WO2008059542A1 - Implant osseux à propriétés de surface améliorées - Google Patents

Implant osseux à propriétés de surface améliorées Download PDF

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Publication number
WO2008059542A1
WO2008059542A1 PCT/IT2006/000805 IT2006000805W WO2008059542A1 WO 2008059542 A1 WO2008059542 A1 WO 2008059542A1 IT 2006000805 W IT2006000805 W IT 2006000805W WO 2008059542 A1 WO2008059542 A1 WO 2008059542A1
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WO
WIPO (PCT)
Prior art keywords
pectin
molar percent
moles
galacturonic acid
bone implant
Prior art date
Application number
PCT/IT2006/000805
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English (en)
Inventor
Clara Cassinelli
Marco Morra
Giovanna Cascardo
Giovanni Agliati
Ignazio Agliati
Original Assignee
Nobil Bio Ricerche S.R.L.
Agliati S.R.L.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nobil Bio Ricerche S.R.L., Agliati S.R.L. filed Critical Nobil Bio Ricerche S.R.L.
Priority to PCT/IT2006/000805 priority Critical patent/WO2008059542A1/fr
Publication of WO2008059542A1 publication Critical patent/WO2008059542A1/fr

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Classifications

    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • 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

  • the present invention relates to a bone implant, in particular for dental, orthopaedic and spinal surgery prosthetics, with improved surface properties with respect to the devices of the prior art.
  • the implanted device is securely fixed in the implant site thanks to the growth of newly formed bone tissue coming into direct contact with the device itself.
  • This phenomenon known as osteointegration, has been comprehensively studied and
  • mesenchymal cells are normally found in an undifferentiated layer in the bone marrow.
  • mesenchymal cells migrate from their normal site and, under a complex series of signals and chemotaxis stimuli, reach the wound site. Once at this site they undergo the so-called differentiation process, i.e. they are transformed from unspecialised progenitor cells into cells specifically tasked with new tissue regeneration.
  • Bone marrow mesenchymal cells can
  • the mechanisms presiding over the transformation along any given lineage in vivo are somewhat poorly understood in the current art. The reason whereby certain surfaces promote cell adhesion but not differentiation is also poorly understood, while it is known that if cells do not adhere to a surface, then they do not differentiate.
  • undifferentiated cells can be directed along the various lineages mentioned by means of soluble agents dissolved in the culture medium, creating the so-called “differentiating media” , as opposed to "basal medium” which maintains the cells in the undifferentiated state.
  • collagen may introduce contamination problems due to it being derived from questionable animal sources (particularly bovine collagen) , or rejection due to the possibility of incompatibility between different species.
  • vitronectin is a protein that is present in low quantities in organisms, and is thus very expensive and has poor stability.
  • the problem underlying the present invention therefore is that of providing a bone implant with improved surface properties with respect to the devices of the known art, particularly capable of increasing osteoblastic differentiation, thus ensuring faster healing for implanted patients.
  • the present invention relates to a bone implant, particularly for dental, orthopaedic and spinal surgery prosthetics, onto the surface of which is immobilised pectin, or a pectin fraction, obtained by v subjecting pectin to enzymatic digestion.
  • pectins were discovered in the 19th century, being initially labelled by the family name "pectic acid” , due to the presence of carboxylic acid groups.
  • Figure 1 shows the typical structure of pectins.
  • Pectins are very complex polysaccharides which can be generally subdivided into three different domains: a domain defined as “smooth” , predominantly composed of polygalacturonan, with very few or no side chains, and two domains defined as “hairy” , which are based on polysaccharides such as Rhamnogalacturonan I and Rhamnogalacturonan II, and which are highly branched.
  • the various pectin domains can be purified or, in any case, modified, by chemical action (hydrolysis, saponification) or by means of the action of enzymes and enzyme preparations, as described in the literature for the sector. Both pectins and the enzyme preparations required for the degradation thereof, are widely available commercially.
  • pectin or fractions thereof obtained by means of enzymatic digestion, when bound to the surface of solid substrates, are capable of stimulating the differentiation of bone marrow mesenchymal cells along the osteogenic lineage pathway in a manner superior to that observed with uncoated substrates.
  • Pectins are extracted from the cell walls of fruit, vegetables, or the by-products thereof, that are rich in cell walls, for example the peel.
  • fruit and vegetables include apple, pear, onion, carrot, leek and potato.
  • Pectins are extracted using the extraction and purification methods known in the sector and which are thus not reported at this time. Alternatively, pectins may be purchased.
  • the pectin fractions used for the purposes of the invention are obtained by subjecting the pectins, fruit, vegetables, or the pectin-containing by-products thereof to enzymatic digestion according to the methods known in the sector and described, for example, in Schols, efc al.; J. Carbohydr. Res. 1990, 206, 117-129.
  • the pectins or fruit, vegetables or the pectin- containing by-products thereof are mixed with water and the pH adjusted to 4-6, preferably at pH of around 5, using a base, for example sodium hydroxide.
  • a base for example sodium hydroxide.
  • One or more enzymes advantageously selected from the group consisting of cellulase, hemi-cellulase, pectinase
  • pectin-lyase Pg lyase
  • pectin methyl esterase polygalacturonase
  • acetyl esterase acetyl esterase
  • rhamnogalacturonan hydrolase lyase
  • galactanase arabinanase
  • xylogalacturonan hydrolase mixtures thereof are added, in quantities of between 0.025 and 0.25% by weight, preferably between 0.05 and 0.15% by weight (the percentage by weight of enzyme is in reference to the quantity of starting product, i.e. pectins, fruit, vegetables or the pectin-containing by-products thereof) .
  • the commercial enzyme preparations Rapidase C600 and Rapidase Liq+ may be used.
  • the mixture is left incubating for 2-4 hours, preferably for approx. 3 hours, at a temperature of between 40 and 50 0 C, preferably approx. 45 0 C and with constant stirring.
  • a second aliquot of one or more enzymes selected from the above-indicated group is added in quantities of between 0.025 and 0.25% by weight, preferably between 0.05 and 0.15% by weight, and the incubation continued for 1-2 hours, preferably approx. 1.5 hours.
  • the enzyme is then inactivated by heating the mixture to approx. 90 0 C for approx. half an hour.
  • the pectin fraction thus obtained is finally purified by high pressure size exclusion chromatography (HPSEC) and lyophilised.
  • HPSEC high pressure size exclusion chromatography
  • the pectin fraction that can be obtained by means of the above described process essentially comprises the pectin "hairy" regions (HRs) , in that the enzymes mainly degrade the smooth regions, ⁇ i.e. the homogalacturonan domain, thus leaving the HRs.
  • HRs pectin "hairy” regions
  • said hairy regions (HRs) obtained by enzymatic digestion of pectins are modified hairy regions (MHRs) .
  • the enzymes in part, modify the composition and quantity of .. the sugar residues constituting the "hairy” regions, thus giving rise to modified "hairy” regions (MHRs) .
  • the modified "hairy" regions comprise between 5 to 30 molar percent rhamnose, preferably 10-20 molar percent; 2 to 60 molar percent arabinose, preferably 5-25 molar percent; 7 to 30 molar percent galactose, preferably 10-25 molar percent; 0 to 35 molar percent xylose, preferably 0-25 molar percent; 5 to 60 molar percent galacturonic acid; preferably 10-50 molar percent; 0 to 60 moles of methyl esters per 100 moles of galacturonic acid, preferably 0-50 moles/100 moles of galacturonic acid; 0 to 80 moles of acetyl esters per 100 moles of galacturonic acid, preferably 0-70 moles/100 moles of galacturonic acid.
  • the rhamnose to galacturonic acid ratio is comprised of between 0.10 and 0.60, preferably 0.20 and 0.40.
  • Immobilisation of the pectin or pectin fraction comprising HRs and/or MHRs onto the surface of the bone implant occurs by means of prior modification of the surface of the implant by means of plasma deposition or polyimine absorption.
  • the surface of the bone implant is functionalised by means of the introduction of amine groups by means of plasma deposition of allylamine.
  • the implant is immersed in an aqueous solution of pectin or pectin fraction, at a concentration of between 0.1% and 1%, using a suitable coupling agent in order to promote the formation of covalent bonds between the functionalised surface and the polysaccharide.
  • suitable coupling agent include: carbodiimmide and N-hydroxysuccinimide .
  • the implant is left immersed in the polysaccharide solution for approx. 2-24 hours and then washed with distilled water and air dried. » ⁇
  • the surface of the implant is functionalised with amine groups, by immersing it in a 0.2% aqueous solution of polyimine, preferably polyethylenimine, for 1-3 hours.
  • the implant is immersed in an aqueous solution of pectin or pectin fraction under the same conditions as described above, in order to give a bone implant coated with an agent capable of stimulating osteoblastic differentiation,, thus enhancing the rate of bone implant osteointegration.
  • the present invention allows greatly reduced post-operative healing times with respect to the use of uncoated implants, all advantageous with respect to the health and quality of life of the patients.
  • the topography of the device metal or polymer surface is altered by performing a roughening or "texturisation” treatment.
  • roughening may be obtained by sanding, plasma spray deposition, acid treatment and electrochemical treatment .
  • the bone implants of the invention include, for example, dental prosthetics, such as implantable titanium screws, implantable blades, orthopaedic prosthetics, such as femoral prosthetics and bone prosthetics in general, plates, fixing screws, spinal prosthetics, such as spinal cages, screws, spinal fusion and fixing devices, artificial intervertebral discs and the parts thereof.
  • dental prosthetics such as implantable titanium screws, implantable blades, orthopaedic prosthetics, such as femoral prosthetics and bone prosthetics in general, plates, fixing screws, spinal prosthetics, such as spinal cages, screws, spinal fusion and fixing devices, artificial intervertebral discs and the parts thereof.
  • the bone implants of the invention are consisting of any biocompatible material suitable for insertion into the human body, for example titanium, steel or biodegradable polymers.
  • the invention relates to the use of pectin or pectin fractions, for the preparation of a medicament for increasing osteoblastic differentiation.
  • said polysaccharides increase mesenchymal cell differentiation.
  • the pectin fractions correspond to those'- described above.
  • a certain quantity of apples are homogenised using a blender.
  • the preparation is split into two parts and treated with the following commercial preparations: A) Rapidase C600, manufactured by DSM Food Specialities B) Rapidase Liq+, manufactured by DSM Food Specialities
  • the juice thus obtained is subjected to ultrafiltration and the retentate lyophilised in order to obtain the modified hairy parts (MHR) .
  • MHR modified hairy parts
  • Example 4 Binding MHRs to the surface of solids [0047]
  • the reaction in the previous example is carried out as reported above. However, the surface aminisation step is not achieved by means of plasma deposition, but by means of the absorption of polyethyleneimine (PEI, Aldrich) from a 0.2% solution in water, for 2 hours.
  • PEI polyethyleneimine
  • the cells used for the test are normal human bone marrow mesenchymal cells l (hMSC) purchased from the Cambrex cell culture bank through Cambrex Bio Science Italia S.r.l.
  • hMSC normal human bone marrow mesenchymal cells l
  • the purity of the cell lineage and the capacity to differentiate along the osteogenic, chondrogenic and adipogenic pathways, have been verified by means of flow cytometry. Tests have been conducted according to the protocols listed in the international bibliography. In particular, half samples have been subjected to evaluation of alkaline phosphatase (ALP) activity, in both osteogenic and non-osteogenic media. The other half samples have been used for the cell proliferation tests, evaluated by means of the MTT test,
  • ALP alkaline phosphatase
  • the MTT test measures the efficiency of the mitochondrial Krebs cycle enzyme succinate dehydrogenase
  • 25 holds 6 samples of polystyrene modified with MHR, 6 samples of unmodified polystyrene and one control, into which is added just culture medium without cells.
  • the containers are subsequently placed in an incubator at 37 0 C, 5% CO 2 and relative humidity of 98%, some for 3 days, others for 7, and others for 10.
  • 3 samples were used for the evaluation of alkaline phosphatase activity, 3 for the evaluation of cell proliferation by means of the MTT test.
  • alkaline phosphatase catalyses the transformation of p-nitrophenylphosphate into paranitrophenol according to the reported scheme: ALP p-nitrophenylphosphate > p-nitrophenol + H 3 PO 4 . (colourless) 405 nm (yellow) 5
  • the value for ALP activity was normalised for the value of the absorbance produced by the samples of the same family subjected to the MTT test and the phosphatase activity expressed as the specific enzyme activity.
  • a solution of 5 mg/mL of a soluble tetrazolium salt (in particular, 3- (4, 5-dimethylthiazol-2yl) -2 , 5 diphenyl tetrazolium bromide) was added.
  • a soluble tetrazolium salt in particular, 3- (4, 5-dimethylthiazol-2yl) -2 , 5 diphenyl tetrazolium bromide
  • the enzyme succinate dehydrogenase induces the transformation of the tetrazolium salts, initially soluble and yellow in colour, into a blue-coloured water insoluble product, the formazan: the greater the quantity of precipitate, the higher the level of enzyme activity and, consequently, the number of metabolically viable cells.
  • the precipitate was solubilised with acidic isopropanol (2% IN HCl) and measured spectrophotometrically at a wavelength of 560 nm by means of a Genios plate reader (TECAN S . r.1. ) .
  • Genios plate reader TECAN S . r.1.
  • PSMHR is significantly different from that recorded on cell culture polystyrene and from that of the control without cells.
  • Cell adhesio ⁇ . occurs on both cell culture polystyrene and on MHR coated polystyrene, but the PSMHR promotes substantially enhanced osteoblastic differentiation with respect to uncoated polystyrene.
  • the PSMHR induces significantly higher alkaline phosphatase expression with respect to uncoated polystyrene, thus confirming the unexpected effect on osteogenic differentiation.
  • control comprising just culture medium without cells.
  • the samples prepared are used for evaluating osteoblastic differentiation by means of measuring specific alkaline phosphatase activity, as for the previous example.
  • the tests are performed in differentiating medium. The following results are obtained (the numbers report the absorbance measured using a Tecan microplate reader. .Obviously, the higher the number, the greater the enzyme activity:
  • the cells grown on PSMHR express much higher levels of alkaline phosphatase with respect to titanium, both whether smooth, or with the topography bestowed by the acid etching treatment .
  • the above data indicate that the ⁇ MHR coating significantly, and unexpectedly, improves the ability of the surface to stimulate osteoblastic differentiation, making it significantly better with respect to that provided by the surface of the current material of choice for devices coming into contact with bone .

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Medicinal Chemistry (AREA)
  • Dermatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Surgery (AREA)
  • Vascular Medicine (AREA)
  • Materials For Medical Uses (AREA)

Abstract

Implant osseux revêtu de pectine et/ou de fractions de pectine résultant du traitement de pectine et/ou de tissus végétaux contenant de la pectine avec une ou plusieurs enzymes pouvant être: cellulase, hémi-cellulase, pectinase (Pgase), pectin-lyase (Pg lyase), pectine méthyli estérase, polygalacturonase, acétyl estérase, rhamnogalacturonanee hydrolase et lyase, galactanase, arabinanase et xylogalacturonane hydrolase.
PCT/IT2006/000805 2006-11-17 2006-11-17 Implant osseux à propriétés de surface améliorées WO2008059542A1 (fr)

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PCT/IT2006/000805 WO2008059542A1 (fr) 2006-11-17 2006-11-17 Implant osseux à propriétés de surface améliorées

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009057174A1 (fr) * 2007-11-02 2009-05-07 Nobil Bio Ricerche S.R.L. Dispositif médical intracorporel

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001059137A1 (fr) * 2000-02-10 2001-08-16 Biologic A/S Procede de remodelage des structures de polysaccharide de paroi cellulaire dans les plantes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001059137A1 (fr) * 2000-02-10 2001-08-16 Biologic A/S Procede de remodelage des structures de polysaccharide de paroi cellulaire dans les plantes

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KEUREN J F W ET AL: "Thrombogenicity of polysaccharide-coated surfaces", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 24, no. 11, May 2003 (2003-05-01), pages 1917 - 1924, XP004412403, ISSN: 0142-9612 *
MORRA M ET AL: "Biomaterials surface characterization and modification", INTERNATIONAL JOURNAL OF ARTIFICIAL ORGANS, MILAN, IT, vol. 29, no. 9, September 2006 (2006-09-01), pages 824 - 833, XP008081984, ISSN: 0391-3988 *
MORRA M ET AL: "Surface engineering of titanium by collagen immobilization. Surface characterization and in vitro and in vivo studies", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 24, no. 25, November 2003 (2003-11-01), pages 4639 - 4654, XP004452457, ISSN: 0142-9612 *
MORRA M. ET AL.: "EFFECTS OF MOLECULAR WEIGHT AND SURFACE FUNCTIONALIZATION ON SURFACE COMPOSITION AND CELL ADHESION TO HYALURONAN COATED TITANIUM", BIOMEDICINE & PHARMACOTHERAPY, vol. 60, no. 8, September 2006 (2006-09-01), pages 365 - 369, XP002445361 *
MORRA M. ET AL.: "Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions.", BIOMACROMOLECULES, vol. 5, 2004, pages 2094 - 2104, XP002445360 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009057174A1 (fr) * 2007-11-02 2009-05-07 Nobil Bio Ricerche S.R.L. Dispositif médical intracorporel

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