WO2012164135A1 - Procédé d'obtention d'une prothèse biodégradable - Google Patents

Procédé d'obtention d'une prothèse biodégradable Download PDF

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Publication number
WO2012164135A1
WO2012164135A1 PCT/ES2012/070400 ES2012070400W WO2012164135A1 WO 2012164135 A1 WO2012164135 A1 WO 2012164135A1 ES 2012070400 W ES2012070400 W ES 2012070400W WO 2012164135 A1 WO2012164135 A1 WO 2012164135A1
Authority
WO
WIPO (PCT)
Prior art keywords
microparticles
cells
biodegradable
prosthesis
obtaining
Prior art date
Application number
PCT/ES2012/070400
Other languages
English (en)
Spanish (es)
Inventor
María del Carmen ARAQUE MONRÓS
Luis GIL SANTOS
Sagrario GIRONÉS BERNABÉ
Jorge MAS ESTELLES
Manuel MONLEÓN PRADAS
Original Assignee
Universidad Politécnica De Valencia
Asociación De Investigación De La Industria Textil
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 Universidad Politécnica De Valencia, Asociación De Investigación De La Industria Textil filed Critical Universidad Politécnica De Valencia
Publication of WO2012164135A1 publication Critical patent/WO2012164135A1/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
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/08—Muscles; Tendons; Ligaments
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/386—Ligaments, tendons
    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body

Definitions

  • the present invention relates to a new method of obtaining a biodegradable prosthesis, in particular of tendons or ligaments, comprising the injection of microparticles with cells that induce cell regeneration inside the prosthesis.
  • Tendon and ligament prostheses have been studied throughout the 20th century, with studies related to the development of permanent prostheses where non-biodegradable fibers have been used, such as polyesters (Mersilene, Terylene, Dracon, etc.) or polyamides (Nylon). These prostheses have a limited duration and over time the materials used gradually lose their characteristics. In the case of ligamentous prostheses, due to the continuous tensions to which it is subjected, they are lengthening and recovering their initial length less and less, thus losing the size that they must maintain so that it can continue to be useful.
  • fiber coatings have been used by the use of a hydrogel, obtaining improvements in mechanical properties.
  • Studies have also been conducted through the use of biodegradable fibers, which have provided promising results.
  • the latest trends point to the use of poly-L-lactic acid (PLLA) and polyglycolic acid (PGA), either separately, well copolymerized, since they have mechanical properties and degradation rates that can be "adjusted” to those of the tissue natural.
  • PLLA poly-L-lactic acid
  • PGA polyglycolic acid
  • these fibers are coated by a protein matrix, such as fibronectin, gelatin or fibrin, an improvement in adhesion and cell growth is achieved.
  • Patent application EP1561481 (Kladakis et al., 2005) describes an implant for the repair of tendons and ligaments comprising a tissue support of biodegradable synthetic polymeric materials and pockets adapted to contain the viable tissue.
  • materials disclosed are haluronates, chitosan and polylactic acids (L and D).
  • L and D haluronates
  • This prior art document also mentions the possibility of including particles of other materials to the braided material that forms the implant.
  • Cell diffusion towards the radial axis of the prosthesis can be problematic (Liu, Fan et al., 2008), so the use of a prosthesis with inductive cells only on its surface may not achieve cell regeneration inside the prosthesis. prostheses efficiently.
  • the present invention solves this problem by providing a new method for introducing cells that induce cell regeneration inside the prosthesis by injection. Description of the invention
  • the present invention provides a new method of obtaining a biodegradable prosthesis comprising a) a braided support material, b) microparticles and, c) a coating, characterized in that the microparticles are introduced inside the braided material. According to a preferred embodiment, the microparticles are introduced into the braided support material by injection.
  • the biodegradable prosthesis of the present invention is characterized in that the microparticles comprise cells that induce cell regeneration, such as, for example, mesenchymal cells or fibroblasts.
  • the cell regeneration inducing cells are mesenchymal cells. More preferably, human mesenchymal cells of the patient, from bone marrow or fat.
  • This regeneration can be initiated by means of a cell carrier, which may or may not be loaded with growth factors such as, for example, BMP-12, TGF- ⁇ , IGF-1, VEGF or bFGF.
  • the implant of the prosthesis in the organism can be done, either immediately after the injection of the microparticles with the cells, or after a period of culture in a bioreactor. Initially, the prosthesis should behave as a resistant element until the new tissue is formed and all the materials that form it are biodegraded.
  • a cell culture is carried out on the microparticles, these are placed inside the braided material by injection and a mechanical stimulus is applied so that these cells generate an extracellular matrix and collagen fibers, which will leave guiding and giving rise to tissue regeneration and the complete disappearance of the prosthesis.
  • the structure of the prosthesis of the present invention comprises a) a braided support material, b) microparticles inside the braid and, c) a coating ( Figures 1 and 2).
  • Braided material is one of the most important parts of the prosthesis, and its main function is to withstand mechanical loads.
  • the braided material part of the structure of the prosthesis of the present invention is a tubular mesh or hollow braid.
  • the braided structure allows the mechanical properties of the resulting braid to be adjusted, depending on the way the braid is made.
  • the braided material may be composed of PLLA (poly-L-lactic acid), PGA (polyglycolic acid), poly-e-caprolactone or copolymers formed by PLLA and PGA.
  • PLLA poly-L-lactic acid
  • PGA polyglycolic acid
  • poly-e-caprolactone copolymers formed by PLLA and PGA.
  • the braided material may consist mainly of PLLA (poly-L-lactic acid).
  • the morphological and mechanical characteristics of this braided material may vary so that it reproduces the tension curve. characteristic deformation of the mechanical behavior of the natural tissue, in particular tendon or ligament, which is intended to regenerate. For small deformations the braid is not very resistant, but as it deforms it becomes increasingly rigid.
  • Young's modulus of the prosthesis of the present invention in the linear behavior zone is similar to the values cited in the literature for natural tendon and ligaments.
  • the braided material is made with 12 threads of 3 or 4 ends of 336 dtex each.
  • the inner core of the braid (figures 3a and 3b) is extracted giving rise to braided material with an inner diameter between 1000-800 ⁇ and is obtained after extracting the inner core from the braid.
  • Mechanical tests performed show that the PLLA braids of the present invention reproduce the typical strain strain curve of a ligament ( Figure 4a and 4b) or tendon.
  • the microparticles are located inside the braided material and have the function of transporting the cells and providing matrix components extracellular These microparticles are characterized by having a diameter smaller than the internal diameter of the braid, preferably between 50 and 100 ⁇ .
  • These microparticles can be formed of different materials, preferably it is chosen from poly-L-lactic acid (PLLA), chitosan (CHT), hyaluronic acid (HA) and mixture thereof, in particular mixtures of PLLA: HA.
  • the coating included in the prostheses of the present invention has as its main function to avoid adhesions with the surrounding tissues, to protect the braided material and, preferably, to be resistant to abrasion.
  • This coating can be made on the braided material itself or by manufacturing a tube or sheath, where the braided material can then be introduced (Figure 5).
  • the coating comprises hyaluronic acid since it is part of the extracellular matrix, so it is completely biocompatible. Even so, the use of other biodegradable materials such as chitosan or mixtures of hyaluronic acid and chitosan is not ruled out.
  • the coating is formed by a cross-linked hyaluronic acid tube.
  • the ends of the tendon or ligament prosthesis can be anchored to the body by suturing or by biodegradable screws in the bone.
  • the ends of the prosthesis can have a flat or ring-shaped shape, so that the suture can be made, and in the second case, the ring-shaped end is most convenient to be able to anchor it to the screw.
  • Figure 1 Structure of the prosthesis obtained by the method of the present invention.
  • Figure 2 Scheme of the structure of the prosthesis comprising a) PLLA hollow braid, b) microparticles and c) coating.
  • Figure 3a Photograph showing a PLLA braid made with 12 threads with 4 ends of 336 dtex adding a total of 1344 dtex / thread. Outside diameter approximately 2 rom.
  • Figure 3b Photograph of a PLLA braid made with 12 threads with 4 ends of 336 dtex adding a total of 1344 dtex / thread to which the internal soul is extracted. Inner diameter after extracting the inner soul between 1000 and 800 ⁇ .
  • Figure 4a Typical strain-strain curve of a ligament.
  • Figure 4b Typical strain-strain curve of a PLLA braid made with 12 strands to 4 ends of 336 dtex totaling 1344 dtex / strand.
  • Figure 5 Braids inside a hyaluronic acid coating made on the braid itself.
  • Figure 6a Culture with L929 cell line, mouse fibroblasts, in microparticles of poly-L-lactic acid (PLLA).
  • PLLA poly-L-lactic acid
  • Figure 6b Culture with L929 cell line, mouse fibroblasts, in chitosan microparticles (CHT).
  • Figure 6c Culture with L929 cell line, mouse fibroblasts, in hyaluronic acid (HA) microparticles.
  • Figure 7 Culture with L929 cell line, mouse fibroblasts, in microparticles of mixtures of PLLA and HA
  • Figure 8 Aggregates of microparticles-cells seen by electron microscope.
  • Figure 10 Micrographs by electron microscope showing the adhered cells on the surface. Examples
  • Poly-L-lactic acid is dissolved in dichloromethane (CH 2 CI 2 ) in a concentration of 5%, (1 gr of PLLA in 20 ml of CH 2 CI 2 ).
  • This solution organic phase
  • PVA polyvinyl alcohol
  • a 2% chitosan solution (CHT) is prepared, for which 2 g of chitosan is weighed and dissolved in 100 ml of a 1% acetic acid solution. This solution is added dropwise at a rate of 10 ml / h on the organic phase composed of 100 ml of mineral oil, plus 1 ml of Tween 80. It is stirred for 30 min at 1750 rpm and then, from here all In the absence of light, the genipin crosslinker with a concentration of 30 mM (0.068 g of genipin in 10 ml of distilled water) is added, at a rate of lml / min. It is stirred for 15 hours with the same revolutions, 1750 rpm, washed and centrifuged repeatedly with ethanol. 100 ml of Mineral OH +
  • a solution of 5% hyaluronic acid (0.5 g of HA in 10 ml of 0.2M NaOH) is prepared and added dropwise, at a rate of 10 ml / h on a solution of 100 ml of isooctane, 1.7 ml of heptanol and 1.34 gr of sodium bis (2-etihexyl) sulfosuccyanate.
  • Example 4 Long time cell culture of the different microparticles (CHT, PLLA and HA) obtained in examples 1, 2 and 3
  • microparticles 50 mg are weighed in the wet state of ETOH 70% / Eppendorf, sterilized with 70% ethanol for 4-5 h and conditioned 24 h in Dulbecco's Modified Eagle Medium, DMEM (Sigma) 4.5 g / 1 D-glucose. After this time they are cultured with the type of cells chosen (cell line L929, mouse fibroblasts) 100,000 cells / 50 mg of microparticles are cultured.
  • DMEM Dulbecco's Modified Eagle Medium
  • Example 5 Long time cell culture of mixtures of different microparticles
  • microparticles are cultured according to the above procedure with L929 cell line cells, mouse fibroblasts, but instead of using a single type of microparticles, the culture is carried out on a mixture of PLLA microparticles and HA microparticles in compositions ( PLLA: HA) (2: 1, 1: 2). The test times 1, 7 and 14 days. The results are shown in Figure 7.
  • Example 6 Short-term cell culture of PLLA microparticles to prevent good injectability
  • a culture test has been carried out at short times: 1, 2, 3 and 6h in order to observe at what time it would be optimal to perform a good injectability and to be able to avoid the obturation of the needle by the aggregates (microparticles + cells) that could be formed
  • the L929 cell line mouse fibroblasts
  • a cell suspension of 100 ⁇ is grown on the microparticles with a density of 5 cells / 50 mg of microparticles, sterilized and preconditioned.
  • the whole cells / microparticles are incubated at short times (1, 2, 3, 6 hours), after that time they are fixed with a 2.5% glutaraldehyde solution and observed under the electron microscope ( Figure 8). It is observed that at 6 h aggregates are formed (cells / microparticles) that could worsen the good injectability by needle sealing.
  • Example 7 Long time cell culture of PLLA microparticles
  • the culture is carried out with the same L929 cell line and the same procedure is followed above but for long times 1, 7, and 14 days in order to observe if the cells grow and proliferate on the support. This is checked by a cell viability and electron microscopy (SEM) and confocal test.
  • SEM cell viability and electron microscopy

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Transplantation (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Zoology (AREA)
  • Vascular Medicine (AREA)
  • Rehabilitation Therapy (AREA)
  • Rheumatology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Developmental Biology & Embryology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Materials For Medical Uses (AREA)

Abstract

L'invention concerne un procédé d'obtention de prothèses biodégradables qui comprennent a) une matière torsadée de support, b) des microparticules, et c) un enrobage caractérisé en ce que les microparticules sont introduites à l'intérieur de la matière torsadée de support. De préférence, les microparticules sont introduites à l'intérieur de la matière torsadée de support au moyen d'une injection. Selon un autre mode de réalisation préféré, les microparticules renferment des cellules induisant la régénération cellulaire.
PCT/ES2012/070400 2011-06-03 2012-05-30 Procédé d'obtention d'une prothèse biodégradable WO2012164135A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP201130919 2011-06-03
ES201130919A ES2392857B1 (es) 2011-06-03 2011-06-03 Procedimiento de obtención de una prótesis biodegradable.

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WO2012164135A1 true WO2012164135A1 (fr) 2012-12-06

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1561481A2 (fr) * 2004-02-09 2005-08-10 DePuy Mitek, Inc. échafaudage contenant du tissu viable
WO2006023803A2 (fr) * 2004-08-20 2006-03-02 Artes Medical, Inc. Methodes d'administration de microparticules combinees a des composants corporels autologues
US20080124400A1 (en) * 2004-06-24 2008-05-29 Angiotech International Ag Microparticles With High Loadings Of A Bioactive Agent
US20090163936A1 (en) * 2007-12-21 2009-06-25 Chunlin Yang Coated Tissue Engineering Scaffold
US20100298937A1 (en) * 2009-05-22 2010-11-25 Soft Tissue Regeneration, Inc. Mechanically competent scaffold for ligament and tendon regeneration
WO2011005493A2 (fr) * 2009-06-22 2011-01-13 Mayo Foundation For Medical Education And Research Procédés et matériels pour réparation de tissu

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1561481A2 (fr) * 2004-02-09 2005-08-10 DePuy Mitek, Inc. échafaudage contenant du tissu viable
US20080124400A1 (en) * 2004-06-24 2008-05-29 Angiotech International Ag Microparticles With High Loadings Of A Bioactive Agent
WO2006023803A2 (fr) * 2004-08-20 2006-03-02 Artes Medical, Inc. Methodes d'administration de microparticules combinees a des composants corporels autologues
US20090163936A1 (en) * 2007-12-21 2009-06-25 Chunlin Yang Coated Tissue Engineering Scaffold
US20100298937A1 (en) * 2009-05-22 2010-11-25 Soft Tissue Regeneration, Inc. Mechanically competent scaffold for ligament and tendon regeneration
WO2011005493A2 (fr) * 2009-06-22 2011-01-13 Mayo Foundation For Medical Education And Research Procédés et matériels pour réparation de tissu

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Publication number Publication date
ES2392857B1 (es) 2013-11-13
ES2392857A1 (es) 2012-12-14

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