WO2003066117A1 - Cold-curing acrylic formulations comprising low toxicity activators derived from diaminodiphenylcarbinol - Google Patents

Cold-curing acrylic formulations comprising low toxicity activators derived from diaminodiphenylcarbinol Download PDF

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WO2003066117A1
WO2003066117A1 PCT/ES2003/000055 ES0300055W WO03066117A1 WO 2003066117 A1 WO2003066117 A1 WO 2003066117A1 ES 0300055 W ES0300055 W ES 0300055W WO 03066117 A1 WO03066117 A1 WO 03066117A1
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cement
bone
composition according
butyl
liquid phase
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PCT/ES2003/000055
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Spanish (es)
French (fr)
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Blanca VÁZQUEZ LASA
Julio SAN ROMÁN DEL BARRIO
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Consejo Superior De Investigaciones Científicas
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Priority to AU2003208725A priority Critical patent/AU2003208725A1/en
Publication of WO2003066117A1 publication Critical patent/WO2003066117A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/06—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K6/00—Preparations for dentistry
    • A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
    • A61K6/884—Preparations for artificial teeth, for filling teeth or for capping teeth comprising natural or synthetic resins
    • A61K6/887—Compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • TECHNICAL FIELD This invention is part of the polymeric bone cements used as controlled release systems for medications in orthopedic surgery for prosthetic fixation and in dental applications as a polymerizable component of "composites”.
  • Bone cements used in orthopedic surgery are predominantly based on poly (methyl methacrylate), PMMA. They are obtained by cold curing of the precursor compositions which generally comprise two phases: a solid phase and a liquid phase.
  • the solid phase typically comprises particles in the form of prepolymerized PMMA beads or their copolymers, together with one or more radical initiators and, optionally, one or more radiopaque agents.
  • a radical type initiator is a compound, for example a peroxide, which is capable of producing free radicals.
  • a radiopaque agent is a compound that, as the name implies, is substantially opaque to radiation, in particular to the radiation used in medical diagnosis, such as X-rays.
  • the liquid phase comprises a polymerizable monomer, usually methyl methacrylate, MMA, along with one or more activators.
  • the activator is a compound, such as an aromatic tertiary amine, that causes decomposition of the initiator at room temperature to give rise to free radical formation.
  • the liquid phase may also comprise one or more inhibitors and / or stabilizers that are added in order to avoid polymerization of the monomer during storage.
  • PMMA-based bone cements A disadvantage of PMMA-based bone cements is the highly exothermic nature of the polymerization reaction and temperatures can be generated. Between 70 and 100 ° C in the center of the cement mass. This high temperature increase can cause necrosis in the surrounding tissue and the consequent bone damage, the references found in the literature in this regard being numerous.
  • Another disadvantage is the hypotension produced mainly by the monomer, methyl methacrylate, which can induce systemic effects if it enters the bloodstream.
  • the unreacted residual monomer can migrate to surrounding tissues producing chemical necrosis.
  • PMMA bone cement is a fragile material and has a low fracture toughness and poor fatigue life.
  • new formulations have been developed by adding a reinforcing agent, producing a low elastic modulus cement or obtaining bioactive cements.
  • new techniques for mixing the precursor components have been developed due to the influence they exert mainly on the porosity of the resulting material, which will subsequently affect its mechanical properties.
  • G. Lewis has published an important review on this subject in his article entitled “Properties of acrylic bone cement: State of the art” (J. Biomed. Mater. Res. (Appl. Biomaterials) 38, 155-182, 1997 ).
  • Aseptic loosening this is the loosening of the implant and the consequent formation of the fibrous membrane at the bone / cement interface over time, is another major problem associated with joint replacements. Its causes, although uncertain, are related to cement fracture and bone necrosis. As mentioned above, an adverse biological response is attributed to low molecular weight residues from the cement precursor composition, especially those components that have sufficient solubility in physiological fluids to be leached from the polymer matrix to surrounding tissues. . Another of the current concerns is the use of the activator N, N-dimethyl-4- toluidine (DMT) or N, N-dimethylaniline since both belong to a class of compounds able to react with DNA.
  • DMT N-dimethyl-4- toluidine
  • N-dimethylaniline since both belong to a class of compounds able to react with DNA.
  • the present invention is related to the development of cold cure acrylic formulations for use as bone cement cement precursor compositions.
  • Polymeric bone cements have been used during the last decades in orthopedic surgery and traumatology for the fixation of joint prostheses, the function of bone cement being immobilization of the prosthesis.
  • certain adverse reactions caused by the medium and long-term cement have been described that relate mainly to the chemical composition of the cement and / or its physical properties.
  • the present invention provides a composition for use as a bone cement precursor composition
  • a composition for use as a bone cement precursor composition comprising one or more compounds of general structure such as that presented in Formula (I) below:
  • Ri is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
  • R 2 is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
  • R 3 is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
  • 1 ⁇ is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
  • Formula (I) The structure presented in Formula (I) was selected based on the structure of the gentian violet compound (Formula (II)), a compound widely used at the hospital level for its antiseptic properties.
  • the compounds of the general formula Formula (I) have greater hydrophobicity than DMT and therefore, a reduction in the necrosis of the surrounding tissue caused by the migration of these cement compounds to adjacent tissues and systemic circulation is expected.
  • the compounds of the general formula Formula (I) offer the advantage of having toxicity values expressed as lethal dose 50 (LD 50 ) much lower than the value of the DMT.
  • the compounds of the general formula Formula (I) offer the advantage of being less cytotoxic than DMT on polymorphonuclear leukocytes.
  • the compounds of the general formula Formula (I) have a higher activity than DMT against different microorganisms.
  • the present invention also provides a cement that has been obtained by curing any of the compositions described above and which are suitable for use as bone cement precursor compositions and / or as self-healing medicament dosing systems.
  • the cements thus obtained have some advantages over the cements that have been described previously.
  • Bone cement precursor compositions which comprise in their liquid phase a compound of general formula Formula (I) as activator cure at temperatures below those of commercial compositions. This could potentially reduce the damage caused to adjacent tissues during bone cement formation "in situ”.
  • the cements of the present invention comprising in the liquid phase an activator of the general formula Formula (I) provide beneficial effects in the bone regeneration process when they have been implanted in their pasty state in the rabbit femur, and cured "in if you".
  • the tissue adjacent to the implanted cement showed greater and faster bone neoformation compared to that observed in the presence of commercial formulations
  • the animals were subsequently stored in air-conditioned rooms at a temperature of 22 ⁇ 1 ° C and 55 + 5% relative humidity. Under these conditions, the behavior of the animals was followed until the seventh day after administration.
  • the average lethal dose and curve was calculated by a programmed probit analysis (Finney) from the percentage of dead animals, observed during the seven-day period after administration.
  • Table I shows the values of lethal dose 50, LD 50 , BZN and DMT, with LD 50 being defined as the minimum dose administered to a population of mice that causes 50% of their mortality.
  • LD 50 being defined as the minimum dose administered to a population of mice that causes 50% of their mortality.
  • the results showed that the compound BZN has a value of LD 50 3.58 times higher than the DMT under the same experimental conditions.
  • Table I Values of lethal dose 50 (with a 95% confidence limit) for the DMT and BZN compounds together with the linear regression of the dose effect.
  • FIG. 1 shows the diagrams obtained from the analysis of the data for three percentages of mortality, 16%, 59%) and 84%.
  • the BZN activator has higher lethal dose values than DMT for any mortality rate.
  • the diagrams in FIG. 1 show the dose range necessary to increase mortality from 16% to 84%). While for DMT the interval is 60 mg / kg, BZN has a range of 100 mg / kg under the same experimental conditions.
  • mHBSS modified Hank's saline solution free of Ca + and Mg 2+
  • Contaminated erythrocytes were isolated by resuspension of the cells in 5 ml. of isotonic buffered solution of tris-ammonium hydrochloride (0.38%, pH 7.2) for 10 min. at 37 ° C. A new centrifugation was performed at 4 ° C at 400g for 10 min., Eliminating the supernatant.
  • the recovered cells were resuspended again in 10 ml. of mHBSS. A sample was taken to count the number of cells using a hemocytometer.
  • the 10 mi. recovered were centrifuged again at 4 ° C at 800g for 10 min., discarding the supernatant.
  • the cells were resuspended at 2.5x10 6 cells / min. in an unmodified HBSS solution containing 1.26 mM Ca 2+ and 0.9 mM Mg 2+ .
  • FIG. 2 shows the cytotoxicity of BZN and DMT obtained at different molar concentrations 0.1, 0.5 and 1.0 M.
  • the cytotoxicity of DMT increases with the concentration reaching a cytotoxicity level of 52.5%> (with respect to the Triton X-100 control used as a positive control) when the DMT concentration is 1.0 M.
  • the cytotoxicity of BZN does not change markedly with the concentration reaching levels below 30% for any concentration.
  • BZN 4,4'-bis-dimethylaminobenzidrol
  • DMT N, N-dimethylamino-4-toluidine
  • Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538 and Candida albicans CECT 1001 were obtained from the Spanish Class Crops Collection. Bacteria were routinely grown in culture medium of Soya-Tripticasa (Difco) and the microorganism C. albicans in YED (Yeast Extract [Difco] 2%, dextrose 2% w / v in distilled water), both media were solidified with agar (0.2% w / v) when necessary. The antimicrobial activity was well tested in Mueller Hinton (Difco) culture medium with bacteria, or in YED with C. albicans. Incubation was done at 37 ° C in all cases.
  • Soya-Tripticasa Difco
  • YED Yeast Extract [Difco] 2%, dextrose 2% w / v in distilled water
  • microculture dilution tests were performed in 96-well microplates with 90 ⁇ l. of the medium and lO ⁇ l. of inoculum in each well.
  • the compounds to be tested were dissolved in dimethylsulfoxide (DMSO) / distilled water (20/80 v / v) in a concentration of 10% (w / v).
  • DMSO dimethylsulfoxide
  • distilled water (20/80 v / v) in a concentration of 10% (w / v).
  • Duplicate dilutions of each compound in each row of wells were prepared, with a concentration range of 5% to 0.02% (w / v).
  • DMSO was included in a row in a concentration range of 10% to 0.04% (v / v), and a row was left only with means to be taken as a positive control.
  • OD optical density
  • the MIC values for DMT and BZN activators together with those obtained for the DMSO vehicle, against different microorganisms are shown in Table II. According to the established protocol, the antimicrobial power of the compounds increases as the value of the MIC decreases. The MIC values of DMSO are considerably higher than those of amines and therefore the effect of DMSO is not significant in these experiments.
  • the BZN / DMT relationship was taken into account. The results shown in Table II indicate that, against the Gram-negative bacteria BZN is twice as active as DMT. The results obtained for the Gram-positive bacterium showed an increase in the activity of the BZN of up to 8.04 times greater than the DMT.
  • the results obtained for the C. albicans fungus are very representative since this microorganism is present in the periodontal cavity and may be responsible for a large number of postoperative traumatic infections. Once again, BZN activity was 8.04 times higher than DMT.
  • Table II Minimum inhibitory concentration (MIC) values of the compounds defined as the dilution that causes an 80% reduction in the optical density of the control.
  • Bone cement precursor compositions were formulated using as an activator the compound 4,4'-bis-dimethylamino benzidrol (BZN) and as solid phase particles of poly (methyl methacrylate) (QL beads) whose morphological characteristics are given in the table of the Table III QL pearls are commercial pearls and were supplied by Levante Surgical Industries.
  • BZN 4,4'-bis-dimethylamino benzidrol
  • QL beads poly (methyl methacrylate)
  • Table IV Composition of commercial bone cement taken as a comparative example together with the bone cement precursor composition formulated with compound BZN.
  • T p co The peak temperature (T p co) is defined as the maximum temperature reached during the polymerization reaction and recorded according to ASTM (F451) standard.
  • the two components of the bone cement precursor composition are mixed and the resulting paste is introduced into a Teflon mold.
  • a thermocouple is placed in the center of the mold at a height of 3 mm in the internal cavity. Time is taken from the beginning of the Mix the two components and record the temperature. An average of two measurements was performed for each formulation. The exotherms were recorded at a temperature of 25 ° C.
  • the time of the pasty state (t pas t 0s o) represents the time in which the cement mass does not adhere to the surgical glove. At this time the cement is implanted in the organism for example, in the femoral cavity.
  • Curing time (t CUADO ) was determined according to ASTM (F451) as the time at which the cement mass temperature is the arithmetic mean of the sum T max + T amb , where T raax is the maximum temperature in ° C and T am b is the ambient temperature, 23 ° C.
  • the residual monomer content was determined by H-NMR spectroscopy.
  • the samples were stored at room temperature in air for seven days before being analyzed. Three samples of each composition were dissolved in deuterated chloroform and the spectra were recorded on a 300MHz Vary spectrophotometer.
  • Table V Cure parameter values, residual monomer and mechanical properties of the cured cements from the precursor compositions shown in Table IV.
  • the peak temperatures of the precursor compositions containing BZN were approximately 10 ° C lower than those obtained with commercial formulations which represents an important and significant benefit from a biological point of view.
  • the residual monomer values of both formulations were of the same order indicating that the conversion of the activated polymerization reaction with the compound BZN is comparable to that achieved in commercial formulations.
  • the cement rods (3 mm diameter x 15 mm length) were introduced by means of a cannula in the dorsal muscle of Wistar female rats of average weight 300 + 10 g.
  • the wound was sutured with a 3/0 silk stitch and Betadine® was applied.
  • Two groups corresponding to 2 and 3 animals were made respectively, which were sacrificed at 4 and 8 weeks after the operation.
  • FIG. 3-A and FIG. 3-B show, respectively, the histological image of the muscle response to the control cement (FIG. 3-A) and to the cured cement from precursor compositions containing BZN (FIG. 3 -B), after 4 weeks of implantation.
  • the cross sections of surrounding tissue showed the formation of a fibrous membrane together with the presence of polymorphonuclear leukocytes, macrophages and eosinophils.
  • no signs of necrosis were detected as expected due to the implantation of a cured cement "in vitro".
  • FIG.3-C and FIG.3-D reflect the tissue response after 8 weeks of implantation.
  • FIG. 3-C shows the response to CMW 3 commercial cement and FIG. 3-D obtained after implantation of the cement containing BZN.
  • the fibrous membrane has grown into a capsule. fibrous rich in oriented collagen fibers.
  • the inflammatory reaction that is observed in short periods of time has been decreasing considerably although the presence of some macrophages and eosinophils was still detected.
  • the response of the tissues to the implantation of the cement prepared with BZN does not differ from that obtained with the implantation of commercial cements and can be considered as normal in the implantation of this type of materials.
  • FIG. 4 The series of photographs presented in FIG. 4 show the surfaces of the rods before implantation and after 4 weeks of being implanted, for commercial cement formulated with DMT (FIG. 4-A and FIG. 4-B ) and for the experimental cement formulated with BZN (FIG. 4-C and FIG. 4-D).
  • the surface before implantation presents some irregularities caused by the curing process such as the formation of bubbles during the handling of the dough and its introduction into the mold .
  • the surfaces of the explanted rods at 4 weeks were smooth, presenting an appearance as if they had undergone an erosion process and the irregularities have disappeared. This erosion may be related to the inflammatory response that occurs in the first days after implantation.
  • mice Twenty adult female rabbits of the New Zealand medium-weight 3,820 kg (3,450-4,260 kg) were operated under aseptic conditions. The animals were distributed in two groups. Ten animals constituted the control group in which the CMW 3 commercial cement compositions were implanted and another ten animals constituted the experimental group in which the compositions containing BZN were implanted. Table VI shows the experimental design and the number of animals implanted in the experiments.
  • Table VI Experimental design and number (N) of animals implanted in the rabbit femur implantation experiments of the bone cement precursor compositions shown in Table IV.
  • control and experimental groups were operated on different surgical acts and, in both cases, implantation was performed in the left rabbit femur.
  • the rabbits were premedicated with atropine sulfate (0.3 mg / kg, IM) and chlorpromazine (10 mg / kg, IM).
  • General anesthesia was administered intramuscularly, ketamine hydrochloride (50mg / Kg, IM) and fentanyl (0.17 mg / Kg, IM).
  • the surgical area was disinfected with iodine and a longitudinal incision was made.
  • Two bone defects were created in the femoral limbs using a low speed drill.
  • the cement in its pasty state was injected with a syringe and allowed to cure inside for a few minutes, so that the exothermic reaction took place "in situ" after implantation.
  • the samples were decalcified by the technique of nitric formalin (950 ml of 10% formalin and 50 ml of nitric acid) prior to inclusion in paraffin for 24 h. Subsequently, cross sections (6 ⁇ m thick) were made with a manual microtome (Minot-Leniz) and the samples were stained using the hematoxylin-eosin technique for histological examination.
  • necrosis can be caused by thermal, chemical causes as well as by the surgical procedure itself.
  • the damage of some local areas of the bone is inevitable in any implantation procedure that involves the drilling of the bone, although it is done at low speed, and also the injection of a material that heals "in situ" (PA Revell, M. Braden, MAR Freeman, Review of the biological response to a novel bone cement containing poly (ethyl methacrylate) and n-butyl methacrylate, Biomaterials, 19, 1579-1586 (1998)).
  • polymorphonuclear leukocytes were observed around the remains of the material, and in other areas farther from the material, mononuclear cells, mainly eosinophils, lymphocytes, plasma cells and macrophages, clear indicators of the inflammation reaction were detected.
  • FIG.5-A and FIG.5-B show representative photomicrographs of the histological sections of the interface between the CMW 3 cement or the experimental cement respectively, and the adjacent tissue 2 days after implantation.
  • the histological analysis at 4 weeks after implantation showed the existence of fewer necrotic areas than in the previous periods as a consequence of the reabsorption process carried out by the macrophages.
  • the inflammatory response continues its reparative process, observing a greater amount of macrophages and multinucleated giant cells surrounding the remains of the two types of cement.
  • FIG.6-A and FIG.6-B illustrate these differences in terms of bone neoformation that accompany the implantation of a commercial cement (CMW 3) and the experimental cement containing BZN, respectively.
  • FIG. 7 shows the area of femoral epiphysis after 12 weeks of the implantation of the experimental cement where an intense bone proliferation can be observed, with fine trabeculae, separated by a labyrinth of interconnected spaces that contain bone marrow.
  • the most relevant in this period is the bone formation that can be seen in the response to the experimental cement, which demonstrates the confluence of bone trabeculae, especially in the diaphysis, so that the thickness of the neoformed bone is more noticeable than that obtained with the control formulation of CMW 3.
  • FIG.8-A and FIG.8-B corresponding to the histological response for this period of time (24 weeks) to the control and experimental cements respectively, illustrate this behavior.
  • FIG. 8-B corresponds to the femoral shaft treated with the experimental cement and shows the laminar disposition that acquires bone formation, with osteocyte-containing lagoons.
  • FIG. 1 is a graph showing the relationship between the dose of BZN or DMT, and the percentage of mortality determined by intravenous injection in mice of a saline solution at
  • FIG.2 is a graph showing the effects of DMT and BZN compounds on the integrity of rat polymorphonuclear leukocytes measured in terms of release of
  • FIG. 3 is a series of photographs showing the histological response to intramuscular implantation in rats of commercial CMW 3 cement rods and experimental cement formulated with the compound BZN.
  • FIG. 4 is a series of photographs showing the surfaces of the rods before and after 4 weeks of implantation, of commercial cement CMW 3 and of experimental cement formulated with the compound BZN.
  • FIG. 5 is a series of photographs showing the histological response two days after the intraosseous implantation of the bone cement precursor compositions shown in the
  • FIG. 6, cured "in situ”.
  • FIG. 6 is a series of photographs showing the histological response 4 weeks after the intraosseous implantation of the bone cement precursor compositions shown in FIG. 6, cured "in situ".
  • FIG. 7 is a photograph showing the histological analysis of bone tissue adjacent to a cement formulated with the BZN activator after 12 weeks of implantation.
  • FIG. 8 is a series of photographs showing the histological reaction to intraosseous implantation of a commercial cement and an experimental cement formulated with BZN after 24 weeks of implantation.

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Abstract

The invention relates to the development of cold-curing acrylic formulations for use in bone cement precursor compositions having low toxicity and good biocompatibility. In recent decades, polymeric bone cements have been used as media for the controlled release of medicaments and, in orthopaedic surgery and traumatology, for fixing joint prostheses, the bone cement serving to immobilise the prosthesis. However, certain adverse reactions which are caused by the cement have been observed in the medium and long term, said reactions being connected primarily to the chemical composition of the cement and/or the physical properties thereof.

Description

FORMULACIONES ACRÍLICAS DE CURADO EN FRÍO CON ACTIVADORES DERIVADOS DE DIAMINODIFENILCARBINOL DE BAJA TOXICIDADACRYLIC FORMULATIONS OF COLD CURING WITH ACTIVATORS DERIVED FROM DIAMINODIFENILCARBINOL OF LOW TOXICITY
Sector de la técnica Esta invención se enmarca dentro de los cementos óseos poliméricos utilizados como sistemas de liberación controlada de medicamentos en cirugía ortopédica para la fijación de prótesis y en aplicaciones dentales como componente polimerizable de "composites".TECHNICAL FIELD This invention is part of the polymeric bone cements used as controlled release systems for medications in orthopedic surgery for prosthetic fixation and in dental applications as a polymerizable component of "composites".
Estado de la técnica Los cementos óseos utilizados en cirugía ortopédica están basados predominantemente en poli(metacrilato de metilo), PMMA. Se obtienen por curado en frío de las composiciones precursoras que generalmente comprenden dos fases: una fase sólida y una fase líquida. La fase sólida normalmente comprende partículas en forma de perlas de PMMA prepolimerizadas o sus copolímeros, junto con uno o más iniciadores radicales y, opcionalmente, uno o más agentes radiopacos. Un iniciador de tipo radical es un compuesto, por ejemplo un peróxido, que es capaz de producir radicales libres. Un agente radiopaco es un compuesto que como su nombre indica es sustancialmente opaco a la radiación, en particular a la radiación que se utiliza en el diagnóstico médico, tal como los rayos X. La fase líquida comprende un monómero polimerizable, normalmente metacrilato de metilo, MMA, junto con uno o más activadores. El activador es un compuesto, tal como una amina terciaria aromática, que produce la descomposición del iniciador a temperatura ambiente para dar lugar a la formación de radicales libres. La fase líquida también puede comprender uno o más inhibidores y/o estabilizadores que se añaden con el fin de evitar la polimerización del monómero durante el almacenamiento. Cuando las dos fases de la composición precursora de cemento óseo se mezclan se produce la reacción de polimerización del monómero obteniéndose un cemento óseo que consiste en partículas del polvo sólido embebidas en una matriz intersticial de polímero formado.State of the art Bone cements used in orthopedic surgery are predominantly based on poly (methyl methacrylate), PMMA. They are obtained by cold curing of the precursor compositions which generally comprise two phases: a solid phase and a liquid phase. The solid phase typically comprises particles in the form of prepolymerized PMMA beads or their copolymers, together with one or more radical initiators and, optionally, one or more radiopaque agents. A radical type initiator is a compound, for example a peroxide, which is capable of producing free radicals. A radiopaque agent is a compound that, as the name implies, is substantially opaque to radiation, in particular to the radiation used in medical diagnosis, such as X-rays. The liquid phase comprises a polymerizable monomer, usually methyl methacrylate, MMA, along with one or more activators. The activator is a compound, such as an aromatic tertiary amine, that causes decomposition of the initiator at room temperature to give rise to free radical formation. The liquid phase may also comprise one or more inhibitors and / or stabilizers that are added in order to avoid polymerization of the monomer during storage. When the two phases of the bone cement precursor composition are mixed, the polymerization reaction of the monomer occurs, obtaining a bone cement consisting of particles of the solid powder embedded in an interstitial matrix of formed polymer.
Una desventaja de los cementos óseos basados en PMMA es el carácter altamente exotérmico de la reacción de polimerización y se pueden generar temperaturas comprendidas entre 70 y 100°C en el centro de la masa del cemento. Este elevado aumento de la temperatura puede originar una necrosis en el tejido circundante y el consiguiente daño óseo, siendo numerosas las referencias encontradas en la bibliografía a este respecto.A disadvantage of PMMA-based bone cements is the highly exothermic nature of the polymerization reaction and temperatures can be generated. between 70 and 100 ° C in the center of the cement mass. This high temperature increase can cause necrosis in the surrounding tissue and the consequent bone damage, the references found in the literature in this regard being numerous.
Otra desventaja es la hipotensión producida principalmente por el monómero, metacrilato de metilo, que puede inducir efectos sistémicos si entra en la corriente sanguínea. Además, el monómero residual no reaccionado puede migrar a los tejidos circundantes produciendo una necrosis química.Another disadvantage is the hypotension produced mainly by the monomer, methyl methacrylate, which can induce systemic effects if it enters the bloodstream. In addition, the unreacted residual monomer can migrate to surrounding tissues producing chemical necrosis.
El cemento óseo de PMMA es un material frágil y presenta una baja tenacidad a la fractura y vida en fatiga pobre. Con el fin de mejorar sus propiedades mecánicas se han desarrollado nuevas formulaciones mediante la adición de un agente de refuerzo, la producción de un cemento de bajo módulo elástico o bien la obtención de cementos bioactivos. Así mismo se han desarrollado nuevas técnicas de mezclado de los componentes precursores debido a la influencia que ejercen principalmente en la porosidad del material resultante lo que posteriormente repercutirá en sus propiedades mecánicas. Recientemente, G. Lewis ha publicado una importante revisión sobre este tema en su artículo titulado "Properties of acrylic bone cement: State of the art" (J. Biomed. Mater. Res. (Appl. Biomaterials) 38, 155- 182, 1997).PMMA bone cement is a fragile material and has a low fracture toughness and poor fatigue life. In order to improve its mechanical properties, new formulations have been developed by adding a reinforcing agent, producing a low elastic modulus cement or obtaining bioactive cements. Likewise, new techniques for mixing the precursor components have been developed due to the influence they exert mainly on the porosity of the resulting material, which will subsequently affect its mechanical properties. Recently, G. Lewis has published an important review on this subject in his article entitled "Properties of acrylic bone cement: State of the art" (J. Biomed. Mater. Res. (Appl. Biomaterials) 38, 155-182, 1997 ).
El aflojamiento aséptico, esto es el aflojamiento del implante y la consiguiente formación de la membrana fibrosa en la interfaz hueso/cemento con el tiempo, es otro de los mayores problemas asociados con los reemplazos de articulaciones. Sus causas, aunque inciertas, se relacionan con la fractura del cemento y con la necrosis ósea. Como se ha mencionado anteriormente, una respuesta biológica adversa se atribuye a los residuos de bajo peso molecular procedentes de la composición precursora del cemento, especialmente aquellos componentes que presentan suficiente solubilidad en los fluidos fisiológicos como para ser lixiviados de la matriz polimérica a los tejidos circundantes. Otra de las preocupaciones actuales se centran en el uso del activador N,N-dimetil-4- toluidina (DMT) o N,N-dimetilanilina ya que ambos pertenecen a una clase de compuestos capaces de reaccionar con el DNA. El análisis de la genotoxicidad ha revelado que la DMT en particular es capaz de inducir alteraciones cromosómicas. Además se ha confirmado la presencia de DMT en cementos que han sido implantados en periodos de 2.5 a 10 años. Como alternativas a la DMT se han utilizado otras aminas de mayor peso molecular o incluso aminas polimerizables. Una revisión sobre este tema ha sido publicada por B. Vázquez y col. En su artículo titulado "Role of amine activators on the curing parameters, properties and toxicity of acrylic bone cements" (Polymer International 46, 241-250, 1998).Aseptic loosening, this is the loosening of the implant and the consequent formation of the fibrous membrane at the bone / cement interface over time, is another major problem associated with joint replacements. Its causes, although uncertain, are related to cement fracture and bone necrosis. As mentioned above, an adverse biological response is attributed to low molecular weight residues from the cement precursor composition, especially those components that have sufficient solubility in physiological fluids to be leached from the polymer matrix to surrounding tissues. . Another of the current concerns is the use of the activator N, N-dimethyl-4- toluidine (DMT) or N, N-dimethylaniline since both belong to a class of compounds able to react with DNA. Genotoxicity analysis has revealed that DMT in particular is capable of inducing chromosomal abnormalities. In addition, the presence of DMT has been confirmed in cements that have been implanted in periods of 2.5 to 10 years. As alternatives to DMT, other amines of higher molecular weight or even polymerizable amines have been used. A review on this topic has been published by B. Vázquez et al. In his article entitled "Role of amine activators on the curing parameters, properties and toxicity of acrylic bone cements" (Polymer International 46, 241-250, 1998).
Breve descripción de la invención La presente invención está relacionada con el desarrollo de formulaciones acrílicas de curado en frío para su uso como composiciones precursoras de cementos óseos. Los cementos óseos poliméricos se vienen empleando durante las últimas décadas en cirugía ortopédica y traumatología para la fijación de prótesis de articulaciones siendo la función del cemento óseo la inmovilización de la prótesis. Sin embargo, se han descrito ciertas reacciones adversas producidas por el cemento a medio y largo plazo que se relacionan principalmente con la composición química del cemento y/o sus propiedades físicas.Brief Description of the Invention The present invention is related to the development of cold cure acrylic formulations for use as bone cement cement precursor compositions. Polymeric bone cements have been used during the last decades in orthopedic surgery and traumatology for the fixation of joint prostheses, the function of bone cement being immobilization of the prosthesis. However, certain adverse reactions caused by the medium and long-term cement have been described that relate mainly to the chemical composition of the cement and / or its physical properties.
Descripción detallada de la invenciónDetailed description of the invention
La presente invención proporciona una composición para su uso como composición precursora de cemento óseo que comprende uno o más compuestos de estructura general como la que se presenta en la Fórmula (I) debajo:The present invention provides a composition for use as a bone cement precursor composition comprising one or more compounds of general structure such as that presented in Formula (I) below:
Figure imgf000004_0001
Figure imgf000004_0001
Fórmula (I) donde:Formula (I) where:
Ri es un radical metilo, etilo, propilo, isopropilo, butilo, isobutilo o tert-butilo.Ri is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
R2 es un radical metilo, etilo, propilo, isopropilo, butilo, isobutilo o tert-butilo.R 2 is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
R3 es un radical metilo, etilo, propilo, isopropilo, butilo, isobutilo o tert-butilo. 1^ es un radical metilo, etilo, propilo, isopropilo, butilo, isobutilo o tert-butilo.R 3 is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical. 1 ^ is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
La selección de la estructura presentada en la Fórmula (I) se realizó en base a la estructura del compuesto violeta de genciana (Fórmula (II)), compuesto muy utilizado a nivel hospitalario por sus propiedades antisépticas.The structure presented in Formula (I) was selected based on the structure of the gentian violet compound (Formula (II)), a compound widely used at the hospital level for its antiseptic properties.
Figure imgf000005_0001
Figure imgf000005_0001
Fórmula (II)Formula (II)
Los compuestos de fórmula general Fórmula (I) poseen mayor hidrofobicidad que la DMT y por tanto, es de esperar una reducción en la necrosis del tejido circundante producida por la migración de estos compuestos del cemento a los tejidos adyacentes y a la circulación sistémica.The compounds of the general formula Formula (I) have greater hydrophobicity than DMT and therefore, a reduction in the necrosis of the surrounding tissue caused by the migration of these cement compounds to adjacent tissues and systemic circulation is expected.
Los compuestos de fórmula general Fórmula (I) ofrecen la ventaja de poseer valores de toxicidad expresados como dosis letal 50 (LD50) muy inferiores al valor que presenta la DMT. Los compuestos de fórmula general Fórmula (I) ofrecen la ventaja de ser menos citotóxicos que la DMT sobre leucocitos polimorfonucleares.The compounds of the general formula Formula (I) offer the advantage of having toxicity values expressed as lethal dose 50 (LD 50 ) much lower than the value of the DMT. The compounds of the general formula Formula (I) offer the advantage of being less cytotoxic than DMT on polymorphonuclear leukocytes.
Los compuestos de fórmula general Fórmula (I) presentan una mayor actividad que la DMT frente a diferentes microorganismos.The compounds of the general formula Formula (I) have a higher activity than DMT against different microorganisms.
La presente invención también proporciona un cemento que ha sido obtenido por curado de cualquiera de las composiciones descritas arriba y que son adecuadas para su uso como composiciones precursoras de cemento óseo y/o como sistemas autocurables de dosificación de medicamentos. Los cementos así obtenidos presentan algunas ventajas sobre los cementos que se han descrito con anterioridad.The present invention also provides a cement that has been obtained by curing any of the compositions described above and which are suitable for use as bone cement precursor compositions and / or as self-healing medicament dosing systems. The cements thus obtained have some advantages over the cements that have been described previously.
Las composiciones precursoras de cemento óseo que comprenden en su fase líquida un compuesto de formula general Fórmula (I) como activador curan a temperaturas inferiores a las de las composiciones comerciales. Ésto podría reducir potencialmente el perjuicio ocasionado en los tejidos adyacentes durante la formación del cemento óseo "in situ".Bone cement precursor compositions which comprise in their liquid phase a compound of general formula Formula (I) as activator cure at temperatures below those of commercial compositions. This could potentially reduce the damage caused to adjacent tissues during bone cement formation "in situ".
Los cementos de la presente invención que comprenden en la fase líquida un activador de fórmula general Fórmula (I) proporcionan efectos beneficiosos en el proceso de regeneración del hueso cuando se han implantado en su estado pastoso en el fémur de conejos, y han curado "in situ". El tejido adyacente al cemento implantado mostró una mayor y más rápida neoformación ósea en comparación a la observada en presencia de las formulaciones comercialesThe cements of the present invention comprising in the liquid phase an activator of the general formula Formula (I) provide beneficial effects in the bone regeneration process when they have been implanted in their pasty state in the rabbit femur, and cured "in if you". The tissue adjacent to the implanted cement showed greater and faster bone neoformation compared to that observed in the presence of commercial formulations
EJEMPLO 1EXAMPLE 1
Análisis comparativo de la toxicidad aguda del 4,4'-bis-dimetilaminobenzidrol (BZN) y de la N,N-dimetilamino-4-toluidina (DMT), activador usado comúnmente en las formulaciones comerciales de cemento óseo. La toxicidad aguda se analizó con ratones machos de raza SPF-NMR1 de peso 25+3 gr. (cada animal se pesó de forma individualizada para calcular la cantidad real administrada). Se eligieron 10 animales por cada dosis y compuesto. Se administraron en la vena caudal del ratón soluciones salinas de los correspondientes clorhidratos, debido a la escasa solubilidad de estas aminas en medio salino acuoso. Esta administración se efectuó por la mañana, entre las 8,00 y las 11,00 h. Posteriormente los animales se guardaron en habitaciones con aire acondicionado a una temperatura de 22±1°C y 55+5% de humedad relativa. En estas condiciones, se siguió el comportamiento de los animales hasta el séptimo día tras la administración. La dosis letal promedio y la curva fue calculada mediante un análisis probit programado (Finney) desde el porcentaje de animales muertos, observados durante el período de siete días tras la administración.Comparative analysis of the acute toxicity of 4,4'-bis-dimethylaminobenzidrol (BZN) and N, N-dimethylamino-4-toluidine (DMT), activator commonly used in commercial bone cement formulations. Acute toxicity was analyzed with SPF-NMR1 male mice weighing 25 + 3 gr. (each animal was weighed individually to calculate the actual amount administered). 10 animals were chosen for each dose and compound. Saline solutions of the corresponding hydrochlorides were administered in the caudal vein of the mouse, due to the poor solubility of these amines in aqueous saline medium. This administration was carried out in the morning, between 8.00 and 11.00 h. The animals were subsequently stored in air-conditioned rooms at a temperature of 22 ± 1 ° C and 55 + 5% relative humidity. Under these conditions, the behavior of the animals was followed until the seventh day after administration. The average lethal dose and curve was calculated by a programmed probit analysis (Finney) from the percentage of dead animals, observed during the seven-day period after administration.
La tabla I muestra los valores de la dosis letal 50, LD50, del BZN y DMT, definiéndose la LD50 como la dosis mínima administrada a una población de ratones que provoca el 50% de la mortalidad de los mismos. Los resultados mostraron que el compuesto BZN presenta un valor de LD50 3.58 veces mayor que la DMT en las mismas condiciones experimentales.Table I shows the values of lethal dose 50, LD 50 , BZN and DMT, with LD 50 being defined as the minimum dose administered to a population of mice that causes 50% of their mortality. The results showed that the compound BZN has a value of LD 50 3.58 times higher than the DMT under the same experimental conditions.
Figure imgf000007_0001
Figure imgf000007_0001
Tabla I: Valores de la dosis letal 50 (con un límite de confianza del 95%) para los compuestos DMT y BZN junto con la regresión lineal del efecto de la dosis.Table I: Values of lethal dose 50 (with a 95% confidence limit) for the DMT and BZN compounds together with the linear regression of the dose effect.
La FIG.l muestra los diagramas obtenidos del análisis de los datos para tres porcentajes de mortalidad, 16%, 59%) y 84%. El activador BZN presenta valores de la dosis letal más elevados que la DMT para cualquier índice de mortalidad. Los diagramas en la FIG.l ponen de manifiesto el intervalo de la dosis necesario para aumentar la mortalidad del 16% al 84%). Mientras que para la DMT el intervalo es 60 mg/kg, BZN presenta un intervalo de 100 mg/kg en las mismas condiciones experimentales. EJEMPLO 2FIG. 1 shows the diagrams obtained from the analysis of the data for three percentages of mortality, 16%, 59%) and 84%. The BZN activator has higher lethal dose values than DMT for any mortality rate. The diagrams in FIG. 1 show the dose range necessary to increase mortality from 16% to 84%). While for DMT the interval is 60 mg / kg, BZN has a range of 100 mg / kg under the same experimental conditions. EXAMPLE 2
Análisis de la citotoxicidad sobre leucocitos polimorfonucleares del activador 4,4'-bis- dimetilaminobenzidrol (BZN) en comparación con N,N-dimetilamino-4-toluidina (DMT).Cytotoxicity analysis on polymorphonuclear leukocytes of the 4,4'-bis-dimethylaminobenzidrol activator (BZN) compared to N, N-dimethylamino-4-toluidine (DMT).
La citotoxicidad de estos compuestos fue estudiada "in vitro" sobre leucocitos polimorfonucleares extraídos de ratas macho Wistar.The cytotoxicity of these compounds was studied "in vitro" on polymorphonuclear leukocytes extracted from male Wistar rats.
Ratas machos Wistar de peso 250+25 gr. fueron inyectadas en peritoneo con 10 mi. de solución de glucógeno al 6%. A las 16 horas de la inyección se sacrificó a los animales mediante luxación cervical. Se despejó la zona abdominal de piel y grasa sin dañar el interior, y se inyectaron 60 mi. de solución salina de Hank's modificada libre de Ca + y Mg2+ (mHBSS) en la cavidad peritoneal. Después de aplicar masaje manual a la cavidad peritoneal durante 90 seg., se retiraron 50 mi. de fluido peritoneal, que posteriormente se centrifugaron a 4°C a 800g en tubos de polipropileno durante 10 min. desechando el sobrenadante. Los eritrocitos contaminados se aislaron mediante la resuspensión de las células en 5 mi. de solución isotónica tamponada de clorhidrato de tris-amónico (0,38%, pH 7,2) durante 10 min. a 37°C. Se realizó una nueva centrifugación a 4°C a 400g durante 10 min., eliminando el sobrenadante. Las células recuperadas fueron resuspendidas de nuevo en 10 mi. de mHBSS. Se tomó una muestra para realizar un contaje del número de células usando un hemocitómetro. Los 10 mi. recuperados se centrifugaron de nuevo a 4°C a 800g durante 10 min., desechando el sobrenadante. Las células fueron resuspendidas a 2,5x106 céls./min. en una solución sin modificar HBSS que contiene 1,26 mM de Ca2+ y 0,9 mM de Mg2+.Wistar male rats weighing 250 + 25 gr. they were injected in peritoneum with 10 ml. of 6% glycogen solution. At 16 hours after the injection, the animals were sacrificed by cervical dislocation. The abdominal area of skin and fat was cleared without damaging the interior, and 60 ml were injected. of modified Hank's saline solution free of Ca + and Mg 2+ (mHBSS) in the peritoneal cavity. After applying manual massage to the peritoneal cavity for 90 sec., 50 ml were removed. of peritoneal fluid, which were subsequently centrifuged at 4 ° C at 800g in polypropylene tubes for 10 min. discarding the supernatant. Contaminated erythrocytes were isolated by resuspension of the cells in 5 ml. of isotonic buffered solution of tris-ammonium hydrochloride (0.38%, pH 7.2) for 10 min. at 37 ° C. A new centrifugation was performed at 4 ° C at 400g for 10 min., Eliminating the supernatant. The recovered cells were resuspended again in 10 ml. of mHBSS. A sample was taken to count the number of cells using a hemocytometer. The 10 mi. recovered were centrifuged again at 4 ° C at 800g for 10 min., discarding the supernatant. The cells were resuspended at 2.5x10 6 cells / min. in an unmodified HBSS solution containing 1.26 mM Ca 2+ and 0.9 mM Mg 2+ .
Se incubó a 37°C durante 10 min. 500μl de células resuspendidas con lOμl de DMT o BZN a tres concentraciones diferentes: 1, 0,5 y 0,1M. Seis muestras de 500μl de solución de células se incubaron con lOμl de solución Tritón X-100 al 0,2%> en 0,9% de CINa. Otras seis muestras de 500μl se incubaron con lOμl de dimetilsulfóxido (DMSO) como control. Después de 10 min. de incubación, la solución se centrifugó a 4°C a 800g durante 10 min. Se añadió 50μl de sobrenadante a cada uno de los 96 pocilios de cada microplaca, que contienen 200μl de solución de piruvato sódico 0,63 mM en 50 mM de fosfato (pH 7,5) y l,6μl de una solución 40 mM de NADH. Posteriormente se realizó la medida espectrofotométrica a 37°C mediante un lector de microplaca Anthos HTIII. Se efectuaron lecturas a 340 nm durante 60 seg., tomándose medidas cada 20 seg.It was incubated at 37 ° C for 10 min. 500μl of cells resuspended with 10 µL of DMT or BZN at three different concentrations: 1, 0.5 and 0.1M. Six samples of 500 μl of cell solution were incubated with 10 μl of 0.2% Triton X-100 solution> in 0.9% CINa. Another six 500μl samples were incubated with 10 µL of dimethylsulfoxide (DMSO) as a control. After 10 min. incubation, the solution was centrifuged at 4 ° C at 800g for 10 min. 50μl of supernatant was added to each of the 96 wells of each microplate, which they contain 200μl of 0.63 mM sodium pyruvate solution in 50 mM phosphate (pH 7.5) and l, 6μl of a 40 mM solution of NADH. Subsequently, the spectrophotometric measurement was performed at 37 ° C using an Anthos HTIII microplate reader. Readings were made at 340 nm for 60 sec., Taking measurements every 20 sec.
La FIG.2 muestra la citotoxicidad de BZN y DMT obtenidas a diferentes concentraciones molares 0.1, 0.5 y 1.0 M. La citotoxicidad de la DMT aumenta con la concentración alcanzando un nivel de citotoxicidad del 52.5%> (con respecto al control Tritón X-100 usado como control positivo) cuando la concentración de DMT es 1.0 M. Sin embargo, la citotoxicidad de BZN no cambia notablemente con la concentración alcanzando niveles menores del 30% para cualquier concentración.FIG. 2 shows the cytotoxicity of BZN and DMT obtained at different molar concentrations 0.1, 0.5 and 1.0 M. The cytotoxicity of DMT increases with the concentration reaching a cytotoxicity level of 52.5%> (with respect to the Triton X-100 control used as a positive control) when the DMT concentration is 1.0 M. However, the cytotoxicity of BZN does not change markedly with the concentration reaching levels below 30% for any concentration.
EJEMPLO 3EXAMPLE 3
Actividad antimicrobiana del 4,4'-bis-dimetilaminobenzidrol (BZN) en comparación con aquella de la N,N-dimetilamino-4-toluidina (DMT).Antimicrobial activity of 4,4'-bis-dimethylaminobenzidrol (BZN) compared to that of N, N-dimethylamino-4-toluidine (DMT).
Por razones prácticas de acuerdo con la aplicación de estos compuestos, se seleccionaron para probar la actividad de las diferentes aminas, dos bacilos Gram negativos diferentes (E. coli y P. aeruginosa) y un coco Gram positivo (S. aureus), presentes habitualmente en la flora del huésped, así como uno de los hongos patógenos más representativos (C. albicans). Se probó igualmente el efecto del dimetilsulfóxido (DMSO) (20% v/v). Los valores obtenidos fueron apreciablemente mayores que los de las aminas, y por lo tanto el efecto de DMSO no fue significativo en estos experimentos.For practical reasons according to the application of these compounds, two different Gram negative bacilli (E. coli and P. aeruginosa) and a Gram positive coconut (S. aureus), usually present, were selected to test the activity of the different amines. in the flora of the host, as well as one of the most representative pathogenic fungi (C. albicans). The effect of dimethylsulfoxide (DMSO) (20% v / v) was also tested. The values obtained were appreciably higher than those of the amines, and therefore the effect of DMSO was not significant in these experiments.
Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538 y Candida albicans CECT 1001 se obtuvieron a partir de la Colección de Cultivos de Clase Española. Las bacterias se cultivaron rutinariamente en medio de cultivo de Soja-Tripticasa (Difco) y el microorganismo C. albicans en YED (Extracto de Levadura [Difco] 2%, dextrosa 2% p/v en agua destilada), ambos medios se solidificaron con agar (0,2% p/v) cuando fue necesario. La actividad antimicrobiana se ensayó bien en medio de cultivo Mueller Hinton (Difco) con bacterias, o en YED con C. albicans. La incubación se hizo a 37°C en todos los casos.Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538 and Candida albicans CECT 1001 were obtained from the Spanish Class Crops Collection. Bacteria were routinely grown in culture medium of Soya-Tripticasa (Difco) and the microorganism C. albicans in YED (Yeast Extract [Difco] 2%, dextrose 2% w / v in distilled water), both media were solidified with agar (0.2% w / v) when necessary. The antimicrobial activity was well tested in Mueller Hinton (Difco) culture medium with bacteria, or in YED with C. albicans. Incubation was done at 37 ° C in all cases.
Los ensayos de dilución de micro-cultivos se realizaron en microplacas de 96 pocilios con 90μl. del medio y lOμl. del inoculo en cada pocilio. Los compuestos a ensayar, se disolvieron en dimetilsulfóxido (DMSO)/agua destilada (20/80 v/v) en una concentración de 10% (p/v). Se prepararon diluciones por duplicado de cada compuesto en cada fila de pocilios, con un intervalo de concentración de 5% a 0,02% (p/v). Se incluyó el DMSO en una fila en un intervalo de concentraciones de 10% a 0,04% (v/v), y una fila se dejó únicamente con medio para ser tomada como control positivo. El tamaño del inoculo se ajustó por espectrofotometría a una densidad óptica (DO) de DO550=0,02, correspondiendo a una densidad de 2xl05 céls./ml. Se añadieron lOμl. de inoculo a cada pocilio y las placas se incubaron durante 24 h. La DO550 se determinó después de cada incubación usando un scanner de microplacas (Organon Teknika, modelo 510), y la concentración mínima inhibitoria (CMI) se definió como la dilución que provoca una reducción del 80% de la densidad óptica con respecto al control positivo.The microculture dilution tests were performed in 96-well microplates with 90μl. of the medium and lOμl. of inoculum in each well. The compounds to be tested were dissolved in dimethylsulfoxide (DMSO) / distilled water (20/80 v / v) in a concentration of 10% (w / v). Duplicate dilutions of each compound in each row of wells were prepared, with a concentration range of 5% to 0.02% (w / v). DMSO was included in a row in a concentration range of 10% to 0.04% (v / v), and a row was left only with means to be taken as a positive control. The inoculum size was adjusted by spectrophotometry to an optical density (OD) of OD 550 = 0.02, corresponding to a density of 2 x 10 5 cells / ml. 10 μl were added. from inoculum to each well and the plates were incubated for 24 h. The OD 550 was determined after each incubation using a microplate scanner (Organon Teknika, model 510), and the minimum inhibitory concentration (MIC) was defined as the dilution that causes an 80% reduction in optical density with respect to the control positive.
Los valores de la CMI para los activadores DMT y BZN junto con los obtenidos para el vehículo DMSO, frente a diferentes microorganismos se muestran en la Tabla II. De acuerdo con el protocolo establecido, el poder antimicrobiano de los compuestos aumenta a medida que el valor de la CMI decrece. Los valores de la CMI del DMSO son considerablemente más altos que aquellos de las aminas y por tanto el efecto del DMSO no es significativo en estos experimentos. Para hacer un estudio comparativo se tuvo en cuenta la relación BZN/DMT. Los resultados mostrados en la Tabla II indican que, frente a las bacterias Gram-negativas BZN es dos veces más activo que la DMT. Los resultados obtenidos para la bacteria Gram-positiva mostraron un aumento en la actividad del BZN de hasta 8.04 veces mayor que la DMT. Los resultados obtenidos para el hongo C. albicans son muy representativos ya que este microorganismo se encuentra presente en la cavidad periodontal y puede ser responsable de una gran cantidad de infecciones traumáticas postoperativas. Una vez más, la actividad de BZN fue 8.04 veces superior que la DMT. The MIC values for DMT and BZN activators together with those obtained for the DMSO vehicle, against different microorganisms are shown in Table II. According to the established protocol, the antimicrobial power of the compounds increases as the value of the MIC decreases. The MIC values of DMSO are considerably higher than those of amines and therefore the effect of DMSO is not significant in these experiments. To make a comparative study, the BZN / DMT relationship was taken into account. The results shown in Table II indicate that, against the Gram-negative bacteria BZN is twice as active as DMT. The results obtained for the Gram-positive bacterium showed an increase in the activity of the BZN of up to 8.04 times greater than the DMT. The results obtained for the C. albicans fungus are very representative since this microorganism is present in the periodontal cavity and may be responsible for a large number of postoperative traumatic infections. Once again, BZN activity was 8.04 times higher than DMT.
Figure imgf000011_0001
Figure imgf000011_0001
Tabla II: Valores de la concentración mínima inhibidora (CMI) de los compuestos definida como la dilución que provoca una reducción del 80% de la densidad óptica del control.Table II: Minimum inhibitory concentration (MIC) values of the compounds defined as the dilution that causes an 80% reduction in the optical density of the control.
EJEMPLO 4EXAMPLE 4
Formulación de cementos óseos utilizando 4,4'-bis-dimetilamino benzidrol (BZN) como activador.Formulation of bone cements using 4,4'-bis-dimethylamino benzidrol (BZN) as activator.
Se formularon composiciones precursoras de cemento óseo utilizando como activador el compuesto 4,4'-bis-dimetilamino benzidrol (BZN) y como fase sólida partículas de poli(metacrilato de metilo) (Perlas QL) cuyas características morfológicas vienen dadas en la tabla de la Tabla III. Las perlas QL son perlas comerciales y fueron suministradas por Industrias Quirúrgicas de Levante.Bone cement precursor compositions were formulated using as an activator the compound 4,4'-bis-dimethylamino benzidrol (BZN) and as solid phase particles of poly (methyl methacrylate) (QL beads) whose morphological characteristics are given in the table of the Table III QL pearls are commercial pearls and were supplied by Levante Surgical Industries.
Figure imgf000011_0002
Tabla III: Características morfológicas de las perlas QL. Así mismo, se ensayó una composición precursora de cemento óseo comercial (CMW 3) como ejemplo comparativo. Las composiciones del cemento comercial y del experimental se recogen en la tabla IV donde se utilizan las siguientes abreviaturas: PMMA = poli(metacrilato de metilo) BPO = Peróxido de benzoilo MMA = metacrilato de metilo DMT = N,N-dimetil-4-toluidina BZN = 4,4'-bis-dimetilamino benzidrol
Figure imgf000011_0002
Table III: Morphological characteristics of the QL pearls. Likewise, a commercial bone cement precursor composition (CMW 3) was tested as a comparative example. The commercial and experimental cement compositions are listed in Table IV where the following abbreviations are used: PMMA = poly (methyl methacrylate) BPO = Benzoyl peroxide MMA = methyl methacrylate DMT = N, N-dimethyl-4-toluidine BZN = 4,4'-bis-dimethylamino benzidrol
Figure imgf000012_0001
Tabla IV: Composición del cemento óseo comercial tomada como ejemplo comparativo junto con la composición precursora de cemento óseo formulada con el compuesto BZN.
Figure imgf000012_0001
Table IV: Composition of commercial bone cement taken as a comparative example together with the bone cement precursor composition formulated with compound BZN.
Para cada ejemplo se utilizó un total de 40 g de fase sólida y 20 mi de la fase líquida. Estas composiciones precursoras de cemento óseo se mezclaron para formar la pasta de cemento que en unos minutos fragua para dar lugar al cemento curado.For each example, a total of 40 g of solid phase and 20 ml of the liquid phase were used. These bone cement precursor compositions were mixed to form the cement paste which in a few minutes set to give the cured cement.
La temperatura pico (Tp¡co) se define como la temperatura máxima alcanzada durante la reacción de polimerización y se registra de acuerdo con la norma ASTM (F451). Los dos componentes de la composición precursora de cemento óseo se mezclan y la pasta resultante se introduce en un molde de teflón. Se coloca un termopar en el centro del molde a una altura de 3 mm en la cavidad interna. Se toma el tiempo desde el comienzo de la mezcla de los dos componentes y se registra la temperatura. Se realizó un promedio de dos medidas para cada formulación. Las exotermas se registraron a una temperatura de 25°C.The peak temperature (T p co) is defined as the maximum temperature reached during the polymerization reaction and recorded according to ASTM (F451) standard. The two components of the bone cement precursor composition are mixed and the resulting paste is introduced into a Teflon mold. A thermocouple is placed in the center of the mold at a height of 3 mm in the internal cavity. Time is taken from the beginning of the Mix the two components and record the temperature. An average of two measurements was performed for each formulation. The exotherms were recorded at a temperature of 25 ° C.
El tiempo del estado pastoso (tpast0so) representa el tiempo en el que la masa de cemento no se adhiere al guante quirúrgico. En este momento el cemento es implantado en el organismo por ejemplo, en la cavidad femoral.The time of the pasty state (t pas t 0s o) represents the time in which the cement mass does not adhere to the surgical glove. At this time the cement is implanted in the organism for example, in the femoral cavity.
El tiempo de curado (tCUrado) se determinó de acuerdo con la norma ASTM (F451) como el tiempo en el que la temperatura de la masa de cemento es la media aritmética de la suma Tmax+Tamb, donde Traax es la temperatura máxima en °C y Tamb es la temperatura ambiente, 23°C.Curing time (t CUADO ) was determined according to ASTM (F451) as the time at which the cement mass temperature is the arithmetic mean of the sum T max + T amb , where T raax is the maximum temperature in ° C and T am b is the ambient temperature, 23 ° C.
El contenido en monómero residual se determinó por espectroscopia de H-NMR. Las muestras se almacenaron a temperatura ambiente en aire durante siete días antes de ser analizadas. Tres muestras de cada composición se disolvieron en cloroformo deuterado y los espectros se registraron en un espectrofotómetro Varían 300MHz.The residual monomer content was determined by H-NMR spectroscopy. The samples were stored at room temperature in air for seven days before being analyzed. Three samples of each composition were dissolved in deuterated chloroform and the spectra were recorded on a 300MHz Vary spectrophotometer.
Los ensayos de tensión y compresión se realizaron en una Máquina de Ensayos Universal Instron. Se utilizó una velocidad de desplazamiento del cabezal de 20 mm/min para el ensayo de compresión según indica la norma ASTM citada anteriormente, mientras que para el ensayo de tracción se utilizó una velocidad de 1 mm/min de acuerdo con las especificaciones de la norma ISO (527-1). Las probetas se prepararon introduciendo la masa de cemento en su estado pastoso en moldes de teflón y ejerciendo una presión de 1.4 MPa durante aproximadamente 30 min. Las probetas se ensayaron después de haber sido almacenadas a temperatura ambiente en aire durante siete días. Se ensayaron un mínimo de siete probetas para cada composición.Tension and compression tests were performed on a Universal Instron Test Machine. A head displacement speed of 20 mm / min was used for the compression test as indicated by the ASTM standard cited above, while for the tensile test a speed of 1 mm / min was used according to the specifications of the standard ISO (527-1). The specimens were prepared by introducing the cement mass in its pasty state in Teflon molds and exerting a pressure of 1.4 MPa for approximately 30 min. The specimens were tested after they were stored at room temperature in air for seven days. A minimum of seven specimens were tested for each composition.
Los valores de los parámetros de curado, el contenido de monómero residual y las propiedades mecánicas de las composiciones de cemento recogidas en la Tabla IV se muestran en la Tabla V. The values of the curing parameters, the residual monomer content and the mechanical properties of the cement compositions listed in Table IV are shown in Table V.
Figure imgf000014_0001
Figure imgf000014_0001
*G. Lewis, Properties of acrylic bone cement: satate of the art review, J. Biomed. Mater. Res., 38, 155-182, 1997.* G. Lewis, Properties of acrylic bone cement: satate of the art review, J. Biomed. Mater. Res., 38, 155-182, 1997.
Tabla V: Valores de los parámetros de curado, monómero residual y propiedades mecánicas de los cementos curados a partir de las composiciones precursoras que se muestran en la Tabla IV.Table V: Cure parameter values, residual monomer and mechanical properties of the cured cements from the precursor compositions shown in Table IV.
Las temperaturas pico de las composiciones precursoras que contienen BZN fueron aproximadamente 10°C inferiores a las obtenidas con las formulaciones comerciales lo que representa un beneficio importante y significativo desde un punto de vista biológico. Los valores de monómero residual de ambas formulaciones fueron del mismo orden indicando que la conversión de la reacción de polimerización activada con el compuesto BZN es comparable a la que se alcanza en las formulaciones comerciales.The peak temperatures of the precursor compositions containing BZN were approximately 10 ° C lower than those obtained with commercial formulations which represents an important and significant benefit from a biological point of view. The residual monomer values of both formulations were of the same order indicating that the conversion of the activated polymerization reaction with the compound BZN is comparable to that achieved in commercial formulations.
Las propiedades mecánicas obtenidas en los ensayos de tracción y compresión de los cementos formulados con BZN fueron comparables a las obtenidas en las formulaciones comerciales, presentando valores un 20% más elevados de la resistencia a tensión y una elongación a rotura un 15% superior al control.The mechanical properties obtained in the tensile and compression tests of the cements formulated with BZN were comparable to those obtained in commercial formulations, presenting 20% higher values of tensile strength and elongation at break 15% higher than the control .
EJEMPLO 5EXAMPLE 5
Implantación intramuscular de varillas de cementos curados a partir de composiciones precursoras formuladas con el activador 4,4'-bis-dimetilamino benzidrol (BZN). Se implantaron varillas de cementos curados a partir de las composiciones precursoras que se muestran en la Tabla IV. Las varillas del cemento comercial se implantaron como control.Intramuscular implantation of cured cement rods from precursor compositions formulated with the 4,4'-bis-dimethylamino benzidrol (BZN) activator. Cured cement rods were implanted from the precursor compositions shown in Table IV. Commercial cement rods were implanted as a control.
Las varillas de cemento (3 mm diámetro x 15 mm longitud) se introdujeron mediante una cánula en el músculo dorsal de ratas hembra Wistar de peso medio 300+10 g. La herida se suturó con un punto de seda 3/0 y se aplicó Betadine®. Se hicieron dos grupos correspondientes a 2 y 3 animales respectivamente, que se sacrificaron a las 4 y 8 semanas de la operación.The cement rods (3 mm diameter x 15 mm length) were introduced by means of a cannula in the dorsal muscle of Wistar female rats of average weight 300 + 10 g. The wound was sutured with a 3/0 silk stitch and Betadine® was applied. Two groups corresponding to 2 and 3 animals were made respectively, which were sacrificed at 4 and 8 weeks after the operation.
Las muestras se fijaron en una disolución tamponada de formol al 10% (tampón fosfato, pH=7,6) y se embebieron en parafina. Se prepararon secciones histológicas que fueron teñidas según la técnica de hematoxilina-eosina. Las muestras se examinaron en un microscopio óptico Nikon Microphot-FXA. Así mismo, la superficie de las varillas explantadas se analizó por microscopía óptica utilizando un microscopio Nikon Eclipse E400 con iluminación estereoscópica utilizando fibra óptica y un filtro de luz.The samples were fixed in a 10% formalin buffered solution (phosphate buffer, pH = 7.6) and embedded in paraffin. Histological sections were prepared that were stained according to the hematoxylin-eosin technique. The samples were examined in a Nikon Microphot-FXA optical microscope. Likewise, the surface of the explanted rods was analyzed by optical microscopy using a Nikon Eclipse E400 microscope with stereoscopic illumination using optical fiber and a light filter.
Las FIG.3-A y FIG.3-B muestran, respectivamente, la imagen histológica de la respuesta del músculo al cemento control (FIG.3-A) y al cemento curado a partir de composiciones precursoras que contienen BZN (FIG.3-B), después de 4 semanas de la implantación. En ambos casos las secciones transversales de tejido circundante mostraron la formación de una membrana fibrosa junto con la presencia de leucocitos polimorfonucleares, macrófagos y eosinófilos. Sin embargo, no se detectaron signos de necrosis como era de esperar debido a la implantación de un cemento curado "in vitro".FIG. 3-A and FIG. 3-B show, respectively, the histological image of the muscle response to the control cement (FIG. 3-A) and to the cured cement from precursor compositions containing BZN (FIG. 3 -B), after 4 weeks of implantation. In both cases the cross sections of surrounding tissue showed the formation of a fibrous membrane together with the presence of polymorphonuclear leukocytes, macrophages and eosinophils. However, no signs of necrosis were detected as expected due to the implantation of a cured cement "in vitro".
Las FIG.3-C y FIG.3-D reflejan la respuesta tisular después de 8 semanas de la implantación. La FIG.3-C muestra la respuesta al cemento comercial CMW 3 y la FIG.3-D la obtenida después de la implantación del cemento que contiene BZN. A este periodo de tiempo se observa que la membrana fibrosa ha crecido convirtiéndose en una cápsula fibrosa rica en fibras de colágeno orientadas. La reacción inflamatoria que se observa en periodos cortos de tiempo ha ido decreciendo considerablemente aunque todavía se detectó la presencia de algunos macrófagos y eosinófilos. En general, puede decirse que la respuesta de los tejidos a la implantación del cemento preparado con BZN no difiere de la obtenida con la implantación de cementos comerciales y puede ser considerada como normal en la implantación de este tipo de materiales.FIG.3-C and FIG.3-D reflect the tissue response after 8 weeks of implantation. FIG. 3-C shows the response to CMW 3 commercial cement and FIG. 3-D obtained after implantation of the cement containing BZN. At this period of time it is observed that the fibrous membrane has grown into a capsule. fibrous rich in oriented collagen fibers. The inflammatory reaction that is observed in short periods of time has been decreasing considerably although the presence of some macrophages and eosinophils was still detected. In general, it can be said that the response of the tissues to the implantation of the cement prepared with BZN does not differ from that obtained with the implantation of commercial cements and can be considered as normal in the implantation of this type of materials.
La serie de fotografías que se presentan en la FIG.4 muestran las superficies de las varillas antes de la implantación y después de 4 semanas de estar implantadas, para el cemento comercial formulado con DMT (FIG.4-A y FIG.4-B) y para el cemento experimental formulado con BZN (FIG.4-C y FIG.4-D). En ambos casos, la superficie antes de la implantación (FIG.4-A y FIG.4-C) presenta algunas irregularidades originadas en el proceso de curado como son la formación de burbujas durante la manipulación de la masa y su introducción en el molde. Las superficies de las varillas explantadas a las 4 semanas fueron lisas, presentando un aspecto como si hubiesen sufrido un proceso de erosión y las irregularidades han desaparecido. Esta erosión puede estar relacionada con la respuesta inflamatoria que tiene lugar en los primeros días después de la implantación. Durante este periodo de tiempo, la presencia de macrófagos e hidroperóxidos junto con las condiciones de pH pueden ser responsables del fenómeno erosivo. Sin embargo, en este caso la superficie de la varilla que contiene BZN (FIG.4-D), además de lo descrito anteriormente, presentó restos de tejido conectivo adheridos al material. Este comportamiento puede ser atribuido a un efecto estimulante del crecimiento tisular que podría estar relacionado con el posible efecto antiséptico intrínseco que parece tener el BZN.The series of photographs presented in FIG. 4 show the surfaces of the rods before implantation and after 4 weeks of being implanted, for commercial cement formulated with DMT (FIG. 4-A and FIG. 4-B ) and for the experimental cement formulated with BZN (FIG. 4-C and FIG. 4-D). In both cases, the surface before implantation (FIG. 4-A and FIG. 4-C) presents some irregularities caused by the curing process such as the formation of bubbles during the handling of the dough and its introduction into the mold . The surfaces of the explanted rods at 4 weeks were smooth, presenting an appearance as if they had undergone an erosion process and the irregularities have disappeared. This erosion may be related to the inflammatory response that occurs in the first days after implantation. During this period of time, the presence of macrophages and hydroperoxides together with the pH conditions may be responsible for the erosive phenomenon. However, in this case the surface of the rod containing BZN (FIG. 4-D), in addition to what was described above, showed connective tissue remains adhered to the material. This behavior can be attributed to a stimulating effect of tissue growth that could be related to the possible intrinsic antiseptic effect that BZN seems to have.
EJEMPLO 6EXAMPLE 6
Implantación intraósea de composiciones precursoras de cemento formuladas con el activador 4,4'-bis-dimetilamino benzidrol (BZN). Composiciones precursoras comerciales de cemento óseo (CMW 3) utilizadas como control y composiciones precursoras que comprenden el compuesto BZN (Tabla IV) se implantaron en la cavidad femoral de conejos de la variedad Nueva Zelanda.Intraosseal implantation of cement precursor compositions formulated with the 4,4'-bis-dimethylamino benzidrol (BZN) activator. Commercial bone cement precursor compositions (CMW 3) used as control and precursor compositions comprising the BZN compound (Table IV) were implanted in the femoral cavity of rabbits of the New Zealand variety.
Se operaron 20 conejos adultos hembra de la variedad Nueva Zelanda de peso medio 3.820 Kg (3.450-4.260 Kg) en condiciones asépticas. Los animales se distribuyeron en dos grupos. Diez animales constituyeron el grupo control en el cual se implantaron las composiciones del cemento comercial CMW 3 y otros diez animales constituyeron el grupo experimental en el cual se implantaron las composiciones que contienen BZN. En la Tabla VI se muestra el diseño experimental y el número de animales implantados en los experimentos.Twenty adult female rabbits of the New Zealand medium-weight 3,820 kg (3,450-4,260 kg) were operated under aseptic conditions. The animals were distributed in two groups. Ten animals constituted the control group in which the CMW 3 commercial cement compositions were implanted and another ten animals constituted the experimental group in which the compositions containing BZN were implanted. Table VI shows the experimental design and the number of animals implanted in the experiments.
Figure imgf000017_0001
Figure imgf000017_0001
Tabla VI: Diseño experimental y número (N) de animales implantados en los experimentos de implantación en el fémur de conejo de las composiciones precursoras de cemento óseo mostradas en la Tabla IV.Table VI: Experimental design and number (N) of animals implanted in the rabbit femur implantation experiments of the bone cement precursor compositions shown in Table IV.
Los grupos control y experimental se operaron en actos quirúrgicos diferentes y, en ambos casos, la implantación se realizó en el fémur izquierdo del conejo.The control and experimental groups were operated on different surgical acts and, in both cases, implantation was performed in the left rabbit femur.
Los conejos fueron premedicados con sulfato de atropina (0.3 mg/Kg, IM) y clorpromacina (10 mg/Kg, IM). Se administró anestesia general por vía intramuscular, clorhidrato de ketamine (50mg/Kg, IM) y fentanilo (0.17 mg/Kg, IM). Después de afeitar el muslo, la zona quirúrgica se desinfectó con yodo y se hizo una incisión longitudinal. Se crearon dos defectos óseos en las extremidades femorales utilizando un taladro de baja velocidad. El cemento en su estado pastoso se inyectó con una jeringa y se dejó curar en el interior durante unos minutos, de manera que la reacción exotérmica tuvo lugar "in situ" después de la implantación. El músculo se suturó con un hilo de vicril y la piel con puntos discontinuos de seda. Después de la cirugía, se permitió a los animales libre actividad en sus jaulas y fueron sacrificados a diferentes tiempos por inyección intravenosa de pentotal®. Los implantes se extrajeron con el tejido adyacente y se fijaron en solución tamponada de formol al 10% (pH=7.4).The rabbits were premedicated with atropine sulfate (0.3 mg / kg, IM) and chlorpromazine (10 mg / kg, IM). General anesthesia was administered intramuscularly, ketamine hydrochloride (50mg / Kg, IM) and fentanyl (0.17 mg / Kg, IM). After shaving the thigh, the surgical area was disinfected with iodine and a longitudinal incision was made. Two bone defects were created in the femoral limbs using a low speed drill. The cement in its pasty state was injected with a syringe and allowed to cure inside for a few minutes, so that the exothermic reaction took place "in situ" after implantation. The muscle was sutured with a vicril thread and the skin with discontinuous silk stitches. After surgery, the animals were allowed free activity in their cages and were sacrificed at different times by intravenous injection of pentotal®. The implants were extracted with the adjacent tissue and fixed in 10% formalin buffered solution (pH = 7.4).
Para el análisis histológico las muestras se descalcificaron mediante la técnica de formol nítrico (950 mi de formol al 10% y 50 mi de ácido nítrico) previo a la inclusión en parafina durante 24 h. Posteriormente se realizaron cortes transversales (6μm de espesor) con un microtomo manual (Minot-Leniz) y las muestras se tiñeron mediante la técnica de hematoxilina-eosina para el examen histológico.For histological analysis, the samples were decalcified by the technique of nitric formalin (950 ml of 10% formalin and 50 ml of nitric acid) prior to inclusion in paraffin for 24 h. Subsequently, cross sections (6μm thick) were made with a manual microtome (Minot-Leniz) and the samples were stained using the hematoxylin-eosin technique for histological examination.
La respuesta del tejido óseo a la presencia de cemento se estudió durante el periodo comprendido entre 2 días y 24 semanas. Durante este tiempo ninguno de los animales mostró signos clínicos de alteración en el área del fémur así como tumores en ningún otro sitio.The response of bone tissue to the presence of cement was studied during the period between 2 days and 24 weeks. During this time none of the animals showed clinical signs of alteration in the area of the femur as well as tumors in any other site.
La evaluación microscópica del material explantado mostró que todas las muestras fueron toleradas bien por el tejido óseo.Microscopic evaluation of the explanted material showed that all samples were well tolerated by bone tissue.
Dos días después de la inyección del cemento la evaluación histológica del tejido adyacente en el lugar de la inyección mostró la presencia de algunas áreas de necrosis para cualquier composición. La causa de esta necrosis no puede ser claramente elucidada. La necrosis puede ser producida por causas térmicas, químicas así como por el propio procedimiento quirúrgico. El daño de algunas zonas locales del hueso es inevitable en cualquier procedimiento de implantación que conlleve el taladro del hueso, aunque se haga a baja velocidad, y también la inyección de un material que cura "in situ"(P.A. Revell, M. Braden, M.A.R. Freeman, Review of the biological response to a novel bone cement containing poly(ethyl methacrylate) and n-butyl methacrylate, Biomaterials, 19, 1579-1586 (1998)). Así mismo, se observaron leucocitos polimorfonucleares alrededor de los restos del material, y en otras áreas más alejadas del material se detectaron células mononucleares, principalmente eosinófilos, linfocitos, células plasmáticas y macrófagos, claros indicadores de la reacción de inflamación.Two days after the cement injection, the histological evaluation of the adjacent tissue at the injection site showed the presence of some areas of necrosis for any composition. The cause of this necrosis cannot be clearly elucidated. Necrosis can be caused by thermal, chemical causes as well as by the surgical procedure itself. The damage of some local areas of the bone is inevitable in any implantation procedure that involves the drilling of the bone, although it is done at low speed, and also the injection of a material that heals "in situ" (PA Revell, M. Braden, MAR Freeman, Review of the biological response to a novel bone cement containing poly (ethyl methacrylate) and n-butyl methacrylate, Biomaterials, 19, 1579-1586 (1998)). Likewise, polymorphonuclear leukocytes were observed around the remains of the material, and in other areas farther from the material, mononuclear cells, mainly eosinophils, lymphocytes, plasma cells and macrophages, clear indicators of the inflammation reaction were detected.
Las FIG.5-A y FIG.5-B muestran microfotografías representativas de las secciones histológicas de la interfaz entre el cemento CMW 3 o el cemento experimental respectivamente, y el tejido adyacente a los 2 días de la implantación.FIG.5-A and FIG.5-B show representative photomicrographs of the histological sections of the interface between the CMW 3 cement or the experimental cement respectively, and the adjacent tissue 2 days after implantation.
La principal diferencia entre la formulación experimental y la control se encuentra en el proceso de oseoformación que se manifiesta por la presencia de una matriz ósea no mineralizada (osteoide). Este proceso fue mucho más acusado para la implantación del cemento experimental, como se refleja en la FIG.5-C.The main difference between the experimental formulation and the control is found in the oseoformation process that is manifested by the presence of a non-mineralized bone matrix (osteoid). This process was much more pronounced for the implantation of experimental cement, as reflected in FIG. 5-C.
El análisis histológico del tejido adyacente al cemento óseo a las 2 semanas reveló características similares para ambos tipos de cemento en lo que respecta a la necrosis y a la respuesta inflamatoria como se describe arriba. Sin embargo, con relación a la osteogénesis la formulación experimental presentó la mejor respuesta manifiesta en una mayor neoformación de trabécula ósea.Histological analysis of the tissue adjacent to the bone cement at 2 weeks revealed similar characteristics for both types of cement in regards to necrosis and the inflammatory response as described above. However, in relation to osteogenesis, the experimental formulation presented the best manifest response in a greater bone trabecula neoformation.
El análisis histológico a las 4 semanas de la implantación mostró la existencia de menos áreas necróticas que en los periodos anteriores como consecuencia del proceso de reabsorción llevado a cabo por los macrófagos. La respuesta inflamatoria continua su proceso reparativo observándose una mayor cantidad de macrófagos y células gigantes multinucleadas rodeando los restos de los dos tipos de cemento.The histological analysis at 4 weeks after implantation showed the existence of fewer necrotic areas than in the previous periods as a consequence of the reabsorption process carried out by the macrophages. The inflammatory response continues its reparative process, observing a greater amount of macrophages and multinucleated giant cells surrounding the remains of the two types of cement.
Sin embargo, como en periodos anteriores de tiempo, el proceso de neoformación ósea difiere en la respuesta a un cemento u otro. El cemento que contiene BZN dio lugar a una mayor y más rápida neoformación caracterizada por la presencia de un ribete eosinófilo más abundante que traduce el depósito osteoide. Esta neoformación ósea adquiere la característica morfológica de englobar los restos del material. Las FIG.6-A y FIG.6-B ilustran estas diferencias en cuanto a la neoformación ósea se refiere que acompañan a la implantación de un cemento comercial (CMW 3) y al cemento experimental que contiene BZN, respectivamente.However, as in previous periods of time, the bone neoformation process differs in response to one cement or another. The cement containing BZN resulted in a greater and faster neoformation characterized by the presence of an eosinophilic edging more abundant that translates the osteoid deposit. This bone neoformation acquires the morphological characteristic of encompassing the remains of the material. FIG.6-A and FIG.6-B illustrate these differences in terms of bone neoformation that accompany the implantation of a commercial cement (CMW 3) and the experimental cement containing BZN, respectively.
La FIG.7 muestra la zona de epífisis femoral después de 12 semanas de la implantación del cemento experimental donde se puede observar una intensa proliferación ósea, con trabéculas finas, separadas por un laberinto de espacios interconexionados que contienen médula ósea.FIG. 7 shows the area of femoral epiphysis after 12 weeks of the implantation of the experimental cement where an intense bone proliferation can be observed, with fine trabeculae, separated by a labyrinth of interconnected spaces that contain bone marrow.
La respuesta histológica a largos periodos de tiempo, 24 semanas, cuando se puede considerar que se ha alcanzado el estado estacionario, presentaba la misma tendencia observada en los periodos anteriores. Lo más relevante en este periodo es la formación ósea que se aprecia en la respuesta al cemento experimental, que demuestra la confluencia de trabéculas óseas, sobre todo en la diáfisis, de tal forma que el espesor del hueso neoformado es más notable que el obtenido con la formulación control de CMW 3.The histological response to long periods of time, 24 weeks, when it can be considered that the steady state has been reached, presented the same tendency observed in the previous periods. The most relevant in this period is the bone formation that can be seen in the response to the experimental cement, which demonstrates the confluence of bone trabeculae, especially in the diaphysis, so that the thickness of the neoformed bone is more noticeable than that obtained with the control formulation of CMW 3.
Las FIG.8-A y FIG.8-B, correspondientes a la respuesta histológica para este periodo de tiempo (24 semanas) a los cementos control y experimental respectivamente, ilustran este comportamiento. Particularmente, la FIG.8-B corresponde a la diáfisis femoral tratada con el cemento experimental y muestra la disposición laminar que adquiere la formación ósea, con lagunas conteniendo osteocitos. FIG.8-A and FIG.8-B, corresponding to the histological response for this period of time (24 weeks) to the control and experimental cements respectively, illustrate this behavior. In particular, FIG. 8-B corresponds to the femoral shaft treated with the experimental cement and shows the laminar disposition that acquires bone formation, with osteocyte-containing lagoons.
Breve descripción de las figurasBrief description of the figures
FIG.l es una gráfica que muestra la relación entre la dosis de BZN o DMT, y el porcentaje de mortalidad determinada por inyección intravenosa en ratones de una solución salina alFIG. 1 is a graph showing the relationship between the dose of BZN or DMT, and the percentage of mortality determined by intravenous injection in mice of a saline solution at
5% del correspondiente clorhidrato. FIG.2 es una gráfica que muestra los efectos de los compuestos DMT y BZN sobre la integridad de leucocitos polimorfonucleares de rata medidos en términos de liberación de5% of the corresponding hydrochloride. FIG.2 is a graph showing the effects of DMT and BZN compounds on the integrity of rat polymorphonuclear leukocytes measured in terms of release of
LDH.LDH.
FIG.3 es una serie de fotografías que muestran la respuesta histológica a la implantación intramuscular en ratas de varillas de cemento comercial CMW 3 y de cemento experimental formulado con el compuesto BZN.FIG. 3 is a series of photographs showing the histological response to intramuscular implantation in rats of commercial CMW 3 cement rods and experimental cement formulated with the compound BZN.
FIG.4 es una serie de fotografías que muestran las superficies de las varillas antes y después de 4 semanas de implantación, de cemento comercial CMW 3 y de cemento experimental formulado con el compuesto BZN.FIG. 4 is a series of photographs showing the surfaces of the rods before and after 4 weeks of implantation, of commercial cement CMW 3 and of experimental cement formulated with the compound BZN.
FIG.5 es una serie de fotografías que muestran la respuesta histológica a los dos días de la implantación intraósea de las composiciones precursoras de cemento óseo mostradas en laFIG. 5 is a series of photographs showing the histological response two days after the intraosseous implantation of the bone cement precursor compositions shown in the
FIG.6, curadas "in situ".FIG. 6, cured "in situ".
FIG.6 es una serie de fotografías que muestran la respuesta histológica a las 4 semanas de la implantación intraósea, de las composiciones precursoras de cemento óseo mostradas en la FIG.6, curadas "in situ". FIG.7 es una fotografía que muestra el análisis histológico del tejido óseo adyacente a un cemento formulado con el activador BZN después de 12 semanas de la implantación.FIG. 6 is a series of photographs showing the histological response 4 weeks after the intraosseous implantation of the bone cement precursor compositions shown in FIG. 6, cured "in situ". FIG. 7 is a photograph showing the histological analysis of bone tissue adjacent to a cement formulated with the BZN activator after 12 weeks of implantation.
FIG.8 es una serie de fotografías que muestran la reacción histológica a la implantación intraósea de un cemento comercial y de un cemento experimental formulado con BZN después de 24 semanas de la implantación. FIG. 8 is a series of photographs showing the histological reaction to intraosseous implantation of a commercial cement and an experimental cement formulated with BZN after 24 weeks of implantation.

Claims

Reivindicaciones l.Una composición para su uso como composición precursora de cemento óseo caracterizada porque comprende una o más aminas terciarias como activadores en una cantidad de hasta un 3%-p con respecto al peso total de la fase líquida y porque el activador es un compuesto cuya estructura química general es:Claims 1. A composition for use as a bone cement precursor composition characterized in that it comprises one or more tertiary amines as activators in an amount of up to 3% -p with respect to the total weight of the liquid phase and because the activator is a compound whose general chemical structure is:
Figure imgf000022_0001
Figure imgf000022_0001
Fórmula (I)Formula (I)
donde:where:
Ri es un radical metilo, etilo, propilo, isopropilo, butilo, isobutilo o tert-butilo.Ri is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
R2 es un radical metilo, etilo, propilo, isopropilo, butilo, isobutilo o tert-butilo.R 2 is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
R3 es un radical metilo, etilo, propilo, isopropilo, butilo, isobutilo o tert-butilo. i es un radical metilo, etilo, propilo, isopropilo, butilo, isobutilo o tert-butilo.R 3 is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical. i is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl radical.
2.Una composición según la reivindicación 1 porque también incluye metacrilato de metilo, MMA, en una cantidad de 95-98 %-p con respecto al peso total de la fase líquida.2. A composition according to claim 1 because it also includes methyl methacrylate, MMA, in an amount of 95-98% -p with respect to the total weight of the liquid phase.
3. Una composición según la reivindicación 1 caracterizado porque también incluye uno o más inhibidores en una cantidad de hasta un 0.01 %-p y/o uno o más estabilizadores en una cantidad de hasta un 0.01 %-p.3. A composition according to claim 1 characterized in that it also includes one or more inhibitors in an amount of up to 0.01% -p and / or one or more stabilizers in an amount of up to 0.01% -p.
4. Una composición según la reivindicación 3 caracterizada porque el inhibidor y/o el estabilizador es un compuesto de la familia de las quinonas. 4. A composition according to claim 3 characterized in that the inhibitor and / or the stabilizer is a compound of the quinone family.
5. Una composición para su uso como composición precursora de cemento óseo caracterizada porque contiene una fase sólida y una fase líquida, donde la fase líquida comprende una composición según una cualquiera de las reivindicaciones 1 a 4.5. A composition for use as a bone cement precursor composition characterized in that it contains a solid phase and a liquid phase, wherein the liquid phase comprises a composition according to any one of claims 1 to 4.
6. Una composición según las reivindicaciones 1 ó 5, caracterizada porque la fase sólida comprende partículas de poli(metacrilato de metilo) prepolimerizado, PMMA, o copolímeros de MMA con otros monómeros, y porque las partículas están presentes en una cantidad comprendida entre el 70 y 95 %-p, preferiblemente entre el 80 y 95 %-p con respecto al peso total de la fase sólida.A composition according to claims 1 or 5, characterized in that the solid phase comprises particles of prepolymerized poly (methyl methacrylate), PMMA, or copolymers of MMA with other monomers, and that the particles are present in an amount between 70 and 95% -p, preferably between 80 and 95% -p with respect to the total weight of the solid phase.
7. Una composición precursora de cemento óseo según las reivindicaciones 1, 4 o 6, caracterizada porque la fase sólida comprende uno o más iniciadores en una cantidad de hasta un 3 %-p y/o uno o más agentes radiopacos en una cantidad de hasta un 20 %-p con respecto al peso total de la fase sólida.7. A bone cement precursor composition according to claims 1, 4 or 6, characterized in that the solid phase comprises one or more initiators in an amount of up to 3% -py / or one or more radiopaque agents in an amount of up to 20% -p with respect to the total weight of the solid phase.
8. Una composición precursora de cemento óseo según la reivindicación 7 caracterizada porque el iniciador es peróxido de benzoilo y/o el/los agentes radiopacos es/son seleccionados entre sulfato de bario, dióxido de zirconio u óxido de tántalo.8. A bone cement precursor composition according to claim 7 characterized in that the initiator is benzoyl peroxide and / or the radiopaque agents are / are selected from barium sulfate, zirconium dioxide or tantalum oxide.
9. Un cemento caracterizado porque se obtiene por curado de una composición según una cualquiera de las reivindicaciones 1 a 8 y a las que se les adiciona cualquier tipo de sales de calcio y muy especialmente fosfatos de calcio.9. A cement characterized in that it is obtained by curing a composition according to any one of claims 1 to 8 and to which any type of calcium salts and especially calcium phosphates are added.
10. Un proceso de obtención de un cemento que comprende una fase sólida y una fase líquida, el mezclado de ambas fases y la consiguiente polimerización de la fase líquida para dar lugar al material curado, donde la fase sólida comprende partículas de polímero y uno o más iniciadores de tipo radical como se detalla en cualquiera de las reivindicaciones 6 a 8, y la fase líquida es una composición como se detalla en cualquiera de las reivindicaciones 1 a 5. 10. A process for obtaining a cement comprising a solid phase and a liquid phase, the mixing of both phases and the consequent polymerization of the liquid phase to give rise to the cured material, where the solid phase comprises polymer particles and one or more radical type initiators as detailed in any of claims 6 to 8, and the liquid phase is a composition as detailed in any of claims 1 to 5.
11. Uso del cemento óseo obtenido según una cualquiera de las reivindicaciones 1 a 10, entre otros, para cirugía ortopédica y traumatología para la fijación de prótesis de articulaciones, para relleno de cavidades óseas o dentales y como soporte de cesión controlada de medicamentos y compuestos bioactivos. 11. Use of bone cement obtained according to any one of claims 1 to 10, among others, for orthopedic surgery and traumatology for the fixation of joint prostheses, for filling of bone or dental cavities and as a support for controlled transfer of medications and compounds. bioactive
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