Resorbable crosslinked form stable membranes
阅读说明：本技术 可再吸收交联形式稳定膜 (Resorbable crosslinked form stable membranes ) 是由 N·施蒂费尔 R·考夫曼 S·斯滕泽尔 于 2018-06-01 设计创作，主要内容包括：本发明涉及一种可再吸收交联形式稳定膜,其包含胶原蛋白材料和无机陶瓷颗粒的复合层,所述无机陶瓷颗粒包含相对于1重量份的胶原蛋白材料为1.5至3.5重量份的无机陶瓷,所述复合层夹在两个弹性预张紧胶原蛋白材料层(已经被拉伸以处于应力-应变曲线的线性/弹性区中的胶原蛋白材料)之间,所述胶原蛋白材料包含50％至100％(w/w)的胶原蛋白和0％至50％(w/w)的弹性蛋白并且具有适合在鼻成形术、后外侧脊柱融合或眼眶重建中用于口腔外的人组织再生的形状和尺寸。(The present invention relates to a resorbable crosslinked form stable membrane comprising a composite layer of collagen material and inorganic ceramic particles comprising 1.5 to 3.5 parts by weight of inorganic ceramic relative to 1 part by weight of collagen material, said composite layer being sandwiched between two layers of elastic pre-tensioned collagen material (collagen material that has been stretched to be in the linear/elastic region of the stress-strain curve), said collagen material comprising 50% to 100% (w/w) collagen and 0% to 50% (w/w) elastin and having a shape and size suitable for use in extraoral human tissue regeneration in rhinoplasty, posterolateral spinal fusion or orbital reconstruction.)
1. A resorbable crosslinked form stable membrane comprising a composite layer of collagen material and inorganic ceramic particles, the composite layer comprising 1.5 to 3.5 parts by weight of inorganic ceramic to 1 part by weight of collagen material and being sandwiched between two layers of elastic pre-stressed collagen material, wherein the elastic pre-stressed collagen material is a collagen material that has been stretched so as to be in the linear/elastic region of the stress-strain curve, the collagen material comprising 50% to 100% (w/w) collagen and 0% to 50% (w/w) elastin and having a shape and size suitable for use in extraoral human tissue regeneration in rhinoplasty, posterolateral spinal fusion or orbital reconstruction.
2. The resorbable crosslinked form stable membrane of claim 1 selected from the group consisting of:
a nasal arch membrane for rhinoplasty, sized to fit the required nasal size,
an oval tubular membrane for posterolateral spinal fusion, the length of which covers more than two vertebrae, and
a membrane for reconstruction of orbital fractures, which is shaped after definition of the bony prominences apt to support the implant, and is of a size to facilitate its insertion into the orbital cavity.
3. A resorbable crosslinked form stable membrane according to claim 2 which is a nasal arch for rhinoplasty as shown in fig. 1(4) having a length l, width i and height h of 40mm to 80mm, 10mm to 15mm and 10mm to 15mm, respectively.
4. The resorbable crosslinked form stable membrane of claim 3, wherein the thickness of the nasal arch membrane is 0.5mm to 2.5 mm.
5. The resorbable crosslinked form stable membrane of claim 3, wherein the wall of the nasal arch has a thickness of 1.0mm to 2.0 mm.
6. The resorbable crosslinked form stable membrane of claim 2 which is a slit oval tubular membrane for posterolateral spinal fusion as shown in fig. 1 (5') with a length l of 60mm to 300mm, an inner diameter k of 5mm to 10mm and an outer diameter j of 15mm to 30 mm.
7. The resorbable crosslinked form stable membrane of claim 6, wherein the membrane has a thickness of 0.5mm to 2.5 mm.
8. A resorbable crosslinked form stable membrane according to claim 1 which is a membrane for reconstruction of orbital fractures as shown in figures 1(6) and (6'), wherein the length m, width o and height c of the membrane are 30mm to 50mm, 20mm to 40mm and 5mm to 25mm, respectively.
9. The resorbable crosslinked form stable membrane of claim 8, wherein the membrane has a thickness of 0.5mm to 2.5 mm.
10. A resorbable crosslinked form stable membrane according to any of claims 1 to 9 wherein the composite layer of collagen material and inorganic ceramic particles comprises 2.0 to 3.0 parts by weight of inorganic ceramic per 1 part by weight of collagen material.
11. A resorbable crosslinked form stable membrane according to any of claims 1 to 9 wherein the collagen material comprises 70% to 90% collagen and 10% to 30% elastin.
12. The resorbable crosslinked form stable membrane of any one of claims 1 to 9, wherein the collagen material is derived from porcine, bovine, or equine peritoneal or pericardial membranes, small intestinal mucosa (SIS), or muscle fascia.
13. A resorbable crosslinked form stable membrane according to any of claims 1 to 9 wherein one or both of the layers of elastic pre-stressed collagen material comprise pores of 5 μ η ι to 1000 μ η ι.
14. A resorbable crosslinked form stable membrane according to any of claims 1 to 9 wherein the inorganic mineral particles have a size of 150 μm to 500 μm.
15. A resorbable crosslinked form stable membrane according to any of claims 1 to 9 wherein the inorganic ceramic is selected from the group consisting of hydroxyapatite or hydroxyapatite bone mineral.
16. A method of regenerating human tissue outside the oral cavity comprising applying the resorbable crosslinked form of the stabilization membrane of claim 1 to a surgical site of a human patient undergoing rhinoplasty, posterolateral spinal fusion, or orbital reconstruction.
Technical Field
The present invention relates to resorbable, cross-linked form-stable compositions for use outside the oral cavity, in particular in the craniofacial area and cervical, thoracic, lumbar and sacral regions of the spine, or in human tissue regeneration in craniotomy reconstructive surgery.
Background
In order to regenerate non-bone-containing defects by bone formation, for example, in horizontal or Vertical enlargement of The maxilla or mandible, mechanical stabilization of The defect is required (Bendkowski, 2005, Space to group, The Dentist; Merli et al, 2007, Vertical edge assessment with automatic bone graft: restricted areas supported by The underlying bone surfaces Vertical titanium-reinforced concrete-A laminated support of a blocked, random controlled locked loop, internal J major Implants; Burger, 2010, user of open-oriented porous polymeric-D-L-composites (Sound insulation) resin and resin, aluminium-reinforced concrete-resin, aluminium-reinforced concrete-D-L-concrete-resin (aluminium-concrete) resin and resin, aluminium-reinforced concrete-resin, aluminium-reinforced concrete-resin, aluminium-concrete-cement, aluminium-alloy, aluminium-reinforced concrete-cement, aluminium-aluminium alloy, aluminium-cement, aluminium-aluminium alloy. Indeed, oral tissue is exposed to complex mechanical forces during chewing, swallowing, tongue movement, speech, tooth movement and orthodontic treatment. Particularly during wound healing after surgery, internal and external forces may be generated, creating pressure, shear and bending moments on the regenerative device and the newly formed tissue.
Form-stabilizing membranes against these forces are a useful means of achieving mechanical stability.
For this purpose it is known to use Ti mesh, Ti plates or Ti reinforced PTFE form stabilizing membranes, which must be removed after bone regeneration during a second operation. One example of a commercially available Ti-enhanced form stable film is sold by Osteogenics
Non-reinforced PTFE membranes were widely used before the introduction of resorbable collagen membranes in 1996, but disappeared soon after the introduction of collagen membranes.
Resorbable form-stable membranes are of interest in order to avoid the need to remove the form-stable membrane or mesh in a secondary operation. Several resorbable form-stable films or meshes have been described, essentially made of PLA (polylactic acid) or PLGA (polylactic-co-glycolic acid). Examples are in particular (1) Sonic from KLS Martin
To overcome PLGA/PLA-related wound healing problems, autologous bone pieces from patients are used as well as partially or fully purified bone pieces, e.g., Geistlich
In order to be able to use autologous bone fragments harvested during surgery, usually in combination with xenogenic bone graft particles, the so-called bone shield (bone shield) technique was developed using autologous cortical bone from the mandible (Khoury et al, 2007, bone augmentation in Oral implantation research). The disadvantage of this procedure is that it is extremely technically sensitive and is associated with morbidity and more pain in the second site. Furthermore, the shield is applied only laterally and therefore does not give mechanical protection from the coronal plane of the defect. The term "bone shield" is used to generalize PLA/PLGA membranes and partially demineralized cortical bone shields (semi-soft or soft thin plates from Tecnoss)
An attempt to combine good healing and form stability is a resorbable form-stable collagen membrane disclosed in US-8353967-B2, which is prepared from a collagen suspension in 5-25% ethanol/water in a mould by freeze-drying and heating at 100 ℃ to 140 ℃. Such membranes are manufactured by Osseous Technologies in the United states and sold by Zimmer under the trade name "Zimmer CurV Preshaped Collagen Membrane". However, the commercial membrane had poor form stability and a thickness of about 1.5mm, increasing to about 2.3mm after incubation in saline; this may lead to a risk of high cracking rates.
In summary, the current solutions are therefore not entirely satisfactory for the dentist or the patient. Or a secondary operation must be performed and/or there is a high risk of poor wound healing. Solutions that are not associated with a high risk of poor wound healing are either not form stable membranes, require secondary surgery or have other drawbacks.
US2013/0197662 discloses a method for manufacturing a biomaterial comprising a) bonding a porous collagen-based material to a non-porous collagen-based material by applying a controlled amount of a gel comprising collagen to a bonding surface of the non-porous collagen-based material and contacting the porous collagen-based material surface with the gel applied to the bonding surface to partially hydrate a portion of the porous material located at an interface between the materials; b) drying the gel to bond the materials together; and c) crosslinking the collagen in the adhesive layer. The manufactured biomaterial obtained combines a porous collagen-based material that can mineralize [0042], [0048] and a non-porous collagen-based material that is mechanically strong, thus providing a scaffold for regenerating load-bearing tissues, in particular meniscus, articular cartilage, tendons and ligaments, with porosity and mechanical strength, i.e. capable of withstanding compressive and tensile forces. There is no disclosure regarding the composition of the porous collagen-based material resistant to bending moments or mineralisation of the combined biomaterial.
US2014/0193477 teaches that stretching collagen before its crosslinking increases its mechanical strength, in particular ultimate tensile strength, stiffness and elastic modulus (young's modulus) when preparing a collagen mat from soluble collagen (see in particular [0109], [0110 ]).
Chachra et al, 1996, Effect of applied systemic stress on rate and biochemical effects of cross-linking in tissue-derived Biomaterials, Biomaterials disclose stretching a pericardium-derived membrane to increase its tensile strength and stiffness prior to crosslinking.
The object of the invention disclosed in EP-a1-3175869 is to provide a resorbable form stable membrane for the oral cavity (resorbable form stable membrane) which is easily resistant to pressure, shear forces and bending moments so as to support bone formation, bone regeneration, bone repair and/or bone replacement at non-containing bone defect sites, in particular at horizontally or vertically enlarged sites of the maxilla or mandible, without the above-mentioned disadvantages.
EP-a1-3175869 reports that this object is achieved by the invention defined by a resorbable crosslinked form of stable film (or composition) for the oral cavity comprising a composite layer of a collagen material and inorganic ceramic particles, said composite layer being sandwiched between two layers of elastic pre-tensioned (pre) collagen material, said composite layer containing 1.5 to 3.5 parts by weight of inorganic ceramic relative to 1 part by weight of collagen material, said collagen material comprising 50-100% (w/w) collagen and 0-50% (w/w) elastin.
The term "collagen material" herein refers to a collagen-based material comprising 50-100% (w/w) collagen and 0-50% (w/w) elastin. Elastin content is measured herein by desmosine/isodesmosine assays according to modifications of known methods involving hydrolysis and RP-HPLC (see, e.g., Guida et al, 1990, Development and evaluation of a high performance chromatography for The determination of desmosine in properties, Journal of chromatography; or Rodriguqe, 2008, Quantification of motion Lung enzyme Dual in Prenat Development, The Open resolution media Journal). To determine the desmosine/isodesmosine content of dry Elastin, the Elastin of the sponge is subjected to an Elastin isolation procedure (Purification and Comparison of elastic from Different Animal specificities, Analytical Biochemistry) as described by Starcher et al, 1976.
The collagen material is suitably derived from a tissue of natural origin containing collagen and elastin in such proportions. Examples of such tissues include peritoneum or pericardium, placental membrane, Small Intestinal Submucosa (SIS), dermis, dura mater, ligaments, tendons, diaphragm (diaphragm), omentum, fascia of a muscle or organ of a vertebrate, particularly a mammal (e.g., pig, cow, horse, sheep, goat, rabbit). Such tissue is preferably porcine, bovine or equine. One tissue of interest is the peritoneum of pigs, cattle or horses.
Typically, the collagen is predominantly type I collagen, type III collagen, or a mixture thereof. The collagen may also comprise a proportion of type II, type IV, type VI or type VIII collagen or any combination thereof or any collagen type.
Preferably, the collagen material comprises 70-90% (w/w) collagen and 30-10% (w/w) elastin.
Examples of suitable starting materials for the preparation of such collagen materials are collagen films from porcine, bovine or equine peritoneum or pericardium prepared by a method similar to that described in the "examples" of EP-B1-1676592, or a film Geistlich prepared from porcine peritoneum by this method
Preferably, the collagen material is derived from porcine, bovine or equine peritoneal or pericardial membranes, small intestinal mucosa (SIS) or muscle fascia.
The collagen material is preferably a fibrous collagen material, either having a natural fibrous structure or as a cut collagen fiber.
However, non-fibrous collagen materials, such as fibrils restored from molecular collagen or cross-linked collagen fragments with sufficient biocompatibility and reabsorbability, may also be used in the composite layer of the collagen material and inorganic ceramic particles, or in the elastic pre-stressed collagen material layer, provided that the collagen material has sufficient mechanical stability in terms of elastic modulus and maximum tensile strength (see below).
The term "resorbable" as used herein means that the cross-linked form of the stable membrane is capable of being absorbed significantly in vivo through the action of collagenase and elastase. Controlled in vivo resorbability of the cross-linked form stable membrane is critical to healing without excessive inflammation or dehiscence. The enzymatic degradation test using collagenase (example 4.3) from clostridium histolyticum (clostridium histolyticum), described in detail below, gives an excellent prediction of in vivo reabsorbability.
All test models (prototypes) tested for the resorbable crosslinked form stable membranes of the invention showed at least 10% collagen degradation (assessed by DC protein assay using type I collagen as standard) after 4 hours, collagen degradation rate (lower than Geistlich)
The term "crosslinked" means that the resorbable form-stable membrane or composition has been subjected to at least one crosslinking step, typically chemical crosslinking (using for example EDC and NHS) or by dehydrothermal treatment (DHT), which is typically carried out on an assembled composite layer of collagen material and inorganic ceramic particles sandwiched between two layers of elastic pre-stressed collagen material, by chemical crosslinking (using for example EDC and NHS) or by dehydrothermal treatment (DHT). Optionally, the composite layer of collagen material and inorganic ceramic particles has been cross-linked, typically by chemical cross-linking or by dehydrothermal treatment (DHT), prior to assembly into the inventive membrane.
The term "form-stable film for use in the oral cavity" refers to a film that resists formation in the oral cavity and other tissues by providing mechanical stabilization of defectsThe resorbable cross-linked membranes are capable of supporting bone formation, bone regeneration, bone repair and/or bone replacement at non-containing (non-contracting) dental bone defects in humans or animals. The form stability of the inventive film was evaluated by the 3-point uniaxial bending test described in detail below (in example 4.2): the test is similar to the method described in EN ISO 178 and ASTM D6272-10, the inventive film being immersed in Phosphate Buffered Saline (PBS) at pH 7.4 and temperature 37 ℃. The test shows that the film of the invention provides a specific PLA film
Typically, the resorbable crosslinked form stable membrane resists a force of at least 0.20N, preferably at least 0.30N, for a strain of 8mm in a 3-point uniaxial bending test.
The term "elastic pre-tensioned collagen material layer" means that the collagen material layer has been subjected to a tensioning action prior to its cross-linking, resulting in an elongation or extension of the original dimension of the collagen material layer from the toe region (toe region) of the stress-strain curve into the linear (also called elastic) region (Roeder et al, 2002, Tensile mechanical properties of the soft-dimensional type I collagen ex-cellular substrates with the variable geometry, J Biomech Eng, especially figure 3, page 216, or figure 5 of this application). In this linear region, the modulus of elasticity is highest and therefore the highest stiffness can be achieved. This tensioning can be performed radially on the collagen material block, for example by means of a spring. The force applied to cause the collagen material to elongate or stretch to the linear region of the stress-strain curve depends on the collagen material. When the collagen material originates from the peritoneum of a pig, cow or horse, the tensioning that results in the linear region of the stress-strain curve of the collagen material may be performed radially on the collagen material mass, for example by a spring with a tension in the range of 1N to 3N such that the initial dimension of the collagen material layer is elongated or stretched by 40% to 100%.
The term "elastic pre-tensioned collagen material" therefore means a collagen material that has been stretched so as to be in the linear (elastic) region of the stress-strain curve.
The elastic modulus (also called young's modulus), i.e. the slope of the linear region of the stress-strain curve expressed in MPa, of the elastic pre-tensioned collagen material is typically from 1 to 1000MPa, preferably from 2 to 150MPa, in particular from 5 to 80 MPa.
The presence of those two "elastic pre-tensioned collagen material" layers sandwiching the composite layer of collagen material and inorganic ceramic particles may be necessary to prevent the composite layer from breaking when the film is subjected to tension, compression, shear forces and bending moments.
It is preferred that one or both of the layers of elastic pre-tensioned collagen material comprises 5-500 μm pores. These pores allow easy invasion of bone forming cells into the inorganic ceramic-collagen composite when the membrane is in a position where the elastic pre-tensioned piercing layer of collagen material will be oriented towards the bone defect.
The inorganic ceramic is a biocompatible material that promotes bone regeneration, such as hydroxyapatite or natural bone minerals.
A well-known natural bone mineral for promoting bone growth in dental, periodontal and maxillary bone defects is Geistlich
Preferably, the inorganic ceramic is a natural bone mineral based on hydroxyapatite, for example, Geistlich
The inorganic ceramic particles generally have a size of from 50 to 600. mu.m, preferably from 150 to 500. mu.m, in particular from 250 to 400. mu.m.
The composite of the collagen material and the inorganic ceramic particles comprises 1.5 to 3.5 parts by weight, preferably 2.0 to 3.0 parts by weight of the inorganic ceramic with respect to 1 part by weight of the collagen material.
In fact, it was unexpectedly found that if less than 1.5 parts by weight of inorganic ceramic per 1 part by weight of collagen material or more than 3.5 parts by weight of inorganic ceramic per 1 part by weight of collagen material, the membrane is not "form stable" as defined above and evaluated by the 3-point uniaxial bending test (in example 4.2) described in detail below. The form stability is particularly high when the composite of the collagen material and the inorganic ceramic particles comprises 2.0 to 3.0 parts by weight of the inorganic ceramic with respect to 1 part by weight of the collagen material.
The resorbable crosslinked form stable membranes of this invention are hydrophilic and are generally completely wetted by PBS within 5 to 15 minutes.
The resorbable crosslinked form stable membranes of the invention have properties similar to Geistlich
This good healing property has been observed when the cross-linked form of the present invention stabilizes the membrane to prevent bone defects that develop in rabbit skull.
The thickness of the resorbable crosslinked form-stable membranes of the invention is generally from 0.5 to 2.5mm, preferably from 1.0 to 2.0mm, in particular from 1.2 to 1.8 mm.
Typical shapes and typical dimensions of resorbable crosslinked form-stable membranes of the invention are shown in FIG. 1 of EP-A1-3175869.
The invention of EP-a1-3175869 also relates to the use of the above resorbable crosslinked form stable membranes as implants to support bone formation, bone regeneration, bone repair and/or bone replacement at sites of non-containing dental bone defects in the oral cavity of a human or animal.
EP-a1-3175869 also describes a method of preparing a resorbable crosslinked form stable membrane as defined above, said membrane comprising a composite layer of collagen material and inorganic ceramic particles sandwiched between two layers of elastic pre-tensioned collagen material, the method comprising the steps of:
(a) preparing a composite layer of collagen material and inorganic ceramic particles, optionally cross-linking the composite layer,
(b) assembling and bonding said composite layer of collagen material and inorganic ceramic particles between two layers of collagen material subjected to a tensioning action causing the collagen material to stretch into the linear region of the stress-strain curve, thereby obtaining a composite layer of collagen material and inorganic ceramic particles sandwiched between two layers of elastic pre-tensioned collagen material, and
(c) the composite layer of collagen material and inorganic ceramic particles sandwiched between two layers of elastic pre-tensioned collagen material is cross-linked and subsequently subjected to a hydrophilization treatment.
Step (a) may be performed as follows:
-obtaining hydroxyapatite bone mineral particles from cortical or cancellous bone as inorganic ceramic particles by A method similar to the method described in US-A-5417975, or by grinding Geistlich Bio-ossmall Granules (available from Geistlich pharmA AG) to smaller particles, and sieving these particles in the desired range (e.g. 150-.
-preparing a fibrous collagen material as follows:
the collagen-rich tissue from porcine, bovine or equine peritoneum or pericardium is subjected to a method similar to the method described in the examples of EP-B1-1676592, or starting from a gerliclichbio-Gide membrane obtained by this method from porcine peritoneum (available from gerlich Pharma AG) or an intermediate obtained before sterilization from the industrial production of gerlich Bio-Gide membrane, herein referred to as unsterilized gerlich Bio-Gide membrane,
o cutting the collagen fiber tissue thus obtained into pieces (for example, with scissors), mixing these cut collagen fiber tissue pieces with dry ice with a knife mill, thereby obtaining cut collagen fibers,
o cutting the collagen fiber tissue block with a cutting mill with a sieve, thereby obtaining a sieved fraction of collagen fiber fragments.
-preparing a composite layer of fibrous collagen material and hydroxyapatite bone mineral particles by:
o mixing and shaking 0 to 40 wt% of the cut collagen fiber and 60 to 100 wt% of the above obtained sieved fraction of collagen fiber pieces in phosphate buffered saline PBS,
omicron 1.5 to 3.5 parts by weight, in particular 2.0 to 3.0 parts by weight of the sieved hydroxyapatite bone mineral particles obtained above are added to 1 part by weight of the fibrous collagen obtained above, centrifuged at 2000 to 6000 xg, preferably 3000 to 5000 xg, the resulting pellets are cast into a rectangle and formed into a flat plate using a spatula. And drying the obtained composite layer of the fibrous collagen material and the hydroxyapatite bone mineral particles in a vacuum oven.
It is not necessary to cross-link the dry composite layer of collagen material and inorganic ceramic particles at the end of (a), but it has the advantage of facilitating the handling of the composite layer during step (b).
The crosslinking can be carried out using chemicals or by dehydrothermal treatment (DHT).
Crosslinking using chemicals can be performed using any pharmaceutically acceptable crosslinking agent capable of imparting the mechanical strength required to stabilize the membrane in a crosslinked form. Suitable such crosslinking agents include glutaraldehyde, glyoxal, formaldehyde, acetaldehyde, 1, 4-butanediglycidyl ether (BDDGE), N-sulfosuccinimidyl-6- (4 '-azido-2' -nitrophenylamino) hexanoate, hexamethylene diisocyanate (HMDC), cyanamide, diphenylphosphoryl azide, genipin (genipin), EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide), and mixtures of EDC and NHS (N-hydroxysuccinimide).
Crosslinking using chemicals can conveniently be performed using a mixture of EDC and NHS.
In this case, the fiber obtained as aboveThe dried composite layer of the collagenous material and hydroxyapatite bone mineral particles can be crosslinked in a solution of 10-400mM EDC and 13-520mM NHS in 0.1M MES (2- (N-morpholino) -ethanesulfonic acid) and 40% ethanol at pH 5.5 at room temperature for 1 to 3 hours. Then the solution can be passed through 0.1M Na at pH9.52HPO4The reaction was terminated by incubating the model in buffer twice for 1 to 3 hours. Polar residues were removed by incubating the model for 1 hour in 1M sodium chloride solution and twice for 1 hour in 2M sodium chloride solution. The chemically crosslinked model can be washed in distilled water for a total of 8 times from 30 to 60 minutes. Drying may then be carried out by: immersion in ethanol for a total of 5 times for 15 minutes, followed by treatment with diethyl ether for three times for 5 minutes, followed by drying at 10mbar and 40 ℃ overnight, or by freeze-drying (freezing to below-10 ℃ and drying by conventional freeze-drying processes).
Alternatively, the crosslinking is carried out by dehydrothermal treatment (DHT) at 0.1-10mbar and 80-160 ℃ for 1-4 days. In this case, a subsequent drying process is not required.
Step (b) may be performed as follows:
-preparing collagen fibrin glue as follows:
o using a high pressure homogenizer at 1500-2000 bar, the above collagen fragment sieve fraction at pH 3.5H3PO4Mixing the aqueous solution at a concentration of 3%, repeating the mixing several times,
o neutralizing the resulting slurry to pH 7.0 by adding sodium hydroxide solution, concentrating the collagen by freeze-drying and homogenizing it by knife mill,
o preparing collagen fibrin glue from a slurry obtained as a 2-10% solution in phosphate buffered saline, PBS, pH 7.4, by heating to 60 ℃ until no other particles are observed, and
subjecting the two pre-wetted collagen material layers to a tensioning action, using for example an apparatus similar to figure 2, causing said collagen material to stretch in the linear region of the stress-strain curve, thus obtaining two wet elastic pre-tensioned collagen material layers,
inserting the composite layer of the collagen material absorbed with the collagen fibril glue and the inorganic ceramic particles obtained in (a) between the two wet elastic pre-tensioned collagen material layers,
laminating the two wet elastic pre-tensioned collagen materials on a composite layer of collagen material absorbed with collagen fibre gel and inorganic ceramic particles using, for example, an apparatus similar to that of figure 3, and
the composite layer of collagen material and inorganic ceramic particles sandwiched between two layers of wet elastic pre-tensioned collagen material is dried at a temperature of 35 to 45 ℃ and under reduced pressure (e.g. 20 to 1 mbar).
In the above process, one or both of the pre-wetted collagen material layers may have been punctured with a needle to include pores of 5-500 μm.
In step (c), the composite layer of collagen material and inorganic ceramic particles sandwiched between two layers of elastic pre-tensioned collagen material may be cross-linked using chemicals (using, for example, EDC and NHS) or by dehydrothermal treatment of DHT.
Chemical crosslinking may be carried out using any pharmaceutically acceptable crosslinking agent capable of imparting the crosslinked form with the mechanical strength required to stabilize the membrane. Suitable such crosslinking agents include glutaraldehyde, glyoxal, formaldehyde, acetaldehyde, 1, 4-butane diglycidyl ether (BDDGE), N-sulfosuccinimidyl-6- (4 '-azido-2' -nitrophenylamino) hexanoate, hexamethylene diisocyanate (HMDC), cyanamide, diphenylphosphoryl azide, genipin, EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide), and mixtures of EDC and NHS (N-hydroxysuccinimide).
Crosslinking using chemicals can conveniently be performed using a mixture of EDC and NHS.
In this case, the composite layer of collagen material and inorganic ceramic particles sandwiched between two layers of elastic pre-tensioned collagen material obtained as above can be crosslinked at room temperature for 1 to 3 hours at pH 5.5 in a solution of 10-400mM EDC and 13-520mM NHS in 0.1M MES (2- (N-morpholino) -ethanesulfonic acid) and 40% ethanol.
This may then be done by 0.1M Na at pH9.52HPO4In a buffer solutionThe model was incubated for 1 to 3 hours twice to terminate the reaction. Polar residues were removed by incubating the model for 1 hour in 1M sodium chloride solution and twice for 1 hour in 2M sodium chloride solution. The chemically crosslinked model can be washed in distilled water for a total of 8 times from 30 to 60 minutes. Drying may then be carried out by: immersion in ethanol for a total of 5 times for 15 minutes, followed by treatment with diethyl ether for three times for 5 minutes, followed by drying at 10mbar and 40 ℃ for 30 minutes, or by freeze-drying (freezing to below-10 ℃ and drying by conventional freeze-drying processes) without solvent treatment.
Alternatively, the crosslinking is carried out by dehydrothermal treatment (DHT) at 0.1-10mbar and 80-160 ℃ for 1-4 days. In this case, a subsequent drying process is not required.
The hydrophilization treatment of step c) generally comprises immersing the crosslinked composite layer of collagen material and inorganic ceramic particles sandwiched between two layers of elastic pre-tensioned collagen material in a physiologically acceptable salt solution, for example a sodium chloride solution, preferably 100-.
It is preferred that the hydrophilisation treatment comprises immersing the crosslinked composite layer of collagen material and inorganic ceramic particles sandwiched between two layers of elastic pre-tensioned collagen material in a sodium chloride solution to render it hydrophilic and subsequently drying as described in any of the methods described above.
The resorbable crosslinked form of the stable membranes of EP-A1-3175869 can be sterilized by X-ray, β ray or gamma ray.
EP-A1-3175869 discloses resorbable crosslinked form-stable films of various shapes for use in the oral cavity. The membrane may be flat (1), (1'), U-shaped straight (2), (2'), or U-shaped curved (3), (3') corresponding to the alveolar space of 1 to 3 teeth (incisors, canines, premolars, or molars) located at the front, left or right side curve or back of the denture.
It has now been found that a resorbable crosslinked form stabilising composition comprising a composite layer of a collagen material and inorganic ceramic particles sandwiched between two elastic pre-tensioned collagen layers as disclosed in EP-a1-3175869 can be used as a resorbable crosslinked form stabilising membrane which can be used for human tissue regeneration outside the oral cavity, in particular in the craniofacial area and the cervical, thoracic, lumbar and sacral areas of the spine, or in craniotomy reconstructive surgery, when its shape and dimensions are adapted for this purpose. The shape and size of the resorbable crosslinked form stable membranes disclosed in EP-a1-3175869 for use in the oral cavity are not practically suitable for such use outside the oral cavity.
Disclosure of Invention
Accordingly, the present invention relates to a resorbable crosslinked form stable membrane comprising a composite layer of collagen material and inorganic ceramic particles comprising 1.5 to 3.5 parts by weight of inorganic ceramic relative to 1 part by weight of collagen material, said composite layer being sandwiched between two elastic pre-tensioned collagen material layers, wherein the pre-tensioned collagen material is a collagen material that has been stretched to be in the linear/elastic region of the stress-strain curve, said collagen material comprising 50% to 100% (w/w) collagen and 0% to 50% (w/w) elastin and having a shape and size suitable for use in extraoral human tissue regeneration in rhinoplasty, posterolateral spinal fusion or orbital reconstruction.
Preferably, the resorbable crosslinked form stable membrane is selected from the group consisting of:
a nasal arch membrane for rhinoplasty, sized to fit the required nasal size,
an oval tubular membrane for posterolateral spinal fusion, the length of which covers more than two vertebrae, and
a membrane for reconstruction of orbital fractures, which is shaped after the definition of the bony processes (bone ridges) apt to support the implant, and is of a size to facilitate its insertion inside the orbital cavity.
All terms of the invention relating to the above definitions have the same meaning as set out above for EP-A1-3175869.
The resorbable crosslinked form stable membrane may be a nasal arch membrane for rhinoplasty as shown in fig. 1, (4) wherein the length l, width i and height h are typically 40mm to 80mm, 10mm to 15mm and 10mm to 15mm, respectively. Typically, the thickness of the wall of the nasal arch is 0.5mm to 2.5mm, preferably 1.0mm to 2.0 mm.
The resorbable crosslinked form of the stabilization membrane may be a slit oval tubular membrane for posterolateral spinal fusion as shown in fig. 1, (5') wherein typically the length l is 60mm to 300mm, the inner diameter k is 5mm to 10mm and the outer diameter j is 15mm to 30 mm. Typically, the wall of the oval tubular membrane used for posterolateral spinal fusion is 0.5 to 2.5mm thick.
The resorbable crosslinked form stable membrane for use outside the oral cavity may also be a slit rectangular tube membrane for posterolateral spinal fusion as shown in 1, (5) having a length l of 60mm to 300mm, a width k of 5mm to 10mm and a height j of 15mm to 30 mm. Typically, the thickness of the wall of a slit rectangular tube membrane for posterolateral spinal fusion is 0.5 to 2.5 mm.
The resorbable crosslinked form stable membrane may be a membrane for reconstruction of orbital fractures as shown in fig. 1, (6 ') and (6'), wherein typically the length m, width o and height c of the membrane are 30mm to 50mm, 20mm to 40mm and 5mm to 25mm, respectively. Typically, the thickness of the wall of the membrane used for reconstruction of orbital fractures is 0.5 to 2.5 mm.
Preferably, the composite layer of the collagen material and the inorganic ceramic particles includes 2.0 to 3.0 parts by weight of the inorganic ceramic with respect to 1 part by weight of the collagen material.
Preferably, the collagen material comprises 70% to 90% collagen and 10% to 30% elastin
The collagen material is conveniently derived from porcine, bovine or equine peritoneal or pericardial membranes, small intestinal mucosa (SIS) or muscle fascia.
Advantageously, one or both of the layers of elastic pre-tensioned collagen material comprises pores of 5 to 1000 μm.
Typically, the inorganic mineral particles are 150 μm to 500 μm in size.
The inorganic ceramic may be selected from the group consisting of hydroxyapatite or hydroxyapatite bone mineral.
The invention also relates to a resorbable crosslinked form-stable membrane comprising a composite layer of collagen material and inorganic ceramic particles comprising 1.5 to 3.5 parts by weight of inorganic ceramic to 1 part by weight of collagen material, the composite layer being sandwiched between two elastic pre-tensioned collagen material layers, wherein the pre-tensioned collagen material is a collagen material that has been stretched to be in the linear/elastic region of the stress-strain curve, the collagen material comprising 50% to 100% (w/w) collagen and 0% to 50% (w/w) elastin and having a shape and size suitable for use in extraoral human tissue regeneration in rhinoplasty, posterolateral spinal fusion or orbital reconstruction, wherein form-stable membranes for use in the oral cavity are excluded, the latter is flat (1), (1'), U-shaped straight (2), (2') or U-shaped curved (3), (3') corresponding to the alveolar space of 1 to 3 teeth (incisors, canines, premolars or molars) located at the front, left or right side curve or back of the denture.
The present invention also relates to a method of regenerating human tissue outside the oral cavity comprising applying the resorbable crosslinked form of the stabilization membrane of
Drawings
The invention will be described in more detail hereinafter with reference to an illustrative example of a preferred embodiment of the invention and the accompanying drawings, in which:
figure 1 shows typical shapes and typical dimensions of resorbable crosslinked form stable membranes, which:
is an invention according to EP-A1-3175869 for use in the oral cavity. These membranes can be flat (1), (1'), straight (2), (2') or curved (3), (3') in a U-shape corresponding to the alveolar spaces of 1 to 3 teeth (incisors, canines, premolars or molars) located in the front, left or right curvature or in the back of the denture. The front product is similar in size to the back product, with a radius of curvature that conforms to the alveolar ridge. Typical dimensions are a 5-20nm, b 8-20mm, c 6-10mm, d 25-40mm, e 15mm, f 20-40 mm.
To regenerate human tissues outside the oral cavity according to the invention:
a nasal arch (4) for rhinoplasty,
-slotted rectangular or elliptical tubular membranes (5), (5'), and
membranes (6) and (6') for reconstruction of orbital fractures (front and plan views, respectively).
Typical dimensions of the nasal arch (4) for rhinoplasty are: l-40-80 mm, i-10-15 mm, and h-10-15 mm. The kiss-ridges of the device may have different shapes.
Typical dimensions of membranes (5 and 5') used for posterolateral spinal fusion are: l-60-300 mm, j-15-30 mm, and k-5-10 mm. Membranes for posterolateral spinal fusion may be rectangular (5) or elliptical (5').
(6) And (6') show front and plan views of representative membranes for reconstruction of orbital fractures. Typical dimensions are: m-30-50 mm, o-20-40 mm, and c-5-25 mm.
Fig. 2 is a schematic illustration of an apparatus suitable for enabling tensioning of a polymer layer prior to assembly of the polymer layer into a flat or U-shaped form stable film made in accordance with the present invention.
Figure 3 shows an assembly of flat form stabilizing membranes wherein (1) is a steel plate, (2) is a compressed polyurethane sponge, (3) is a polyamide mesh, (4) is a layer of elastically pre-tensioned collagen, and (5) is a cross-linked hydroxyapatite-collagen white plate.
FIG. 4 shows a PLA film
FIG. 5 shows the stress-strain curves of some commercially available moist sterile collagen materials that can be used in the elastic pre-tensioned collagen material layer of a resorbable cross-linked form stable membrane according to the invention, i.e. Geistlich from porcine peritoneal membrane
FIG. 6 is for Geistlich
The following examples are intended to illustrate the invention without limiting its scope.
Detailed Description
Rhinoplasty
Nasal reshaping changes the geometry of the nose for various reasons, such as aesthetics, trauma, or cancer. If the nose is to be enlarged, an augmentation material is used, for example autologous cartilage, skin and bone material or artificial material, in particular polytetrafluoroethylene (ptfe), silicone or polyethylene implants. For small augmentations, collagen or hyaluronic acid fillers are commonly used (Jasin, 2013, nasal cartilage using collagen filters, Facial plant Surg Clin North Am; and Malone et al, 2015, nasal Augmentation in nasal plant: ASurvey and Review, Facial plant Surg). The film for rhinoplasty prepared according to the invention is made for large scale augmentation and therefore does not need to be directly compared to a bulking agent.
The dimensions of the membrane for rhinoplasty prepared according to the invention are adapted to the desired nasal dimensions. Representative membranes for rhinoplasty are shown in fig. 1, (4). The length l, width i and height h of the film for rhinoplasty prepared according to the present invention are generally 40-80mm, 10-15mm and 10-15mm, respectively. The thickness of the wall of the resorbable crosslinked membrane for rhinoplasty is generally from 0.5 to 2.5mm, preferably from 1.0 to 2.0mm, in particular from 1.2 to 1.8 mm. The membrane for rhinoplasty prepared according to the invention may contain pores of 5 to 1000 μm to promote liquid absorption and bone formation within the wall of the product. The surgeon may cut the device to the desired size using scissors or a scalpel. Furthermore, it may be secured using, for example, resorbable threads, pins or screws.
The advantages of the membrane for rhinoplasty prepared according to the present invention over the use of autologous bone, cartilage or skin include: (I) its unlimited availability, (II) reduced surgical time, (III) neglect donor site morbidity, (IV) overall flexibility in selecting shape, and (V) absorption or tissue integration time is more predictable.
Advantages of the film for rhinoplasty prepared according to the present invention over the use of non-natural and non-resorbable materials such as polytetrafluoroethylene, silicone or polyethylene include: (I) excellent tissue adhesion to the membrane, thus avoiding product extrusion during wound healing and long-term use, and (II) less complicated tissue integration due to the use of natural materials.
Posterolateral spinal fusion
Posterolateral spinal fusion is a common procedure for reducing back pain, particularly in The lumbar region (Jacobs et al, 2013, The evidence on scientific interventions for low back disorders, an overview of systematic reviews, Eur Spine J). During surgery, two or several vertebrae are fused by placing two pedicle screws on each vertebra to be fused and connecting the vertebrae using steel rods. Between the transverse vertebrae and around the steel rods, autologous bone, allogeneic or xenogeneic bone blocks, bone substitute slurry (bone tissue poty), demineralized bone matrix, BMP with a carrier, or a sponge containing collagen and hydroxyapatite or any other suitable material is placed. Mechanical stabilization by steel rods and pedicle screws subsequently allows new bone formation to occur in the graft area and between the vertebrae (Cheng et al, 2009, Posterior lung interbody fusion in spinal fusion: a proactive controlled study in the Han national issue, Int ortho).
Membranes for posterolateral spinal fusion prepared according to the present invention are generally slotted rectangular or elliptical tubes. Figures 1, 5 and 5' depict representative membranes for posterolateral fusion. The length of the device is such that it covers more than 2 vertebrae. Therefore, its length l is usually at least 6 cm. Typically, the inner diameter k of an elliptical device is 5 to 10mm, while the outer diameter j is 15 to 30 mm. Typically, for rectangular devices, the width is 5 to 10mm and the height is 15 to 30 mm. The thickness of the wall of the membrane for posterolateral fusion prepared according to the invention is generally from 0.5 to 2.5 mm. The membrane may contain pores of 5 μm to 1000 μm to promote bone formation inside the product.
The surgeon may use scissors or a scalpel to cut the device to the desired size. Before or after application, the device may be filled with autologous bone particles, allogeneic or xenogeneic bone particles, replacement bone slurry, demineralized bone matrix, BMP with a carrier, or a sponge containing collagen and hydroxyapatite, BMA, a blood fraction (e.g. platelet rich plasma or platelet rich fibrin) or any other suitable material obtained during surgery instead of at a second site. The membrane is then fixed to the pedicle or spinous process (process of the verterbra) with a screw, k-wire or resorbable wire. In another variant of this procedure, the hollow membrane for posterolateral fusion prepared according to the invention is fixed with pedicle screws and the remaining space in the tube is filled with the above-mentioned material after the fixation of the steel rod.
The advantages of the membrane for posterolateral spinal fusion prepared according to the invention with respect to the use of autologous bone mass include: (I) the device can be filled with the selected material (see list above), (II) there is no donor site morbidity, and (III) the surgical procedure is simplified and accelerated due to the fact that only one device needs to be fixed per pedicle.
The advantages of the membrane for posterolateral spinal fusion prepared according to the present invention over the use of allogeneic bone blocks include: (I) the device may be filled with the selected material (see list above), and (II) the surgical procedure is simplified and accelerated due to the fact that only one device needs to be fixed per pedicle.
The advantages of the membrane for posterolateral fusion prepared according to the invention over the use of a slurry (putty) material include: (I) simplified and accelerated procedures since the device has been shaped, (II) the ability to use autologous bone particles, BMA, platelet rich plasma or fibrin, (III) improved mechanical stability so that more bone formation can be achieved, (IV) the ability to use screws, k-wires or resorbable wires for fixation, whereby a mechanically more stable interface between the device and the vertebrae can be achieved, which promotes bone formation.
Advantages of the membrane for posterolateral fusion prepared according to the invention over the use of collagen/hydroxyapatite sponges, collagen sponges with or without growth factors or demineralized bone matrix include: (I) the device can be filled with the selected material (see above list), (II) the mechanical stability is improved, so that more bone formation can be achieved.
Reconstruction of orbital fractures
Orbital fractures are most common due to vehicle accidents, assaults, and sports-related injuries. Orbital fractures are usually repaired immediately or after two weeks after the fracture, and the repair procedure involves the use of autologous tissue or different types of implants, such as: (I) autologous bone or cartilage, (II) non-customized but conformable Materials such as titanium mesh or porous polyethylene, (III) resorbable sheets made of, for example, polylactic acid, polyglycolic acid, polylactic glycolic acid (plga) or polydioxanone (polydioxanone), (IV) resorbable and conformable xenografts, made for example of dura mater or demineralized human bone, (V) patient-specific non-resorbable devices, made for example of titanium, polyetheretherketone or glass bioceramics (Boyette et al 2015, Management of organic reactions: scales and solutions, Clinical opthalmology; and Avashia et al 2012, Materials used for structural bone graft flow reactor, J crystal fracture Surg).
Different approaches may be used to achieve access to the orbital fracture. The fracture site is prepared to define bony processes that are readily supported by the implant. The implant is then shaped and placed, typically using screws for fixation; implants on minor defects were not fixed (Kunz, 2012, Orbital fractions. principles of the Craniomaxillof scientific Skeleton, Trauma and orthophonthic Surgery, Ehrenfeld et al, AOFoundation).
The size of the membrane for reconstruction of orbital fractures prepared according to the present invention facilitates its insertion into the orbit. Representative membranes for orbital fracture reconstruction of the right orbital floor and medial wall are depicted in fig. 1, (6) and (6'). The length m, width o and height c of the membrane used for reconstruction of orbital fractures may be, for example, 30 to 50mm, 20 to 40mm and 5 to 25mm, respectively. Depending on the fracture and size of the orbit, different sizes of membranes for orbital reconstruction can be manufactured. The left orbital floor and the inner sidewall reconstruction device have the same mirror image dimensions. Depending on the fracture situation, it is possible to manufacture products with additional protrusions to allow fixation, for example, on the front of the zygomatic bone.
The thickness of the wall of resorbable crosslinked membranes prepared according to the invention for reconstruction of orbital fractures is typically 0.5 to 2.5 mm. The membrane for orbital fracture reconstruction may contain 5 to 1000 μm pores to promote liquid absorption and bone formation within the product wall. The surgeon may use scissors or a scalpel to cut the device to the desired size. Furthermore, it may be secured using, for example, resorbable threads, pins or screws. The advantages of the membrane for reconstruction of orbital fractures prepared according to the present invention compared to autologous bone or cartilage include: (I) its unlimited availability, (II) reduced surgical time, (III) neglecting donor site morbidity, (IV) better fitting of the contour of the orbital fracture to the time of complete ossification is predictable and at least for the bone, and (V) is radio-opaque compared to cartilage, thus making it possible to control the position of the implant by computed tomography.
The advantages of the membrane for reconstruction of orbital fractures prepared according to the present invention over non-customized but compliant materials such as titanium mesh or porous polyethylene include: (I) its design to gradually ossify and resorb or osseointegrate over time, (II) not to compromise the smooth edges of the surrounding soft tissue, (III) its radiopacity allows the position of the implant to be controlled by computer tomography compared to non-radiopaque materials such as porous polyethylene.
Advantages of the membrane for reconstruction of orbital fractures prepared according to the present invention over resorbable sheets made of, for example, polylactic acid, polyglycolic acid, polylactic glycolic acid, or polydioxanone include: (I) its design of progressive ossification and resorption or osseointegration (osteoconduction) over time, (II) the fact that during resorption no acidic degradation products are actually released which are liable to cause inflammation and local bone resorption, and (III) radiopacity, thus making it possible to control the position of the implant by computer tomography.
The advantages of the membrane for reconstruction of orbital fractures prepared according to the present invention over resorbable and adaptable xenogeneic or allograft implants made of, for example, dura mater or demineralized human bone, include: (I) high form stability when bent, making it useful for treating large fractures, and (II) radiopacity, thus allowing the position of the implant to be controlled by computed tomography.
Attractive features of resorbable cross-linked form stable membranes for regeneration of human tissue outside the oral cavity include those disclosed in EP-a1-3175869, in particular:
-the composite layer of collagen material and inorganic ceramic particles comprises 2.0 to 3.0 parts by weight of inorganic ceramic relative to 1 part by weight of collagen material,
-the collagen material comprises 70-100% (w/w) collagen and 10-30% (w/w) elastin,
-the collagen material is derived from tissue of natural origin selected from the group consisting of mammalian peritoneum or pericardium, placental membrane, Small Intestinal Submucosa (SIS), dermis, dura mater, ligament, tendon, diaphragm (diaphragm), omentum, and fascia of a muscle or organ,
-the collagen material is derived from porcine, bovine or equine peritoneal or pericardial membranes, small intestinal mucosa (SIS) or muscle fascia,
elastic Pre-tensioned collagen Material has an elastic modulus of 2 to 150MPa, in particular 5 to 80MPa,
one or both of the layers of elastic pre-tensioned collagen material comprise pores of 5 to 1000 μm,
-the size of the inorganic mineral particles is from 150 to 500 μm,
-the inorganic ceramic is a hydroxyapatite,
-the inorganic ceramic is a hydroxyapatite bone mineral,
the form-stable film is chemically cross-linked, and/or
The shape-stable film is crosslinked by dehydrothermal treatment of DHT.
The form-stable membrane may be used in combination with growth factors and/or chondrogenic or bone forming cells.
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