WO2015189266A1 - Dosage de biocompatibilité - Google Patents

Dosage de biocompatibilité Download PDF

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Publication number
WO2015189266A1
WO2015189266A1 PCT/EP2015/062932 EP2015062932W WO2015189266A1 WO 2015189266 A1 WO2015189266 A1 WO 2015189266A1 EP 2015062932 W EP2015062932 W EP 2015062932W WO 2015189266 A1 WO2015189266 A1 WO 2015189266A1
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WIPO (PCT)
Prior art keywords
sample
amniotic membrane
medium
biocompatibility
cells
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PCT/EP2015/062932
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English (en)
Inventor
Veronika HRUSCHKA
Heinz Redl
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Ludwig Boltzmann Gesellschaft
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Publication date
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Publication of WO2015189266A1 publication Critical patent/WO2015189266A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells

Definitions

  • the present invention relates to methods for assessment of biocompatibility .
  • Biocompatibility testing is the assessment of the ability of a given substance or product to be in contact with a living system without producing an adverse effect to this living system.
  • biocompatibility in biomedical therapy defines biocompatibility as "the ability of a material to perform with an appropriate host response in a specific application”.
  • biomaterials in vitro cells are incubated either with the extract of the biomaterial or in direct contact with the biomaterial (according to the guideline ISO 10993-5; see also e.g. Hillegass et al . , Interdicip. Rev. Nanomed. Nanobiol. 2 (2010), 219-231).
  • the biocompatibility is subsequently evaluated using morphological analysis and/or quantitative assays (cell- count, enzyme-release, reduction of vital stainings, etc.).
  • the present invention provides a method for testing biocompatibility of a sample comprising the following steps :
  • an improved test system for the assessment of biocompatibility is provided.
  • the present assay does not rely on test animals, the results provided have high relevance for the in vivo situation.
  • the present assay allows the evaluation of the biocompatibility of any test substance or article (especially biomaterials) by a 3D (three dimensional) sensor membrane that - due to its intact, viable and healthy tissue nature - closely resembles the in vivo situation.
  • the method according to the present invention is specifically suitable to assess biocompatibility of a given material or substance (including substance compositions) that is planned to be applied to a human patient and may, e.g. require an assessment of biocompatibility for health authorities.
  • the present system can indeed be regarded as a 3D system as the cells in the membrane are provided as a tissue material - in contrast to a 2D system usually applied for biocompatibility testing using cell cultures with isolated cells that are not provided in a tissue context.
  • the amniotic membrane is a suitable tissue material for biocompatibility evaluations.
  • Amniotic membrane constitutes a pre-formed sheet of stem cells. With the present invention also methods for in situ differentiation of these stem cells into various tissues without their prior isolation were used.
  • Amnion is the innermost of the fetal membranes and is usually discarded after birth as a part of the placenta.
  • increasing attention is paid to this tissue, since the membrane as a whole and isolated cells thereof show great promise for regenerative medicine.
  • Amnion tissue has many beneficial properties besides its nearly unlimited availability, the easy procurement and the low processing costs for therapeutic application: It is bacteriostatic, antiangiogenic, reduces pain, suppresses inflammation, inhibits scarring and promotes wound healing and epithelialization . Furthermore amniotic membrane shows low or no immunogenicity and acts as an anatomical and vapor barrier. Because of these characteristics, amnion has been applied in surgery and wound treatment e.g. for burned skin, bedsore, ulcers, ophthalmology (see for instance Schwab, Ivan R.
  • the EP 2 664 337 Al discloses amniotic membrane preparations and purified compositions and methods of use.
  • the compositions can be used to treat various diseases, such as wound healing, inflammation and angiogenesis-related diseases.
  • the WO 2010/133853 Al relates to synthetic grafts.
  • a plastically-compacted collagen gel as a substrate for the growth of corneal cells, particularly limbal corneal epithelial stem cells.
  • Cells grown on such a substrate can be cultured to produce artificial ocular epithelia which can be used in ocular toxicity testing or for transplantation.
  • Example 10 (p. 19) of the document teaches the expansion of limbal corneal epithelial cells on compressed collagen gels or denuded amniotic membrane. For denuding, the amniotic membrane is treated with trypsin and the original epithelial cells (i.e. the amniotic epithelial cells) are scraped off.
  • the amniotic membrane is disclosed to be "cell-free” after denuding. Only then are limbal stem cells (LSCs) seeded onto the cell-free denuded amniotic membrane, or onto the compressed collagen gels, to obtain artificial ocular corneal epithelium.
  • LSCs limbal stem cells
  • Example 19 of the document discloses testing of an artificial ocular epithelium for ocular toxicity. However, it is not specified in Example 19 of the document whether artificial ocular epithelial cell cultures on cell-free denuded amniotic membrane are used at all for toxicity testing, e.g. epithelial cell cultures on compressed collagen gels may have been used instead.
  • the present invention does not teach denuding of the amniotic membrane to obtain a cell-free amniotic membrane.
  • the amniotic membrane within the context of the present invention differs in many testable ways from the artificial ocular epithelium grown on cell-free denuded amniotic membranes (see e.g. next paragraph) .
  • testing for ocular toxicity is not the same as testing for biocompatibility .
  • the method of the present invention is simpler (e.g. by not relying on a denuding step) and closer to the in-vivo situation. Therefore, the WO 2010/133853 cannot take away novelty or inventive step from the present invention.
  • Amniotic membrane is composed of a single layer of epithelial cells that reside on a basement membrane and an underlying avascular stromal layer containing stromal cells.
  • cells isolated from both the epithelial and stromal layers express markers of mesenchymal and embryonic stem cells (Parolini et al . , Stem Cells 26 (2008), 300-311).
  • the "epithelial cells” within the context of the present invention are amniotic epithelial cells, having distinct characteristics compared to other epithelial cells such as ocular epithelial cells.
  • the "stromal cells” within the context of the present invention are amniotic stromal cells.
  • the amniotic membrane within the context of the present invention comprises amniotic epithelial cells.
  • the amnion also contains an extracellular matrix with collagen.
  • the amnion is therefore a truly 3D system with various cells embedded in a tissue context. These cells can be differentiated along different lineages, including adipogenic, osteogenic, chondrogenic, hepatic, cardiomyogenic, and neurogenic reviewed in (Parolini et al . , 2008). Allogenic application seems to be feasible due to immunomodulatory characteristics of these cells.
  • amniotic cells are able to suppress proliferation of stimulated allogenic blood cells and several clinical trials in humans proved that allogenic transplantation of amniotic membrane or amniotic cells does not cause acute immune rejection even without immunosuppressive treatment.
  • amniotic membrane within the context of the present invention is not denuded.
  • biocompatibility according to the present invention is defined according to the general IUPAC definition, i.e. in the "ability [of a given material, substance or composition] to be in contact with a living system without producing an adverse effect” (Vert et al., Pure Appl . Chem., 84 (2012), 377-410). With the present invention, however, also the biocompatibility with regard to biomedical therapy can be tested, i.e. the "ability of a material to perform with an appropriate host response in a specific application”.
  • biocompatibility is "the quality of not having toxic or injurious effects on biological systems", “comparison of the tissue response produced through the close association of the implanted candidate material to its implant site within the host animal to that tissue response recognised and established as suitable with control materials” (American Society for Testing and Materials (ASTM) ) , “the ability of a biomaterial to perform its desired function with respect to a medical therapy, without eliciting any undesirable local or systemic effects in the recipient or beneficiary of that therapy, but generating the most appropriate beneficial cellular or tissue response in that specific situation, and optimising the clinically relevant performance of that therapy” (Williams et al., Biomaterials 29 (2008), 2941-2953), “the capability of a prosthesis implanted in the body to exist in harmony with tissue without causing deleterious changes", etc..
  • the sample is preferably a pharmaceutical material, a medical implant, a textile product, a synthetic material, a plastic material, a plant material, a food product, an aerosol, a household product, a cosmetic product, or mixtures thereof.
  • the amniotic membrane is a human amniotic membrane.
  • Human placenta provides a source for human amniotic membranes with nearly unlimited availability, since it is usually not further used after birth.
  • human material is preferred, the method according to the present invention can also be performed with other amniotic material, preferably from larger mammals, especially from pig, sheep, cattle or horse.
  • the method according to the present invention is preferably performed with at least two different amniotic membranes from different donors to exclude (or at least detect) donor-specific reactions. Accordingly, the test is performed with at least two membranes from different individuals, preferably at least three, even more preferred at least four, especially at least five different membranes from different donors.
  • a control sample is compared with the sample when assessing biocompatibility.
  • the control should have a proven or verified status with respect to the assay (e.g. as being biocompatible or not) .
  • a control sample with ascertained biocompatibility showing no cytotoxic activity (“negative control") .
  • Such control samples are usually those wherein the material or substance to be tested is planned to be provided to the (human) patient (e.g. the control medium, i.e. as an "empty control" .
  • examples of such (negative) control samples are biocompatible standard control media, preferably a medium comprising Dulbecco ' s Modified Eagle's Medium (DMEM) ; a medium comprising fetal calf serum (FCS) ; a medium comprising amino acids; a medium comprising a buffer, especially a Tris buffer, a phosphate buffer or a carbonate buffer; a medium comprising a carbohydrate, especially glucose or sucrose; a medium comprising a biocompatible antibiotic, especially Penicillin or Streptomycin; or mixtures thereof.
  • DMEM Dulbecco ' s Modified Eagle's Medium
  • FCS fetal calf serum
  • a medium comprising amino acids a medium comprising a buffer, especially a Tris buffer, a phosphate buffer or a carbonate buffer
  • a medium comprising a carbohydrate especially glucose or sucrose
  • a biocompatible antibiotic especially Penicillin or Streptomycin
  • control substances with proven cytotoxicity may be applied as "positive" controls, e.g. latex or polyvinylchloride (PVC) .
  • PVC polyvinylchloride
  • the cells contained in the human amnion have stem cell potential, and can be induced to acquire the phenotype of differentiated cells also in the membrane tissue.
  • a predifferentiation is beneficial if the influence of a biomaterial should be tested on cells of a differentiated tissue type. As an example, the influence on osteogenic cells differentiated from bone substitute materials (such as Ostim ® ) could be evaluated.
  • the differentiation capabilities of the amnion is therefore used for a "tailor made" test system for the biocompatibility with the tissue to which the substance/composition/material is planned to be applied to human patients.
  • the amniotic membrane is subjected to a predifferentiation before the contacting step.
  • the predifferentiation may e.g. an adipogenic, osteogenic, chondrogenic, hepatic, cardiomyogenic, or neurogenic predifferentiation, preferably an osteogenic predifferentiation or a chondrogenic predifferentiation .
  • Osteogenic differentiation is e.g. exemplified by Lindenmair et al . , 2010: "Osteogenic differentiation was induced with osteogenic stimulatory kit (OKit, Stem Cell Technologies, Cologne, Germany) or osteogenic medium (OM) described by Pittenger et al .
  • DMEM modified Eagle's medium
  • FCS fetal calf serum
  • PAA penicillin/streptomycin
  • L-glutamine PAA
  • 50 mMascorbate-2-phosphate Sigma, Vienna, Austria
  • 0.1 mM dexamethasone Sigma
  • 10 nM 1,25- dihydroxy-vitamin D3 Sigma
  • 10 mM b-glycerophosphate Stem Cell Technologies
  • Chondrogenic differentiation is exemplified by Lindenmair et al . , 2014: "Chondrogenic differentiation was induced with hMSC Mesenchymal Stem Cell Chondrocyte Differentiation Medium (C; Lonza, Verviers, Belgium) ; not described concerning concentration but containing sodium pyruvate, ITS , ascorbate, dexamethasone, proline, L-glutamine, antibiotics and 10 ng/ml TFG-b3) , with or without supplementing 10 ng/ml basic fibroblast growth factor
  • FGF2 chondrocyte redifferentiation medium based on Tallheden et al . (2004) [T; Dulbecco's modified Eagle's medium (DMEM, PAA) , ITS-G (Gibco, Vienna, Austria) , 5 yg/ml linoleic acid (Sigma, Vienna, Austria) , 1 mg/ml human serum albumin (Sigma) , 10 ng/ml TGF-b3 (Lonza) , 14 yg/ml ascorbat-2-phosphate (Sigma), 1 % penicillin/streptomycin
  • PAA 1 % L-glutamine
  • PAA 10 "7 M dexamethasone (Sigma); Osteoarthritis Cartilage 12 (2004), 525-535 ].
  • CM control medium
  • DMEM 10 % FCS
  • PAA penicillin/streptomycin
  • L-glutamine 1 % L-glutamine
  • viability tests there is a wide variety of viability tests available for use within the present method. Depending on the cytotoxicity of the material to be tested, damage of the cells of the membrane is visible or measurable by e.g. optical methods or methods comprising staining. Quantitative as well as qualitative methods may be applied.
  • a preferred technique for viability assessment comprises a quantitative assay determining the metabolic activity of cells (and correlating such metabolic activity to the number of viable cells) .
  • the viability of the amniotic membrane is analysed by measuring metabolic activity of the cells in the membrane, by staining the cells in the membrane, especially with 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide, calcein-acetoxymethylester, 2, 3-bis- (2-methoxy-4-nitro-5- sulfophenyl) -2H-tetrazolium-5-carboxanilide (XTT, EZ4U ), 3- (4, 5- dimethylthiazol-2-yl) -5- ( 3-carboxymethoxyphenyl ) -2- (4- sulfophenyl ) -2H-tetrazolium (MTS) and phenazine methosulfate
  • MTT assay is a colorimetric assay for assessing cell viability.
  • NAD ( P) H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3- (4, 5-dimethylthiazol-2-yl) - 2 , 5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple colour.
  • Such test can e.g. carried out as disclosed by Ovsianikov et al . (Langmuir 30 (2014), 3787-3794): MTT reagent
  • Double staining for Ki-67 and osteocalcin was carried out using an immunohistochemical double staining kit (Envision Doublestain System, Dako, Glostrup, Denmark) according to the manufacturer's protocol except for primary antibody incubation times.
  • First primary anti-Ki67 1:200
  • second primary antibody second primary antibody
  • EZ4U (based on MTT assay): somehowTo quantify cell viability, an EZ4U-Nonradioactive Cell Proliferation and Cytotoxicity Assay (Biomedica, Vienna, Austria) was performed according to the manufacturer' s instructions and viability calculated in % of fresh (dO) AM.”
  • viability assays or staining methodes comprise MTS, Alamarblue, BrdU ELISA, CalceinAM/propidium iodide staining, etc ..
  • viability assessment according to the present invention is preferably performed by MTT assay (or a variant thereof, such as XTT, MTS or WSTs) .
  • WSTs are WST-1, -3, -4, -5, -8, -9, -10, -11 (chemical formulae according to FT- F98881 of InterBioTech - Interchim) .
  • the present invention relates to a method for the evaluation of the suitability of a sample for supporting differentiation of cells of the amniotic membrane comprising the following steps:
  • a differentiation assessment of a given substance, composition or material may be made, especially whether a supportive effect of a given differentiation direction is achieved by such substance, etc..
  • two amniotic membranes may be provided in an osteogenic differentiation medium (see e.g. Lindenmair et al . , 2010) with a sample substance between the membranes.
  • the differentiation capacity of the sample substance (or composition, material) may then be assessed by comparison with the control (i.e. differentiation without the sample substance) and other comparable substances approved for clinical usage if available.
  • predifferentiation as used herein can be understood as the differentiation of the cells of the amniotic membrane prior to the contact to the sample in the method according to the present invention.
  • lineage differentiation is exchangeable herein with the term “differentiation”.
  • the membrane may also be differentiated in the presence of the sample .
  • Assessing suitability of a sample for supporting lineage differentiation according to the present method may e.g. be performed by evaluation of expression for lineage specific markers (e.g. RT-PCR for Osteocalcin, Osteopontin, ALP, Osteonectin, etc.
  • lineage specific markers e.g. RT-PCR for Osteocalcin, Osteopontin, ALP, Osteonectin, etc.
  • cartilage oligomeric matrix protein (COMP) , aggrecan (AGC1), versican (CSPG2), COL1A1, COL9A2, melanoma inhibitory activity (MIA) , and cartilage-linking protein 1 (CRTL1) Collagen Type (chondrogenic differentiation) ; stainings such as Alcian blue staining to visualize accumulation of glycosaminoglycans (GAGs) (chondrogenic differentiation) or von Kossa or Alizarin Red S staining (osteogenic differentiation) , immunohistochemical stainings such as Collagen I, II and X (chondrogenic differentiation) or Osteocalcin (osteogenic differentiation) (see e.g. Lindenmair et al . , 2010 for osteogenic differentiation and Lindenmair et al . , 2014 for chondrogenic differentiation) ) .
  • stainings such as Alcian blue staining to visualize accumulation of glycosaminoglycans (GAGs
  • the present invention also relates to a kit for performing the method according to the present invention comprising:
  • the kit comprises a control sample with proven biocompatibility or with proven differentiation capacity.
  • the kit according to the present invention further comprises single or combined components of the test sample.
  • C2C12 cells were cultured in Dulbecco's minimum essential medium (DMEM) supplemented with 5% fetal calf serum (FCS) , 1% L- Glutamine, 1% Penicillin/Streptomycin (all from Sigma-Aldrich, Austria) in an incubator which was maintained at 37°C and 5% CO 2 . Cells were seeded with a cell density of 10 4 cells/well (48-well plate) . The cells were allowed to settle for 24 hours before the exposure to the materials.
  • DMEM Dulbecco's minimum essential medium
  • FCS fetal calf serum
  • Penicillin/Streptomycin all from Sigma-Aldrich, Austria
  • DMEM-LG 10% FCS, 1% Penicillin/Streptomycin and 1% L-Glutamin
  • Figure 2D system The assessment according to this cell-based 2D system showed a concentration-dependent cytotoxic effect of both, Ostim ® and HAP, although both materials are indeed well tolerated and fully biocompatible without inflammatory effects (as assessed e.g. in Busenlechner et al . , Biomaterials 29 (2008) 3195-3200).
  • the 3D method according to the present invention resembles the in vivo situation (as evidenced by Busenlechner et al . , 2008) much closer than the cell-based 2D system of example 1.2.
  • the method according to the present invention is applying a system that resembles a valid tissue structure with the cells of the membrane being interconnected in a tissue environment, whereas cell culture systems, even with layered structure cannot mimick tissue properties in such a manner.
  • 3 cm biopsies of amniotic membrane were prepared as described for cytotoxicity evaluation. Osteogenic stimulation was performed with the medium DMEM containing 10% FCS, 50 ⁇ ascorbate-2-phosphate, 0.1 ⁇ dexamethasone, 10 nM 1,25- dihydroxy-vitamin D3, and 10 mM ⁇ -glycerophosphate . After three weeks in culture, central punch biopsies (8mm in diameter) were taken and bone-specific mineral deposition was demonstrated by von Kossa staining only in amniotic membrane cultivated in osteogenic stimulation medium and not in control medium (DMEM 10% FCS), or in fresh amniotic membrane. Bone-specific marker genes were quantified using quantitative RT-PCR.
  • Example osteogenic lineage As described in Example 2.1, biopsies of the amiotic membrane were taken. Osteogenic differentiation was induced by exposure to osteogenic medium DMEM containing 10% FCS, 50 ⁇ ascorbate-2 -phosphate , 0.1 ⁇ dexamethasone, 10 nM 1 , 25-dihydroxy-vitamin D3, and 10 mM ⁇ -glycerophosphate . After three weeks of differentiation, the materials were placed between two layers of the membrane as described in Example 1. After 48 hours of incubation central punch biopsies (8mm in diameter) were taken and their viability analysed using a quantitative MTT assay .

Abstract

L'invention concerne un procédé permettant de tester la biocompatibilité d'un échantillon et comprenant les étapes suivantes : - la fourniture d'un échantillon, - la fourniture d'une membrane amniotique, - la mise en contact de l'échantillon avec la membrane amniotique, - l'évaluation de la biocompatibilité de l'échantillon par l'analyse de la viabilité de la membrane amniotique après le contact avec l'échantillon. L'invention concerne également un kit permettant d'exécuter le procédé.
PCT/EP2015/062932 2014-06-10 2015-06-10 Dosage de biocompatibilité WO2015189266A1 (fr)

Applications Claiming Priority (2)

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EP14171816.3 2014-06-10
EP14171816 2014-06-10

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WO2015189266A1 true WO2015189266A1 (fr) 2015-12-17

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010133853A1 (fr) * 2009-05-22 2010-11-25 University Of Reading Greffe synthétique
EP2664337A1 (fr) * 2005-09-27 2013-11-20 TissueTech, Inc. Preparations de membrane amniotique et compositions purifiees, et procedes d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2664337A1 (fr) * 2005-09-27 2013-11-20 TissueTech, Inc. Preparations de membrane amniotique et compositions purifiees, et procedes d'utilisation
WO2010133853A1 (fr) * 2009-05-22 2010-11-25 University Of Reading Greffe synthétique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCHWAB I R: "Cultured corneal epithelia for ocular surface disease", MEDLINE, 31 December 1999 (1999-12-31), XP002240243 *

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