CN101296712A - Biodegradable scaffold with ecm material - Google Patents

Abstract

We add discontinuous regions of Extra Cellular Matrix (ECM) to a biodegradable scaffold. Hereby it is possible to combine the range of physical properties the scaffold can offer with the reconstructive properties of the ECM. The optimal amount of discrete ECM material for each application is disclosed and this concentration is equally distributed in the dressing hence avoiding unnecessary high concentrations of ECM. In addition to the effect of the ECM, the porous structure of the base material provides the cells with a structure for in-growth.

Description

Biodegradable scaffold with ECM material

Invention field

The present invention relates to comprise the support of the biodegradable layer with ECM material, described ECM material is incorporated biodegradable layer into lamellar, fibrous, graininess, form such as Powdered.

Background technology

Support is to be used for the structure of tissue, growth and differentiation of cell guiding in forming newborn functional organization process.

For reaching the target of tissue reconstruction, support must meet some specific requirements.High porosity rate and enough apertures are to promote that cell growth and cell and the diffusion of nutritional labeling in total are necessary.Because support need be absorbed by surrounding tissue and needn't remove with operation, so biodegradability is absolutely necessary.

Many different materials (natural with synthetic, biodegradable and permanent) had been studied as support.Before organizational project occurred as research topic, major part was known by medical domain in these materials, and as biological absorbable stitching thread.The example of this class material is collagen protein or fats polyester that some are linear.

Yet, when the made support of test laboratory in vivo, often find that cell is difficult for growing in these supports, this may be because do not have bio signal molecule (for example somatomedin) in the synthetic support.

For biological property and the accelerating wound healing that improves support, some laboratorys have added somatomedin in the synthetic support and have found that it has beneficial effect to wound healing.In all these published thesis, single somatomedin had been merged in thin layer or the hydrogel.The somatomedin of having tested comprises FGF-2 (1; 2), test concentrations is 25 μ g/cm ²β-FGF-2 (2), FGF-1 (3; 4), EGF (5) (14) or test concentrations are 2 μ g/cm ²TGF-β (6; 7).Extracellular matrix from the acellular of warm-blooded vertebrate is widely used in organizational project and plastic surgery (8).Found that the acellular extracellular matrix has many somatomedin (9-11).ECMs comprises many biological molecule and has found that cell is easy to fill these spissated ECM layers (12,13).ECM in the market is people source or pig source.Cell is separated from tissue, will organize lyophilizing and section then.The tissue lamellar in pig source varies in size.The price of these lamellas is very high.This lamella is harder when aquation not.For example from the lamella of Acell company.They sell the ECM lamella can promote wound healing (bladder substrate, UBM).(7 * 5cm) weight are about 100mg to such lamella, and density is about 190mg/cm ³

ECMs or the application of ECM protein in Wound healing and bone regeneration are widely known by the people.This series products is lamella or form of hydrogels.The example of lamella series products is as from the OASIS (freeze dried pig source ECM lamella) of Healthpoint with from the Graftjacket (freeze dried people source ECM lamella) of Wright medical.These lamellas can offer support and protein complex for the cell of wound.The example that contains the proteinic non-support series products of ECM on the market as from

Xelma, this is a kind of hydrogel that comprises the protein extract of developmental pig tooth ECM.

Summary of the invention

This application discloses incorporates ECM into growth-promoting effect that support can keep ECM.We have proved that when use comprised the support of ECM material, the ECM of high concentration unexpectedly can not produce better cellular morphology.Be lower than 60% concentration and just be enough to obtain best cellular morphology and distribution.In addition, prove that the physical characteristic of described support can change thereupon by changing EMC concentration of material discrete in the support, but this change is the material that depends on support.The application is applied to this understanding on one's body the patient by showing a kind of bioactive sterilization strategy that can keep the ECM material after sterilization.

Detailed content

The present invention relates to comprise the discrete particulate provisional compound rest of ECM.

Add on the support by discontinuity zone, may make up the physical characteristic (for example intensity, pliability, motility, ruggedness) of the available certain limit of described support, with the reconstitution properties of ECM with ECM.In addition, the price of this support is lower than other ECM support, both because powder had been from the waste material of producing acellular ECM lamella, thereby also because can determine at the optimised quantity of the discrete ECM raw material of each application and can be distributed to the high concentration ECM that avoids unnecessary in the dressing equably.Except the effect of ECM, the loose structure of host material offers the ingrown structure of cell.In one embodiment, by adding the discontinuity zone that discrete ECM raw material (such as graininess, lamellar, fibrous or Powdered) obtains ECM.

The discrete ECM material that on its form and density, is different from their embedded basic material that is meant mutually of ECM material.Scanning electron microscope (SEM) among tissue slice among the embodiment 5 or the embodiment 6 is observed can prove this point.By adding the discontinuity zone of ECM, we can control the concentration of ECM.Show that as embodiment (for example embodiment 2 and 3) it is vital that control concentration is optimized the cell growth.

The preceding ECM material is added in the support of rack forming (for example lyophilizing) is preferred.In this way, the ECM material is distributed in the support equably.That is to say that the density of the ECM of (for example refrigerating process) support one end may be slightly higher than the other end in the support solidification process.Yet homogeneous distributes and allows the interior density gradient of this support in this article, as long as density＞0 of carriage center.Like this, preferred embodiment relate to provisional successive support, it comprises the discontinuity zone of the ECM of homogeneous distribution, and wherein the concentration of the discontinuity zone of ECM is between 20% (w/w) and 60% (w/w).

In this article, interim support be meant can disappear, be hydrolyzed, disintegrate, biodegradation/biology absorbs again/biological support absorbable, soluble or that disappear from the wound by other modes.Can remove anything from wound is a huge clinical advantages.Organizing of like this, newly forming can be because of not removing the destroyed and compressing of falsework.The support that preferably can in 1 day-10 week (depending on application conditions), disintegrate traditionally.Use for open wound, preferably can be at 1-10 days, such as the support of disintegrating in 2-7 days.In aspect of this invention, described support is biodegradable.

In one embodiment, support is the seriality support.The support of continuous phase just.Seriality support with discontinuity zone produces composite.As other composites, this is the engineering material of being made by two kinds or more kinds of composition material, and it has significantly different physics or chemical property and can keep separation and different in the structure of finishing.

Extracellular matrix (ECM) is the acellular part in animal or the people's tissue.Thereby ECM is exactly pericellular composite.So preferably, the ECM discontinuity zone is acellular zone.Acellular zone can obtain by using physics, enzyme and/or chemical method.Cellular layer can be removed to physical property with for example crossing the erasing tissue.Can use detergent and enzyme that the cell in the tissue is disconnected from each other.Also can make water or other hypisotonic solutions, thereby because hypotonicly can impel the cell rupture in the tissue to promote to take off cell processes.

The another kind of method that obtains acellular zone is that ECM powder (discontinuity zone of ECM) is added in the backing substrate.Because cell component can cause immunoreation, so in case after implanting, acellular product can minimize the risk of any immunity rejection.

In broad terms, ECMs has three kinds of main components: fiber element (particularly collagen protein, elastin laminin or reticulin), connect albumen (for example fibronectin, laminin) and space filling molecule (normally mucopolysaccharide).The storehouse that known ECMs can serve as somatomedin and cytokine attracts cell and promotes cell proliferation (9; 10).Can be by from the cell at wound edge and come the cell of self-loopa blood to fill when the falsework that comprises graininess BCMs is used in the wound.When cell intrusion support, timbering material can be degraded and support finally can be substituted by new organization.

The concentration of ECM discontinuity zone preferably is higher than 15% (w/w), is higher than 20% (w/w), such as being higher than 30% (w/w).The concentration of ECM discontinuity zone preferably is lower than 95% (w/w), is lower than 90% (w/w), such as being lower than 80% (w/w), or is lower than 70% (w/w).In particularly preferred embodiment of the present invention, this concentration is between 20% (w/w) and 60% (w/w), such as between 20% (w/w) and 40% (w/w).

People's skin comprises the upper strata epidermis that is formed by wherein horn cell.Be corium under the epidermis, form by wherein fibroblast and endotheliocyte.

When promoting fibroblastic growth, the embodiment of this paper (for example embodiment 3) shows that ECM concentration is increased to about 60% (w/w) from 0% (w/w) and can causes the remarkable increase of rack surface cell quantity and the remarkable improvement of cellular morphology.Like this, one aspect of the present invention relates to and comprises 40% (w/w)-60% (w/w) ECM to promote the Wound care device of fibroblastic growth.

When promoting Keratiocyte growth, the embodiment of the use gelatin support of this paper shows that the concentration of ECM is increased to ability (doing as horn cell) and cellular morphology and the cell total amount that about 25% (w/w) can significantly improve the cell aggregation growth from 0% (w/w).40% (w/w) is above can to reduce the cell growth-promoting effect of representing with cell quantity, cellular morphology and the cell number on surface but the concentration of ECM is increased to.Thus, an aspect of this invention is the Wound care device that contains 20% (w/w)-30% (w/w) ECM about, and it can promote the growth of horn cell.

Wound dressing of the present invention can comprise a lot of layers.These layers can comprise one or more layers Biodegradable material, and these materials all can randomly comprise ECM.If ECM is merged in above in one deck, its dosage can change at interlayer.In one embodiment, ground floor comprises the ECM of 40% (w/w)-60% (w/w); The second layer comprises the ECM of 20% (w/w)-30% (w/w).

In another embodiment, design the growth stimulation that described support is used for different cell types.Just, when being used for the growth of fibroblasts stimulation, optium concentration is 40% (w/w)-60% (w/w) ECM, and the optium concentration that is used for endotheliocyte is 30% (w/w)-60% (w/w) ECM, and being used for the growth stimulation of horn cell, optium concentration is 20% (w/w)-30% (w/w) ECM.An embodiment of the invention relate to the Wound care device that comprises two kinds of supports, first kind of support is used for stimulating fibroblastic growth and contains the concentration of ECM discontinuity zone of 40% (w/w)-60% (w/w) and second kind of support is used for stimulating Keratiocyte growth and contains the concentration of the ECM discontinuity zone of 20% (w/w)-30% (w/w).Can add the third support to this Wound care device, it contains the concentration of the ECM discontinuity zone of 20% (w/w)-30% (w/w).

The data of this paper make the support of the discontinuity zone that comprises 40% (w/w)-60% (w/w) ECM can be used for stimulating fibroblastic growth.The data of this paper also can make the support of the ECM discontinuity zone that comprises 20% (w/w)-30% (w/w) be used to stimulate Keratiocyte growth.

Our experience is, when promoting fibroblastic growth, the fibroblast of growth can secrete somatomedin and induce the growth of horn cell.Like this, preferred aspect of the present invention relates to the wherein support of concentration between 40% (w/w) and 50% (w/w) of the discontinuity zone of ECM.Therefore, thereby promoted that growth of fibroblasts has promoted the growth of horn cell subsequently, and the wound that healed.

With the ECM concentration in the w/w percentage calculation supporting structure.Promptly be: concentration (w/w)=M _ECM/ (M _ECM+ M _Support) x100%.M wherein _ECMBe ECM quality with the gram expression, and M _SupportIt is quality (not comprising ECM) with the support of gram expression.

In soluble support (for example MPEG-PLGA), you can be dissolved in this support in the solvent and filter ECMs.After lyophilizing, this material is weighed.

In the non-solubility support, described material is embedded in the suitable embedded material (for example paraffin), and with statistics (statically) representative number section and with suitable dyeing, described dyestuff only dyes and timbering material do not dyeed ECMs.Use graphical analysis, calculate the amount of the relative support of ECMs.

Preferred ECM comprises the tissue-derived bioactive ECM component from described material.For example, they can comprise fibroblast growth factor-2 (basic FGF), transforming growth factor-beta (TGF-β) and VEGF (VEGF).Also preferably, ECM basic material of the present invention comprises additional biological active component, comprises, for example one or more collagen protein, mucopolysaccharide, glycoprotein and/or Dan Baijutang.Described ECM can comprise basement membrane, and its major part is made by IV collagen type, laminin and Dan Baijutang.ECM material of the present invention is preferably used the tissue preparation of collecting with animal from meat product, and described animal is including, but not limited to pig, cattle and sheep.Other warm-blooded vertebrates also can be used as tissue-derived, but make that with the more high availability of the tissue of animal this tissue is preferred from meat product.The pig that genetic engineering is removed galactosyl (galacatosyl), α 1,3 galactose (GAL epi-position) can be used as the tissue-derived of production ECM material.In preferred embodiment, described ECM is the pig source.

Described ECM material can obtain from any animal.It can from, but be not limited to intestinal tissue, bladder, liver, spleen, stomach, lymph node or skin.Particularly preferably be the ECM of skin, Vesica sus domestica tela submucosa (UBS), Vesica sus domestica substrate (UBM) or pig intestinal mucosa lower floor (SIS) from people's corpse.

The preferred people's tissue of avoiding is to minimize the transfer of disease.Like this, in preferred embodiment, the discontinuity zone of ECM is from animal tissue.Since species similarity, the preferred ECM that uses from warm-blooded mammals.

In particularly preferred embodiments, the discontinuity zone of ECM is UBM (bladder substrate) granule.Described UBM material comprises the proteinic specific mixture of ECM, and some of them are quantified as: TGF-β 293 ± 8pg/g, b-FGF 3862 ± 170pg/g and VEGF 475 ± 22pg/g (promptly being pg VEGF/g UBM).It is 3mg/cm that the ECM lamella has average density ², concentration is about TGF-β: 0, and 9pg/cm ², b-FGF:11.6pg/cm ²And VEGF1.4pg/cm ²

The form of ECM

One aspect of the present invention provides the support with lasting somatomedin administration.A characteristic of support used herein is to disperse the discontinuity zone of described ECM in the porous basic material, thereby described ECM can be near cell.When cell moved in backing substrate, the discontinuity zone of described ECM was exposed to proteinase activity and is degraded, and it is believed that this has caused biological active component to discharge (14) from the discontinuity zone of ECM.Like this, can continue to keep the release of biological active component during use to a certain extent, thereby the administration that continues to a certain extent to wound kitchen range and cell is provided.In one embodiment, the discontinuity zone of described ECM uniform distribution in falsework.

ECM has several miniature forms: for example graininess, lamellar, fibrous or Powdered.All these is considered to the discontinuity zone of ECM, promptly discrete ECM material.

The preferred form of the discontinuity zone of ECM is the ECM granule.It is about 150 μ m that preferred granule has average diameter.It is average to measure volume weighting by the Mastersizer 200 from Malvern Instrument.For example, weighted average is that to have smallest particles be 3 μ m on the surface of 100 μ m, and the largest particles is 750 μ m.In the case, volume weighting on average is 250 μ m.

By in porous support, disperseing the discontinuity zone of ECM, may optimize the physical characteristic (for example intensity, pliability, motility, ruggedness) of this support, and not to the macrolesion of the discontinuity zone of ECM.

Be to obtain beneficial effect and the available physical characteristic of porous support of ECMs, specific ECM can be included in the wound dressing (as support) and be used for organizational project (for example reconstruction of soft tissue engineering, skeleton, cartilage, ligament and tendon) or dental applications.This porous support should preferably biodegradable material.Described falsework is lyophilized form, fibers form (weave or do not weave), form of foam or thin film.In form of ownership, the discontinuity zone of ECM is come-at-able to outer surface and the cell on the inner surface that is in porous/fibre structure.

The material that is used for this support is any Biodegradable material, can be from synthetic and natural raw material.In the support that natural material makes up, the particularly preferred material that is based on the extracellular matrix derivant.The example of this material is a protein material, for example collagen protein or fibrin, and polysaccharide material, for example chitosan or mucopolysaccharide (GAGs).

In one embodiment, described Biodegradable scaffold is made by comprising proteinic material.This can carry out degraded by protein lyase.Such support is preferably made by following protein: collagen protein, keratin, fibrin, elastin laminin, laminin, Vimentin, vitronectin, fibronectin, Fibrinogen and these proteic derivant and analog, perhaps denatured protein, for example gelatin.

By using polymeric material, for example gelatin, fibrin, hyaluronic acid, collagen protein, chitin, chitin, keratin, Sargassum salt, PLA and PLGA make support, may and modify the physical characteristic (intensity, pliability, motility) that changes support by combination.

In another embodiment, described Biodegradable scaffold is made by the material that comprises carbohydrate/polysaccharide.This makes and can degrade by hydrolysis and this polysaccharide of enzymatic degradation.Such support is preferably by polysaccharide, and for example Heparan sulfate, chondroitin sulfate, dermatan sulfate, heparin, keratan sulfate and these derivant, alginate, HSC cellulose and cellulose derivative (CMC), some alginate, chitosan, chitin, pectin and pectin derivant, hyaluronic acid and Dan Baijutang (mucopolysaccharide) and these derivant are made.

On the other hand, described falsework is synthetic.Such support is mainly by degrading with the combined hydrolysis of enzymatic digestion.These supports are preferably made by being selected from following material: PLA (polyactide), PGA (poly-Acetic acid, hydroxy-, bimol. cyclic ester), the PLGA (copolymer of poly-(lactide-co-glycolide), MPEG-PLGA, PCL (polycaprolactone), poe, PPDO, poly-anhydride, polyhydroxy-alkanoate and above these materials.

The example of known natural support/gel is collagen-base (3,6), fibrin base (4), chitosan-based (1) or gelatin-based (2,7).

Synthetic material commonly used is the PLA-polylactic acid.It is a kind of polyester, and degraded forms lactic acid in human body, and the latter is that natural chemical substance is easy to remove from human body.Similar material is polyglycolic acid (PGA) and polycaprolactone (PCL): their degradation mechanism is similar to PLA, but they are compared with PLA and show faster and slower degradation rate respectively.Can synthesize such MPEG-PLGA polymer as follows: the stannous octoate in MPEG, DL-lactide, Acetic acid, hydroxy-, bimol. cyclic ester and 4% (w/v) toluene is added in the bottle of nitrogen cover, seal this bottle, and heating, vibration, homogeneous transparent up to content, place it in the 120-200 ℃ of stove 1 minute-24 hours thereafter.Also can in the solution of suitable solvent (for example dioxane), carry out this and synthesize, be beneficial to purification thereafter.The inferior stannum of MPEG, DL-lactide, Acetic acid, hydroxy-, bimol. cyclic ester and 4% (w/v) diethyl caproic acid is added in the bottle of nitrogen cover and as above-mentioned processing.

This polymer can following purification: with this polymer dissolution in suitable solvent (for example dioxane, oxolane, chloroform, acetone), and in solvent-free (for example water, methanol, ethanol, 1-propanol or 2-propanol), under-40 ℃ of-40 ℃ of temperature precipitation while stirring.Make this polymer precipitation, abandon solvent, then this polymer dried overnight in 40 ℃-120 ℃ vacuum oven.

Example is described as mentioned, and the basic material of support can be made by the material of synthetic and/or natural origin--comprising their combination.Therefore, this support can comprise the combination of protein, polysaccharide and synthetic polymer.

A function of the support that uses among the present invention provides the substrate that promotes the cell growth.A standard that promotes cell to grow in support is that support at room temperature is solid-state.That is to say that this support has fixed physical arrangement, bicontinuous structure.Utilize this structure, help cell to pass through stent migration and form new tissue.

Another promotes that the standard of cell growth is the support with perforate, or has the cell migration of permission at least to porous.

Porous is defined as P=1-ρ (V/M).

Wherein P is the porous of support, and ρ is the density of the polymer system that uses, and M is a weight, and V is the volume of the support of manufacturing.

An embodiment of the invention relate to and comprise the porous support of the discontinuity zone of ECM as described herein.As institute is illustrational in an embodiment, the porous above 50% makes cell grow.Like this, in a preferred implementation, described support comprises the porous greater than 50%, for example＞80%, even greater than 90%, or 95% porous.

Preferred porous support has open interconnected hole.

In preferred implementation of the present invention, described falsework has 0.1-8mm, preferred 0.3-3mm, preferred 0.5-2mm thickness.In particularly preferred embodiment of the present invention, the thickness of biodegradable layer is about 1mm.In preferred implementation of the present invention, described biodegradable layer directly contacts with wound.

Purpose according to support of the present invention is as wound dressing.

Factor in wound dressing of keeping firmly in mind is to make it soft and comfortable.Soft and comfortable implication be its when being used for open wound, can fit or pain because the edge can not cut and oppress responsive wound around, and the dressing meeting is crooked along with the curvature of wound.This has also guaranteed wound area and direct contact that contains between the ECM support.

An example of soft and comfortable substrate is a chitosan like this, and it prepares in 1-2% (w/w) solution and lyophilizing.Gained is softish open substrate, promptly has the support in open interconnective hole.This substrate also has enough perforates makes cell grow and migration.

In one group of embodiment, dressing according to the present invention is used for acute wounds, burn, chronic wounds and/or surgical wound.

In another embodiment, dressing according to the present invention is used for the plastic surgery.

In a relevant embodiment, the support that comprises the ECM discontinuity zone is used for organizational project (for example, reinventing soft tissue, skeleton, cartilage, ligament and tendon) or dental applications.

In many these are used, need be sterilized by dressing according to the present invention.An embodiment of the invention relate to sterilization, interim, the continuous support that comprises the ECM discontinuity zone.Be typically expressed as the support that comprises the ECM discontinuity zone interim, successive, fungi-proofing packing, this product of description of symbols is sterilized on the packaging.As in embodiment 4 illustrated, kept biological action to ECM dependency cantilever type by for example radiating sterilization.Fungi-proofing material is well known by persons skilled in the art.

List of references

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Description of drawings

Fig. 1: with 3 kinds of variable concentrations (cell/cm ²) LDH of inoculation people elementary fibroblastic gelatin support measures.The bar shaped post is illustrated in the 3rd day growth, measures with Abs.

Fig. 2: with 3 kinds of variable concentrations (cell/cm ²) LDH of inoculation people elementary fibroblastic MPEG-PLGA support measures.The bar shaped post is illustrated in the 3rd day growth, measures with Abs.

Fig. 3: with 3 kinds of variable concentrations (cell/cm ²) LDH of gelatin support of inoculation people elementary horn cell measures.The bar shaped post is illustrated in the 3rd day growth, measures with Abs.

Fig. 4: with 3 kinds of variable concentrations (cell/cm ²) LDH of MPEG-PLGA support of inoculation people elementary horn cell measures.The bar shaped post is illustrated in the 3rd day growth, measures with Abs.

Fig. 5: with 3 kinds of variable concentrations (cell/cm ²) LDH of gelatin support of inoculation Human umbilical vein endothelial cells measures.The bar shaped post is illustrated in the 3rd day growth, measures with Abs.

Fig. 6: with 3 kinds of variable concentrations (cell/cm ²) LDH of MPEG-PLGA support of inoculation Human umbilical vein endothelial cells measures.The bar shaped post is illustrated in the 3rd day growth, measures with Abs.

Fig. 7: the digital picture of ECM distribution of particles in the MPEG-PLGA support.

Fig. 8: the SEM picture of MPEG-PLGA support (250x amplification).

Fig. 9: the SEM picture (250x amplification) that comprises the particulate MPEG-PLGA of 40%ECM.

Figure 10: the digital picture of endothelial cell growth in the MPEG-PLGA support.

Figure 11: the digital picture that comprises endothelial cell growth in the particulate MPEG-PLGA support of 23%ECM.

Figure 12: the digital picture that comprises endothelial cell growth in the particulate MPEG-PLGA support of 23%ECM is shown enlarged in the capillary tube sample form of the more deep layer of support.

The specific embodiment

Embodiment 1: the primary human fibroblast is having and is not having inside growth in the particulate synthetic support of ECM

The support of being made by the biodegradable polyesters that comprises 40% (w/w) UBM (Acell) granule (average diameter is about 150 μ m) is in the test of cellular morphology and 3D growth and do not have to compare between the particulate support of ECM.

Metoxy-polyethylene glycol-(lactide-co-glycolide) (Mn 2.000-30.000, L: G 1: 1) is dissolved in 1, forms 1.5% solution in the 4-dioxane.For the support that comprises UBM, add 0.03g UBM to 3ml polymer solution (40%w/w dry), mixed at high speed is also poured in the mould of 3 * 3cm.With this solution-5 ℃ freezing, and-20 ℃ of lyophilizing 5 hours, placed about 60 hours at 20 ℃.This sample be placed in the exsiccator stretch (hydraulic pamp) 5 hours thereafter.

Assessment is seeded in elementary growth of fibroblasts and the form test on two kinds of rack surfaces.

To be classified into 1-5 from the 1st, 3 and 7 day result, wherein the poorest situation of 1 correspondence and 5 be best.In mixing the particulate support of ECM, the distribution of cell and all be 5 grades in whole these days of growing, and (all deciding grade and level is in these days to be better than crt bracket

).

Conclusion: the biological activity of powder ECM substrate has kept its activity after incorporating synthetic support into, and compares with independent support and to cause obviously better growth on support.

Embodiment 2: cellular morphology in gelatin-ECM composite of the ECM that comprises 5 kinds of variable concentrations and 3D growth.

Be seeded in the elementary fibroblastic cellular morphology and the 3D Study on Growth of gelatin-ECM rack surface.This gelatin support is crosslinked by heating, and comprises the UBM (0,12% (w/w), 26% (w/w), 41% (w/w), 51% (w/w) and 58% (w/w)) of the concentration that raises gradually.Concentration is calculated as the amount of UBM than solid total amount, and 58% (w/w) support that promptly comprises 0.05g polymer and 0.07g UBM is corresponding to 13.8mg UBM/cm ³

Will (A type, Blume value be 175, Sigma) are dissolved among milli-Q water and the t-BuOH (95: 5), form 1% solution from the gelatin of Corii Sus domestica.For the sample that comprises UBM, this UBM is added in this solution while stirring (0,12,26,41,51,58%w/w:0,0.007,0.018,0.035,0.053,0.07g/ support).The gelatin solution that 5ml is comprised UBM is poured (D=5cm) in the mould into.The mould that will contain solution was placed 1 hour at+5 ℃, then-20 ℃ freezing ,-20 ℃ of lyophilizing 5 hours, placed 36 hours at 20 ℃ again.Thereafter with this sample in vacuum oven 120 ℃ crosslinked 15 hours.

This studies show that by improving the UBM concentration in the described compound rest, does not compare with there being UBM, and it is better and have a higher multiplication rate that fibroblast distributes.In preceding 2 days of research, finding is not having on the support of UBM, and cell only is grown in the zone that they are employed, and cell UBM concentration greater than the rack surface of 26% (w/w) distribute better more even.Since the 14th day, clearly find to compare with the compound rest that comprises UBM, do not have on the gelatin support of UBM cell quantity still less.About cellular morphology, fibroblast has circle but the form of adhering on smooth gelatin support, but along with the rising of UBM amount, observes more fibroblast form.Since 41% (w/w) UBM, observe best fibroblast form and distribution.UBM concentration is elevated to greater than 41% (w/w) can not be created on the compound rest better cellular morphology and distribution.

Embodiment 3: contain preparation and the cellular morphology wherein and the 3D growth of the compound rest of the particulate MPEF-PLGA of ECM of 6 kinds of variable concentrations or gelatin.

The preparation of the compound rest of gelatin and ECM: will be dissolved among milli-Q water and the t-BuOH (95: 5) from the gelatin (A type, Gelitapharmagrade 832) of Corii Sus domestica, form 1% solution.For the sample that comprises UBM, this UBM is added in this solution while stirring; 0,0.006,0.013,0.021,0.033,0.05,0.075g/ support (0,10,20,30,40,50,60%w/w).The gelatin solution that 5ml is comprised UBM is poured (D=5cm) in the mould into.The mould that will contain solution was placed 1 hour at+5 ℃, then-20 ℃ freezing ,-20 ℃ of lyophilizing 5 hours, placed 18 hours at 20 ℃ again.Thereafter with this sample in vacuum oven 130 ℃ crosslinked 15 hours.

The preparation of the compound rest of MPEG-PLGA and ECM: Metoxy-polyethylene glycol-(lactide-co-glycolide) (Mn 2.000-30.000, L: G 1: 1) is dissolved in 1, forms 1.5% solution in the 4-dioxane.For the sample that comprises UBM, this UBM is added in this solution while stirring; 0,0.017,0.038,0.064,0.1,0.15,0.225g/ support (0,10,20,30,40,50,60%w/w), mixed at high speed is also poured 10ml in the mould of 7.3 * 7.3cm.With this solution-5 ℃ freezing ,-20 ℃ of lyophilizing 5 hours, placed about 15 hours at 20 ℃ again.This sample be placed in the exsiccator stretch (hydraulic pamp) 24 hours thereafter.

Be the cellular morphology and the 3D growth of assessment compound rest, take out biological biopsy material from every type support punching, and on the surface of this support with 2.5 * 10 ⁴Cell/cm ²Density inoculation primary human fibroblast (going down to posterity 3 times), Human umbilical vein endothelial cells (HUVEC, go down to posterity 4 times) or elementary horn cell (going down to posterity 5 times), describedly be seeded in that (elementary fibroblast is in the DMEM of 10%FCS in a small amount of growth medium that comprises antibiotic (penicillin, streptomycin and amphotericin B); HUVEC ' s is in EGM-2; Elementary horn cell is in KGM-2).Before adding additional growth medium, with this support at 37 ℃, 5%CO ₂Incubation.Assessed cell adhesion, form, growth and the group of this support at the 1st, 3 and 7 day, it re-uses the Leica DMIRE2 inverted microscope of having equipped the cold color camera of Evolution MP (Media Cybernetics) and assesses by using dimethyl diaminophenazine chloride dyeing.Use Image Pro Plus 5.1 softwares (Media Cybernetics) capturing digital image.Use Cytotoxicity Detection test kit (LDH, Roche Diagnostics GmbH) to calculate cell quantity.Cell is with 3 kinds of different concentration (1.25 * 10 ⁴, 2.5 * 10 ⁴With 5 * 10 ⁴Cell/cm ²) be inoculated into the top of different support type according to above-mentioned identical method.The the 1st, 3 and 7 day assessment of stent, it carried out lysis with 0.5%CHAPS thereafter by wash this support with PBS on 4 ℃ of flat agitators.Supernatant is transferred on the microplate, measured the amount of LDH according to operating instruction.

Fibroblast

Fibroblastic quantitative measurement shows the increase along with the time, and cell quantity also increases, but does not see influence (Fig. 1) between the gelatin support of variable concentrations UBM.

In the MPEG-PLGA support, cell in time with support in the amount of UBM and raise (Fig. 2).Cellular morphology in the gelatin support of no UBM and 3D growth is presented at first day of research, and adherent cell is with the rounded form growth and rest on the position of its application.The a little spindle that these cells become during remaining research.In this support, add 10% (w/w) UBM and can not change this kind pattern, but under 20% (w/w) UBM situation, increasing cell becomes the fusiform cell with normal fibroblast form, and these cells begin to expand widely on the surface.At 30-40% (w/w), observe the fusiform cell of maximum ratio, and see in the growth of the cell of rack surface to cell with the transformation of the cellular morphology of 3D more in the growth of support deep layer.In MPEG-PLGA, observe identical pattern, and have exception: rising UBM concentration is created on the support and the cell of its interior elevated amounts.Between 20-30% (w/w) UBM, see the cell and the cell with spindle form of expansion gradually more.More than 40% (w/w), cell begins with 3D form growth, and is substituted in the growth on surface and grows into the deep layer of support.

Horn cell

Inoculate elementary horn cell to the gelatin support that contains 10% (w/w) UBM, show that comparing cell quantity with the support that does not have UBM raises.At first day that studies, the cell of visible maximum quantity in 10-20% (w/w) UBM, but in the research of 20-30% (w/w) UBM, show maximum efficiency afterwards.The rising of UBM concentration causes the reduction (Fig. 3) of cell quantity on the support.

On the MPEG-PLGA support, there be not visible maximum cell quantity on the support of UBM.Add the reduction that UBM causes cell quantity in the support that increases UBM quantity.In the final stage of research, this effect is more obvious.Usually, because overlapping on the concentration observes big relatively variation (Fig. 4) between repeated trials.Cellular morphology and 3D growth show good single growth of the horn cell that adheres to the gelatin rack surface.In 10% (w/w) UBM support, the cell of elevated amounts closely connects growth each other, as little lamella.As if the development in time of this effect more obvious.Concentration is elevated to 20-30% (w/w) UBM still to be produced and adheres to growth but tight during not as 10% (w/w) UBM.Greater than 20-30% (w/w) UBM, visible more unicellular growth has the cell expansion of rising simultaneously, and the decline of cell quantity.In not having the MPEG-PLGA support of UBM, cell is assembled at the center of support, and closely growth is almost as lamella.The cell that raising UBM concentration causes raising is expanded, and the decline of cell quantity.Under 2 maximum concentrations, find that dead cell is absorbed in the supporting structure.

Endotheliocyte

Use the gelatin support of Huvec ' s inoculation to show such trend, the suitableeest is that about 20-30% (w/w) UBM quantity raises, and as seen reduces thereafter (Fig. 5).The MPEG-PLGA that contains Huvec ' s shows that the concentration rising of UBM causes the rising of cell quantity.Usually, as seen big variation (Fig. 8) in the contiguous concentration of 2 kinds of cantilever type of using Huvec ' s overlapping.Cellular morphology and 3D growth show that UBM is elevated to the raising that 30% (w/w) UBM generation Huvec ' s adheres to the ability on surface in the gelatin support, and with flat form and normal short enation.From 40% (w/w) and more than, cell is with the growth of round form, and cell quantity descends.In not having the MPEG-PLGA support of UBM, Huvec ' s quantity is low and with rounded form growth, thereby causes there was not cell at the 7th day.In this support, add the celliferous expansion effect of 10-20% (w/w) UBM, but to not effect of form.Concentration is elevated to the cellular morphology that produces the best greater than 30-40% (w/w), and rising concentration further produces higher cell 3D growth.Usually the variation of visible cell form and 3D growth in the gelatin that comprises UBM and two kinds of supports of MPEG-PLGA.Sum up and improve of the effect of UBM concentration elementary fibroblast, elementary horn cell and human endothelial cell:

Embodiment 4:ECM+/-sterilization of incorporating support into is to the effect of elementary fibroblastic cellular morphology and 3D growth.

The Metoxy-Polyethylene Glycol--poly-(lactide-co-glycolide) (Mn 2.000-30.000, L: G 1: 1) is dissolved in 1, forms 1.5% solution in the 4-dioxane.For the sample that comprises UBM, add the UBM of the non-sterilization of 0.045g in the 10ml polymer solution (23%w/w dry), mixed at high speed is also poured in the mould of 7 * 7cm.With this solution-5 ℃ freezing ,-20 ℃ of lyophilizing 5 hours, placed about 18 hours at 20 ℃ again.This sample be placed in the exsiccator stretch (hydraulic pamp) 15 hours thereafter.

The sample that has and do not have a UBM with 0,1 * 25kGy and 2 * 25kGy carry out the β radiation.Prepare another sample with identical method, but use the UBM (0.045g/5ml solution) of pre-sterilized (2 * 25kGy β radiation), and this sample is unsterilised after preparation.

Will (A type, Blume value be 175, Sigma) are dissolved among milli-Q water and the t-BuOH (95: 5), form 1% solution from the gelatin of Corii Sus domestica.Add the UBM of the non-sterilization of 0.015g in the 5ml solution (23%w/w dry) while stirring, and pour (D=5cm) in the mould into.The mould that will contain solution was placed 2 hours at+5 ℃, then-20 ℃ freezing ,-20 ℃ of lyophilizing 5 hours, placed 20 hours at 20 ℃ again.Thereafter with this sample in vacuum oven 120 ℃ crosslinked 15 hours.The sample that has and do not have a UBM with 0,1 * 25kGy and 2 * 25kGy carry out the β radiation.Prepare the sample that another does not have UBM with identical method, this sample after preparation with 0,1 * 25kGy and 2 * 25kGy sterilization.

Will (A type, Blume value be 175, Sigma) are dissolved among milli-Q water and the t-BuOH (95: 5), form 1% solution from the gelatin of Corii Sus domestica.(1 * 25kGy) adds (23%w/w dry) in the 5ml solution to the UBM that 0.015g is pre-sterilized while stirring, and pours (D=5cm) in the mould into.The mould that will contain solution was placed 1 hour at+5 ℃, then-20 ℃ freezing ,-20 ℃ of lyophilizing 5 hours, placed 50 hours at 20 ℃ again.Thereafter with this sample in vacuum oven 130 ℃ crosslinked 15 hours.

Cellular morphology and 3D increment study show that the radiation that improves the UBM lamella is reduced in the cell quantity on the UBM lamella but the pair cell form does not have influence.At gelatin support and containing in the gelatin of 30% (w/w) UBM, visible cell quantity and the change that reduces from typical fibroblast to round cellular morphology, effect is the most obvious in the gelatin support.Before incorporating the gelatin support into to UBM granule sterilization with before the sterilization support, incorporate the UBM granule into and compare, produce better cellular morphology and 3D growth.In MPEG-PLGA, the cell quantity that the radiation of increase causes having the fibroblast form increases, and this is because increased moistening to support.The MPEG-PLGA support radiation that contains 30% (w/w) UBM and UBM granule compared by radiating support before incorporating support into cause higher cell quantity and better fibroblast 3D form.

Originally studies show that in non-radiative gelatin support to obtain the highest biological activity, and radiation reduces this activity.Opposite, when the UBM granule is incorporated the MPEG-PLGA support into and sterilized subsequently, obtain the highest biological activity.It is believed that radiation reduces the biological activity of UBM.Radiation can be passive or be influenced timbering material energetically, and this depends on the material relevant with biological activity.There is sign to show that timbering material (for example MPEG-PLGA) has the protective effect to UBM during sterilizing.

The discontinuous granule of ECM among the embodiment 5:MPEG-PLGA.

With elementary fibroblast with 2.5 * 10 ⁴Cell/cm ²Density, inoculation contains the particulate MPEG-PLGA support of UBM of 41% (w/w) in small size growth medium (10%FCS is in the DMEM that contains antibiotic (penicillin, streptomycin and amphotericin B)).Before adding the appositional growth culture medium with this support incubation at 37 ℃, 5%CO ₂In.After 7 days, should prop up to be placed on and fix 3 days among the Lillys, be embedded in then in the paraffin, and be sliced into 8 μ m thin slices and use Meyer ' s hematoxylin Yihong (HE) dyeing.Use is equipped with the BX-60 Olympus microscope of the cold color camera of Evolution MP (Media Cybernetics) and gathers digital image (4x and 20x amplify), and uses Image Pro Plus 5.1 softwares to obtain the number image.

The digital picture of the particulate distribution of ECM shows the discrete UBM granule that is incarnadined by HE in the MPEG-PLGA support, and distinguishes with timbering material.The fibroblast that grows in the support is dyed blueness (Fig. 7).

UBM discrete particle among the MPEG-PLGA that embodiment 6:SEM shows.

As the preparation support of describing among the embodiment 1.

The SEM picture shows (Fig. 9) and does not have the particulate MPEG-PLGA support of (Fig. 8) UBM.This picture is taken from the top skin of support, and 250 times of amplifications.Described SEM picture is obtained in Denmark technical research institute (2005-160).

Embodiment 7: contain in the particulate support of ECM three-dimensional endothelial cell growth and differentiation.

Metoxy-polyethylene glycol-(lactide-co-glycolide) (Mn 2.000-30.000, L: G 1: 1) is dissolved in 1, in the 4-dioxane, forms 1.5% solution.For the sample that comprises UBM, add the UBM of the non-sterilization of 0.045g in the 10ml polymer solution (23%w/w dry), mixed at high speed is also poured in the mould of 7 * 7cm.With this solution-5 ℃ freezing ,-20 ℃ of lyophilizing 5 hours, placed about 16 hours at 20 ℃ again.This sample be placed in the exsiccator stretch (hydraulic pamp) 15 hours thereafter.

To cultivate altogether at MPEG-PLGA support and the rack surface that contains 23% (w/w) UBM with the primary human dermal fibroblast from the primary human endotheliocyte of umbilical cord.Described structure is immersed in the defined endothelial cell growth culture medium cultivated 6-10 days, thereafter with its air transport and cultivated other 9 days.In the last day of cultivating, fix these structures with 4% formalin buffer, cut and paraffin embedding.

By the immunohistochemistry peroxidase dyeing of CD31/PECAM (PECAM), endotheliocyte is manifested in 5 μ m section.In identifying fibroblast, parallel slices is dyeed with the painted PECAM peroxidase of the combination sub-background of Lignum Sappan.Because endothelial cell growth and differentiation are subjected to the fibroblast Effect on Performance, all timbering materials are tested with two kinds of former groups of different fibers, but do not produce different results.

All MPEG-PLGA supports are supported fibroblast and endothelial cell growth.Find that fibroblast is in the whole volume of all MPEG-PLGA supports.In the training period, distribution of UBM granule homogeneous and support are kept perfectly.Yet cultivating, endotheliocyte on the MPEG-PLGA support and fibroblast have only the endothelial cell surface growth--the substrate internal breeding that endotheliocyte produces the adjacent fibroblast by a top of the trellis.Add the UBM granule and promote fibroblast and the growth of endotheliocyte in the support deep layer, and endotheliocyte has adopted capillary tube sample form.Endotheliocyte is guided along the particulate surface of UBM, rather than is transferred in these granules.Therefore, we find to comprise that in support the UBM granule causes the obvious raising of endothelial cell growth and differentiation.Different fibroblast groups do not produce different results.

Contain 23% (w/w) UBM (Figure 11) among MPEG-PLGA support (Figure 10) and the MPEG-PLGA and show the growth of endotheliocyte on the surface of MPEG-PLGA support, wherein this grows into and contains the particulate deep layer of UBM (endotheliocyte dye redness (being shown as black)--fibroblast is invisible).

The capillary tube sample form (Figure 12) of visible endotheliocyte in the more deep layer of the MPEG-PLGA support that contains 23% (w/w) UBM.These structures are not seen in the MPEG-PLGA support.

Embodiment 8: the physics and the mechanical property that comprise the particulate support of variable concentrations ECM.

Prepared sample:

The lyophilizing support that contains gelatin substrate

Contain the particulate lyophilizing support of gelatin substrate and 40w/w UBM

Contain the particulate lyophilizing support of gelatin substrate and 80w/w UBM

To be dissolved among milli-Q water and the t-BuOH (95: 5) from the gelatin (A type (PG-832-6Gelita)) of Corii Sus domestica, form 1% solution.For the sample that comprises UBM, UBM is added in the solution while stirring (40%w/w:0.033g/5ml, 80%w/w:0.2g/5ml).The gelatin solution that 5ml is comprised UBM is poured (D=5cm) in the mould into.The mould that will contain solution was placed 2.5 hours at+5 ℃, then-20 ℃ freezing ,-20 ℃ of lyophilizing 5 hours, placed 100 hours at 20 ℃ again.

Prepared sample:

The lyophilizing support that contains PLGA substrate

Contain the particulate lyophilizing support of PLGA substrate and 40w/w UBM

Contain the particulate lyophilizing support of PLGA substrate and 80w/w UBM

The Metoxy-Polyethylene Glycol--poly-(lactide-co-glycolide) (Mn 2.000-30.000, L: G 1: 1) is dissolved in 1, forms 1.5% solution in the 4-dioxane.For the sample that comprises UBM, UBM added to (40% (w/w): 0.1g/10ml, 80% (w/w): 0.6g/10ml), mixed at high speed is also poured in the mould of 7 * 7cm in the polymer solution.This solution is chilled in 1 at-5 ℃, on the 4-dioxane layer,, placed about 100 hours at 20 ℃ again-20 ℃ of lyophilizing 5 hours.This sample be placed in the exsiccator stretch (hydraulic pamp) 15 hours thereafter.

Physical characteristic and mechanical test

Depend on the amount of host material and add to UBM, can obtain different physics and engineering properties.

● along with the amount of the UBM that is added, porous reduces, thereby density raises.

● if described substrate is hydrophobic, and then UBM provides the wetting power of raising.

● when height arrived at least 40% (w/w) UBM, the gelatin support had kept its hot strength, and intensity reduces thereafter, and the PLGA support is slightly strengthened by the UBM granule.Low material concentration produces low hot strength with the lyophilizing program in the compositions, and it also is the situation in the sample in the present embodiment.

Use the vernier cursor measuring height.

Following bulk density:

Density=quality/(area x height)

Following calculating porous:

Porous=(density polymer-sample rate)/density polymer

This density polymer is according to the UBM (3mg/cm that is added ³) weight adjusted.

Calculated wetting power by the time span of absorption of sample fully with a water, monitor with picture.

On fabric analysis instrument, carry out extension test from stable Micro Systems.

Claims (10)

1. comprise the discontinuity zone of ECM, provisional successive support, the concentration of the discontinuity zone of wherein said ECM is between 20% (w/w) and 60% (w/w).

2. the temporary stent of claim 1, wherein said support is biodegradable.

3. claim 1 or 2 temporary stent, the discontinuity zone of wherein said ECM is that homogeneous distributes.

4. each temporary stent of claim 1-3, wherein said Biodegradable scaffold is made by comprising proteinic material.

5. each temporary stent of claim 1-3, wherein said Biodegradable scaffold is made by the material that comprises polysaccharide.

6. each temporary stent of claim 1-3, wherein said Biodegradable scaffold is made by the material that comprises synthetic polymer.

7. the temporary stent of above each claim, wherein said Biodegradable scaffold is made by the combination in any of claim material that 4-6 gives.

8. the temporary stent of above each claim, wherein said support has open interconnective hole.

9. the temporary stent of above each claim, wherein said support has the thickness of 0.1-8mm.

10. the temporary stent of above each claim, wherein said support is fungi-proofing packing, underlined this product that shows is sterilized on its packing.

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