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WO2013068723A1 - Echafaudages de tissu poreux - Google Patents

Echafaudages de tissu poreux Download PDF

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Publication number
WO2013068723A1
WO2013068723A1 PCT/GB2012/052706 GB2012052706W WO2013068723A1 WO 2013068723 A1 WO2013068723 A1 WO 2013068723A1 GB 2012052706 W GB2012052706 W GB 2012052706W WO 2013068723 A1 WO2013068723 A1 WO 2013068723A1
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Prior art keywords
sample
gelatin
porous
tissue scaffold
porous tissue
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PCT/GB2012/052706
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English (en)
Inventor
Huibert Van Boxtel
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Fujifilm Imaging Colorants Ltd
Fujifilm Manufacturing Europe BV
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Fujifilm Imaging Colorants Ltd
Fujifilm Manufacturing Europe BV
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Publication of WO2013068723A1 publication Critical patent/WO2013068723A1/fr
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Ceased legal-status Critical Current

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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
    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24

Definitions

  • the present invention relates to porous tissue scaffolds with a distinct pore geometry and to biocompatible articles produced from these scaffolds.
  • Biomaterials are designed to replace injured or diseased tissue. Ideally, they are scaffolds for tissue regeneration with properties similar to those of the healthy tissue that they replace. Designed to cover a two-dimensional surface or to fill a three-dimensional void, they should, in parallel to healing, gradually be absorbed so that, ultimately, the site of injury becomes almost indistinguishable from the surrounding tissue. To achieve these goals, the biomaterial must fulfil several design requirements: it has to possess a sufficiently large porosity, its surface chemistry and topography must be suited for cell adhesion, proliferation and differentiation; it needs to possess an appropriate architecture to guide tissue regeneration; and it should allow for controlled absorption when the scaffold is no longer required.
  • the scaffold must, despite a high overall porosity that considerably weakens its mechanical properties, possess sufficient stiffness, strength and toughness to perform the natural tissue's function while the wound is healing.
  • the currently available tissue scaffolds comprise different unabsorbable biocompatible polymers such as polyethylene terephthalate; fluorinated polymers, such as polytetrafluoroethylene (PTFE) and fibers of expanded PTFE; and polyurethanes.
  • Some available tissue scaffolds do comprise absorbable polymers such as poly-lactic acid, hyaluronic acid, collagen and gelatin. However, their pore geometry ranges are not optimal.
  • Typical methods for preparing such three-dimensional porous polymer scaffolds include: a solvent-casting and particle-leaching technique comprising mixing a polymer with single-crystal salt particles, drying the mixture and then immersing the dried material to leach the salt particles (A.G. Mikos et al., Polymer,
  • the material be highly uniform (e.g. the material density, pore size and pore orientation or mechanical properties should be have a limited variation throughout the material).
  • the current biopolymer porous tissue scaffolds lack sufficient uniformity.
  • the biopolymers from which the porous tissue scaffold is prepared have enhanced properties for cell attachment and growth.
  • the object of the current invention is to provide highly uniform tissue scaffolds made from recombinant gelatin-like proteins.
  • the present invention provides a porous tissue scaffold comprising a recombinant gelatin-like protein which has a uniform columnar porous structure where the average equivalent circular diameter (ECD) of the columnar porous structure is in the range of from between about 10 to about 1000 microns.
  • ECD average equivalent circular diameter
  • the average equivalent circular diameter of the columnar porous structure is in the range of from about 100 to 500 microns (depending on targeted cell type and tissue type and location).
  • the porous tissue scaffold of the present invention preferably has a small ECD standard deviation (ECD SD )- Typical values obtained by the current invention for ECD SD are in the range of 20 to 60% of the ECD value. Preferred ECDS D values are 40% or less.
  • the porous tissue scaffold according to this invention has an improved uniformity with respect to its material density, pore size and pore orientation over the materials described in the prior-art. This renders it particularly suitable for use in biocompatible articles.
  • Term 'porous tissue scaffold' and 'porous scaffold' are used interchangeably and are to be interpreted as used herein as a three dimensional molecular matrix of biocompatible polymers which acts as a microenvironment to which tissue cells are attracted and can attach.
  • uniform and "uniformity” as used herein is to be interpreted as a limited variation of parameters such as, but not limited to pore-size, the circular diameter, pore-shape, the range of observed angles between individual directed pores, the straightness of pores and the mechanical properties such as but not limited to rigidness, brittleness, compressibility.
  • columnar pore as used herein is to be interpreted as a pore geometry of the pore lumen that approximates a cylinder or elliptic cylinder in which a cylinder is defined as a body the surface of which is formed by the points at a fixed distance from a given line segment, the axis of the cylinder. The solid enclosed by this surface and by two planes perpendicular to the axis is also called a cylinder.
  • An elliptic cylinder is a cylinder whose directrix is an ellipse.
  • a cross section perpendicular to the longitudinal columnar pore direction as used herein can have an irregular shape with a roundness of 0.5 or more. In one embodiment the roundness error is 40 or less.
  • R roundness
  • a perfect circle has a roundness of 1 .
  • R is calculated from the area of the pore (A), and the maximum diameter (dmax) according to the formula:
  • ECD Equivalent Circular Diameter
  • in-vitro culturing system as used herein is to be interpreted as any kit, apparatus or compounds used for the growth of cells, tissues, organs or parts of organs ex-vivo.
  • biocompatible article as used herein is to be interpreted as any material used for the treatment of a medical condition or for a cosmetic correction where the material is placed on or in the body of a human or animal and which do not evoke an adverse immunologic response. This includes materials which may degrade and be absorbed by the body over time. This material can be in any form such as, but not limited to: bandages, powders, sponges, hemostats, and sutures, implants of any kind, injectable particles, microspheres, microcarriers, gels or putties.
  • pluripotent As used herein, “pluripotent,” “pluripotency,” “pluripotent cells” and equivalent expressions refer to cells that are capable of both proliferation and self-renewal in cell culture and differentiation towards a variety of cell populations that include those that exhibit multipotent properties, for example, pluripotent ES cells can give rise to each of the three embryonic cell lineages. Pluripotent cells, however, cannot give rise to extra-embryonic tissues such as the amnion, chorion, and other components of the placenta, and may not be capable of producing an entire organism, i.e. pluripotent cells are not "totipotent".
  • Pluripotency can be demonstrated by providing evidence of stable developmental potential, to form derivatives of all three embryonic germ layers from the progeny of a single cell and to generate a teratoma after injection into an immuno-suppressed mouse.
  • Other indications of pluripotency include expression of genes known to be expressed in pluripotent cells and, characteristic morphology.
  • the pluripotent cells of the present invention can be derived using any method known to those skilled in the art.
  • Pluripotent cells include but are not limited to stem cells, induced pluripotent cell (iPS cell) such as an induced pluripotent stem cell (iPSC), e.g., a human induced pluripotent stem cell (hiPSC), or a human embryonic stem cell (hESC), parthenogenic cells and the like.
  • iPS cell induced pluripotent cell
  • hiPSC human induced pluripotent stem cell
  • hESC human embryonic stem cell
  • parthenogenic cells and the like parthenogenic cells and the like.
  • Totipotent refers to the ability of a cell to develop into all types of cells, including extraembryonic tissues (e.g. placenta) and to give rise to an entire organism (e.g. a mouse or human).
  • Self-renewal refers to the ability of a stem cell to divide and form more stem cells with properties identical to the parent stem cell, thereby allowing the population of stem cells to be replenished indefinitely.
  • particle as used herein is to be interpreted as any particle of solid matter of any shape irregular or discrete with a "smallest dimension size" of at least 20 to 50 nm this includes microspheres, any type of granules, any type of fibers or filaments.
  • particle free means that the solution is essentially free of particles of a size greater than 50nm and preferably it is free of particles of a size greater than 20 nm.
  • the size of any particles may be determined by electron microscopic inspection or laser light scattering techniques, dynamic (PCS) or static (SLS).
  • the presence of particles can be detected by means of various independent methods, such as elemental mapping (EDAX) of sample cross-sections to observe locally enhanced densities of specific elements, or (optical or electron) microscopic inspection of sample cross-sections to detect embedded particles, or using specific enzymes (e.g. trypsin for collagens or gelatins) to hydrolyse the sample polymer network until completion and using light scattering techniques to detect particles.
  • EDAX elemental mapping
  • specific enzymes e.g. trypsin for collagens or gelatins
  • freeze-casting and "thermally induced phase separation” are used interchangeably and refer to methods that create porous structures by solidifying a solvent within a, solution, sol-gel or dispersion by lowering the temperature of the solution, sol-gel or dispersion in such a way that the solvent separates from the dissolved and dispersed materials.
  • removing the solidified solvent by a second process a porous structure of the dissolved material remains.
  • the temperature change dissipates throughout the dispersion, solution or sol-gel the geometry of the pores can be adjusted.
  • the temperature gradient is applied in one direction (also called the freeze front travel direction) it is known as 'unidirectional freeze-casting'
  • perpendicular as used herein is to be interpreted as a line or plane which forms an angle of about 80 to1 10 degrees with another line or plane.
  • protein or “polypeptide” or “peptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, three-dimensional structure or origin.
  • Gelatin and "gelatin-like” as used herein refers to any gelatin, whether extracted by traditional methods or recombinant or biosynthetic in origin, or to any molecule having at least one structural and/or functional characteristic of gelatin.
  • the term encompasses both the composition of more than one polypeptide included in a gelatin product, as well as an individual polypeptide contributing to the gelatin material.
  • gelatin as used in reference to the present invention encompasses both a gelatin material comprising gelatin polypeptides, as well as an individual gelatin polypeptide.
  • Polypeptides from which gelatin can be derived are polypeptides such as collagens, procollagens, and other polypeptides having at least one structural and/or functional characteristic of collagen.
  • Such a polypeptide could include a single collagen chain, or a collagen homotrimer or heterotrimer, or any fragments, derivatives, oligomers, polymers, or subunits thereof, containing at least one collagenous domain (Gly-Xaa-Yaa region, wherein Xaa and Yaa are independently any amino acid).
  • the term specifically contemplates engineered sequences not found in nature, such as altered collagen sequences, e.g. a sequence that is altered, through deletions, additions, substitutions, or other changes, from a naturally occurring collagen sequence.
  • Such sequences may be obtained from, for example, suitable altered collagen polynucleotide constructs as described by applicant in EP0926543, EP1014176, WO01/34646, WO04/085473, EP1894945, WO08/103041 , WO08/103044, WO08/103043 and also specifically the examples of EP0926543 and EP1014176 which are hereby incorporated by reference.
  • cross-linking agent refers to a composition comprising a cross-linker.
  • Cross-linker refers to a reactive chemical compound that is able to introduce covalent intra- and intermolecular chemical bonds in organic molecules.
  • Figure 1 shows the temperature/time profile for producing a porous material according to the invention.
  • Figure 2 shows the temperature/time for producing a porous material according to the invention when smaller columnar pores are required.
  • Figure 3 shows scanning electron microscope (SEM) images of the range of columnar pore sizes according the invention.
  • the upper row are images of cross-sections cut laterally/vertically in the center sample part and the bottom row is cut transversally/horizontally 2 ⁇ 3mm from the top down
  • Figure 4 shows an optical micrograph of a lateral cross-section of the entire Comparative Example 2.
  • the sample height is 12 mm and the sample circular diameter is 4.5 cm.
  • Figure 5 shows a SEM image of a lateral (vertical) cross-section of Comparative Example 2 cut in the upper sample region
  • Figure 6 shows a SEM image of a transversal (horizontal) cross-section of Comparative Example 3 cut in the bottom sample region
  • Figure 7 shows a SEM image of a transversal (horizontal) cross-section of Comparative Example 2 cut in the top sample region
  • Figure 8 shows an optical optical micrograph of a lateral cross-section of
  • Example height is 12 mm sample circular diameter is 4.5 mm.
  • Figure 9 shows a SEM image of a lateral (vertical) cross-section of the full sample height of a sample prepared according to Inventive Example 1 showing a thin dense nudeation layer at the bottom and uniform parallel pores from this layer to the top of the sample.
  • Figure 10 shows a SEM transversal (horizontal) cross-section of Inventive Examplel cut in the lower region, just above the nudeation layer.
  • Figure 1 1 shows a SEM image of a lateral (vertical) cross-section of Comparative Example 2 cut near the top of the sample.
  • Figure 12 shows a SEM image of a transversal (horizontal) cross-section of
  • Comparative Example 2 cut in a region near the top of the sample.
  • Figure 13 shows an optical micrograph of a lateral cross-section of entire Comparative Example 3. Sample height is 12 mm sample circular diameter is 4.5 mm.
  • Figure 14 shows a SEM image of a lateral (vertical) cross-section of
  • Figure 15 shows a SEM image of a transversal (horizontal) cross-section of Comparative Example 3 cut near the bottom region of the sample just above the nudeation layer
  • Figure 16 shows a SEM image of a transversal (horizontal) cross-section of
  • Comparative Example 3 cut near the top of the sample.
  • Figure 17 shows an optical micrograph of a lateral cross-section of entire Comparative Example 6. Sample height is 12 mm sample circular diameter is 4.5 mm.
  • Figure 18 shows a SEM image of a lateral (vertical) cross-section of
  • Comparative Example 6 cut near the top of the sample.
  • Figure 19 shows a SEM image of a transversal (horizontal) cross-section of Comparative Example 6. cut near the bottom region of the sample just above the nudeation layer.
  • Figure 20 Shows a SEM image of a transversal (horizontal) cross-section of Comparative Example 6 cut near the top of the sample.
  • gelatin-like proteins are of also of medical benefit in comparison to the conventionally produced gelatins from animal sources.
  • safety issues with natural gelatins such as the concern over potential immunogenic, e.g., antigenic and allergenic, responses.
  • immunogenic e.g., antigenic and allergenic
  • the inability to completely characterize, purify, or satisfactorily reproduce naturally derived gelatin mixtures is of ongoing concern in the pharmaceutical and medical communities.
  • Recombinant technology allows the design of gelatin-like proteins with superior characteristics such as, for example, low immunogenicity, improved cell attachment and controlled biodegradability.
  • EP0926543, EP1014176, WO01/34646, WO04/085473, EP1894945, WO08/103041 , WO08/103044, WO08/103043 and also specifically the examples of EP0926543 and EP1014176 describe recombinant gelatins and their production methods, using methylotrophic yeasts, in particular Pichia pastoris and the recombinant gelatin-like proteins disclosed in these references are incorporated herein by reference.
  • the a recombinant gelatin-like protein used in the porous tissue scaffold of the present invention comprises at least one RGD motif. More preferably the recombinant gelatin-like protein is further enriched in RGD motifs.
  • RGD-enriched gelatin-like proteins in the context of this invention are described in WO04/085473 and WO08/103041 and the RGD-enriched gelatins disclosed in these references are incorporated herein by reference.
  • the percentage of RGD motifs related to the total number of amino acids is at least 0.4% and if the RGD- enriched gelatin comprises 350 amino acids or more, then each stretch of 350 amino acids contains at least one RGD motif. More preferably the percentage of RGD-motifs related to the total number of amino acids is at least 0.6%, especially at least 0.8%, more especially at least 1 .0%, particularly at least 1 .2% and more particularly at least 1 .5%.
  • the recombinant gelatin-like protein has a reduced level of hydroxyproline residues.
  • Hydroxylation of proline is a requirement for the formation of triple helices in collagen which is an unfavorable characteristic for the porous scaffold material formed by the current invention as it leads to particulate aggregates and fibers or filaments of proteinacious material.
  • less than 10%, more preferably less than 5% of the amino acid residues of the recombinant gelatin-like proteins are hydroxyprolines, preferably the recombinant gelatin-like protein is free from hydroxyprolines.
  • a further benefit described in WO2002/070000 of recombinant gelatin-like proteins which are free from hydroxyprolines is that they do not show immune reactions involving IgE in contrast to natural gelatin.
  • the recombinant gelatin-like protein is free from hydroxylated amino acids.
  • the gelatin-like proteins are functionalized for enhanced cell binding and/or with minimal immunogenicity such as, for example, those gelatin-like proteins disclosed in EP1608681 and EP1368056 and the gelatin like proteins disclosed in these references are incorporated herein by reference.
  • Functionalized recombinant gelatin-like proteins can be designed to have improved cell-binding properties that stimulate cellular infiltration of tissues surrounding the medical device after implantation.
  • the recombinant gelatin-like proteins used in the present invention are recombinant gelatin-like proteins with a calculated isoelectric point above 5, preferably a calculated iso-electric point above 6 and most preferably a calculated iso-electric point above 7.
  • recombinant gelatin-like proteins used in the present invention have a molecular weight of at least 20kDa, more preferably 25 kDa, especially of at least 35 kDa and more especially of at least 50 kDa.
  • the recombinant gelatin-like proteins used in the present invention have a molecular weight in the range of from 20 kDa to 75 kDa.
  • porous material comprising gelatin-like protein
  • more than one form of gelatin may be used.
  • the gelatin-like protein used to make the porous tissue scaffolds of the present invention should be biodegradable and so not require invasive surgical methods for its removal after stimulation/tissue regeneration. Moreover biodegradability is another important stimulatory factor in the regeneration of tissue.
  • a priori it is not obvious whether recombinant gelatins will be broken down by the same mechanisms causing degradation of natural gelatins.
  • MMP matrix-metalloproteinases
  • Matrix metalloproteinases are zinc-dependent endopeptidases. The MMP's belong to a larger family of proteases known as the metzincin superfamily.
  • MMP's are capable of degrading all kinds of extracellular matrix proteins, but also can process a number of bioactive molecules.
  • An important group of MMP's are the collagenases. These MMP's are capable of degrading triple-helical fibrillar collagens into distinctive 3/4 and 1/4 fragments. These collagens are the major components of bone and cartilage, and MMP's are the only known mammalian enzymes capable of degrading them. Traditionally, the collagenases are: MMP-1 (interstitial collagenase), MMP-8 (neutrophil collagenase), MMP-13 (collagenase 3) and MMP-18 (collagenase 4). Another important group of MMP's is formed by the gelatinases.
  • the main substrates of these MMP's are type IV collagen and gelatin, and these enzymes are distinguished by the presence of an additional domain inserted into the catalytic domain.
  • This gelatin-binding region is positioned immediately before the zinc binding motif, and forms a separate folding unit which does not disrupt the structure of the catalytic domain.
  • the two members of this sub-group are: MMP-2 (72 kDa gelatinase, gelatinase-A) and MMP-9 (92 kDa gelatinase, gelatinase-B).
  • porous tissue scaffolds may be prepared using any method which would be known to a skilled person.
  • the scaffold is prepared by a process that comprises the following steps:
  • gelatin-like protein solution (and optional additives) into a thermally insulated container with a single thermally conducting surface, d. optionally allowing at least part of the gelatin-like protein solution to gel by cooling the container to a temperature in the range of from 1 to 25°C;
  • step (iii) gradually lowering the temperature of the cooling device so as to induce a constant unidirectional growth rate of ice-crystals in the gelatin-like protein gel/solution, initiated from the frozen layer formed in step c (i);
  • step e (i) optionally removing the material which corresponds to the thin dense layer formed in step e (i) that has no columnar pores;
  • porous tissue scaffold material with a wide range of columnar pores having a narrow ECD standard deviation.
  • Freezing/temperature control in this method may be achieved using any suitable cooling device which would be known to a person skilled in the art.
  • the cooling device is a chill bath.
  • Figures 1 and 2 show the gelatin-like protein solution being introduced into a thermally insulated container with a single thermally conducting surface in a chill bath (T 0 , t 0 ).
  • Additives such as ethanol, methanol or acetic acid or other non-toxic or easily removable compounds can optionally be added to alter the average pore size of the final freeze-dried sample.
  • the gelatin-like protein solution is cooled and at least a fraction of it forms a gel (1 volume percent or more with the gel typically occupying the volume closest to the chilled surface) (Ti,ti).
  • the temperature is then rapidly dropped.
  • the chill bath is cooled within 5 minutes (t 2 -t-i) to a temperature (T 2 ) and the gelatin-like protein gel/solution is allowed to form a thin layer of frozen gelatin-like protein solution at the thermally conductive side of the container (T 3 , t 3 ).
  • the chill bath temperature is then rapidly raised to temperature (T 4 ) within 5 minutes (t 4 -t 3 ) wherein T 4 is closer to Tm than T 2 but still below it.
  • the temperature parameters T 0 , T-,, T 2 , T 3 , T 4 and T 5 and the time parameters, t 2 , t 3 , t 4 , and t 5 to obtain a targeted pore structure and average ECD, need to be optimized for each type and concentration of gelatin-like protein and depending on what additives are present.
  • the sample columnar pore height requires optimization of the parameters T 5 and t 5 , with respect to T 4 and t 4. Higher samples will require a longer duration of slow temperature ramping from T 4 to T 5 to complete the freezing process.
  • the material which corresponds to the thin frozen layer formed in step c i, which has no columnar pores, is removed.
  • This dense bottom layer typically measures 1 to 2 mm thickness but, if desired, can be reduced to less than 1 mm by optimizing the chill bath temperature profile. Specifically the parameters t 2 -t-i , T 2 , t 3 -t 2 , T 3 , T 4 and t 4 -t 3 .
  • the temperature T 2 is in the range of from about minus 10 °C to about minus 50°C.
  • the chill bath temperature drop from T-, to T 2 is preferably reached within 3 minutes and is more preferably reached in less than 2 minutes.
  • the temperature profile for this temperature change may have any shape or form. This includes both linear and non linear profiles and may be optimized to achieve a specific uniform pore-size.
  • the temperature T 4 is about minus 4°C but always above temperature T 3 .
  • the raise in temperature of the chill bath from T 3 to T 4 is preferably reached within 2 minutes and is more preferably reached in less than 1 minute.
  • the temperature profile for this temperature change may have any shape or form. This includes both linear and non linear profiles and can be optimized to achieve a specific uniform average pore-size.
  • the temperature profile T 4 to T 5 may have any shape or form. This includes both linear and non linear profiles and may be optimized to achieve a specific uniform pore-size.
  • T 4 is set and the greater the slope of the temperature drop T 4 to T 5 the smaller the pore size.
  • the thermally insulated container can have any shape or form and can be made from any suitable material.
  • the thermally conductive surface may be made from any suitable thermally conductive material such as, for example, metals like copper and aluminum and include thermally conductive ceramics.
  • the thermally insulated container is PTFE or a teflon-like plastic, optionally in combination with a foamed material such as polyurethane or a ceramic foam, and the thermally conductive surface is a metal, particularly good thermal conductors like aluminum or copper.
  • step d any suitable freeze drying methods known in the art may be used.
  • the gelatin-like protein solution is degassed by lowering the atmospheric pressure below 50 mbar for at least 10 minutes.
  • gelatin-like protein solution is filter sterilized before use.
  • Cross-linking may utilize any cross-linking agent and technique known to one skilled in the art.
  • the porous material is cross-linked by a process which comprises chemical cross-linking.
  • Suitable chemical cross-linking agents include: aldehydes or dialdehydes, such as formaldehyde and glutaraldehyde, carbodiimides, diisocyanates, diketones, such as diacetyl and chloropentanedion, bis (2-chloroethylurea), 2-hydroxy-4,6-dichloro-1 ,3,5-triazine, reactive halogen-containing compounds disclosed in US 3,288,775, carbamoyl pyridinium compounds in which the pyridine ring carries a sulphate or an alkyl sulphate group disclosed in US 4,063,952 and US 5,529,892, divinylsulfones, and the like and S-triazine derivatives such as 2-hydroxy-4,6-dichloro-s-triazine. It also includes photo-activated cross-linking techniques.
  • Preferred chemical cross-linking agents are 1 -ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and hexamethylene diisocyanate (HMDIC).
  • porous material is cross-linked by a process which comprises dehydrothermal cross-linking.
  • a second aspect of the invention provides an in-vitro cell culturing system comprising a porous tissue scaffold according to the first aspect of the invention.
  • a third aspect of the invention provides an implantable biocompatible article comprising a porous tissue scaffold material according to the first aspect of the invention.
  • a fourth aspect of the invention provides the use of an implantable biocompatible article comprising the porous tissue scaffold material according to the first aspect of the invention as a bone filling material or bone filler, preferably a dental bone filler.
  • a fifth aspect of the invention provides the use of an implantable biocompatible article comprising the porous tissue scaffold material according to the first aspect of the invention as a microcarrier for cells preferably pluripotent cells.
  • the sample was subsequently filtered through a sterilizing (0.2 micron) syringe filter and deposited in a cylindrical thermally conducting Teflon-coated thin-walled aluminium container the sides of which are thermally insulated with a Teflon bush and insulating polymer foam between the Teflon bush and the aluminium sides.
  • the sample was optionally gelled at a reduced temperature T 0 to T-,, preferably at a temperature between the sample Tm and +25°C.
  • (c) gradually lower the temperature experienced by the thermally conducting surface, the rate of temperature drop being dictated by the desired pore structure and pore size (T 4 , t 4 to T 5 , t 5 ).
  • the T-profile T 4 to T 5 is not necessarily linear but can be optimised by a person skilled in the art.
  • the frozen samples are dried under a vacuum as is common in the art to produce dried and porous scaffolds.
  • the scaffold is then cross-linked in the absence of water by one of the many commonly used agents (HMDIC, EDC, glutaraldehyde, etc.) or a process (heating under a vacuum condition).
  • the basic collagen suspension used in this example was prepared from commercially available calf skin collagen type I, Sigma-Aldrich, prod, nr C351 1 . An amount was weighed in a flask and water was added to make a 2% (mass percent) dispersion. Then HCI was added to adjust the pH value to 3.2. According to (Schoof, Apel et al. 2001 ) acetic acid should be added to make directional pore structures. So, 2.5wt% acetic acid was added to the sample.
  • This collagen preparation is a fibrillar, insoluble type I collagen which is isolated from bovine skin. According to Schoof this suspension is a polydisperse system containing low concentrations of molecules, fibrils, and fibres.
  • the length and the width of the collagen aggregates vary in a wide range. Subsequently the dispersion was centrifuged to remove bubbles and an amount of 204 grams was deposited into a freezing container. The sample was allowed to cool to 2°C in a refrigerator for 2 hours and then placed in a chill bath at -5°C to allow complete freezing. Results
  • the sponge structure after drying showed directional pores.
  • EP0926543 or Example 1 of EP1014176 which example are herein incorporated by reference was used for the sample.
  • the ethanol (abs) was from J.T. Baker.
  • the water used was ultrafiltrated and deionized to the same specifications as pharmaceutical grade Water For Injection (WFI).
  • the solution was filtered by passing through a Whatman PURADISC® 25 AS (PES) syringe filter 0.2 ⁇ , using a 10ml NORM-JECT® luer syringe.
  • the freeze dryer used was a Zirbus 3x4x5 Sublimator.
  • An AZ 8852 dual input type K/J/T thermometer with a Thermo-Electra handheld probe nr. 80106 was used for solution temperature measurements.
  • Optical sponge inspection was done using an Olympus SZX12 microscope equipped with an Olympus digital camera C-3040ZOOM and DP-Soft V3.2 software.
  • Sample lighting was done using either the internal light source or a FOSTECTM DCR (DDL) external light source.
  • a Jeol JSM-6330F Field Emission Scanning Electron Microscope was used for generating SEM images. Sample cutting was done manually using GEM stainless steel razor blades (uncoated).
  • sample solutions were gelled by putting them in a refrigerator at 2°C for at least 3 hours.
  • the (pre-gelled) sample was then subjected to freezing condition of -10°C in the thermostatic circulator bath (2 to 3mm deep) for 45 minutes until completely frozen.
  • the frozen sample was then dried under a vacuum for 2 days.
  • Cross-linking by DeHydroThermal (DHT) treatment was carried out with the samples cut to the desired size by heating them in a vacuum oven at 160°C at a pressure less than 2 mbar for 2 days.
  • DHT DeHydroThermal
  • the frozen sample was then dried under a vacuum for 2 days.
  • Cross- linking by DeHydroThermal (DHT) treatment was carried out with the samples cut to the desired size by heating them in a vacuum oven at 160°C at a pressure less than 2 mbar for 2 days.
  • the sample solution was gelled in a refrigerator set at 2°C for 45 minutes.
  • the (pre-gelled) sample was then subjected to a freezing condition of -10°C in a chill bath (2 to 3mm deep) for 45 minutes until completely frozen.
  • the frozen sample was then dried under a vacuum for 2 days.
  • Cross-linking by DeHydroThermal (DHT) treatment was carried out with the samples cut to the desired size in a vacuum oven at 160°C and a pressure less than 2 mbar for 2 days.
  • DHT DeHydroThermal
  • the resulting pore structure was columnar over the sample volume with columnar pores of narrow size distribution and large average size of approx. 350 micron in the upper sample half. Columnar pore widening from the lower to the upper sample volume was also seen, see Figures 15 and 16. It is believed that this was due to the use of a constant chill bath temperature instead of a gradual temperature ramp. Thus, as the freezing front moves away from the cooled container, the local freezing temperature was expected to be ever higher and due to this the pores were growing larger.
  • PE-foil was used as a cover to exclude airborne dust and prevent the evaporation of water or ethanol from the top of the sample.
  • the sample solution was gelled by placing in a refrigerator at 2°C for 1 day. The sample was then placed in a chill bath set at 18°C and pump A was started, pumping very cold (-72 ⁇ 2°C) liquid (ethanol) into the chill bath with vigorous mixing at such a rate that the chill bath temperature dropped at a rate of 22°C/min until the chill bath temperature reached -52°C. During this T-ramping the first appearance of ice formation was observed at a chill bath temperature of -16°C.
  • DHT DeHydroThermal
  • the sample solution was gelled in the chill bath at 10°C for 20 minutes.
  • the chill bath was then rapidly cooled by pumping very cold (-72 ⁇ 2°C) liquid (ethanol) into the chill bath with vigorous mixing at such a rate that the chill bath temperature droped at a rate of 28°C/min until the temperature reached -30°C.
  • very cold (-72 ⁇ 2°C) liquid (ethanol) into the chill bath with vigorous mixing at such a rate that the chill bath temperature droped at a rate of 28°C/min until the temperature reached -30°C.
  • the cold liquid pump A was stopped and the chill bath temperature remained fairly constant within the time it took for the full contacting sample container to become covered with a frozen sample layer.
  • DHT DeHydroThermal
  • the resulting pore structure was very homogeneous over the sample volume with columnar pores of narrow size distribution and large average size of approx. 350 micron.
  • the pore structure in the lower sample region was very similar to the structure in the upper sample region ( Figure 10) in contrast to Comparative Example 6 ( Figures 19 and 20).
  • the roundness of the pores was higher than 0.5, as preferred.
  • the dense nucleated bottom layer See Figures 8 and 9) can be cut and discarded at will.
  • the average pore ECD for this example was 300 micron with an ECD SD of 90 micron.

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  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Epidemiology (AREA)
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Abstract

L'invention concerne un échafaudage de tissu poreux à base de gélatine recombinante ayant une structure de pore définie régulière.
PCT/GB2012/052706 2011-11-07 2012-10-31 Echafaudages de tissu poreux Ceased WO2013068723A1 (fr)

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GBGB1119182.2A GB201119182D0 (en) 2011-11-07 2011-11-07 Porous tissue scaffolds

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114904058A (zh) * 2021-12-22 2022-08-16 广州锐澄医疗技术有限公司 一种规则多孔支架及其制备方法与其在人工角膜中的应用
CN119455067A (zh) * 2025-01-14 2025-02-18 四川大学 一种复合孔结构胶原蛋白止血海绵及其制备方法
US12268713B2 (en) * 2015-04-03 2025-04-08 Biochange Ltd. Methods of generating cross-linked protein foams in situ

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12268713B2 (en) * 2015-04-03 2025-04-08 Biochange Ltd. Methods of generating cross-linked protein foams in situ
CN114904058A (zh) * 2021-12-22 2022-08-16 广州锐澄医疗技术有限公司 一种规则多孔支架及其制备方法与其在人工角膜中的应用
CN114904058B (zh) * 2021-12-22 2024-04-16 广州锐澄医疗技术有限公司 一种规则多孔支架及其制备方法与其在人工角膜中的应用
CN119455067A (zh) * 2025-01-14 2025-02-18 四川大学 一种复合孔结构胶原蛋白止血海绵及其制备方法

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