathogen-Specific Therapy

[0085] A. Staphylococcus aureus (MSSA & MRSA)

[0086] . MSSA:

[0087] o Nafcillin or Oxacillin (IV)

[0088] o Cefazolin (IV)

[0089] o Rifampin (if implant retained, always combined with another antibiotic)

[0090] . MRSA:

[0091] o Vancomycin (IV)

[0092] o Daptomycin (IV)

[0093] o Linezolid (Oral or IV)

[0094] o Rifampin (adjunct for biofilm eradication)B. Coagulase-Negative Staphylococci (CNS)

[0095] • Vancomycin (IV) (if methicillin-resistant)

[0096] • Cefazolin (IV) (if methicillin-sensitive)

[0097] • Rifampin (in combination for biofilm-related infections)

[0098] C. Gram-Negative Bacteria (e.g., Pseudomonas aeruginosa, Enterobacteriaceae)

[0099] • Cefepime (IV)

[0100] • Meropenem (IV) (if resistant strains suspected)

[0101] • Ciprofloxacin or Levofloxacin (Oral, for suppressive therapy in susceptible strains)

[0102] D. Anaerobic Infections (e.g., Bacteroides spp.)

[0103] • Metronidazole (IV or Oral)

[0104] • Clindamycin (if Clostridium spp. suspected)

[0105] 3. Long-Term Suppressive Therapy

[0106] Used when implant removal is not an option or as maintenance therapy after surgical debridement. Common agents include:

[0107] • Doxycycline (Oral)

[0108] • Trimethoprim-Sulfamethoxazole (Bactrim) (Oral)

[0109] • Ciprofloxacin (Oral, if Gram-negative infection)

[0110] • Rifampin (always in combination, never as monotherapy)

[0111] 4. Duration of therapy

[0112] • With prosthesis retention: 6 weeks IV therapy, followed by 3-6 months of oral suppressive therapy.

[0113] • After prosthesis removal: 4-6 weeks IV therapy, with possible suppressive therapy if reimplantation is planned.

[0114] • For chronic suppressive therapy: May be lifelong if surgery is not possible.

[0115] For locally administered antibiotics for prosthetic joint infections (PJIs), it should be mentioned that in addition to systemic (IV / oral) antibiotics, local antibiotic therapy plays a crucial role in treating prosthetic joint infections (PJIs) by achieving high drug concentrations at the infection site while minimizing systemic toxicity.1. Local antibiotic delivery methods

[0116] A. Antibiotic-loaded bone cement (PMMA)

[0117] • Used in two-stage revision surgeries or as a spacer in infected joint replacements.

[0118] • Slowly releases antibiotics to maintain local therapeutic levels.

[0119] • Commonly used antibiotics in PMMA cement today:

[0120] o Vancomycin (for MRSA, MSSA, and Gram-positive coverage) o Gentamicin (for Gram-negative coverage, especially Pseudomonas)

[0121] o Tobramycin (alternative aminoglycoside for Gram-negative coverage)

[0122] o Clindamycin (for Staphylococcus and anaerobes) B. Antibiotic-loaded beads (PMMA or calcium sulfate)

[0123] • Used in surgical debridement and dead space management.

[0124] • Calcium sulfate beads dissolve over time, eliminating the need for removal.

[0125] • Common antibiotics used:

[0126] o Vancomycin

[0127] o Gentamicin

[0128] o Tobramycin

[0129] o Daptomycin (for MRSA, experimental use)

[0130] C. Intra-articular antibiotic injections

[0131] • Used in severe infections as adjunct therapy.

[0132] • Commonly injected antibiotics:

[0133] o Vancomycin (for Gram-positive infections)

[0134] o Tobramycin or Gentamicin (for Gram-negative infections) o Daptomycin (for MRSA and resistant Gram-positives) D. Hydrogel or Nanoparticle-Based Local Antibiotics (Experimental and Advanced Techniques)

[0135] • Newer biodegradable antibiotic carriers are being developed for sustained drug release.

[0136] Some promising agents are:

[0137] o Vancomycin-loaded hydrogelso Gentamicin-impregnated scaffolds

[0138] o Silver nanoparticles (antimicrobial coatings on implants) 2. Choosing the right local antibiotic - see the below

[0139] Bacteria Preferred local antibiotics

[0140] MSSA / MRSA Vancomycin, Daptomycin, Clindamycin Pseudomonas aeruginosa Tobramycin, Gentamicin

[0141] Enterobacteriaceae Gentamicin, Tobramycin

[0142] Anaerobes Clindamycin, Metronidazole

[0143] Key benefits of local antibiotic therapy are:

[0144] - High local concentration of antibiotics

[0145] - Reduced systemic toxicity

[0146] - Better biofilm penetration (especially when combined with systemic therapy)

[0147] - Useful in chronic & recurrent infections

[0148] Furthermore, according to one embodiment of the present invention, the polymerization of the present bone cement occurs at a lower temperature (<40°C) compared to standard PMMA based bone cement (>60°C), hence reducing the amount of temperature induced tissue damages.

[0149] According to one embodiment, the bone cement formulation is arranged to promote a released linoleic acid concentration of at least 4 mg / L saline solution, preferably at least 6 mg / L saline solution. Here the released linoleic acid concentration is measured in saline solution, such as Phosphate Buffered Saline (PBS) solution, which is used to maintain a stable pH. These type of levels of released linoleic acid concentrations may e.g. be obtained with a bone cement formulation comprising 12 vol% linoleic acid of the liquid component in the bone cement formulation according to the present invention.

[0150] It should further be noted that the combination of linoleic acid and antibiotics may provide a combined effect against both Gram-positive andGram-negative bacteria. In line with this, according to one embodiment, the present invention describes the use of a bone cement formulation where the bone cement formulation comprises at least one antibiotic agent, to provide for antibacterial effect against both Gram-positive and Gram-negative bacteria.

[0151] The bone cement formulation according to the present invention comprises polymethyl methacrylate (PMMA). Moreover, according to one embodiment of the present invention, the polymethyl methacrylate (PMMA) is a dry powder component, and wherein the bone cement formulation also comprises a liquid component comprising methyl methacrylate (MMA) monomer. According to one embodiment, the liquid component comprises 90 - 99.9 vol.% methacrylic acid methyl ester, preferably the liquid component comprises 0.1 - 5 vol.% benzenamine, N,N,4-trimethyl and 30 - 85 ppm 1,4-dihydoroxy benzene. According to yet another embodiment, the powder component comprises 50 - 60 wt.% PMMA, preferably the powder component comprises 40 - 55 wt.% zirconium dioxide, more preferably the powder component also comprises 0.5 - 1.5 wt.% dibenzoyl peroxide.

[0152] According to one further embodiment, the powder component comprises 60 -80 wt.% PMMA, preferably the powder component comprises 20 - 40 wt.% barium sulfate, more preferably the powder component also comprises 0.5 -1.5 wt.% dibenzoyl peroxide.

[0153] Moreover, according to yet another embodiment, the powder component comprises a copolymer, preferably polystyrene.

[0154] Furthermore, according to one embodiment, the elastic modulus of the bone cement formulation is maximum 2000 MPa, preferably maximum 1500 MPa, most preferably in a range of 200 - 1400 MPa, preferably wherein the compressive stress of the bone cement formulation is maximum 50 MPa, preferably in a range of 10 - 40 MPa. With reference to the elastic modulus of the bone cement formulation, this is decreasing with an increasing concentration of the linoleic acid. According to one specific embodiment of the present invention, the elastic modulus of the bone cement formulation is maximum 1400 MPa at a concentration of linoleic acid above 6 vol.% of the liquid component in the bone cement formulation. According to yet anotherembodiment, the elastic modulus of the bone cement formulation is maximum 1200 MPa at a concentration of linoleic acid of at least 8 vol.% of the liquid component in the bone cement formulation.

[0155] According to yet another embodiment, the elastic modulus of the bone cement formulation is at least 700 MPa and the concentration of linoleic acid is in a range of 6 - 14 vol.% of the liquid component in the bone cement formulation, and wherein the use is for spinal surgery, such as vertebroplasty, discoplasty, tumor, defect repair, or prosthetic joint infections treatment, trauma or defects repair. As supported by table 3, with examples according to the present invention, according to one embodiment for these applications, the elastic modulus of the bone cement formulation is 700 - 1300 MPa and the concentration of linoleic acid is in a range of 6 - 14 vol.% of the liquid component in the bone cement formulation.

[0156] Furthermore, according to one embodiment, the elastic modulus of the bone cement formulation is maximum 700 MPa and the concentration of linoleic acid is at least 14 vol.% of the liquid component in the bone cement formulation, and wherein the use is for cranial application, local antibacterial treatment, e.g. in PMMA bone cement beads, trauma or defects repair. As supported by table 3, with examples according to the present invention, according to one embodiment for these applications, the elastic modulus of the bone cement formulation is 200 - 700 MPa and the concentration of linoleic acid is in a range of 14 - 22 vol.% of the liquid component in the bone cement formulation.

[0157] As said above, the present invention is directed to use of linoleic acid of at least 6 vol.% of the liquid component in a bone cement formulation, said bone cement formulation comprising polymethyl methacrylate (PMMA), a liquid component and a bone cement softener comprising linoleic acid, optionally in combination with at least one antibiotic agent, for promoting an increased level of at least released linoleic acid and thus antibacterial effect in the bone cement formulation, preferably the concentration of linoleic acid is at least 7 vol.% of the liquid component in the bone cement formulation, more preferably at least 8 vol.%, more preferably at least 9 vol.%, most preferably at least 12 vol.% of the liquid component in the bone cement formulation.According to one embodiment, at least one antibiotic agent is used in combination with linoleic acid of at least 6 vol.% of the liquid component in the bone cement formulation according to the present invention.

[0158] According to yet another embodiment, the use of the bone cement formulation is performed for injecting into at least one vertebra or at least one vertebral disc of a subject, and for promoting an increased level of at least released linoleic acid and thus antibacterial effect in the bone cement formulation. Examples are the treatment of one or more vertebral fractures, infections, tumors, angioma or degenerated disc, or a combination thereof, such as e.g. spinal infection and fractures (vertebral osteomyelitis, spondylodiscitis).

[0159] The present invention is also directed to a process for the production of a bone cement formulation according to the present invention, wherein the process comprises sterilizing a powder component comprising polymethyl methacrylate (PMMA), a liquid component and a bone cement softener comprising linoleic acid, where all components are sterilized individually, and then mixing sterilized components for providing a mixed bone cement formulation with a concentration of linoleic acid of at least 6 vol.% of the liquid component in the bone cement formulation. Also in this case, the concentration of linoleic acid is preferably at least 7 vol.% of the liquid component in the bone cement formulation, more preferably at least 8 vol.%, more preferably at least 9 vol.%, and most preferably at least 12 vol.% of the liquid component in the bone cement formulation. Moreover, according to one embodiment, at least one antibiotic agent is used in combination with linoleic acid of at least 6 vol.% of the liquid component in the bone cement formulation according to the present invention.

[0160] According to one embodiment, the bone cement softener is added to the liquid component before mixing the powder component with the liquid component.

[0161] Moreover, according to yet another embodiment, the step of sterilizing for linoleic acid is performed by autoclaving. Suitably, the other components are sterilized by other means. For example, the powder component may be sterilized by gamma irradiation or ethylene oxide, and the liquid componentmay be sterilized by using sterile filtering. Furthermore, an ampoule used may be sterilized by using ethylene oxide.

[0162] Example and detailed description of the drawing

[0163] Bone cement formulations having different linoleic acid (LA) concentrations were investigated.

[0164] Methods

[0165] Bone cement specimen preparation

[0166] The unmodified cement, VS, was prepared. The modified cements (VS+3vol%LA, VS+6vol%LA, VS+9vol%LA, VS+12vol%LA) were prepared by adding the additive before mixing the powder and the liquid. As should be understood, the concentrations are provided based on the liquid component in the cement formulations. The liquid component was added with 3vol%LA, 6vol%LA, 9vol%LA, and VS+12vol%LA were shaken and mixed for 5 seconds. After mixing, the modified cement liquid and the powder were mixed manually according to the procedure described for the unmodified cement. The resulting paste of both modified and unmodified cement was then prepared into the peg shape to use the FlexiPeg device for evaluation.

[0167] Bacterial strains and growth conditions

[0168] A clinical gentamicin-susceptible S. aureus isolate and a clinical gentamicin-resistant S. aureus isolate were used in the test. Bacterial cultures were grown in Lysogeny Broth (LB). Bacterial inoculums for biofilm formation were prepared by diluting overnight cultures grown at 37°C shaking at 190 rpm a 10,000-fold with LB (approximately 105cells per well) for the incubation of biofilm on bone cement samples. Biological replicates of biofilms started from independent liquid cultures that were inoculated from independent colonies. In all microbiological experiments, three biological replicates were used.Biofilm growth analysis by cell viability counting

[0169] For a direct quantification of biofilm growth on the bone cement pegs, colony forming units (CFLI) from each peg were counted. At the end of the incubation (24h), the pegs were washed 3x1 min by transferring the lid with pegs to a 96-well plate containing 250 pL of sterile phosphate-buffered saline (PBS). Then the peg lid was put on the tube rack containing sterile glass tubes (15-16 mm diameter) and the pegs were pushed down into the tubes containing 600 pL PBS. Tubes with pegs in PBS were vortexed for 2 min at full speed to disperse the biofilms. CFU counts were performed by preparing serial dilutions of the bacterial dispersion in PBS and serial dilutions of the LB media exposed to bacteria and pegs. Then, appropriate dilutions will be plated on LB agar plates. The experiments were done with three biological replicates and each of them had two technical replicates for plating and counting CFU per peg and CFU per well. For this method, if the CFU per peg was less than 102, which is the detection level, the CFU value would not be determined. When the CFU was above the detection level, the average CFU per peg can be calculated.

[0170] In fig. 1 there is provided the viable cell count of two bacterial strains after 24h of biofilm growth on the VS, VS+3vol%LA, VS+6vol%LA, VS+9vol%LA, and VS+12vol%LA bone cement pegs. The concentrations provided here are based on concentrations of the liquid component.

[0171] Left: Gentamicin-susceptible S. aureus. Right: Gentamicin-resistant S. aureus. CFU / Peg indicates if the bacteria attach and grow on the cement surface. CFU / Well indicated if the released LA can inhibit the growth of bacteria. The black line indicates the mean and the long grey line (<102CFU per peg) indicates the detection limit, i.e. no colonies were observed on agar plates after plating the lowest dilution.

[0172] For both types of S. aureus, with 9vol% and 12vol% LA-loaded cement, no bacterial growth was observed on the cement peg surface or in the well. In this case, 9vol % of LA was enough to be released to kill the bacteria in the well and prevent the bacteria attachment and the biofilm growth on the cement peg. With 6vol% LA-loaded cement, it was observed that there were bacteria found on the peg cultured with gentamicin-resistant S. aureus andthere were bacterial found in the well cultured with both types of S. aureus, which means the released LA from the peg may be exerting a bacteriostatic effect, but was not sufficient enough to eliminate bacteria. In addition, more bacteria growth was observed on the peg and in the well with VS+3vol%LA.

[0173] Further experiments in relation to mechanical properties, handling properties, and LA release rate

[0174] Below further experiments have been conducted to further evaluate mechanical properties, handling properties, and LA release rate

[0175] for technical applications intended according to the present invention.

[0176] Materials

[0177] • Linoleic acid (LA)

[0178] • V-steady (VS):

[0179] o Powder component - 1 sachet of 26 g powder contains: PMMA Poly (methyl methacrylate) 14.08 ± 0.70 g. Zr02 (Zirconium dioxide) 11.70 ± 0.59 g. BPO (Benzoyl peroxide) 0.22 ± 0.03 g. o Liquid component - 1 ampoule of 10 ml liquid contains: MMA (Methyl methacrylate) 9.26 ± 0.46 g. DMPT (N.N-dimethyl-p- toluidine) 0.13 ± 0.01 g. HQ (Hydroquinone) 50 ± 5 ppm.

[0180] • Mendec Spine (MS):

[0181] o Powder (20 g): Polymethyl methacrylate (68,40 % w / w) ; Barium sulphate (30,00 % w / w): Benzoyl peroxide (1,60 % w / w).

[0182] o Liquid (9,2 g): Methyl methacrylate (99,10 % w / w); N.N- dimethyl-p-toluidine (0.90 % w / w): Hydroquinone (75 ppm).

[0183] Test method and results

[0184] • Cement preparation

[0185] Use the reverse pipetting technique to take 0.638 ml LA from the vial. Mix the LA with the monomer liquid in the glass container by shaking 5s. After mixing, the cement liquid and the powder were mixed manuallyaccording to the procedure described for VS or MS. The resulting paste was then injected into customized molds for further evaluation.

[0186] • Mechanical property

[0187] VS and MS with 6 vol% LA specimens were prepared by using cylindrical molds of around 6 mm in diameter and 12 mm in height for compression testing. Each cement formulation was prepared in 6 specimens fortesting by using a universal testing machine (Shimadzu, AGS-X). Specimen compression testing was performed at a crosshead speed of 20 mm / min.

[0188] The testing was conducted after conditioning specimens in phosphate-buffered saline(37°C) for 24 h. The compressive elastic modulus and compressive strength of specimens were determined from load-versus-displacement curves. The compressive strength was calculated from the 2% offset load or the upper yield-point load, whichever occurred first.

[0189] The results are as below: there is no big difference in mechanical properties between VS + 6 vol% LA and MS+6 vol% LA.

[0190] Results are provided in table 1 below.

[0191]

[0192] Table 1

[0193] • Handling properties

[0194] The maximum polymerization temperature (Tmax) and the setting time (tsetting) were determined according to the standard procedures followed the ISO 5833. Tmax is defined as the maximum temperature reachedduring the exothermic polymerization reaction, betting is defined as the time taken to reach a temperature midway between the ambient and the maximum temperature.

[0195] The results are as below presented in table 2. As seen, there is no big difference in handling properties between VS + 6 vol% LA and MS+6 vol% LA.

[0196]

[0197] Table 2

[0198] • LA release test

[0199] Bone cement was prepared into disc shapes (diameter: 12.95 ± 0.1 mm, height: 2.00 mm ± 0.1 mm) by injecting the cement paste into metal disc molds. The size and weight of all the specimens were recorded. After 30 min ± 1 min from the onset of mixing at 37°C, the specimens were immersed in 5 mL pre-heated PBS and then transferred into a 37°C oven for the following durations, 1h, 8h,1 day, 3 days, and 7 days. There were five specimens for each time point. After the required immersion time, the supernatant was collected from each sample. The Free Fatty Acid Assay Kit (ab65341 , Abeam, Cambridge, United Kingdom) was used to quantify the concentration of LA in the supernatant. Following the instructions from the kit producer, 50 pL from each supernatant was directly added to a 96-well plate, and palmitic acid standards with different concentrations from the kit were also prepared and transferred to the 96 well plate. First, the acyl-CoA synthesis was performed by adding 2 pL ACS reagent into each sample followed by an incubation at 37°C for 30 minutes. Then, the Reaction Mix of 50 pL (45.6 pL assay buffer, 0.4 pL fatty acid probe, 2 pL enzyme mix, 2 pL enhancer) was prepared and added to each well. After incubation at 37°C for 30 minutes in the dark, the fluorescent intensity was immediately measured with Ex / Em = 535 / 587 in a microplate reader (Infinite M200, Tecan, Switzerland). A standard curve was drawn basedon the concentration of the standard solution and its fluorescent intensity. Then, the LA concentration (mg / L) of each supernatant was calculated based on the standard curve.

[0200] For each sampling time point, differences among the three experimental groups (MS+6% LA, VS+6% LA, and VS+12% LA) were evaluated using a one-way analysis of variance (one-way ANOVA).

[0201] Whenever the ANOVA indicated a statistically significant group effect (p < 0.05), Tukey’s Honestly Significant Difference (Tukey HSD) test was performed for post-hoc pairwise comparisons. All statistical analyses were performed in R Studio.

[0202] The results are shown in fig. 2. Significant differences among the three cements were observed at 1h and 7-day time points, driven primarily by differences between VS+12% LA and VS+6% LA (p=0.009 and 0.03, separately). While no significant group differences occurred at other time points. These results indicated that LA can be released from different types of PMMA bone cement. And the LA release levels were quite similar, no matter the loading ratio between 12vol% and 6vol%.

[0203] In table 3 below there is provided different possible technical applications according to the present invention, together with preferred levels of elastic modulus and concentration of linoleic acid according to the present invention. It should be noted that load-bearing applications in the case of using a linoleic acid concentration below 6 vol.% of the liquid component in the bone cement formulation should not be regarded as part of the scope of the present invention. For load-bearing applications in which an antibacterial effect is to be provided, that is by having a linoleic acid concentration of at least 6 vol.% of the liquid component, is however part of the scope of the present invention.

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[0206]

[0207] Table 3Summary

[0208] For the antibacterial properties, with 6 vol% LA in VS, it can inhibit the S. aureus growth, while with 9 vol% or above vol% LA in VS, it has a stronger effect in preventing the Gram-positive bacteria, especially S. aureus growth and killing the bacteria. In terms of mechanical properties, with 12 vol% LA in VS, it has the best ability to modify the mechanical properties of cement. From 14 to 20 vol% LA in VS, it can decrease the elastic modules even more.

Claims

Claims1. A use of linoleic acid of at least 6 vol.% of the liquid component in a bone cement formulation, said bone cement formulation comprising polymethyl methacrylate (PMMA), a liquid component and a bone cement softener comprising linoleic acid, optionally in combination with at least one antibiotic agent, for promoting an increased level of at least released linoleic acid and thus antibacterial effect in the bone cement formulation, preferably the concentration of linoleic acid is at least 7 vol.% of the liquid component in the bone cement formulation, more preferably at least 8 vol.%, more preferably at least 9 vol.%, most preferably at least 12 vol.% of the liquid component in the bone cement formulation.

2. The use according to claim 1 , wherein the bone cement formulation comprises at least one antibiotic agent, to provide for antibacterial effect against both Gram-positive and Gram-negative bacteria.

3. The use of the bone cement formulation according to claim 1 or 2, for injecting into at least one vertebra or at least one vertebral disc of a subject, and for promoting an increased level of at least released linoleic acid and thus antibacterial effect in the bone cement formulation.

4. The use according to any of claims 1-3, wherein the use is performed for the treatment of one or more vertebral fractures, infections, tumors, angioma or degenerated disc, or a combination thereof.

5. The use according to any of claims 1-4, wherein the concentration of linoleic acid is at least at least 7 vol.%, more preferably at least 8 vol.%, more preferably at least 9 vol.% of the liquid component in the bone cement formulation, most preferably at least 12 vol.% of the liquid component in the bone cement formulation.

6. The use according to any of claims 1-5, wherein the bone cement formulation also comprises at least one antibiotic agent to further promote the antibacterial effect.

7. The use according to claim 6, wherein said at least one antibiotic agent is gentamicin, vancomycin, tobramycin, or clindamycin, or a combination thereof.

8. The use according to any of claims 1-7, wherein the bone cement formulation is arranged to promote a released linoleic acid concentration of at least 4 mg / L saline solution, preferably at least 6 mg / L saline solution.

9. The use according to any of claims 1-8, wherein the polymethyl methacrylate (PMMA) is a dry powder component, and wherein the bone cement formulation also comprises a liquid component comprising methyl methacrylate (MMA) monomer.

10. The use according to any of claims 1-9, wherein the liquid component comprises 90 - 99.9 vol.% methacrylic acid methyl ester, preferably the liquid component comprises 0.1 - 5 vol.% benzenamine, N,N,4-trimethyl and 30 -85 ppm 1 ,4-dihydoroxy benzene.

11. The use according to any of claims 1 -10, wherein the powder component comprises 50 - 60 wt.% PMMA, preferably the powder component comprises 40 - 55 wt.% zirconium dioxide, more preferably the powder component also comprises 0.5 - 1.5 wt.% dibenzoyl peroxide.

12. The use according to any of claims 1-11, wherein the powder component comprises 60 - 80 wt.% PMMA, preferably the powder component comprises 20 - 40 wt.% barium sulfate, more preferably the powder component also comprises 0.5 - 1.5 wt.% dibenzoyl peroxide.

13. The use according to any of claims 1-12, wherein the powder component comprises a copolymer, preferably polystyrene.

14. The use according to any of claims 1-13, wherein the elastic modulus of the bone cement formulation is maximum 2000 MPa, preferably maximum 1500 MPa, most preferably in a range of 200 - 1400 MPa, preferably wherein the compressive stress of the bone cement formulation is maximum 50 MPa, preferably in a range of 10 - 40 MPa.

15. The use according to any of claims 1-14, wherein the elastic modulus of the bone cement formulation is at least 700 MPa, preferably 700 - 1300 MPa, and wherein the concentration of linoleic acid is in a range of 6 - 14 vol.% of the liquid component in the bone cement formulation, and wherein the use is for spinal surgery, such as in vertebroplasty / kyphoplasty in osteoporotic fractures, discoplasty with degenerative disc, disc infection (discitis / spondylodiscitis), screw augmentation, chest wall defect filler or infection treatment, anterior column reconstruction in infected segments, or tumor cavity reconstruction requiring immediate stability.

16. The use according to any of claims 1-14, wherein the elastic modulus of the bone cement formulation is maximum 700 MPa, preferably 200 - 700 MPa, and wherein the concentration of linoleic acid is at least 14 -22 vol.% of the liquid component in the bone cement formulation, and wherein the use is for cranial application, local antibacterial treatment, such as in vertebroplasty / kyphoplasty in osteoporotic fractures, discoplasty with degenerative disc, disc infection (discitis / spondylodiscitis), cancellous defect fillers or infection spacers (discitis after debridement), anterior column reconstruction in infected segments, or local antibacterial treatment, e.g., as PMMA bone cement beads.

17. A process for the production of a bone cement formulation comprising polymethyl methacrylate (PMMA), a liquid component and a bone cement softener comprising linoleic acid, wherein the bone cement formulation isloaded with linoleic acid and wherein a concentration of linoleic acid is at least 6 vol.% of the liquid component in the bone cement formulation to provide for an antibacterial effect of the bone cement formulation, wherein the process comprises sterilizing a powder component comprising polymethyl methacrylate (PMMA), a liquid component and a bone cement softener comprising linoleic acid, where all components are sterilized individually, and then mixing sterilized components for providing a mixed bone cement formulation with a concentration of linoleic acid of at least 6 vol.% of the liquid component in the bone cement formulation.

18. The process according to claim 17, wherein the bone cement softener is added to the liquid component before mixing the powder component with the liquid component.

19. The process according to claim 17 or 18, wherein the step of sterilizing for the linoleic acid is performed by autoclaving.

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